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This month on Episode 59 of Discover CircRes, host Cindy St. Hilaire highlights original research articles featured in the April 12 and April 26th issues of Circulation Research. This Episode also includes a discussion with Dr Craig Morrell and Chen Li from University of Rochester about their study, Thrombocytopenia Independently Leads to Changes in Monocyte Immune Function.
Article highlights:
Arkelius, et al. LOX-1 and MMP-9 Inhibition Improves Stroke Outcomes
Cruz, et al. C122Y Disrupts Kir2.1-PIP2 Interaction in ATS1
Blaustein, et al. Environmental Impacts on Cardiovascular Health and Biology: An Overview
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This month on Episode 58 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the March 1 and March 15th issues of Circulation Research. This Episode also includes a discussion with Drs Frank Faraci, Tami Martino, and Martin Young about their contributions to the Compendium on Circadian Mechanisms in Cardiovascular and Cerebrovascular Disease.
Article highlights:
Yan, et al. GCN2 in Ponatinib-Induced Cardiotoxicity
Wang, et al. Activating v-ATPase Ameliorates Cardiac Cardiomyopathy
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This month on Episode 57 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the February 2nd and February 19th issues of Circulation Research. This Episode also includes a discussion with Dr Kathryn Howe and Dr Sneha Raju from University of Toronto, about their manuscript titled Directional Endothelial Communication by Polarized Extracellular Vesicle Release.
Article highlights:
Ren, et al. ZBTB20 Regulates Cardiac Contractility
Faleeva, et al. Sox9 Regulates Vascular Extracellular Matrix Aging
Bai, et al. PKA Is Critical for Cardiac Growth
Wang, et al. Indole-3-Propionic Acid Protects Against HFpEF
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This month on Episode 56 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the January 5th and January 19th issues of Circulation Research. This Episode also includes a discussion with Dr Julie Freed and Gopika Senthilkumar from the Medical College of Wisconsin about their study, Necessary Role of Ceramides in the Human Microvascular Endothelium During Health and Disease.
Article highlights:
He, et al. T Cell LGMN Deficiency Prevents Hypertension
Salyer, et al. TnI-Y26 Phosphorylation Improves Relaxation
Jacob, et al. MFN2 in Megakaryocyte and Platelet Function
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This month on Episode 55 of Discover CircRes, host Cynthia St. Hilaireaire highlights two original research articles featured in the December 8th issue of Circulation Research. This Episode also includes a discussion with Dr José Luis de la Pompa and Dr Luis Luna-Zurita from the National Center for Cardiovascular Research in Spain about their study, Cooperative Response to Endocardial NOTCH Reveals Interaction With Hippo Pathway.
Article highlights:
Shi, et al. Nat10 Mediated ac4C in Cardiac Remodeling
Knight, et al. CDK4 Oxidation Attenuates Cell Proliferation
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This month on Episode 54 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the October 27th and November 10th issues of Circulation Research. This Episode also includes a discussion with Dr Sophie Susen and Dr Caterina Casari about their study, Shear Forces Induced Platelet Clearance Is a New Mechanism of Thrombocytopenia, published in the October 27th issue.
Article highlights:
Pass, et al. Single Nuclei Transcriptome of PAD Muscle
Liu, et al. Myocardial Recovery in DCM: CDCP1 and Fibrosis
Grego-Bessa, et al. Neuregulin-1 Regulates Chamber Morphogenesis
Agrawal, et al. A New Model of PH due to HFpEF
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This month on Episode 53 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the September 29th and October 13th issues of Circulation Research. This Episode also includes a discussion with Dr Margaret Schwarz and Dr Dushani Ranasinghe about their study, Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension, published in the September 29 issue.
Article highlights:
Serio, et al. p300/CBP-Upregulated Glycolysis and Cardiac Aging
Sharifi, et al. ADAMTS-7 and TIMP-1 in Atherosclerosis
Zhang, et al. TMEM215 Represses Endothelial Apoptosis
Perike, et al. PPP1R12C Promotes Atrial Hypocontractility in AF
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This month on Episode 52 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the September 1 and September 15th issues of Circulation Research. This Episode also includes a discussion with Dr Manuel Mayr about the study, Proteomic Atlas of Atherosclerosis, the Contribution of Proteoglycans to Sex Differences, Plaque Phenotypes and Outcomes, published in the September 15 issue.
Article highlights:
Sun, et al. CCND2 modRNA Remuscularization Hearts with AMI
Ho, et al. Lymphatic Genes Prevent Cardiac Valve Disease
Shanks, et al. Cardiac Vagal Activity Increases During Exercise
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This month on Episode 51 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the August 4th and August 18th issues of Circulation Research. This Episode also includes a discussion with Dr Eric Small and Dr Xiaoyi Liu from the University of Rochester Medical Center about their article p53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricular Pressure Overload, published in the July 21st issue of the journal.
Article highlights:
Régnier, et al. CTLA-4 Pathway Is Pivotal in Giant Cell Arteritis
Zarkada, et al. Chylomicrons Regulate Lacteal Permeability
Schuermans, et al. Age at Menopause, Telomere Length, and CAD
Bayer, et al. T-cell MyD88 Regulates Fibrosis in Heart Failure
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This month on Episode 50 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the June 23, July 7, and July 21 issues of Circulation Research. This Episode also includes a discussion with BCVS Outstanding Early Career Investigator Award Qiongxin Wang from University of Washington St. Louis, Haobo Li from Massachusetts General Hospital, and Asma Boukhalfa from Tufts Medical Center.
Article highlights:
Tong, et al. The Role of DRP1 in Mitophagy
Abe, et al. ERK5-NRF2 Axis and Senescence-Associated Stemness
Dai, et al. Therapeutic Targeting of Endocytosis Defects in DCM
Weng, et al. PDCD5 Suppresses Cardiac Fibrosis
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This month on Episode 49 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the May 26th issue and provides an overview of the June 9th Compendium on Early Cardiovascular Disease of Circulation Research. This Episode also includes a discussion with Dr Tejasvi Dudiki and Dr Tatiana Byzova about their study, Mechanism of Tumor Platelet Communications in Cancer.
Article highlights:
Nichtová, et al. Mitochondria-SR Tethering and Cardiac Remodeling
Ferrucci, et al. Muscle Transcriptomic and Proteomic in PAD
Compendium on Early Cardiovascular Disease.
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This month on Episode 48 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the April 28th issue of Circulation Research. This Episode also includes a discussion between Dr Mina Chung, Dr DeLisa Fairweather and Dr Milka Koupenova, who all contributed to manuscripts to the May 12th Compendium on Covid-19 and the Cardiovascular System.
Article highlights:
Heijman, et al. Mechanisms of Enhanced SK-Channel Current in AF
Chen, et al. IL-37 Attenuates Platelet Activation
Enzan, et al. ZBP1 Protects Against Myocardial Inflammation
Compendium on Covid-19 and the Cardiovascular System.
Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. Today, I'm going to be highlighting articles from our April 28th and May 12th issues of Circulation Research. I'm also going to have a chat with Dr Mina Chung, Dr DeLisa Fairweather and Dr Milka Koupenova, who all contributed to articles in the May 12th COVID Compendium. But before we have that interview, let's first talk about some highlights.
The first article I want to present is titled Enhanced Calcium-Dependent SK-Channel Gating and Membrane Trafficking in Human Atrial Fibrillation. This article is coming from the University of Essen by Heijman and Zhou, et al. Atrial fibrillation is one of the most common forms of heart arrhythmia in humans and is characterized by irregular, often rapid heartbeats that can cause palpitations, dizziness and extreme fatigue.
Atrial fibrillation can increase a person's risk of heart failure, and though treatments exist such as beta blockers, blood thinners and antiarrhythmia medications, they can have limited efficacy and side effects. A new family of drugs in development are those blocking small-conductance calcium-activated potassium channels called SK channels, which exhibit increased activity in animal models of AF and suppression of which attenuates the arrhythmia. In humans however, the relationship between SK channels and atrial fibrillation is less clear, at least in terms of SK channel mRNA levels. Because mRNA might not reflect actual channel activity, this group looked at just that and they found indeed that channel activity was increased in cardiomyocytes from atrial fibrillation patients compared to those from controls even though the mRNA and protein levels themselves were similar. The altered currents were instead due to changes in SK channel trafficking and membrane targeting.
By confirming that SK channels play a role in human atrial fibrillation, this work supports the pursuit of SK channel inhibitors as possible new atrial fibrillation treatments.
The next article I want to present is titled IL-37 Attenuates Platelet Activation and Thrombosis Through IL-1R8 Pathway. This article comes from Fudan University by Chen and Hong, et al. Thrombus formation followed by the rupture of a coronary plaque is a major pathophysiological step in the development of a myocardial infarction. Understanding the endogenous antithrombotic factors at play could provide insights and opportunities for developing treatments. With this in mind, Chen and Hong, et al. investigated the role of interleukin-1 receptor 8, or IL-1R8, which suppresses platelet aggregation in mice, and of IL-37, a newly discovered human interleukin that forms a complex with IL-1R8 and is found at increased levels in the blood of patients with myocardial infarction.
Indeed, the amount of IL-37 in myocardial infarction patients negatively correlates with platelet aggregation. They also show that treatment of human platelets in vitro with IL-37 suppresses the cell's aggregation and does so in a concentration-dependent manner. Moreover, injection of the protein into the veins of mice inhibits thrombus development and better preserves heart function even after myocardial infarction. Such effects were not seen in mice lacking IL-1R8. This suggests IL-37's antithrombotic action depends on its interaction with the receptor. Together, the results suggest IL-37 could be developed as a antithrombotic agent for use in MI patients or indeed perhaps other thrombotic conditions.
The last article I want to present before our interview is titled ZBP1 Protects Against Mitochondrial DNA-Induced Myocardial Inflammation in Failing Hearts. This article is coming from Kyushu University and is by Enzan, et al. Myocardial inflammation is a key factor in the pathological progression of heart failure and occurs when damaged mitochondria within the stricken cardiomyocyte release their DNA, triggering an innate inflammatory reaction. In a variety of cells, DNA sensors such as Z-DNA-binding protein 1 or ZBP1 are responsible for such mitochondrial DNA-induced inflammation. In theory then, it's conceivable that therapeutic suppression of ZBP1 might reduce myocardial inflammation in heart failure and preserve function. But as Enzan and colleagues have now discovered to their surprise, mice lacking ZBP1 exhibited worse, not better heart inflammation and more failure after induced myocardial infarction.
Indeed, the test animals' hearts had increased infiltration of immune cells, production of inflammatory cytokines and fibrosis together with decreased function compared with the hearts of mice with normal ZBP1 levels. Experiments in rodent cardiomyocytes further confirmed that loss of ZBP1 exacerbated mitochondrial DNA-induced inflammatory cytokine production while overexpression of ZBP1 had the opposite effect. While the reason behind ZBP1's opposing roles in different cells is not yet clear, the finding suggests that boosting ZBP1 activity in the heart might be a strategy for mitigating heart inflammation after infarction.
Cindy St. Hilaire: The May 12th issue of Circulation Research is our COVID compendium, which consists of a series of 10 reviews on all angles of COVID-19 as it relates to cardiovascular health and disease. Today, three of the authors of the articles in this series are here with me. Dr Mina Chung is a professor of medicine at the Cleveland Clinic. She and Dr Tamanna Singh and their colleagues wrote the article, A Post Pandemic Enigma: The Cardiovascular Impact of Post-Acute Sequelae of SARS-CoV-2. Dr DeLisa Fairweather, professor of medicine, immunology and clinical and translational science at the Mayo Clinic, and she and her colleagues penned the article, COVID-19 Myocarditis and Pericarditis. Dr Milka Koupenova is an assistant professor of medicine at the UMass Chan School of Medical and she led the group writing the article, Platelets and SARS-CoV-2 During COVID-19: Immunity, Thrombosis, and Beyond. Thank you all for joining me today.
DeLisa Fairweather: Thank you so much for having us.
Mina Chung: Thank you.
Milka Koupenova: Thank you for having us, Cindy.
Cindy St. Hilaire: In addition to these three articles, we have another seven that are on all different aspects of COVID. Dr Messinger's group wrote the article, Interaction of COVID-19 With Common Cardiovascular Disorders. Emily Tsai covered cell-specific mechanisms in the heart of COVID-19 patients. Mark Chappell and colleagues wrote about the renin-angiotensin system and sex differences in COVID-19. Michael Bristow covered vaccination-associated myocarditis and myocardial injury. Jow Loacalzo and colleagues covered repurposing drugs for the treatment of COVID-19 and its cardiovascular manifestations. Dr Stephen Holby covered multimodality cardiac imaging in COVID, and Arun Sharma covered microfluidic organ chips in stem cell models in the fight against COVID-19.
Cindy St. Hilaire As of today, worldwide, there have been over six hundred million individuals infected with the virus and more than six and a half million have died from COVID-19. In the US, we are about a sixth of all of those deaths.
Obviously now we're in 2023, the numbers of individuals getting infected and dying are much, much lower. As my husband read to me this morning, one doctor in Boston was quoted saying, "People are still getting wicked sick." In 75% of deaths, people have had underlying conditions and cardiovascular disease is found in about 60% of all those deaths. In the introduction to the compendium, you mentioned that the remarkable COVID-19 rapid response initiative released by the AHA, which again is the parent organization of Circ Research and this podcast, if I were to guess when that rapid response initiative started, I would've guessed well into the pandemic, but it was actually March 26th, 2020. I know in Pittsburgh, our labs have barely shut down. So how soon after we knew of SARS-CoV-2 and COVID, how soon after that did we know that there were cardiovascular complications?
Mina Chung: I think we saw cardiovascular complications happening pretty early. We saw troponin increases very early. It was really amazing what AHA did in terms of this rapid response grant mechanism. You mentioned that the RFA was announced, first of all, putting it together by March 26th when we were just shutting down in March was pretty incredible to get even the RFA out. Then the grants were supposed to be submitted by April 6th and there were 750 grants that were put together and submitted. They were all reviewed within 10 days from 150 volunteer reviewers. The notices were distributed April 23rd, less than a month out.
Cindy St. Hilaire: Amazing.
Mina Chung: So this is an amazing, you're right, paradigm for grant requests and submissions and reviews.
DeLisa Fairweather: For myocarditis, reports of that occurred almost immediately coming out of China, so it was incredibly rapid.
Cindy St. Hilaire: Yeah, and that was a perfect lead up to my next question. Was myocarditis, I guess, the first link or the first clue that this was not just going to be a respiratory infection?
DeLisa Fairweather: I think myocarditis appearing very early, especially it has a history both of being induced by viruses, but being strongly an autoimmune disease, the combination of both of those, I think, started to hint that something different was going to happen, although a lot of people probably didn't realize the significance of that right away.
Cindy St. Hilaire: What other disease states, I guess I'm thinking viruses, but anything, what causes myocarditis and pericarditis normally and how unique is it that we are seeing this as a sequelae of COVID?
DeLisa Fairweather: I think it's not surprising that we find it. Viruses around the world are the primary cause of myocarditis, although in South America, it's the parasite Trypanosoma cruzi. Really, many viruses that also we think target mitochondria, including SARS-CoV-2, have an important role in driving myocarditis. Also, we know that SARS-CoV-1 and MERS also reported myocarditis in those previous infections. We knew about it beforehand that they could cause myocarditis.
Cindy St. Hilaire: Is it presenting differently in a COVID patient than say those South American patients with the... I forget the name of the organism you said, but does it come quickly or get worse quickly or is it all once you get it, it's the same progression?
DeLisa Fairweather: Yeah. That's a good question. Basically, what we find is that no matter what the viral infection is, that myocarditis really appears for signs and symptoms and how we treat it identically and we see that with COVID-19. So that really isn't any different.
Cindy St. Hilaire: Another huge observation that we noticed in COVID-19 patients, which was the increased risk of thrombic outcomes in the patients. Dr Koupenova, Milka, you are a world expert in platelets and viruses and so you and your team were leading the writing of that article. My guess is knowing what you know about platelets and viruses, this wasn't so surprising to you, but could you at least tell us the state of the field in terms of what we knew about viruses and platelets before COVID, before Feb 2020?
Milka Koupenova: Before Feb 2020, we actually knew that influenza gets inside in platelets. It leads to not directly prothrombotic events, but it would lead to release of complement 3 from them. That complement 3 would actually increase the immunothrombosis by pushing neutrophils to release their DNA, forming aggregates. In cases when you have compromised endothelium and people with underlying conditions, you would expect certain thrombotic outcomes. That, we actually published 2019 and then 2020 hit. The difference between influenza and SARS-CoV-2, they're different viruses. They carry their genome in a different RNA strand. I remember thinking perhaps viruses are getting inside in platelets, but perhaps they do not. So we went through surprising discoveries that it seemed like it is another RNA virus. It also got into platelets. It was a bit hard to tweak things surrounding BSL-3 to tell you if the response was the same.
It is still not very clear how much SARS or rather what receptor, particularly when it gets inside would induce an immune response. There are some literature showing the MDA5, but not for sure, may be responsible. But what we found is that once it gets in platelets, it just induces this profound activation of programmed cell death pathways and release of extracellular vesicles and all these prothrombotic, procoagulant form of content that can induce damage around, because platelets are everywhere. So that how it started in 2019 and surprisingly progressed to 2021 or 2020 without the plan of really studying this virus.
Cindy St. Hilaire: How similar and how different is what you observe in platelets infected, obviously in the lab, so I know it's not exactly the same, but how similar and how different is it between the flu? Do you know all the differences yet?
Milka Koupenova: No offense here, they don't get infected.
Cindy St. Hilaire: Okay.
Milka Koupenova: Done the proper research. The virus does not impact platelets, but induces the response.
Cindy St. Hilaire: Okay.
Milka Koupenova: That goes back to sensing mechanism. Thank goodness platelets don't get infected because we would be in a particularly bad situation, but they remove the infectious virus from the plasma from what we can see with function.
Cindy St. Hilaire: Got it. So they're helping the cleanup process and in that cleaning up is where the virus within them activates. That is a really complicated mechanism.
Milka Koupenova: Oh, they're sensing it in some form to alert the environment. It's hard to say how similar and how different they are unless you study them hint by hint next to each other. All I can tell is that particularly with SARS-C, you definitely see a lot more various kinds of extracellular vesicles coming out of them that you don't see the same way or rather through the same proportion with influenza. But what that means in how platelet activates the immune system with one versus the other, and that goes back to the prothrombotic mechanisms. That is exactly what needs to be studied and that was the call for this COVID compendium is to point out how much we have done as a team. As scientists who put heads together, as Mina said, superfast response, it's an amazing going back and looking at what happened to think of what we achieved.
There is so much more, so much more that we do not understand how one contributes to all of these profound responses in the organs themselves, such as myocarditis. We see it's important and that will be the problem that we're dealing from here on trying to figure it out and then long COVID, right?
Cindy St. Hilaire: Yeah. Related to what you just said about the mechanism, this cleanup by the platelets or the act of cleaning up helps trigger their activation, is that partly why the antiplatelet and anticoagulant therapies failed in patients? Can you speculate on that? I know the jury's still out and there's a lot of work to be done, but is that part of why those therapies weren't beneficial?
Milka Koupenova: The answer to that in my personally biased opinion is yes. Clearly, the antiplatelet therapies couldn't really control the classical activation of a platelet. So what I think we need to do from here on is to look at things that we don't understand that non-classically contribute to the thrombotic response downstream. If we manage to control the immune response in some way or the inflammation of the infection or how a platelet responds to a virus, then perhaps we can ameliorate a little bit of the downstream prothrombotic effect. So it's a lot more for us to trickle down and to understand in my personal opinion.
DeLisa Fairweather: There is one thing that was really remarkable to me in hearing your experience, Milka, is that I had developed an autoimmune viral model of myocarditis in mice during my postdoc. So I've been studying that for the last 20 years. What is unique about that model is rather than using an adjuvant, we use a mild viral infection so it doesn't take very much virus at all going to the heart to induce it. I also, more recently, started studying extracellular vesicles really as a therapy, and in doing that, inadvertently found out that actually, the model that I'd created where we passage the virus through the heart to induce this autoimmune model, we were actually injecting extracellular vesicles into the mice and that's what was really driving the disease. This is really brought out. So from early days, I did my postdoc with Dr Noel Rose. If you've heard of him, he came up with the idea of autoimmune disease in the '50s. We had always, in that environment, really believed that viruses were triggering autoimmune disease and yet it took COVID before we could really prove that because no one could identify them.
Here we have an example and I think the incidence rates with COVID were so high for myocarditis because for the first time, we had distinguished symptoms of patients going to the doctor right at the beginning of their infection having an actual test to examine the virus, knowing whether it's present or not, whether PCR or antibody test, and then being able to see when myocarditis happened.
Cindy St. Hilaire: Yeah. I think one thing we can all appreciate now is just some of the basic biology we've learned on the backend of this. Actually, those last comments really led well to the article that your team led, Dr Chung, about what we call long COVID, which I guess I didn't realize has an actual name, post-acute sequelae of SARS-CoV-2 or PASC is the now more formal name for long COVID. But what is it? We hinted at it that there's these bits about autoimmune and things like that. What counts as long COVID?
Mina Chung: Yeah. Our article was led by Tamanna Singh. She did a fantastic job of putting this together. We've had, and others, theorized that the huge palette of symptoms that you can experience post-COVID, they can affect all these organ systems with brain fog, these atypical chest pains, postural orthostatic tachycardia, a lot of palpitations, atrial fibrillation, many weakness and fatigue. To us, really, you can get GI symptoms. We've been very interested in, is this an autoimmune phenomenon directed against nerves and all those things. It's also very interesting because many of the non-COVID syndromes that existed pre-COVID like POTS and chronic fatigue syndrome and a lot of other syndromes are associated with autoantibodies. So that is a very interesting area to explore. Is there a persistence of viral fragments. Is there autoimmunity? Is it also a component of persistence of the damage from the initial infection? So it's an area that still needs a lot of work and a lot of work is going into it, but this is like a post or inter pandemic of itself, so hopefully we'll get more insights into that.
Cindy St. Hilaire: Yeah, it's really interesting. I have a friend who has very debilitating long COVID and one of her doctors had said, "If I didn't know any better, I would just describe this as a autoimmune type X." What do we know, I guess, about the current hypothesis of the pathogenesis of PASC? Are there any prevailing theories right now as to why it's occurring? Is the virus still active or is it these domino effects that are leading to multi-organ collapse of some sort?
Mina Chung: Yeah. In some people, persistent viral particles can be identified for months, but whether or not that's what's triggering it, it's hard to know. We see more autoimmune disease that's been reported and various antibodies being reported. So those are clearly processes to be investigated. The microthrombosis is still up there in terms of potentially playing a role in long COVID.
Milka Koupenova: Mina, you probably know better because you see patients, but to all I have been exposed to, long COVID does not really have a homogeneous symptom presentation and then a few theories as to what may be going on in these patients. Not everybody has a microthrombosis. Not everybody have a D-dimer elevated, but some people do. Some people have, as you pointed out, these spectacularly profound brain fog. People can't function. It's probably your friend, Cindy, right?
Cindy St. Hilaire: Yeah.
Milka Koupenova: So one of the theories that I have been, from a viral perspective, very interested in is that a lot of the symptoms in certain individuals such as fatigue, brain fog, sensitivity to light and skin can very well be explained by a flare-up of Epstein-Barr virus that may be what SARS-CoV-2 somehow is inducing. I don't know, DeLisa, what your experience with long COVID is as a scientist. I hope only. But I would like to hear your perspective too because it's so heterogeneous and it is amazing what happens.
DeLisa Fairweather: I have a very interesting perspective from a number of different directions. One, as I mentioned before, my long history with Dr Rose and I've written many articles theorizing how viruses could cause autoimmune disease. This has grown and really, I think this has been extremely revealing during COVID for many of those theories. One thing that I write about in the review for this article is that mast cells, from all the research I've done with myocarditis in our model, mast cells are central to what is driving everything. We show they're the first innate immune cell acting as an antigen-presenting cell, completely driving the response in a susceptible pattern. One of the things that's very important in autoimmune disease is both sex and race. I'd say one of the big weaknesses we have in myocarditis pre-COVID and post-COVID has been ignoring what's going on with race.
In the United States, myocarditis is 90%, 95% white men that are under 50 years of age and most of the cases are under 40 or some of the ones really associated with sudden cardiac death are under 30. So it's very specific. I've been studying sex and race differences and we see those exact differences in our animal models. In animal models, whether you're susceptible or not depends on how many mast cells you have.
Well, I've proposed from the beginning, looking, I've written a lot of different sex difference reviews looking at viruses and autoimmune disease with different autoimmune diseases and hypothesizing and really seeing that mast cells do a lot of the things we're talking about. They have all of the receptors, the whole group of them that have been related to SARS-CoV-2 so they can be activated or stimulated by the virus itself. They act as a antigen-presenting cell. They're critical in the complement pathway as well as macrophages. We see the dominant immune phenotype really being macrophages. Mast cells just are usually not counted anywhere. And of course, these receptors, a lot of them have to do with enzymes and things that are all related to mast cells pathways. Then how they activate the immune response and lead it towards the pathway that leads to chronic autoimmune disease with increased autoantibodies in females, mast cells are very different by sex.
This has to do also when we talked in the Review about myocarditis and pericarditis. It's both those appearing. Although clinically, we have really boxed them as separate things, because there is some definite clinical pericarditis phenotypes that are different, myocarditis in animal models is always myopericarditis. It always then, in that outer pericardial areas where mast cells sit, they sit around the vascular area in most concentrated. So when they degranulate, we see inflammation coming in the vessel, but really concentrated with fibrosis there and along the pericardium. So that's very typical of what's going on. When we shift anything that shifts that, it changes whether you have more pericarditis or less pericarditis and the vascular inflammation by altering anything that affects the mast cells. I talk a little bit about in the review, I think there's only been a few recent things looking at it in COVID, but I think mast cells and certain susceptibility to autoimmune diseases that occur more often in women can really predispose.We need to pay more attention to mast cells and what they might indicate for all these pathways.
Milka Koupenova: I think we should study the platelet mast cell access at this point.
DeLisa Fairweather: Yes.
Milka Koupenova: Because as you're talking about these sex differences, which is spectacular, these things to me are so mind-boggling how one, the infection itself would be more prevalent in men, but then long COVID is more prevalent in women. All of these things and why we understand so very little, what we found about a few years ago in the Framingham Heart Study in the platelets from those people is that all toll-like receptors are expressed at the higher level in women and they associate with different things between men and female. For instance, toll-like receptors in women will associate more with a prothrombotic response while in male with pro-inflammatory response. I think they grossly underestimate the amount of our sex differences from cell to cell.
DeLisa Fairweather: It is, yeah.
Mina Chung: One other thing that I learned about the sex differences from this compendium is Mark Chappell also notes, you mentioned TLR and TLR7 and ACE2 are X chromosome in an area that he says escapes X-linked inactivation. So it could very well be involved in further.
DeLisa Fairweather:
Further, yeah. And ACE2 is expressed more highly in male cells for what's been researched because of the sex difference in COVID, both the COVID infection
Cindy St. Hilaire: So a variety of organ systems are impacted in patients with PASC, also referred to as long COVID, the lungs, the heart, the pancreas, the GI system, pretty much any system, the brain, nervous system. We've just been talking about the mast cell impact. I was really thinking in my head, well, the one thing that connects all of it is the vasculature. I'm a vascular biologist, so I have certain biases, I'm sure, but how much of the sequelae that we see is a function of vascular phenotypes?
Milka Koupenova: I do think the vasculature is super important. It's clear that not all endothelial cells, for instance, will pick up the virus and respond to it. That's why you have this patchy breakage when you look at autopsies. Hence, platelets will respond according to what's local. That's why you find these micro thrombotic events at certain places. Why does it happen in each organ? How does the virus get to each organ to respond? Or is it just inflammation, but why is it in specific places? That's what we don't understand. That's where we need to go. Perhaps, as DeLisa points out, perhaps it's a lot more complicated than how we traditionally think of thrombosis. Actually, my personal bias, again 100% sure that it is a lot more complicated than the traditional mechanisms that we have understood, and that's where the immune system comes and autoimmunity perhaps stems from and they probably speak to each other, right? It's not just one thing.
DeLisa Fairweather: Yeah. I think really, EVs are bringing lots of understanding. A lot of things we used to just think were maybe free-floating and the serum are inside EVs. I think that the immune response is perhaps even more specific than we ever thought and more regulated than we ever understood.
When an EV comes through a cardiomyocyte, whether it's from the mitochondria or through a lysosome, is part of what goes into its outer membrane, something that tells the immune system that that came from the heart, so it knows to go. This will solve a lot of our questions with autoimmune disease if it's very specific like that. It doesn't just have to be the release of free-floating cardiac myosin. We know cardiac myosin is the driver of the autoimmune response in myocarditis, but they're probably much more fine-tuned.
Cindy St. Hilaire: Yeah. I just would love to end with hearing from each of you. You each have your own domain of specialty. If I gave you a massive pot of money, what would be the question you would want to tackle? What's the gap you would love to answer?
Milka Koupenova: We still don't understand specifically what kind of vesicles are coming out, what are their contents in addition to those vesicles. We don't understand. When it comes to platelets, what comes from their granules? We see these breakages of the membrane. Those are non-granule proteins, and non-granule proteins, they serve as dangerous associated molecular pattern signals and can be profoundly inflammatory to the surrounding environment, can be procoagulant. What are those? How are they affecting the surrounding environment? Ultimately, why is there a microthrombi? Why is there not a profound thrombosis everywhere? Thank goodness there isn't, but why isn't? That's what I would do with my money.
DeLisa Fairweather: I think I would do something very similar. All of our research in our animal model, on the one side, we are looking in this viral myocarditis animal model and finding the EVs that come from that are driving myocarditis. On the other hand, we're using EVs that come from healthy human plasma or fat, and we're seeing a profound downregulation of everything if you give it early and we're trying to see how late you can give it and still get an effect. So looking at those and really understanding the components in the context of COVID and COVID vaccines to understand those components, I really think that's the future of where we're going to find what's causing disease and also how we can find therapies. They may be able to reverse this.
Mina Chung: Yeah, I'm interested very much in the autoimmunity and the autoantibodies that are and how they may react with those microthrombi. Perhaps there's autoantibodies within a lot of that material. We're looking at using human and pluripotent stem cell-derived cell models to study the effects of those. That is what I would use our money for.
Cindy St. Hilaire: Well, Dr Mina Chung, Dr DeLisa Fairweather, Dr Milka Koupenova, thank you all so much for joining me today and talking about not only the articles that you wrote and with your colleagues, but also other articles in this amazing compendium. I do think this is one of the first all-encompassing compendiums or group of articles that focus specifically on COVID and cardiovascular disease. So thank you all so much.
Mina Chung: Thank you.
DeLisa Fairweather: Thank you.
Milka Koupenova: You're welcome.
Cindy St. Hilaire: That's it for highlights from the April 28th and May 12th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @circres and #DiscoverCircRes. Thank you to our guests, Dr Mina Chung, Dr DeLisa Fairweather and Dr Milka Koupenova. This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2023. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.
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This month on Episode 47 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the March 31 issue of Circulation Research. We’ll also provide an overview of the Compendium on Increased Risk of Cardiovascular Complications in Chronic Kidney Disease published in the April 14 issue. Finally, this episode features an interview with Dr Elizabeth Tarling and Dr Bethan Clifford from UCLA regarding their study, RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels.
Article highlights:
Shi, et al. LncRNAs Regulate SMC Phenotypic Transition
Chen, et al. Bilirubin Stabilizes Atherosclerotic Plaque
Subramaniam, et al. Mapping Non-Obvious cAMP Nanodomains by Proteomics
Compendium on Increased Risk of Cardiovascular Complications in Chronic Kidney Disease
Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to share three articles selected from our March 31st issue of Circulation Research and give you a quick summary of our April 14th Compendium. I'm also excited to speak with Dr Elizabeth Tarling and Dr Bethan Clifford from UCLA regarding their study, RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels.
So first the highlights. The first article we're going to discuss is Discovery of Transacting Long Noncoding RNAs that Regulates Smooth Muscle Cell Phenotype. This article's coming from Stanford University and the laboratory of Dr Thomas Quertermous. Smooth muscle cells are the major cell type contributing to atherosclerotic plaques. And in plaque pathogenesis, the cells can undergo a phenotypic transition whereby a contractile smooth muscle cell can trans differentiate into other cell types found within the plaque, such as macrophage-like cells, osteoblast-like cells and fibroblast-like cells. These transitions are regulated by a network of genetic and epigenetic mechanisms, and these mechanisms govern the risk of disease.
The involvement of long non-coding RNAs, or Lnc RNAs as they're called, has been increasingly identified in cardiovascular disease. However, smooth muscle cell Lnc RNAs have not been comprehensively characterized and the regulatory role in the smooth muscle cell state transition is not thoroughly understood. To address this gap, Shi and colleagues created a discovery pipeline and applied it to deeply strand-specific RNA sequencing from human coronary artery smooth muscle cells that were stressed with different disease related stimuli. Subsequently, the functional relevancy of a few novel Lnc RNAs was verified in vitro.
From this pipeline, they identified over 4,500 known and over 13,000 unknown or previously unknown Lnc RNAs in human coronary artery smooth muscle cells. The genomic location of these long noncoding RNAs was enriched near coronary artery disease related transcription factor and genetic loci. They were also found to be gene regulators of smooth muscle cell identity. Two novel Lnc RNAs, ZEB-interacting suppressor or ZIPPOR and TNS1-antisense or TNS1-AS2, were identified by the screen, and this group discovered that the coronary artery disease gene, ZEB2, which is a transcription factor in the TGF beta signaling pathway, is a target for these Lnc RNAs. These data suggest a critical role for long noncoding RNAs in smooth muscle cell phenotypic transition and in human atherosclerotic disease.
Cindy St. Hilaire: The second article I want to share is titled Destabilization of Atherosclerotic Plaque by Bilirubin Deficiency. This article is coming from the Heart Research Institute and the corresponding author is Roland Stocker. The rupture of atherosclerotic plaque contributes significantly to cardiovascular disease. Plasma concentrations of bilirubin, a byproduct of heme catabolism, is inversely associated with risk of cardiovascular disease, but the link between bilirubin and atherosclerosis is unknown.
Chen et el addressed this gap by crossing a bilirubin knockout mice to a atherosclerosis prone APOe knockout mouse. Chen et el addressed this gap by crossing the bilirubin knockout mouse to the atherosclerosis-prone APOE knockout mouse, and used the tandem stenosis model of plaque instability to address this question. Compared with their litter mate controls, bilirubin-APOE double knockouts showed signs of increased systemic oxidative stress, endothelial dysfunction, as well as hyperlipidemia. And they had higher atherosclerotic plaque burden.
Hemeatabolism was increased in unstable plaques compared with stable plaques in both of these groups as well as in human coronary arteries. In mice, the bilirubin deletion selectively destabilized unstable plaques and this was characterized by positive arterial remodeling and increased cap thinning, intra plaque hemorrhage, infiltration of neutrophils and MPO activity. Subsequent proteomics analysis confirmed bilirubin deletion enhanced extracellular matrix degradation, recruitment and activation of neutrophils and associated oxidative stress in the unstable plaque. Thus, bilirubin deficiency generates a pro atherogenic phenotype and selectively enhances neutrophil-mediated inflammation and destabilization of unstable plaques, thereby providing a link between bilirubin and cardiovascular disease risk.
Cindy St. Hilaire: The third article I want to share is titled Integrated Proteomics Unveils Regulation of Cardiac Monocyte Hypertrophic Growth by a Nuclear Cyclic AMP Nano Domain under the Control of PDE3A. This study is coming from the University of Oxford in the lab of Manuela Zaccolo. Cyclic AMP is a critically important secondary messenger downstream from a myriad of signaling receptors on the cell surface. Signaling by cyclic AMP is organized in multiple distinct subcellular nano domains, regulated by cyclic AMP hydrolyzing phosphodiesterases or PDEs.
The cardiac beta adrenergic signaling has served as the prototypical system to elucidate this very complex cyclic AMP compartmentalization. Although studies in cardiac monocytes have provided an understanding of the location and the properties of a handful of these subcellular domains, an overview of the cellular landscape of the cyclic AMP nano domains is missing.
To understand the nanodynamics, Subramanian et al combined an integrated phospho proteomics approach that took advantage of the unique role that individual phosphodiesterases play in the control of local cyclic AMP. They combined this with network analysis to identify previously unrecognized cyclic AMP nano domains associated with beta adrenergic stimulation. They found that indeed this integrated phospho proteomics approach could successfully pinpoint the location of these signaling domains and it provided crucial cues to determine the function of previously unknown cyclic AMP nano domains.
The group characterized one such cellular compartment in detail and they showed that the phosphodiesterase PDE3A2 isoform operates in a nuclear nano domain that involves SMAD4 and HDAC1. Inhibition of PDE3 resulted in an increased HDAC1 phosphorylation, which led to an inhibition of its deacetylase activity, and thus derepression of gene transcription and cardiac monocyte hypertrophic growth. These findings reveal a very unique mechanism that explains the negative long-term consequences observed in patients with heart failure treated with PDE3 inhibitors.
Cindy St. Hilaire: The April 14th issue is our compendium on Increased Risk of Cardiovascular Complications in Chronic Kidney Disease. Dr Heidi Noels from the University of Aachen is our guest editor of the 11 articles in this issue. Chronic kidney disease is defined by kidney damage or a reduced kidney filtration function. Chronic kidney disease is a highly prevalent condition affecting over 13% of the population worldwide and its progressive nature has devastating effects on patient health. At the end stage of kidney disease, patients depend on dialysis or kidney transplantation for survival. However, less than 1% of CKD patients will reach this end stage of chronic kidney disease. Instead, most of them with moderate to advanced chronic kidney disease will prematurely die and most often they die from cardiovascular disease. And this highlights the extreme cardiovascular burden patients with CKD have.
The titles of the articles in this compendium are the Cardio Kidney Patient Epidemiology, Clinical Characteristics, and Therapy by Nicholas Marx, the Innate Immunity System in Patients with Cardiovascular and Kidney Disease by Carmine Zoccali et al. NETs Induced Thrombosis Impacts on Cardiovascular and Chronic Kidney disease by Yvonne Doering et al. Accelerated Vascular Aging and Chronic Kidney Disease, The Potential for Novel Therapies by Peter Stenvinkel et al. Endothelial Cell Dysfunction and Increased Cardiovascular Risk in Patients with Chronic Kidney Disease by Heidi Noels et al. Cardiovascular Calcification Heterogeneity in Chronic Kidney Disease by Claudia Goettsch et al. Fibrosis in Pathobiology of Heart and Kidney From Deep RNA Sequencing to Novel Molecular Targets by Raphael Kramann et al. Cardiac Metabolism and Heart Failure and Implications for Uremic Cardiomyopathy by P. Christian Schulze et al. Hypertension as Cardiovascular Risk Factor in Chronic Kidney Disease by Michael Burnier et al. Role of the Microbiome in Gut, Heart, Kidney crosstalk by Griet Glorieux et al, and Use of Computation Ecosystems to Analyze the Kidney Heart Crosstalk by Joachim Jankowski et al.
These reviews were written by leading investigators in the field, and the editors of Circulation Research hope that this comprehensive undertaking stimulates further research into the path flow of physiological kidney-heart crosstalk, and on comorbidities and intra organ crosstalk in general.
Cindy St. Hilaire: So for our interview portion of the episode I have with me Dr Elizabeth Tarling and Dr Bethan Clifford. And Dr Tarling is an associate professor in the Department of Medicine in cardiology at UCLA, and Dr Clifford is a postdoctoral fellow with the Tarling lab. And today we're going to be discussing their manuscript that's titled, RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels. So thank you both so much for joining me today.
Elizabeth Tarling: Thank you for having us.
Bethan Clifford: Yeah, thanks for having us. This is exciting.
Cindy St. Hilaire: I guess first, Liz, how did you get into this line of research? I guess, before we get into that, I should disclose. Liz, we are friends and we've worked together in the ATVB Women's Leadership Committee. So full disclosure here, that being said, the editorial board votes on these articles, so it's not just me picking my friends. But it is great to have you here. So how did you enter this field, I guess, briefly?
Elizabeth Tarling: Yeah, well briefly, I mean my training right from doing my PhD in the United Kingdom in the University of Nottingham has always been on lipid metabolism, lipoprotein biology with an interest in liver and cardiovascular disease. So broadly we've always been interested in this area and this line of research. And my postdoctoral research was on atherosclerosis and lipoprotein metabolism. And this project came about through a number of different unique avenues, but really because we were looking for regulators of LDL biology and plasma LDL cholesterol, that's sort of where the interest of the lab lies.
Cindy St. Hilaire: Excellent. And Bethan, you came to UCLA from the UK. Was this a topic you were kind of dabbling in before or was it all new for you?
Bethan Clifford: It was actually all completely new for me. So yeah, I did my PhD at the same university as Liz and when I started looking for postdocs, I was honestly pretty adamant that I wanted to stay clear away from lipids and lipid strategy. And then it wasn't until I started interviewing and meeting people and I spoke to Liz and she really sort of convinced me of the excitement and that the interest and all the possibilities of working with lipids and well now I won't go back, to be honest.
Cindy St. Hilaire: And now here you are. Well-
Bethan Clifford: Exactly.
Cindy St. Hilaire: ... congrats on a wonderful study. So LDLR, so low density lipoprotein receptor, it's a major determinant of plasmid LDL cholesterol levels. And hopefully most of us know and appreciate that that is really a major contributor and a major risk for the development of atherosclerosis and coronary artery disease. And I think one thing people may not really appreciate, which your study kind of introduces and talks about nicely, is the role of the liver, right? And the role of receptor mediated endocytosis in regulating plasma cholesterol levels. And so before we kind of chat about the nitty-gritty of your study, could you just give us a brief summary of these key parts between plasma LDL, the LDL receptor and where it goes in your body?
Elizabeth Tarling: Yeah. So the liver expresses 70% to 80% of the body's LDL receptor. So it's the major determinant of plasma lipoprotein plasma LDL cholesterol levels. And through groundbreaking work by Mike Brown and Joe Goldstein at the University of Texas, they really define this receptor mediated endocytosis by the liver and the LDL receptor by looking at patients with familial hypercholesterolemia. So those patients have mutations in the LDL receptor and they either express one functional copy or no functional copies of the LDL receptor and they have very, very large changes in plasma LDL cholesterol. And they have severe increases in cardiovascular disease risk and occurrence and diseases associated with elevated levels of cholesterol within the blood and within different tissues. And so that's sort of how the liver really controls plasma LDL cholesterol is through this receptor mediated endocytosis of the lipoprotein particle.
Cindy St. Hilaire: There's several drugs now that can help regulate our cholesterol levels. So there's statins which block that rate limiting step of cholesterol biosynthesis, but there's this new generation of therapies, the PCSK9 inhibitors. And can you just give us a summary or a quick rundown of what are those key differences really? What is the key mechanism of action that these therapies are going after and is there room for more improvement?
Bethan Clifford: Yeah, sure. So I mean I think you've touched on something that's really key about the LDR receptor is that it's regulated at so many different levels. So we have medications available that target the production of cholesterol and then as you mentioned this newer generation of things like PCSK9 inhibitors that sort of try and target LDL at the point of clearance from the plasma.
And in response to your question of is there room for more regulation, I would say that given the sort of continual rate of increased cholesterol in the general population and the huge risks associated with elevated cholesterol, there's always capacity for more to improve that and sort of generally improve the health of the population. And what we sort of found particularly exciting about RNF130 is that it's a distinct pathway from any of these regulatory mechanisms. So it doesn't regulate the level of transcription, it doesn't regulate PCSK9. Or in response to PCSK9, it's a completely independent pathway that could sort of improve or add to changes in cholesterol.
Cindy St. Hilaire: So your study, it's focusing on the E3 ligase, RNF130. What is an E3 ligase, and why was this particular one of interest to you? How did you come across it?
Elizabeth Tarling: is predTates Bethan joining the lab. This is, I think, again for the listeners and those people in training, I think it's really important to note this project has been going in the lab for a number of years and has really... Bethan was the one who came in and really took charge and helped us round it out. But it wasn't a quick find or a quick story. It had a lot of nuances to it. But we were interested in looking for new regulators of LDL cholesterol and actually through completely independent pathways we had found the RNF130 locus as being associated with LDL cholesterol in animals. And then it came out in a very specific genome-wide association study in the African American care study, the NHLBI care study. And so really what we started looking at, we didn't even know what it was.
Elizabeth Tarling: So we asked ourselves, well what is this gene? What is this protein? And it's RNF, so that's ring finger containing protein 130 and ring stands for really interesting new gene. Somebody came up with the glorious name. But proteins that contain this ring domain are very characteristic and they are E3 ubiquitin ligases. And so they conjugate the addition of ubiquitin to a target protein and that signals for that protein to either be internalized and/or degraded through different decorative pathways within the cell. And so we didn't land on it because we were looking at E3 ligases, we really came at it from an LDL cholesterol perspective. And it was something that we hadn't worked on before and the study sort of blossomed from there.
Cindy St. Hilaire: That's amazing and a beautiful, but also, I'm sure, heartbreaking story because these long projects are just... They're bears. So what does this RNF130 do to LDLR? What'd you guys find?
Bethan Clifford: As Liz said, this is a long process, but one of the key factors of RNF130 is it's structurally characteristically looked like E3 ligase. So the first thing that Liz did and then I followed up with in the lab is to see is this E3 ligase ubiquitinating in vitro. And if it is going to ubiquitinate, what's it likely to regulate that might cause changes in plasma cholesterol that would explain these human genetic links that we saw published at the same time.
And so because the LDL cholesterol is predominantly regulated by the LDL receptor and the levels of it at the surface of the parasites in the liver, the first question we wanted to see is does RNF130 interact in any way with that pathway? And I'm giving you the brief view here of the LDL receptor. We obviously tested lots of different receptors. We tested lots of different endocytose receptors and lipid regulators, but the LDL receptor is the one that we saw could be ubiquitinated by RNF130 in vitro. And so then we wanted to sort of go on from there and establish, okay, if this E3 ubiquitin ligase, is it regulating LDL receptor? What does that mean in an animal context in terms of regulating LDL cholesterol?
Cindy St. Hilaire: Yeah, and I guess we should also explain, ubiquitination, in terms of this receptor, and I guess related to Goldstein and Brown and receptor mediated endocytosis, like what does that actually mean for the liver cell and the cholesterol in the LDLR that is binding the receptor?
Bethan Clifford: \So yes, ubiquitination is a really common regulatory mechanism actually across all sorts of different cells, all sorts of different receptors and proteins. And basically what it does is it signals for degradation of a protein. So a ubiquitin molecule is conjugated to its target such as in our case the LDL receptor and that ubiquitin tells the cell that this protein is ready for proteasomal degradation. And that's just one of the many things ubiquitination can do. It can also signal for a trafficking event, it can signal for a protein to protein interaction, but it's most commonly associated with the proteasomal degradation.
Cindy St. Hilaire: So in terms of... I guess I'm thinking in terms of PCSK9, right? So those drugs are stemming from observations in humans, right? There were humans with gain and loss of function mutations, which caused either more or less of this LDLR receptor internalization. How is this RNF130 pathway different from the PCSK9 activities?
Elizabeth Tarling: Yeah, so PCSK9 is a secreted protein, so it's made by hepatocyte and actually other cells in the body and it's secreted and it binds to the LDL particle, LDL receptor complex, and signals for its internalization and degradation in the proteasome. So this is not ubiquitination event, this is a completely different trafficking event. And so the RNF130, actually what Bethan showed, is it directly ubiquitinates the LDL receptor itself, signaling for an internalization event and then ultimately degradation of the LDR receptor through a decorative pathway, which we also define in the study.
So these are two unique mechanisms and actually some key studies that we did in the paper were to modulate RNF130 in animals that do not have PCSK9. And so in that system where in the absence of PCSK9 you have a lot of LDR receptor in the liver that's internalizing cholesterol. What happens when you overexpress RNF130? Do you still regulate at the LDL receptor? And you absolutely do. And so that again suggests that they're two distinct mechanisms and two distinct pathways.
Cindy St. Hilaire: That was one thing I really loved about your paper is every kind of figure or section, the question that would pop up in my head, even ones that didn't pop in my head were beautifully answered with some of these really nice animal models, which is never an easy thing, right? And so one of the things that you brought up was difficulty in making one of the animal models. And so I'm wondering if you could share a little bit for that challenge. I think one thing that we always tend to hide is just science is hard and a lot of what we do doesn't work. And I really think especially for the trainees and really everyone out there, if we kind of share these things more, it's better. So what was one of the most challenging things in this study? And I guess I'm thinking about that floxed animal.
Elizabeth Tarling: Yeah, so I'll speak a bit about that and then I'll let Bethan address because she was really the one on the ground doing a lot of the struggles. But again, we actually weren't going to include this information in the paper. And upon discussion and actually prompted by the reviewers of the paper and some of the questions that they asked us, we realized, you know what? It's actually really important to show this and show that this happens and that there are ways around it.
And so the first story is before Bethan even arrived in the lab, we had purchased embryonic stem cells that were knockout first condition already. And so this is a knockout strategy in which the exon of interest is flanked with lots of P sites so that you can create a flox animal, but also so you can create a whole body knockout just by the insertion of this knockout first cassette.
Elizabeth Tarling: And so we got those mice actually in the first year of Bethan joining the lab. We finally got the chimeric mice and we were able to stop reading those mice. And at the same time we tried to generate our flox animals so that we could move on to do tissue-specific studies. And Bethan can talk about the pain associated with this. But over two years of breeding, we never got the right genotypes from the different crosses that you need to do to generate the flox animal.
And it was actually in discussions with Bethan where we decided we need to go back. We need to go back to those ESLs that we purchased five years ago and we need to figure out if all of the elements that the quality control step had told us were in place are actually present. And so Bethan went back and sequenced the whole locus and the cassette to figure out what pieces were present and we found that one of the essential locks P sites that's required for every single cross from the initial animal was absent and therefore we could actually never make the mouse we wanted to make.
And so that's sort of just a lesson for people going down that route and making these tools that we need in the lab to answer these questions is that despite paying extra money and getting all of the sort of QCs that you can get before you receive the ESLs, we should have gone back and done our own housekeeping and sort of a long journey told us when we went back that we didn't have what we thought we had at the beginning. And that was a real sticking point as Bethan can-
Cindy St. Hilaire: Yeah. And so you know you're not alone. My very first postdoc that I did, I went with a mouse that they had also bought and were guaranteed that it was a knockout and it was not. And it is a painful lesson, but it is critical to... You get over it.
So Bethan, maybe you can also tell us a little bit about what are the other kind of next things you tried? You pivoted and you pivoted beautifully because all the models you used I thought were quite elegant in terms of exactly asking the question you wanted to ask in the right cells. So can you maybe explain some of the in vivo models you used for this study?
Bethan Clifford: Sure, there are definitely a lot. So I mean I think Liz sort of encapsulated the trouble we have with the knockout really succinctly, but actually I want to just take this moment to sort of shout out to another postdoc in the Tarling lab, Kelsey Jarrett, who was really instrumental in the pivoting to a different model. So for the knockouts when we sort of established we didn't have exactly what we thought we did and then to compound that we also weren't getting the DeLiAn ratios breeding this whole body knockout.
We wanted to sort of look at a more transient knockout model. And that's where Kelsey really stepped in and sort of led the way and she generated AAV-CRISPR for us to target RNF130 specifically in the liver. And that had the added beauty of, one, not requiring breeding to get over this hurdle of the knockout being somewhat detrimental to breeding. But it also allowed us to ask the question of what RNF130 is doing specifically in the liver where the liver regulates LDL receptor and LDL cholesterol.
And so that was one of the key models that really, really helped get this paper over the finish line. But we did a whole barrage of experiments, as you've seen. We wanted to make sure... One of the key facets of the Tarling lab is whenever you do anything, no matter what you show Liz, it will always be, "Okay, you showed it to me one way, now show it to me a different way." Can you get the same result coming at it from different ways? And if you can't, why is that? What is the regulation behind that? And so that's really what the paper is doing is asking the same question in as many ways as we can accurately and appropriately probe what RNF130 does to the LDR receptor.
So we tried gain of function studies without adenovirus overexpression. We tried transient knockdown with antisense oligonucleotides, and then we did, as I said, the AAV-CRISPR knockdown with the help of Kelsey and our whole body knockout. And then we also repeated some of these studies such as the adenovirus and the ASO in specific genetic backgrounds. So in the absence of PCSK9, can we still regulate the LDL receptor? And then we also, just to really confirm this, in the absence of the LDL receptor, do we see a difference? And the answer is no, because this effect was really dependent on that LDL receptor being present. So there was a big combination.
Cindy St. Hilaire: It was really nice, really a beautiful step-wise progression of how to solidly answer this question. But a lot of, I think, almost all you did was in mice. And so what is the genetic evidence for relevancy in humans? Can you discuss a little bit about those databases that you then went to to investigate, is this relevant in humans?
Bethan Clifford: I think Liz might be better off answering that question.
Elizabeth Tarling: And I think this sort of pivots on what Bethan was saying. So when we had struggles in the lab, it was a team environment and a collaboration between people in the lab that allowed us to make that leap and make those next experiments possible to then really answer that question. And to be able to include the antisense oligonucleotides required a collaboration with industry. We were very lucky to have a longstanding collaboration with Ionis, who provided the antisense oligonucleotides.
And for the human genetics side of things, that also was a collaboration with Marcus Seldin, who was a former postdoc with Jake Lusis and is now our PI at UC Irvine. And what he helped us do is dive into those summary level databases and ask from that initial study in the NHLBI care population, do we see associations of RNF130 expression in humans with LDL cholesterol with cardiovascular outcomes. And so one database which I would recommend everybody use, it's publicly available, is the StarNet database. And it's in the paper and the website is there. And that allowed us to search for RNF130.
Elizabeth Tarling: And what it does is it asks how RNF130 expression in different tissues is associated with cardiometabolic outcomes and actual in CAD cases and controls, so people with and without heart disease. And we found that expression of RNF130 in the liver was extremely strongly correlated with the occurrence of cardiovascular disease in people with CAD. So in cases versus controls. And then we were also able to find many other polymorphisms in the RNF130 locus that were associated with LDL cholesterol in multiple different studies.
And I think the other message from this paper is this, unlike PCSK9 and unlike LDR receptor itself, which are single gene mutations that cause cardiovascular disease, there are many sub genome-wide significant loci that contribute to this multifactorial disease, which is extremely complex. And I think RNF130 falls within that bracket that those sort of just on the borderline of being genome-wide significant still play significant biological roles in regulating these processes. And they don't come up as a single gene hit for a disease, but combinatorialy they are associated with increased risk of disease and they have a molecular mechanism that's associated with the disease. And so that's what Marcus helped us do in terms of the human genetics is really understand that and get down to that level of data.
Cindy St. Hilaire: Yeah. Yeah, it really makes you want to go back and look at those. Everyone always focuses on that really high peak and those analyses, but what are all those other ones above the noise, right? So it's really important.
Elizabeth Tarling: I think it's really hard to do that. I think that's one where people... Again, it comes down to team science and the group of people that we brought together allowed us to ask that molecular question about how that signal was associated with the phenotype. I think by ourselves we wouldn't have been able to do it.
Cindy St. Hilaire: Yeah. So your antisense oligonucleotide experiments, they were really nice. They showed, I think it was a four-week therapy, they showed that when you injected them expression of RNF130 went down by 90%. I think cholesterol in the animals was lowered by 50 points or so. Is this kind of a next viable option? And I guess related to that, cholesterol's extremely important for everything, right? Cell membrane integrity, our neurons, all sorts of things. Is it possible with something that is perhaps really as powerful as this to make cholesterol too low?
Elizabeth Tarling: I think that what we know from PCSK9 gain and loss of function mutations is that you can drop your plasma cholesterol to very low levels and still be okay because there are people walking around with mutations that do that. I think RNF130 is a little different in that it's clearly regulatory in a homeostatic function in that it's ubiquitously expressed and it has this role in the liver to regulate LDL receptor availability, but there are no homozygous loss of function mutants people walking around, which tells us something else about how important it is in potentially other tissues and in other pathways. And we've only just begun to uncover what those roles might be.
So I think that as a therapy, it has great potential. We need to do a lot more studies to sort of move from rodent models into more preclinical models. But I do think that the human data tell us that it's really important in other places too. And so yeah, we need to think about how best it might work as a therapy. If it's combinatorial, if it's dosed. Those are the types of things that we need to think about.
Cindy St. Hilaire: Yeah, it's really exciting. Do you know, are there other protein targets of RNF130? Is that related to my next question of what is next?
Elizabeth Tarling: I mean, so I should point out, so Bethan unfortunately left the lab last year for a position at Amgen where she's working on obesity and metabolic disease. But before she left, she did two very, very cool experiments searching for new targets or additional targets of RNF130. Starting in the liver, but hopefully we'll move those into other tissues. And so she did gain of function RNF130 versus what loss of function we have of RNF130, and she did specific mass spec analysis of proteins that are ubiquitinated in those different conditions.
And by overlaying those data sets, we're hoping to carve out new additional targets of RNF130. And there are some, and they're in interesting pathways, which we have yet to completely test, but definitely there are additional pathways, at least when you overexpress and reduce expression. Now, whether they turn out to be, again, bonafide in vivo, actual targets that are biologically meaningful is sort of the next step.
Cindy St. Hilaire: Yeah. Well, I'm sure with your very rigorous approach, you are going to find out and hopefully we'll see it here in the future. Dr Elizabeth Tarling and Dr Bethan Clifford, thank you so much for joining me today. I really enjoyed this paper. It's a beautiful study. I think it's a beautiful example, especially for trainees about kind of thoroughly and rigorously going through and trying to test your hypothesis. So thanks again.
Elizabeth Tarling: Thank you.
Bethan Clifford: Thank you very much.
Cindy St. Hilaire: That's it for the highlights from the March 31st and April 14th issues of Circulation Research. Thank you for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes, and #DiscoverCircRes. Thank you to our guests, Dr Liz Tarling and Dr Bethan Clifford.
This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, you're on-the-go source for the most exciting discoveries in basic cardiovascular research.
This program is copyright of the American Heart Association 2022. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.
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This month on Episode 46 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the March 3 and March 17th issues of Circulation Research. This episode also features an interview with Dr Andrew Hughes and Dr Jessilyn Dunn about their review, Wearable Devices in Cardiovascular Medicine.
Article highlights:
Delgobo, et al. Deep Phenotyping Heart-Specific Tregs
Sun, et al. Inhibition of Fap Promotes Cardiac Repair After MI
Sun, et al. Endosomal PI3Kγ Regulates Hypoxia Sensing
Johnson, et al. Hypoxemia Induces Minimal Cardiomyocyte Division
Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to share four articles selected from the March 3rd and March 17th issues of CircRes. I'm also going to have a discussion with Dr Andrew Hughes and Dr Jessilyn Dunn about their review, Wearable Devices in Cardiovascular Medicine. And the Review is also featured in our March 3rd issue.
Cindy St. Hilaire: First, the highlights. The first article I'm going to present is Myocardial Milieu Favors Local Differentiation of Regulatory T-Cells. The first author is Murilo Delgobo and the corresponding author is Gustavo Campos Ramos. After myocardial infarction, the release of autoantigens from the damaged heart cells activates local and infiltrating immune cells such as the T-cell. Studies in mice have shown that fragments of the muscle protein myosin can act as autoantigens, and these myosin fragments are the dominant driver of the T-cell response.
But how do these myosin specific T-cells behave in the damaged heart to drive inflammation and repair is unknown. To find out, Delgobo and colleagues studied endogenous myosin specific T-cells, as well as those transferred into recipient mice. They found, whether exogenously supplied or endogenously created, the myosin specific T-cells that accumulated in the animals' infarcted hearts tended to adopt an immunosuppressive T-regulatory phenotype.
Strikingly, even if the exogenous cells were differentiated into inflammatory TH-17 cells prior to transfer, a significant proportion of them were still reprogrammed into T-regs within the heart. Although cells pre-differentiated into an inflammatory TH-17 phenotype were less inclined to change after the transfer, the results nevertheless indicate that, by and large, the infarcted heart promotes T-cell reprogramming to quell inflammation and drive repair. Yet exactly how the heart does this is a question for future studies.
Cindy St. Hilaire: The next article I'm going to present is titled Inhibition of FAP Promotes Cardiac Repair by Stabilizing BNP. The first authors of the study are Yuxi Sun and Mengqiu Ma, and the corresponding author is Rui Yue, and they are from Tongji University. After myocardial infarction, there needs to be a balance of recovery processes to protect the tissue. Fibrosis, for example, acts like an immediate bandaid to hold the damaged heart muscle together, but fibrosis can limit contractile function.
Similarly, angiogenesis and sufficient revascularization is required to promote survival of cardiomyocytes within the ischemic tissue and protect heart function. To better understand the balance between fibrotic and angiogenic responses, Sun and colleagues examined the role of fibroblasts activated protein, or FAP, which is dramatically upregulated in damaged hearts, and brain natriuretic peptide, or BNP, which promotes angiogenesis in the heart.
In this study, they found that genetic deletion or pharmacological inhibition of FAP in mice reduces cardiac fibrosis and improves angiogenesis and heart function after MI. Such benefits are not seen if BNP or its receptor, NRP-1, are lacking. The in vitro experiments revealed that FAP's protease activity degrades BNP, thus inhibiting the latter's angiogenic activity. Interestingly, while FAP is upregulated in the heart, its levels drop in the blood, showing that BNP inhibition is localized. Together, these results suggest that blocking FAP's activity in the heart after MI could be a possible strategy for protecting the muscle's function.
Cindy St. Hilaire: The next article I want to present is Hypoxia Sensing of Beta-Adrenergic Receptor is Regulated by Endosomal PI-3 Kinase Gamma. The first author of this study is Yu Sun, and the corresponding author is Sathyamangla Naga Prasad. Hypoxia is the most proximate acute stress encountered by the heart during an ischemic event. Hypoxia triggers dysfunction of the beta-adrenergic receptors, beta-1AR and beta-2AR, which are critical regulators of cardiac function.
Under normoxic conditions, activation of PI3K-gamma by beta-adrenergic receptors leads to feedback regulation of the receptor by hindering its dephosphorylation through inhibition of protein phosphatase 2A or PP2A. Although it is known that ischemia reduces beta-adrenergic receptor function, the impact of hypoxia on interfering with this PI3K feedback loop was unknown.
Using in vitro and in vivo techniques, this group found that activation of PI3K-gamma underlies hypoxia sensing mechanisms in the heart. Exposing PI3K-gamma knockout mice to acute hypoxia resulted in preserved cardiac function and reduced beta-adrenergic receptor phosphorylation. And this was due to a normalized beta-2AR associated PP2A activity, thus uncovering a unique role for PI3K-gamma in hypoxia sensing and cardiac function.
Similarly, challenging wild-type mice post hypoxia with dobutamine resulted in an impaired cardiac response that was normalized in the PI3K-gamma knockout mice. These data suggests that preserving beta-adrenergic resensitization by targeting the PI3K-gamma pathway would maintain beta-adrenergic signaling and cardiac function, thereby permitting the heart to meet the metabolic demands of the body following ischemia.
Cindy St. Hilaire: The last article I want to highlight is Systemic Hypoxia Induces Cardiomyocyte Hypertrophy and Right Ventricle Specific Induction of Proliferation. First author of this study is Jaslyn Johnson, and the corresponding author is Steven Houser, and they're at Temple University.
The cardiac hypoxia created by myocardial infarction leads to the death of the heart tissue, including the cardiomyocytes. While some procedures such as reperfusion therapy prevent some cardiomyocyte death, true repair of the infarcted heart requires that dead cells be replaced. There have been many studies that have attempted new approaches to repopulate the heart with new myocytes. However, these approaches have had only marginal success.
A recent study suggested that systemic hypoxemia in adult male mice could induce cardiac monocytes to proliferate. Building on this observation, Johnson and colleagues wanted to identify the mechanisms that induced adult cardiomyocyte cell cycle reentry and wanted to determine whether this hypoxemia could also induce cardiomyocyte proliferation in female mice.
Mice were kept in hypoxic conditions for two weeks, and using methods to trace cell proliferation in-vivo, the group found that hypoxia induced cardiac hypertrophy in both the left ventricle and the right ventricle in the myocytes of the left ventricle and of the right ventricle. However, the left ventricle monocytes lengthened while the RV monocytes widened and lengthened.
Hypoxia induced an increase in the number of right ventricular cardiomyocytes, but did not affect left ventricular monocyte proliferation in male or in female mice. RNA sequencing showed upregulation of cell cycle genes which promote the G1 to S phase transition in hypoxic mice, as well as a downregulation of cullen genes, which are the scaffold proteins related to the ubiquitin ligase complexes. There was significant proliferation of non monocytes in mild cardiac fibrosis in the hypoxic mice that did not disrupt cardiac function.
Male and female mice exhibited similar gene expression patterns following hypoxia. Thus, systemic hypoxia induced a global hypertrophic stress response that was associated with increased RV proliferation, while LV monocytes did not show increased proliferation. These results confirm previous reports that hypoxia can induce cardiomyocyte cell cycle activity in-vivo, and also show that this hypoxia induced proliferation also occurs in the female mice.
Cindy St. Hilaire: With me today for our interview, I have Dr Andrew Hughes and Dr Jessilyn Dunn, and they're from Vanderbilt University Medical Center. And they're here to discuss the review article that they helped co-author called Wearable Devices in Cardiovascular Medicine. And just as a side note, the corresponding author, Evan Brittain, unfortunately just wasn't able to join us due to clinical service, but they're going to help dissect and discuss this Review with us. Thank you both so much for joining me today. Andy, can you just tell us a little bit about yourself?
Andy Hughes: Yeah, thank you, Cindy. I'm Andy Hughes. I'm a third year medicine resident at Vanderbilt University who is currently on an NIH supported research year this year. And then will be applying to cardiology fellowships coming up in the upcoming cycle.
Cindy St. Hilaire: Great, thank you. And Jessilyn, I said you are from Vanderbilt. I know you're from Duke. It was Evan and Andy at Vanderbilt. Jessilyn, tell us about yourself.
Jessilyn Dunn: Thanks. I am an Assistant Professor at Duke. I have a joint appointment between biomedical engineering and biostatistics and bioinformatics. The work that my lab does is mainly centered on digital health technologies in developing what we call digital biomarkers, using data from often consumer wearables to try to detect early signs of health abnormalities and ultimately try to develop interventions.
Cindy St. Hilaire: Thank you. We're talking about wearable devices today, and obviously the first thing I think most of us think about are the watch-like ones, the ones you wear on your wrists. But there's really a whole lot more out there. It's not just Apple Watches and Fitbits and the like. Can you just give us a quick summary of all these different types of devices and how they're classified?
Jessilyn Dunn: Yeah, absolutely. We have a wide variety of different sensors that can be useful. A lot of times, we like to think about them in terms of the types of properties that they measure. So mechanical properties like movement, electrical properties like electrical activity of the heart. We have optical sensors. And so, a lot of the common consumer wearables that we think about contain these different types of sensors.
A good example that we can think about is your consumer smartwatch, like an Apple Watch or a Fitbit or a Garmin device where it has something called an accelerometer that can measure movement. And oftentimes, that gets converted into step counts. And then it may also have an optical sensor that can be used to measure heart rate in a particular method called PPG, or photoplethysmography. And then some of the newer devices also have the ability to take an ECG, so you can actually measure electrical activity as well as the optical based PPG heart rate measurement. These are some of the simpler components that make up the more complex devices that we call wearables.
Cindy St. Hilaire: And how accurate are the measurements? You did mention three of the companies, and I know there's probably even more, and there's also the clinical grade at-home ECG machines versus the one in the smartwatch. How accurate are the measurements between companies? And we also hear recent stories about somebody's Apple Watch calling 911 because they think they're dead, things like that. Obviously, there's proprietary information involved, but how accurate are these devices and how accurate are they between each other?
Jessilyn Dunn: This is a really interesting question and we've done quite a bit of work in my lab on this very topic, all the way from what does it mean for something to be accurate? Because we might say, "Well, the more accurate, the better," but then we can start to think about, "Well, how accurate do we need something to be in order to make a clinical decision based off of that?" And if it costs significantly more to make a device super, super accurate, but we don't need it to be that accurate to make useful decisions, then it actually might not be serving people well to try to get it to that extreme level of accuracy.
So there are a lot of trade-offs, and I think that's a tough thing to think about in the circumstances, is these trade-offs between the accuracy and, I don't know, the generalizability or being able to apply this to a lot of people. That being said, it also depends on the circumstances of use. When we think about something like step counts, for example, if you're off by a hundred step counts and you're just trying to get a general view of your step counts, it's not that much of a problem.
But if we're talking about trying to detect an irregular heart rhythm, it can be very bad to either miss something that's abnormal or to call something abnormal that's not and have people worried. We've been working with the Digital Medicine Society to develop this framework that we call V3, which is verification, analytic validation and clinical validation. And these are the different levels of analysis or evaluation that you can do on these devices to determine how fit for purpose are they.
Given the population we're trying to measure in and given what the goal of the measurement is, does the device do the job? And what's also interesting about this topic is that the FDA has been evolving how they think about these types of devices because there's, in the past, been this very clear distinction between wellness devices and medical devices. But the problem is that a lot of these devices blur that line. And so, I think we're going to see more changes in the way that the FDA is overseeing and potentially regulating things like this as well.
Cindy St. Hilaire: These consumer-based devices have started early on as the step counters. When did they start to bridge into the medical sphere? When did that start to peak the interest of clinicians and researchers?
Jessilyn Dunn: Yeah, sure. What's interesting is if we think back to accelerometers, these have been used prior to the existence of mobile phones. These really are mechanical sensors that could be used to count steps. And when we think about the smartwatch in the form that we most commonly think of today, probably looking back to about 2014 is when ... maybe between 2012, 2014 is when we saw these devices really hitting the market more ... Timing for when the devices that we know as our typical consumer smartwatch today was around 2012 to 2014.
And those were things that were counting steps and then the next generation of that
added in the PPG or photoplethysmography sensor. That's that green light when we look on the back of our watch that measures heart rate. And so, thinking back to the early days, probably Jawbone, there was a watch called Basis, the Intel Basis watch. Well, it was Basis and then got acquired by Intel. Fitbit was also an early joining the market, but that was really the timing.
Cindy St. Hilaire: How good are these devices at actually changing behavior? We know we're really good at tracking our steps now and maybe monitoring our heartbeat or our oxygen levels. How good are they at changing behavior though? Do we know yet?
Andy Hughes: Yeah, that's a great question and certainly a significant area of ongoing research right now with physical activity interventions. Things that we've seen right now is that simple interventions that use the wearable devices alone may not be as effective as multifaceted interventions. And what I mean by that is interventions that use the smartwatch but may be coupled with another component, whether that is health education or counseling or more complex interventions that use gamification or just in time adaptive interventions.
And gamification really takes things to another level because that integrates components, competition or support or collaboration and really helps to build upon features of behaviors that we know have an increased likelihood of sustaining activity. With that being said, that is one of the challenges of physical activity interventions, is the sustainability of their improvements over the course of months to years.
And something that we have seen is the effects do typically decrease over time, but there is work on how do we integrate all of these features to develop interventions that can help to sustain the results more effectively. So we have seen some improvement, but finding ways to sustain the effects of physical activity is certainly an area of ongoing research.
Cindy St. Hilaire: I know it's funny that even as adults we love getting those gold stars or the circle completions. All of these devices, whether it's smartwatches like we're just talking about, or the other things for cardiac rehabilitation, they're generating a ton of data. What is happening with all this data? Who's actually analyzing it? How is it stored and what's that flow through from getting from the patient's body to the room where their physician is looking at it?
Andy Hughes: And that is certainly a challenge right now that is limiting the widespread adoption of these devices into routine clinical care is, as Jessilyn mentioned. The wearables generate a vast amount of data, and right now, we need to identify and develop a way as clinicians to sort through all of the noise in order to be able to identify the information that is clinically meaningful and worthy of action without significantly increasing the workload.
And a few of the barriers that will be necessary in order to reach that point is, one, finding ways to integrate the wearables' data into the electronic health record and also developing some machine learning algorithms or ways with which we can use the computational power of those technologies to be able to identify when there is meaningful data within all of the vast data that comes from wearables. So it's somewhere that certainly we need to get to for these devices to reach their full clinical potential, but we are limited right now by a few of those challenges.
Jessilyn Dunn: I was just going to say, I will add on to what Andy was saying about this idea behind digital biomarkers because this fits really nicely with this idea that giving people this huge data deluge is not helpful, but if we had a single metric where we can say, "Here's the digital biomarker of step count, and if you're above some threshold, you're good to go. And if you're below some threshold, some intervention is needed." That's a lot of the work that we've been doing, is trying to develop what are these digital biomarkers and how can they be ingested in a really digestible way?
Cindy St. Hilaire: Yeah, that's great. Regarding the clinical and the research grade devices, I know a Fitbit or Apple Watch can sometimes be used for those, but I guess I'm talking also about the other kind of more clinically oriented devices, how good is compliance and how trustable is that data? Everybody's on probably their best behavior when they're in the office with the physician or if they're on the treadmill in the cardiac lab, but home is a different story. And what don't we know about compliance when people are out of the office and the reliability of that data that's generated in that space?
Andy Hughes: I think you touched on a really important point right here, and one of the potential advantages of these wearable devices is that they provide continuous long-term monitoring over the course of weeks to months to years as opposed to those erratic measurements that we get from the traditional office visits or hospitalizations where, for example, the measurements we're taking are either in a supervised environment with a six-minute walk distance, for example, or self-reported or questionnaires.
So we build upon that information, but then additionally, we go beyond the observer effect where many individuals, the first week or two that you're wearing this new device, you may be more prone to increase your activity because you know that you're being monitored or you have this novel technology, but as you wear it for months to years, you outgrow those potential biases and you really can garner more comprehensive information.
In terms of compliance, we can speak to some of the research studies that have either really struggled with compliance and that limits the interpretability of their results and something we'll need to address in the future, but I think that's something that can be addressed with future studies keeping in mind all of the advantages that these devices offer compared to some of the traditional measures that we have used in the past.
Cindy St. Hilaire: With all this data we're collecting, whether it be biological data or even just behavioral data, have we actually learned anything new? And I mean that in terms of All Of Us study this, I don't know, it was like 5,000 patients I think, and lo and behold, it found out that higher step count correlated with lower risk for a ton of diseases, which is not exactly groundbreaking. So are we, at this point in time, learning anything new from the use of these at-home devices, or are they really just able to help us enforce what we thought we knew regarding behavior?
Andy Hughes: I think these devices have certainly provided some novel insights that build upon our understanding of physical activity. Many of us can hypothesize that decreased activity would have poor outcomes on health, which the studies have demonstrated in many facets. But in reference to All Of Us study that you mentioned, I think it's interesting to look as well at some of the diagnoses or conditions that were associated with decreased activity.
For example, reflux disease was also highlighted in that study, which may not have been identified if we didn't have the vast data and ability to really look for associations with diseases that have not been previously studied or thought to be related to physical activity. So I think that's one of the strong features of that database, is the wealth of knowledge that really will be hypothesis generating and help to inform future studies as we look even beyond cardiovascular conditions.
Cindy St. Hilaire: One question, and you did bring it up in a bit of the discussion in your piece, is the bias that is in these devices. We know from COVID at-home pulse oximeters do not work as efficiently on darker skin. We actually know that going into bathrooms with the hand sensors that spit out the paper towels. So what kind of disparities or biases do these devices create or reinforce in the population?
Jessilyn Dunn: This is such a critical topic because a lot of these issues had been discovered retrospectively because the people who were developing the technologies were not the representative of the people who were using the technologies. I think that's something that across the board we've been looking at from device development to AI implementation, which is having people who are going to be using the devices in the process of developing the technology and having voices heard from across the board.
We did a detailed look when we were evaluating devices for their accuracy at this exact question of where the heart rate sensors in smartwatches use optical based technology. And there was some evidence that was also an issue for people with varying skin tones, for people with wrist tattoos or more hair or freckles. And so, we did a deep dive and the generation of devices that we looked at which would meet this study was probably about three years ago.
We didn't see any discrepancies. And so, that's just one study and there are many more to be done, but I think prior to the technology development as well as once the technology comes out, keeping an eye on how that technology is doing, whether there are continued reports of failure of the technologies is really important. And there are a lot of ways that we can be vigilant about that.
Cindy St. Hilaire: Yeah, that's great. And so, Andy, regarding patient populations, I can also see perhaps socioeconomic implications of this because smartwatches are not cheap. So how do we see that in terms of helping our patients? Are we going to be able to get a smartwatch through our insurance company?
Andy Hughes: I think that's one of the really important next steps, is finding ways to make sure that as we advance the field of wearable devices in clinical care, that we recognize some of the existing inequities in terms of access to care, access to digital technologies that currently exist, and find ways by partnering with health insurance companies and the industry and providers and members of that community, finding ways to not only advance wearables, but use it in a way that we can decrease health disparities by really helping to increase access for these digital technologies to the underserved communities.
Jessilyn Dunn: Yeah, the beauty of these technologies is that truthfully, at their core, they're very cheap. They're not difficult to develop, they're not difficult to build and disseminate. So a lot of what we think about is the infrastructure that goes around these devices. Does it require a smartphone to transfer data? Does it require internet access? What are the other pieces that need to be in place for these devices to work within an ecosystem? So this starts to get to questions beyond the devices themselves, but there's certainly a lot to think about and be done in the area of equity and ensuring that these devices can help everyone.
Cindy St. Hilaire: And there's also the, I guess, ethical considerations of who owns this data. Obviously, if it's a consumable that you went and bought at Target, that's probably different than the one you're getting from your cardiologist. But who owns the data? Who has access to it? And are there any cases in the literature where an individual who's had certain measurements taken, have those measurements come back to bite them?
And I guess I'm thinking of something like cardiac rehab. If a patient doesn't get up and move enough or doesn't follow their physical therapy enough or lose weight quick enough, could their insurance coverage get cut? Could their premiums go up? What safeguards are in place for these very tricky situations? Are there safeguards in place?
Andy Hughes: And on the clinical side, I think it will be important to treat this information just like any other protected health information that we have as part of the electronic health record. And so, there will be inherently safeguards around that in a similar manner for how we treat other protected health information.
But I think another important component of that will be a very clear consent policy when we reach the point that patients are consenting to include this information and their electronic health record, in terms of what the proposed benefits are and the potential risks associated with it, because it really is a vast amount of unique data that needs to be protected and safeguarded. And part of that comes by treating it as protected health information, but we will also need to make sure that there's a very clear consent policy that goes with it.
Cindy St. Hilaire: Yeah. What do we see as the next steps in wearable devices? What do you guys see as the next big thing? I know one's coming from the actual AI and device side of things, and the other one is coming from the clinical side of things. What do each of you see as the next thing in this field?
Jessilyn Dunn: I think on the device and AI side of things, I think we're thinking toward improving battery life, increasing the suite of sensors that are being added to these devices so we have a wider variety of measurements that are more representative of physiology, and then better algorithms to have better detection of sleep or activity or certain types of activity or certain types of arrhythmias. This combination of hardware and software and algorithms, I think coming together as all of these different pieces evolve will show us some really cool technology in the years to come.
Andy Hughes: And I think from a clinical side, it's really twofold moving forward. I think as Jessilyn mentioned, there's a lot of novel sensor technologies that have a lot of exciting and evolving potential that we can hopefully integrate into the clinical space, but on the other hand, it's how can we use these wearable devices to enhance traditional therapies that we're already using?
For example, if we take the heart failure population, is there a way that we can use the wearable devices and the existing measurements with heart rate and physical activity and blood pressure to find a way to improve remote management and safely up-titrate guideline directed medical therapy, which are medications that we know have clinical benefit. But can we augment their clinical benefit and their utility by using some of the existing technologies that we already have?
And then lastly, building upon the initial studies with larger trials in more diverse generalizable populations to really enhance our understanding of the benefits that these devices may have for different cardiovascular conditions.
Cindy St. Hilaire: Well, this was wonderful. Dr Andrew Hughes and Dr Jessilyn Dunn, thank you so much for joining me. The review, Wearable Devices in Cardiovascular Medicine, will be out in our March 3rd issue of Circulation Research. I forget which one, so I'll have to edit that out. Thank you so much for joining us, and I learned a ton. This was great.
Jessilyn Dunn: Thank you.
Andy Hughes: Thank you.
Cindy St. Hilaire: That's it for our highlights from the March 3rd and March 17th issues of Circulation Research. Thank you for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Andrew Hughes and Dr Jessilyn Dunn.
This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy texts for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, you're on-the-go Source for the most exciting discoveries in basic cardiovascular research.
This program is copyright of the American Heart Association, 2023. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.
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This month on Episode 45 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the February 3rd and February 17th issues of Circulation Research. This episode also features an interview with Dr Hind Lal and Dr Tousif Sultan from the University of Alabama at Birmingham about their study Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation.
Article highlights:
Pi, et al. Metabolomic Signatures in PAH
Carnevale, et al. Thrombosis TLR4-Mediated in SARS-CoV-2 Infection
Cai, et al. Macrophage ADAR1 in AAA
Koide, et al. sEVs Accelerate Vascular Calcification in CKD
Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cynthia St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting the articles from our February 3rd and 17th issues of Circulation Research. I'm also going to have a chat with Dr Hind Lal and Dr Tousif Sultan from the University of Alabama at Birmingham about their study, Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. But before I get to the interviews, here are a few article highlights.
Cindy St. Hilaire: The first article I want to highlight comes from the laboratory of Dr Peter Leary at the University of Washington, and the title is Metabolomic Signatures Associated With Pulmonary Arterial Hypertension Outcomes. Pulmonary Arterial Hypertension or PAH is a rare but life-threatening disease in which progressive thickening of the walls of the lung’s blood vessels causes increased blood pressure and that increased blood pressure ultimately damages the heart's right ventricle.
Interestingly, progression to heart failure varies considerably among patients, but the reasons why there is variability are not well understood. To find out, this group turned their attention to patient metabolomes, which differ significantly from those of healthy people and thus may also change with severity. Blood samples from 117 PAH patients were analyzed for more than a thousand metabolites by mass spectrometry and the patient's progress was followed for the next three years. 22 patients died within a three-year period and 27 developed significant right ventricle dilation. Other measures of severity included pulmonary vascular resistance, exercise capacity and levels of BNP, which is a metric of heart health. Two metabolic pathways, those relating to polyamine and histidine metabolism, were found to be linked with all measures of severity suggesting a key role for them in disease pathology. While determining how these pathways influence disease as a subject for further study, the current findings may nevertheless lead to new prognostic indicators to inform patient care.
Cindy St. Hilaire: The next article I want to discuss is coming from our February 3rd issue of Circulation Research and this is coming from the laboratory of Dr Francisco Violi at the University of Rome and the title is Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection. Thrombosis can be a complication of COVID-19 and it is associated with poor outcomes, including death. However, the exact mechanism by which the virus activates platelets, which are the cells that drive thrombosis, is not clear. For one thing, platelets do not appear to express the receptor for SARS-CoV-2. They do however, express the TLR4 receptor and that's a receptor that mediates entry of other viruses as part of the immune response. And TLR4 is ramped up in COVID-19 patient platelets. This group now confirms that, indeed, SARS-CoV-2 interacts with TLR4, which in turn triggers thrombosis.
The team analyzed platelets from 25 patients and 10 healthy controls and they found that the platelet activation and thrombic activity were both boosted in the patient samples and could not be blocked using a TLR4 inhibitor. Additionally, immunoprecipitation and immunofluorescent experiments further revealed colocalization between the virus protein and the TLR4 receptor on patient platelets. The team went on to show that the signaling pathway involved reactive oxygen species producing factors p47phox and Nox2, and that inhibition of phox 47, like that of the TLR4 receptor itsel,f could prevent platelet activation. As such, this study suggests that inhibiting either of these proteins may form the basis of an antithrombotic treatment for COVID-19.
Cindy St. Hilaire: The third article I want to highlight is coming from the lab of Shi-You Chen at University of Missouri and the title of this article is ADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic Aneurysm. Macrophage activation plays a critical role in abdominal aortic aneurysm development, or AAA development. Inflammation is a component of this pathology; however, the mechanisms controlling macrophage activation and vascular inflammation in AAA are largely unknown. The ADAR1 enzyme catalyzes the conversion of adenosine to inosine in RNA molecules and thus this conversion can serve as a rheostat to regulate RNA structure or the gene coding sequence of proteins. Several studies have explored the role of ADAR1 in inflammation, but its precise contribution is not fully understood, so the objective of this group was to study the role of ADAR1 in macrophage activation and AAA formation.
Aortic transplantation was conducted to determine the importance of nonvascular ADAR1 in AAA development and dissection and angiotensin II infusion of ApoE knockout mice combined with a macrophage specific knockout of ADAR1 was used to study the role of ADAR1 macrophage specific contributions to AAA formation and dissection. Allograft transplantation of wild type abdominal aortas to ADAR1 haploinsufficient recipient mice significantly attenuated AAA formation. ADAR1 deficiency in hematopoietic stem cells also decreased the prevalence and the severity of AAA and it also inhibited macrophage infiltration into the aortic wall. ADAR1 deletion blocked the classic macrophage activation pathway. It diminished NF-κB signaling and it enhanced the expression of a number of anti-inflammatory microRNAs. Reconstitution of ADAR1 deficient but not wild type human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries. Together these results suggest that macrophage ADAR1 promotes aneurysm formation in both mouse and human arteries through a novel mechanism of editing the microRNAs that target NF-κB signaling, which ultimately promotes vascular inflammation in AAA.
Cindy St. Hilaire: The last article I want to highlight is also from our February 17th issue of Circulation Research and it is coming from the lab of Shintaro Mandai at Tokyo Medical and Dental University and the title of the article is Circulating Extracellular Vesicle Propagated MicroRNA signatures as a Vascular Calcification Factor in Chronic Kidney Disease. Chronic Kidney Disease or CKD accelerates vascular calcification in part by promoting the phenotypic switching of vascular smooth muscle cells to osteoblast like cells. This study investigated the role of circulating small extracellular vesicles or SUVs from the kidneys in promoting this osteogenic switch. CKD was induced in rats and in mice by an adenine induced tubular interstitial fibrosis and serum from these animals induced calcification in in vitro cultures of A-10 embryonic rat smooth muscle cells. Intraperitoneal administration of a compound that prevents SEV biosynthesis and release inhibited thoracic aortic calcification in CKD mice under a high phosphorus diet. In Chronic Kidney Disease, the microRNA transcriptome of SUVs revealed a depletion of four microRNAs and the expression of the microRNAs inversely correlated with kidney function in CKD patients.
In vitro studies found that transected microRNA mimics prevented smooth muscle cell calcification in vitro. In silico analyses revealed that VEGF-A was a convergent target of all four microRNAs and leveraging this, the group used in vitro and in vivo models of calcification to show the inhibition of the VEGF-A, VEGFR-2 signaling pathway mitigated calcification. So in addition to identifying a new potential therapeutic target, these SUV propagated microRNAs are a potential biomarker that can be used for screening patients to determine the severity of CKD and possibly even vascular calcification.
Cindy St. Hilaire: Today I have with me Dr Hind Lal who's an associate professor of medicine at the University of Alabama Birmingham and his post-doctoral fellow and the lead author of the study Dr Tousif Sultan. And their manuscript is titled Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. And this article is in our February 3rd issue of Circulation Research. So thank you both so much for joining me today.
Tousif Sultan: Thank you.
Hind Lal: Thank you for taking time.
Cindy St. Hilaire: So ponatinib, it's a tyrosine kinase inhibitor and from my understanding it's the only treatment option for a specific group of patients who have chronic myelogenous leukemia and they have to harbor a specific mutation. And while this drug helps to keep these patients alive essentially, it's extremely cardiotoxic. So cardiotoxicity is somewhat of a new field. So Dr Lal, I was wondering how did you get into this line of research?
Hind Lal: So I was fortunate enough to be in the lab of Dr Tom Force and he was kind of father of this new area, now is very developed, it's called cardio-oncology. On those days there were basically everything started in cardio-oncology. So I just recall the first tyrosine kinase approved by FDA was in 2000 and that was... Imagine and our paper came in Nature Medicine 2005 and discovering there is... so to elaborate it a little bit, the cancer therapy broadly divided in two parts. One is called non-targeted therapy like chemotherapy, radiations, et cetera, and then there are cytotoxic drugs. So those cytotoxic drugs because they do not have any targeted name on it so they are, cardiotoxic are toxic to any organ was very obvious and understanding. When these targeted therapy came, which is mainly kinase inhibitor are monoclonal antibodies. So these are targeted to a specific pathway that is activated only in the cancer cells but not in any other cells in the body so they were proposed as like magic bullets that can take off the cancer without any cardiotoxity or minimal side effects. But even in the early phase like 2005 to 2010, these came out, these so-called targeted, they are not very targeted and they are not also the magic bullets and they have serious cardiotoxicity.
Cindy St. Hilaire: And so what's the mechanism of action of ponatinib in the leukemia and how does that intersect with the cardiovascular system?
Hind Lal: Yeah, so this is very good question I must say. So what we believe at this point because, so leukemia if you know is driven by the famous Philadelphia chromosome, which is a translicational gene, one part of human chromosome nine and one part of human chromosome 22 and they translocate make a new gene which is BCR-ABL gene. And because it was discovered in Philadelphia UPENN, is named that Philadelphia chromosome, which is very established mechanism, that's how CML is driven. But what we have discovered that the cardiotoxicity driven by totally, totally different from the ponatinib is one of the inflammatory So it's kind of goodening. So this question is so good. One kind of toxicity is called on-target, when toxicity is mediated by the same mechanism, what is the mechanism of the drug to cure the cancer? So in that case your absolute is minimal because if you manipulate that, the drug's ability to cure the cancer will be affected but if the toxicity and the efficacy is driven by two different mechanism, then as in case of ponatinib seems like it's NLRP3 and inflammasome related mechanism. So this can be managed by manipulating this pathway without hampering the drug efficacy on the cancer.
Cindy St. Hilaire: So what exactly is cardiotoxicity and how does it present itself in these patients?
Hind Lal: So these drugs like ponatinib, they call broader CVD effects. So it's not just cardiac, so they also in hypertensives and atherosclerosis and thrombosis, those kind of thing. But our lab is primarily focused on the heart. So that's why in this paper we have given impresses on the heart. So what we believe at this point that ponatinib lead to this proinflammatory pathway described in this paper, which is just 108A9-NLRP3-IL-1β and this inflammatory pathway lead to a cytokine storm very much like in the COVID-19 and these cytokine storms lead to excessive myocarditis and then finally cardiac dysfunction.
Cindy St. Hilaire: Is the cytokine storm just local in the cardiac tissue or is it also systemic in the patients? Is cardiotoxicity localized only or is it a more systemic problem?
Tousif Sultan: I would like to add in this paper we have included that we look this cytokine things and explain blood circulation, bone marrow. So the effect is everywhere, it's not local. So we didn't check other organs, maybe other organs also being affected with the ponatinib treatment.
Cindy St. Hilaire: And what's the initial phenotype of a patient has when they start to get cardiotoxicity, what's kind of like a telltale symptom?
Hind Lal: So good thing that in recent years cardio-oncology developed. So initially the patient that were going for cancer treatment, they were not monitored very closely. So they only end up in cardiology clinic when they are having some cardiac events already. So thanks to the lot of development and growth in the cardio-oncology field, now most patients who going for a long-term cancer treatment, they are closely monitored by cardiology clinics.
Cindy St. Hilaire: Got it. So they can often catch it before a symptom or an event. That's wonderful.
Hind Lal: Yeah, so there's a lot of development in monitoring.
Cindy St. Hilaire: Wonderful. So you were really interested in figuring out why ponatinib induces cardiotoxicity and you mentioned that really up until now it's been very difficult to study and that's because of the limitation of available murine models. If you just inject a wild type mouse with ponatinib, nothing happens really. So what was your approach to finding relatively good murine models? How did you go about that?
Hind Lal: So this is the top scientific question you can ask. So like science, the field is try and try again. So initially this is the first paper with the ponatinib toxicity using the real in vivo models. Any paper before this including ours studies published, they were done on the cellular model in hiPSC, that isolated cardiomyocytes. So you directly putting the ponatinib directly the isolated cells. So this is first case when we were trying to do in vivo, maybe other attempt in vivo but at least not published. So first we also treated the animals with ponatinib and that failed, we don't see any cardiotoxic effect. And then when we going back to the literature, the clinical data is very, very clear from pharmacovigilance that ponatinib is cardiotoxic in humans. So when we're not able to see any phenotype in mouse, we realize that we are not mimicking what's happening in the humans.
So we certainly missing something. Now once again I quote this COVID-19, so many people get infected with COVID-19 but people are having preexisting conditions are on high risk to developing CVD. So there was some literature on that line. So we use this very, very same concept that if there is preexisting conditions, so likely who'd have developing future cardiac event will be more. So we use two model in this paper one atherosclerosis model which is APoE null mice mice, another is tag branding which is pressure overload model for the heart and as soon as we start using what we call comorbidity model like patient is having some preexisting conditions and we very clearly see the robust defect of ponatinib on cardiac dysfunction.
Cindy St. Hilaire: Yeah, it's really, really well done and I really like that you use kind of two different models of this. Do you think it's also going to be operative in maybe like the diabetic mirroring models? Do you think if we expand to other comorbidities, you might also recapitulate the cardiotoxicity?
Hind Lal: So you got all the best questions.
Cindy St. Hilaire: Thank you. I try.
Hind Lal: So because this is CML drug and lot of the risk factor for cardiovascular and cancer are common and even metabolic disease. So most of the time these patients are elderly patients and they're having metabolic conditions and most of the time they have blood pressure or something CVD risk factors. So I agree with you, it'll be very relevant to expand this to the diabetes or metabolic models, but these were the first study, we put all our focus to get this one out so news is there then we can expand the field adding additional models et cetera. But I agree with you that will be very logical next step to do.
Cindy St. Hilaire: Yeah. And so I guess going back to what you know from the human study or the clinical trials or the human observations, are different populations of patients with CML more predisposed to cardio toxicity than others or is that not known yet?
Hind Lal: So one other area called pharmacovigilance. So what pharmacovigilance does patient all over the world taking these drugs. So WHO have their own vigilance system and FDA have their own, so it's called BG-Base for the WHO and it's called the FAERS for the FDA. So one can go back in those data sets and see if X patient taking this Y drug and what kind of symptoms or adverse effect they are seeing and if these symptoms are associated with something else. So there is data that if patients having CVD risk factor, they are more prone to develop ponatinib induced cardiac events. But it needs more polish like you asked the just previous question, diabetes versus maybe blood pressure means hypertension, atherosclerosis, or thrombosis. So it has not been delineated further but in a one big bucket if patients are having CVD risk factor before they are more prone and more likely to develop the cardiac events.
Cindy St. Hilaire: So after you established that these two murine models could pretty robustly recapitulate the human phenotype, what did you do next? How did you come upon the S100A8/A9-NLRP3-IL-1β signaling circuit? How did you get to that?
Hind Lal: So in basic science work, whenever we do mouse is called until we get there is cardiac dysfunction, it's called phenotype, right? So mouse had a cardiac phenotype. So next step is, "Why? What is leading to that phenotype?" That's what we call mechanism. So there the best idea to fit the mechanism is using one of the unbiased approaches like you do unbiased proteomics, unbiased RNC analysis, something like this that will analyze the entire transcript like RNC and say, "Okay, these pathway are," then you can do further analysis that will indicate these pathway are different, are altered. So in this case we used RNC analysis and it came out that this yes A8 and yes A9, 100A8 and nine, they were the most upregulated in this whole set. And thereafter we were very lucky. So we started this study at Vanderbilt, where my lab was and thereafter we very lucky to move here and found Sultan who had a lot of experience with this inflammation and immune system and then Sultan may add something on this so he'll be the better person to say something on this.
Tousif Sultan: So after our RNC analysis, so we got this S100A8 and nine as top hit with the ponatinib treatment. So then we validated this finding with our flow cytometric, qRT PCR aand then we started which pathway is going to release cytokine and all that. So we found that is NLRP3 inflammasome.
Cindy St. Hilaire: Yeah and well and I guess maybe step back, what is S100A8/A9? What are those?
Tousif Sultan: Yeah, S10A8/A9 is a calcium binding protein. So that's also called alarmin and they basically binds with the pathogen associated pattern and other TLR2 like receptors and then start inflammatory pathway to release cytokine and all that and it's stable in heterodimer form. So S100A8 heterodimer with A9 and then bind with TLR and a start in this inflammatory pathway.
Cindy St. Hilaire: And what type of cell is that happening in? Is that happening in the immune cells only or is it also in the cardiomyocyte, or...?
Tousif Sultan: Yeah, we have included all this data. So from where this alarmin is coming with ponatinib treatment, so literature also suggested that neutrophils and monocytes, those cells are the potential to release the alarmin. So here we also found these two type of cells, neutrophils and monocytes. They release huge alarmin with the treatment of ponatinib.
Cindy St. Hilaire: And so really taking this really neat mechanism to the next level, you then tried attenuating it by using broad anti-inflammatory steroid dexamethasone but also by targeting these specific components, the NLRP and the S100A specific inhibitors and they worked well. It worked really nicely. Does your data show that any of these therapies work better than the other and then are these viable options to use in humans?
Hind Lal: Yeah, we have some data in the paper. Are very broad which help a lot in COVID patients, far very acute infections. So in this case, situation is very different cause most of CML patients will going to take ponatinib for lifelong, there is no remission, right? So in those case, its certainly not a very attractive option. We have shown data in the paper that dexamethasone help with the heart but lead to some metabolic changes. So we have compared those with the NLRP3 inhibitors, those metabolic alterations, dexa versus the NLRP3 inhibitors, CY-09. And we demonstrated that targeting is specifically with paquinimod, our NLRP3 inhibitor CY-09, feel better. It can still rescue the cardiac phenotype without having those adverse effect on metabolic parameters.
Cindy St. Hilaire: That's wonderful. Do you think though that because you have to take ponatinib for life, that long-term NLRP inhibition would also cause problems or...?
Hind Lal: So because not every patient who taking ponatinib would develop the cardiac phenotype, right? Which is like a 10%, 12%, patient developing cardiac dysfunction. So I think someone like I strongly believe paquinimod, which is inhibitor of S100A9, will be really good option or at least we have enough data that make us nail for at least a small clinical trial. And we quickly moving on that. At UAB we have our clinical cardio-oncology program and we are already in touch with the director for the clinical cardio-oncology program. So what we trying to do in that small trial is if one of the standard therapy for heart like beta blocker or ARBs inhibitor, is there any preference like one work better than the other in the standard care? So first we doing that project, then we obviously looking forward if one small clinical trial can be done with paquinimod. I strongly believe it should be helpful.
Cindy St. Hilaire: That is wonderful. And so do you think... There's other chemotherapeutic agents or probably even other non-cancer drugs that cause cardiotoxicity, do you think this mechanism, this pathway, this S100A-NLRP-IL-1β axis is operative in all cardiotoxicities or do you think it's going to be very specific to the ponatinib?
Hind Lal: So it's certainly not all, but it'll be certainly more than ponatinib. So in our lab we are using another kinase inhibitor, which is osimertinib and it's not published yet, but now we know that it's also cardiotoxic because it's taking metabolic root or energetics disruption but not this pro-inflammatory part, but we're doing another project which is strep pneumonia induced cardiac dysfunction, which is called pneumonia. So strep pneumoniae, which leads to the pneumonia ,and lot patient die because of the failing heart we see here in the hospitals and we see these pathways operational over there and we gearing up to do clinical trial on that aspect as well, but it's not generalized like all kind of heart will have the same mechanism.
Cindy St. Hilaire: It's wonderful to see you're already taking those next steps towards really kind of bringing this to a translational/clinical study. So what was the most challenging aspect of this study?
Tousif Sultan: The challenging aspect, ponatinib is a kinase inhibitor and that was surprising for us how it's activating immune cells. Generally kinase inhibitors, inhibits all the cells like that. So that was challenging. So we repeated it many times did in vitro experiment to confirm that. So we just added, just treated in vitro immune cells with the ponatinib and confirmed it. So that was little challenging.
Cindy St. Hilaire: So what's next? You mentioned you're going to try some clinical trials, early stage clinical trials. What's next mechanistically, what do you want to go after?
Hind Lal: So what we are doing next and we are very, very eagerly trying to do that. So what it was done, we used the cardiac comorbidity models, but as you know, anybody who will take ponatinib will have cancer, right? So we strongly believe that we miss one factor. There was no cancer on these. So that is very logical next step. What that will allow us to do, what rescue experiment we'll have done in this paper. So we saw, "Okay, this rescue the cardiac phenotype, which is taken care of now," but very same time, we not able to demonstrate that this is happening without hurting the cancer efficacy. So if we have the dual comorbid mouse, which have CML a real thing and we have cardiac thing, then that will allow us to demonstrate, "Okay, we got something that can take care of the cardiac problem without hurting the efficacy on the cancer." And it will be best if you also help little bit to more potentiate the cancer efficacy.
Cindy St. Hilaire: Yes. Excellent. Well, congratulations on a beautiful study, really exciting findings. Dr Lal and Dr Sultan, thank you so much for taking the time to talk with me today.
Tousif Sultan: Thank you so much.
Hind Lal: Well thank you, Cynthia. We really appreciate your time. Thank you for having us.
Cindy St. Hilaire: Yeah, it was great.
Cindy St. Hilaire: That's it for our highlights from the February 3rd and February 17th issues of Circulation Research. Thank you so much for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Hind Lal and Dr Tousif Sultan. This podcast is produced by Ishara Ratnayake, edited by Melissa Stoner and supported by the editorial team at Circulation Research. Some of the copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover CircRes, you're on-the-go source for most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2023. And the opinions expressed by the speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.
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This month on Episode 44 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the January 6th and January 20th issue of Circulation Research. This episode also features an interview with Dr Timothy McKinsey and Dr Marcello Rubino about their study, Inhibition of Eicosanoid Degradation Mitigates Fibrosis of the Heart.
Article highlights:
Prasad, et al. ACE2 in Gut Integrity and Diabetic Retinopathy
Cui, et al. Epsins Regulate Lipid Metabolism and Transport
Li, et al. Endothelial H2S modulates EndoMT in HF
Luo, et al. F. plautii Attenuates Arterial Stiffness
Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's Journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to be highlighting articles from our January 6th and January 20th issues of Circulation Research. I'm also going to have a chat with Dr Timothy McKinsey and Dr Marcello Rubino about their study, Inhibition of Eicosanoid Degradation Mitigates Fibrosis of the Heart. But before the interview, I want to get to a few articles to highlight.
Cindy St. Hilaire: The first article is titled, Maintenance of Enteral ACE2 Prevents Diabetic Retinopathy in Type 1 Diabetes. The first authors are Ram Prasad and Jason Floyd, and the corresponding author is Maria Grant, and they are from the University of Alabama.
Type 1 Diabetes has a complex etiology and pathology that are not entirely understood. In addition to the destruction of insulin-producing cells, a recently discovered feature of the disease in both humans and in rodent models is that the levels of angiotensin converting enzyme 2 or ACE2 can be unusually low in certain tissues. ACE2 is a component of the renin angiotensin system controlling hemodynamics and interestingly, genetic deficiency of ACE2 in rodents exacerbates aspects of diabetes such as gut permeability, systemic inflammation and diabetic retinopathy, while boosting ACE2 has been shown to ameliorate diabetic retinopathy in mice. This study shows that ACE2 treatment also improves gut integrity and systemic inflammation as well as retinopathy. Six months after the onset of diabetes in a mouse model, oral doses of a bacteria engineered to express humanized ACE2 led to a reversal of the animal's gut barrier dysfunction and its retinopathy. Humans with diabetic retinopathy also displayed evidence of increased gut permeability in low levels of ACE2. This study suggests they may benefit from a similar probiotic treatment.
Cindy St. Hilaire: The next article I want to highlight is titled, Epsin Nanotherapy Regulates Cholesterol Transport to Fortify Atheroma Regression. The first authors are Kui Cui, Xinlei Gao and Beibei Wang, and the corresponding authors are Hong Chen and Kaifu Chen and they're from Boston Children's Hospital. Epsins are a family of plasma membrane proteins that drive endocytosis. They're expressed at varying levels throughout the tissues of the body, and recent research shows that they are unusually abundant on macrophages within atherosclerotic lesions. In mice, macrophage specific Epsin loss results in a reduction in foam cell formation and atherosclerotic plaque development. This study now shows that this effect on foam cells is because Epsins normally promote the internalization of lipids into macrophages through their endosytic activity.
But that's not all. The proteins also impede cholesterol efflux from macrophages to further exacerbate lipid retention. It turns out out Epsins regulate the endocytosis and the degradation of a cholesterol efflux factor called ABCG1. Importantly, these pro atrogenic activities of Epsins can be stopped. Using macrophage targeted nanoparticles carrying Epson specific silencing RNA, the team could suppress reduction of the protein in cultured macrophages and could reduce the size and number of plaques in atherosclerosis prone mice. Together these results suggest blocking Epsins via nanotherapy or other means could be a therapeutic approach to stopping or slowing atherosclerotic plaque progression.
Cindy St. Hilaire: The third article I want to highlight is coming from our January 20th issue of Circ Res and is titled, Hydrogen Sulfide Modulates Endothelial-Mesenchymal Transition in Heart Failure. The first author is Zhen Li, and the corresponding author is David Lefer and they're from Cedars-Sinai. Hydrogen sulfide is a critical endogenous signaling molecule that exerts protective effects in the setting of heart failure. Cystathionine γ-lyase, or CSE, is one of the three hydrogen sulfide producing enzymes, and it's predominantly localized in the vascular endothelium. Genetic deletion of CSE, specifically in the endothelium, leads to reduced nitric oxide bioavailability, impaired vascular relaxation and impaired exercise capacity, while genetic over-expression of PSE in endothelial cells improves endothelial cell dysfunction, and attenuates myocardial infarction following myocardial ischemia-reperfusion injury.
In this study, endothelial cell specific CSE knockout mice and endothelial cell specific CSE overexpressing transgenic mice were subjected to transverse aortic constriction to induce heart failure with reduced ejection fraction. And the goal was to investigate the contribution of the CSE hydrogen sulfide access in heart failure. Endothelial specific CSE knockout mice exhibited increased endothelial to mesenchymal transition and reduced nitric oxide bioavailability in the myocardium. And this was associated with increased cardiac fibrosis, impaired cardiac and vascular function, and it worsened the vascular performance of these animals. In contrast, genetic overexpression of CSE in endothelial cells led to increased myocardial nitric oxide, decreased EndoMT and decreased cardiac fibrosis. It also improved exercise capacity. These data demonstrate that endothelial CSE modulates endothelial mesenchymal transition and ameliorated the severity of pressure overload induced heart failure , in part through nitric oxide related mechanisms. This data further suggests that endothelium derived hydrogen sulfide is a potential therapeutic for the treatment of heart failure with reduced ejection fraction.
Cindy St. Hilaire The last article I want to highlight is titled, Flavonifractor plautii Protects Against Elevated Arterial Stiffness. The first authors are Shiyun Luo and Yawen Zhao, and the corresponding author is Min Xia, and they are at Sun Yat-sen University. Dysbiosis of gut microbiota contributes to vascular dysfunction and gut microbial diversity has been reported to be inversely correlated with arterial stiffness. However, the causal role of gut microbiota in the progression of arterial stiffness and the specific species along with the molecular mechanisms underlying this change remain largely unknown. In this study, the microbial composition in metabolic capacities were compared in participants with elevated arterial stiffness and in normal controls free of medication. And these groups were age and sex match.
Human fecal metagenomic sequencing identified a significant presence of Flavonifractor plautii or F. plautii in normal controls, which was absent in the subjects with elevated arterial stiffness. The microbiome of normal controls exhibited an enhanced capacity for glycolysis and polysaccharide degradation, whereas individuals with increased arterial stiffness exhibited increased biosynthesis of fatty acids and aromatic amino acids. Additionally, experiments in the angiotensin II induced and humanized mouse model show that replenishment with F. plautii or its main effector cis-aconitic acid or CCA improved elastic fiber network and reversed increased pulse wave velocity through the suppression of matrix metalloproteinase-2 and through the inhibition of monocyte chemoattractant protein-1. And this was seen in both the angiotensin II induced and humanized models of arterial stiffness. This study now identifies a novel link between F. plautii and arterial function and raises the possibility of sustaining vascular health by targeting the gut microbiota.
Cindy St. Hilaire: Today with me I have Dr Tim McKinsey and Dr Marcello Rubino from the University of Colorado Anschutz Medical Campus, and we're here to talk about their paper Inhibition of Eicosanoid Degradati`on Mitigates Fibrosis of the Heart. And this article is in our January 6th issue of Circulation Research, so thank you both so much for joining me today.
Timothy McKinsey: Thank you for inviting us.
Marcello Rubino: Yeah, thank you for the opportunity.
Cindy St. Hilaire: And so Dr McKinsey, you're a professor at the University of Colorado. How long have you been investigating cardiac fibrosis?
Timothy McKinsey: Oh, a long time. Before I started the lab here in 2010, I was in industry working in biotech with Myogenic Gilead, and we were very interested in cardiac fibrosis all the way back then.
Cindy St. Hilaire: Oh wow, so you actually made an industry to academia transfer.
Timothy McKinsey: Yes.
Cindy St. Hilaire: Good topic for another podcast. That is really great.
Timothy McKinsey: Yeah, it's of interest to a lot of people, including trainees.
Cindy St. Hilaire: Yeah, I bet. Dr Rubino, you were or are a postdoc in the McKinsey lab?
Marcello Rubino: Yeah, I was a postdoc in Timothy McKinsey lab. I spent four years in Tim's lab. It was my first time studying cardio fibrosis, so it was a little bit difficult at the end, but I think I was right choosing Tim, so I'm really happy now.
Cindy St. Hilaire: Nice and are you sticking with fibrosis or are you moving on?
Marcello Rubino: Yeah, so now I'm back in Milan where I did my PhD student and postdoc. I am like an independent researcher, but it's still not a principal investigator, so I want to become one of the that, studying cardiac fibrosis. Yeah. And inflammation and epigenetics, so yeah, I'm going try to go to my way, thanks to Tim, I think that I find my own way.
Cindy St. Hilaire: I'm sure you will. I mean, based on the great work in this study, right. Building upon that, I'm sure you'll be a success.
Timothy McKinsey: No doubt about it.
Cindy St. Hilaire: So your manuscript, this study, it's investigating whether eicosanoid availability can attenuate fibrosis in the heart. But before we kind of jump into this study, why is fibrosis in the heart a bad thing? Is it always detrimental? Is there some level of fibrosis that's necessary or even helpful?
Timothy McKinsey: I mean, a certain level of extracellular matrix is deposited in your heart and that maintains the structure of the heart. Fibrosis can also be good after you have a myocardial infarction and a big piece of the muscle of your heart has died, it needs to be replaced with a fibrotic scar, essentially to prevent rupture of the ventricle. So fibrosis isn't always bad, but chronic fibrosis can be really deleterious to the heart and contribute to stiffening of the heart and cause diastolic dysfunction. It can create substrates for arrhythmias and sudden cardiac death. So we're really trying to block the maladaptive fibrosis that occurs in response to chronic stress.
Cindy St. Hilaire: Yeah, yeah. And what about eicosanoids? What are they and what role do they play in cardiac fibrosis or what was known about their role in this process before your study?
Timothy McKinsey: Eicosanoids are lipids, they're basically fatty acids, 20 carbon in length and a lot is known about them. It's a very complex system. There are many different eicosanoids, but they're produced from arachidonic acid through the action of cyclooxygenase enzymes like COX-2. And so you're probably familiar with the literature showing that non-steroidal anti-inflammatory drugs that target the COX enzymes can actually increase the risk of cardiac disease, so there was a lot known about what produces eicosanoids in the heart, but our study is really the first to address how they're degraded and how that controls cardiac fibrosis.
Cindy St. Hilaire: What I thought you did really well in the introduction and what I guess I didn't really fully appreciate until I had read your study, was that your goal was to identify compounds that could attenuate fibrosis. And you spent some time emphasizing the differences between a targeted small molecule screen and a phenotype based screen. And I was wondering if you could just expand on this difference for the audience and maybe just explain why in your case you went with the latter.
Timothy McKinsey: Well, we wanted to use an unbiased approach and some people call this a chemical biology approach where we took a targeted library, meaning we took compounds with known activities, meaning compounds that with known targets and we screened that library using a phenotypic assays that we developed in the lab. And the phenotypic assay is an unbiased assay, right? We're just screening for compounds that have the ability to block the activation of fibroblasts. And we monitor activation by looking at markers of fibroblast activation such as alpha smooth muscle Actin. And we can do this in a very quantitative and high throughput manner using this imaging system, high content imaging system that we have in the lab.
It was an unbiased screen looking for inhibitors of fibroblasts activation across organ systems. We not only studied cardiac fibroblasts, but we also studied lung and renal fibroblasts looking for compounds with a common ability to block the activation state of each of those cell types.
One of the things that I get asked frequently is how do we maintain the cardiac fibroblasts in a quiescent state? Because you may know this, but when fibroblasts are plated on cell culture plastic, which has a very high 10 cell strength, they tend to spontaneously activate, so we actually spent a couple of years working out the conditions to maintain the cells in quiescent state, and I think that will also be of great interest to the field.
Cindy St. Hilaire: Probably even the smooth muscle cell biology field where I hang out and even valve interstitial cells that we study. All of those, I guess basic things related to cell culture, we have taken for granted that plastic is not physiological.
Timothy McKinsey: Right.
Cindy St. Hilaire: And so I think with this really nice phenotypic or chemical screen that you conducted, you first identified nine compounds, but what made you zero in on this one, SW033291?
Timothy McKinsey: When we got the hits, we were intrigued by the SW compound SW033291 because there was only one paper describing its action and there was a paper published in Science showing that SW or inhibition of this enzyme 15-PGDH could enhance organ regeneration.
Cindy St. Hilaire: Oh, okay.
Timothy McKinsey: And there's a very interesting interplay between fibrosis and organ regeneration where fibrosis inhibits regeneration and if you can stimulate regenerative pathways, they can actually block fibrosis, so there's this back and forth. And so that's really the main reason we were interested in pursuing SW just because of the novelty and the potential. And also it was a compound that behaved beautifully in our cell culture models with beautiful dose-dependent inhibition of each of the fibroblast types.
Cindy St. Hilaire: It's kind of like the cleanest thing to start with. Also, if there's nothing known, it's ripe for investigation, so that's great. You just said this SW compound acts on 15-PGDH, so what is the role of that protein in fibroblasts and what if any known effects are there on this protein's inhibition in other cell types or disease states?
Marcello Rubino: In fibroblasts team, I would like to say that this was really the first article that was published. Maybe there was just one published in Pulmonary Fibrosis, but like last year, but I didn't really talk about 15-PGDH, so you need to consider that 15-PGDH is an inhibitor, an enzyme that degrades prostaglandin, so if you inhibit the inhibitor, the release increase production, a lot of prostaglandin. And so a lot of paper were talking about this effect, so they will see we are just using SW in order to increase Prostaglandin E2 level and that was why we had this like anti-inflammatory or whatever effect. I would like to say that until now, maybe this can be the first really paper talking about no more than not just prostaglandin but 15-PGDH. Its action total level, a global level at particularly on fibroblasts.
To answer your question, I would like to say that this was also our question first and we checked by level other browser to try to find the answer to your question. We figured out that it was known that 15-PGDH was increasing a pathology condition in different organ, not just related by fibroblasts, not just related to cardiac disease, about the function with discover a function in macrophage that interested us because it can regulate maybe the polarization macrophage, so still involving the prostaglandin production inflammation, so that's why also we decide to take a look because it was still novel in fibrolbasts and we still know that it was doing something important and we were trying not to put the piece together and find something new in that we were lucky for this.
Timothy McKinsey: 15-PGDH is actually expressed at very low levels in fibroblasts. It's much more highly expressed in macrophage, just as Marcello pointed out, so in the future we're very interested in knocking out or inhibiting 15-PGDH in different cell types to see how that contributes to inhibition of cardiac fibrosis.
Cindy St. Hilaire: Really interesting. Related to that, you used a couple different animal models for fibrosis. They're all different or special in their own way. How well did these recapitulate what we observe in humans. Are there any limitations of benefits?
Timothy McKinsey: They're always limitations to animal models. We started out with a very robust commonly used model of cardiac fibrosis, which relies on Angiotensin II infusion in mice. We like that model because it's robust and quick so we can get answers quickly. And then we transitioned into a model of diastolic dysfunction that we've been working with in a lab where we remove a kidney from a mouse and we implant something called DOCA, which is an aldosterone memetic. And so the animals develop hypertension that leads to a mild but significant diastolic dysfunction with preserved ejection fraction.
And that's a model that we like a lot. It has something that we call hidden fibrosis, so if you just do standard histochemical staining of the hearts from the DOCA unit, nephrectomy model, that diastolic dysfunction model, you really can't see robust fibrosis. It's only when you dive more deeply with more sensitive assays like mass spectrometry or atomic force microscopy that you can detect this fibrosis and stiffening of the heart, so we usually lead with a robust model of fibrosis, cardiac fibrosis, and then transition into a slightly more complex model but more physiologically relevant model or disease relevant model.
Cindy St. Hilaire: Obviously you showed some really nice robust results with this SW compound. So in the continuum of heart failure in human, what do you think or what would you speculate would be the ideal timeframe for administration of this compound?
Timothy McKinsey: Wouldn't want to give it immediately after someone's had a heart attack. As we discussed earlier, you need that reparative scar to form so you don't want to block that fibrotic remodeling. We believe that there's kind of smoldering fibroblast activation in the heart, even in someone who's had heart disease for many, many years. And if we can dampen that, we can either prevent further progression of heart failure or perhaps reverse it. We don't really know if we can reverse really established fibrosis in the heart yet. But I would want to try to catch fibrosis fairly early on in the disease process in someone who has chronic hypertension or obesity or a variety of different comorbidities and then start delivering an antifibrotic therapy at that point.
Marcello Rubino: I would like to add that, so it is really tricky when we talk about clinical trials because a lot of molecules that maybe they can work hopefully in a preclinical model don't work at the end in the clinical model. That's because can be some off target also like you just asked what is really important is when you do the administration of the molecule and talk about this in SW, like things say we don't want to prevent the fibrosis because there is something like called a kneeling at the beginning, so it is the good fibrosis we like to say, but the good thing of SW compound is that is affecting in a good way the proliferation of fibroblast that is different for all the other. I would like to say all the other inhibitor that we saw so far, because I remember the first time that I presented this work, there was an expert told me that he didn't believe that all my data because the compound was inhibiting fibrosis, it was inhibiting proliferation.
And I show him, no, this is contrary, so oh okay, I like it. We need to consider this that the action seems to be not like the retire for the cell, so because the cells continue to proliferate, they can proliferate more. But the good thing and we need to investigate more is that SW action seems to increase when the cell are more fibrotic, because we show just few human fibroblasts isolating from a human patient and we saw a higher positive effect of SW compound when the cell were more fibrotic. That can be interesting. I think that it's worth to try to test in the future like in different preclinical models and maybe in patients at the end because if we really can find something like maybe SW that can be specific for the state of pathology, that will be wonderful. I don't really know if we can really do it, but we need some therapy like this, so that's why we were really excited about what we discovered for this compound.
Timothy McKinsey: We have a lot more to learn about this pathway and about fibrosis in general.
Cindy St. Hilaire: Yeah.
Timothy McKinsey: It's a very exciting time to be doing science because of the amazing technologies that we have at our disposal to address detailed mechanisms of disease.
Cindy St. Hilaire: What was the most challenging aspect of the study?
Timothy McKinsey: This was an incredibly difficult study. I can't even stress to you how much work went into this. Spearheaded by Marcello's awesome leadership. There was huge input from a big team. Keith Cook and I worked together in industry and we were able to recruit him over here for a few years as part of our fibrosis center called the CFReT. It's an advertisement. And Keith was able to implement some of the drug discovery approaches that we used in biotech and create this imaging system that we initially employed for the screens. That was challenging. Maintaining the cells in a quiescent state was very challenging as I mentioned. That took a couple of years and then just following up on SW and trying to figure out its mechanism of action was really challenging as well because as Marcello mentioned, most people have attributed SW's effects to an increase in PGE2 levels, so PGE2 is an eicosanoid that is degraded by 15-PGDH.
And definitely when you inhibit 15-PGDH with SW, you see increased PGE2. But surprisingly we couldn't find that PGE2 was doing anything in our cell culture systems, meaning when we added it exogenously it was not blocking fibroblast activation, so then Marcello set out to identify which eicosanoid that is regulated by 15-PGDH is actually the antifibrotic eicosanoid. And that led him to something called 12(S)-HETE. That was challenging. And then just determining at the molecular level what was going on was also challenging. And that led Marcello to this kind of paradoxical discovery that it activating ERK signaling was actually blocking fibroblast activation.
Cindy St. Hilaire: And of course ERK does everything right?
Marcello Rubino: It does. Everything.
Timothy McKinsey: And sort of the dogma is that ERK is promoting fibrosis in the heart, but Marcello's data suggests otherwise.
Timothy McKinsey: And then other shout outs, Josh Travers, who's the second author of the paper provided huge input, especially after Marcello left. Josh helped get this across the finish line. We have an amazing in vivo team conducting the animal model studies. Maria Cavasin and Elizabeth Hardy. I could go on and on. There are a lot of authors and if I didn't mention one of them, it doesn't mean that they weren't key contributors. I just wanted to throw that out there. We also had great collaborators. I think another component of this paper that is of great interest to us, and initially I was against doing any of this, is that Marcello and Josh created this biobank of human cardiac fibroblasts that we obtained from explanted hearts from individuals undergoing heart transplantation.
And initially I thought it was going to be a waste of time and money for Marcello and Josh to do that, but they were persistent and they started isolating these cells. And the cells are really fascinating because even after you take them out of that failed human heart and culture them, they maintain this constituently active state, which is different than the cells we were using for screening where we kept them quiescent and then we stimulated them with TGF-β to activate them. These human cardiac fibroblasts from the failed human hearts are just on all the time.
Cindy St. Hilaire: Wow.
Timothy McKinsey: And SW does a really amazing job of reversing that activated state.
Cindy St. Hilaire: Very cool and excellent resource I'm sure for future studies. So my last question is what's next? You know, you discovered a lot in this paper. What's the next thing you want to tackle?
Timothy McKinsey: Cell type specific roles for 15-PGDH in the heart, in the control of cardiac homeostasis and disease. Basically we want to knock it out in fibroblasts. We want to knock it out in our macrophages and see what the consequences are. That's one thing. We want to really pursue the whole GPR31 12(S)-HETE pathway in the heart. That's something that has never been studied. And so GPR31 is a G protein coupled receptor that is bound by this eicosanoid called 12(S)-HETE. And that seems to be blocking fibroblast activation, so we're going to further pursue that pathway. And then we think that this paradoxical finding related to ERK signaling in the heart is also worthy of pursuit. Why is it that stimulating ERK in a cardiac fibroblast is actually blocking the activation state of that cell?
Marcello Rubino: I'm interested in this like Tim says, but also interested in the role of the interaction of the cell because it's important to study like a specific gene inhibitor, whatever role in a specific cell, but what happened to the other cell, the interaction the other cell when you do knocking in some specific cell, so that's what I'm trying to do in general. Now I move back in Italy, like I told you, I'm like a kind of independent research and I'm studying a lot single cell sequencing right now. Try to do also try to see what happened to interaction, understand during pathology.
The idea is to study like inhibitor treatment and to see what really happened because gene expression is important, but we need to consider also of course the protein shape, the protein interaction, the cell interaction, so I try to grow in this field and see what really happened because the problem of the cell, they're just cell in vitro. They can mimic what happened, but it's not what really happened in vivo, so can we use this novel technology to improve our knowledge, that's what I want to try to do.
Cindy St. Hilaire: Well that's great. Dr McKinsey, Dr Rubino, thank you so much for taking the time to speak with me today. Title of their article was Inhibition of Eicosanoid Degradation Mitigates Fibrosis of the Heart. It's in our January 6th issue of Circ Res. And thank you both so much for joining me today and thank you to you and all of your colleagues who worked so hard on this for this amazing study.
Timothy McKinsey: Thank you. We really enjoyed this visit and we're grateful to have our work published in Circulation Research.
Cindy St. Hilaire: That's it for highlights from the January 6th and 20th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes or #DiscoverCircRes. Thank you to our guests, Dr Tim McKinsey and Dr Marcello Rubino. This podcast is produced by Ishara Rantayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. The opinions expressed by the speakers of this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.
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This month on Episode 43 of Discover CircRes, guest host Nicole Purcell highlights two original research articles featured in the December 2 issue of Circulation Research. This episode also features an interview with Drs Aaron Phillips and Kevin O'Gallagher about their study, The Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans.
Article highlights:
Akerberg, et al. RBPMS2 Regulates RNA Splicing in Cardiomyocytes
Lv, et al. Cardiac Protection by MG53-S255A Mutant
Nicole Purcell: Hi and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I am your host, Dr Nicole Purcell, from the Huntington Medical Research Institutes in Pasadena, California, and today I will be highlighting two articles from our December 2 issue of Circulation Research. I'll also have a chat with Drs Aaron Phillips and Kevin O'Gallagher about their study, The Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans.
Nicole Purcell: But before I get to the interview, here are a few article highlights. The first article we're going to highlight is RBPMS2 Is a Myocardial Enriched Splicing Regulator Required for Cardiac Function. This comes from Boston Children's Hospital with first author Dr Alexander Akerberg, and corresponding author Dr Jeffrey Burns. RNA splicing, along with transcription control and post-translational modifications, is a mechanism for fine tuning the expression of a gene for a particular purpose in a particular tissue. Factors that control splicing are thus often enriched in certain cell types. The factor, RBPMS2, for example, is enriched in the myocytes of amphibians, fish, birds and mammals. This conserve tissue specificity suggesting essential role of RBPMS2 in heart function.
Akerberg and colleagues now confirm this is indeed the case. They generated zebra fish embryos and human cardiomyocytes lacking RBPMS2, and found the fish suffered early cardiac dysfunction by 48 hours post fertilization. The animal's hearts had reduced ejection fractions, compared with the hearts of controlled fish. At the cellular level, the RBPMS2 lacking fish cardiomyocytes displayed malformed sarcomere fibers and disrupted calcium handling, both of which were also seen in the RBPMS2 deficient human cardiomyocytes. Furthermore, RNA sequencing experiments revealed a conserve set of 29 genes in the RBPMS2-lacking fish and human cells that were incorrectly spliced. In revealing the essential cardiac role of RBPMS2 and its RNA targets, the work provides new molecular details for understanding vertebrate heart function and disease, say the team.
Nicole Purcell: Our second article being highlighted is Blocking MG53 Serine 255 Phosphorylation Protects Diabetic Heart from Ischemic Injury. This comes from Peking University with first authors, Fengxiang L, Yingfan Wang and Dan Shan, as well as corresponding author Dr Rui-Ping Xiao. Midsegment 53, or MG53, is a recently discovered muscle-specific protein that is an essential component of the cell membrane repair machinery with cardioprotective effects. MG53 thus has therapeutic potential, but for patients whose heart disease is linked to type 2 diabetes, there's a problem. MG53 also tags certain cellular proteins for destruction, including the insulin receptor and the insulin signaling factor, IRS1. Loss of these factors could worsen insulin resistance. lev and colleagues therefore investigate whether MG53 could be tweaked to provide protection without the diabetes downside.
Nicole Purcell: They discovered the phosphorylation of MG53 at serine 255 is required for its role in protein destruction, and that a mutant version of MG53, incapable of this phosphorylation, MG53 serine to 255 alanine mutant, could still promote cardiomyocyte survival, and protect the cells from membrane damaging insults. Importantly, when a diabetic mouse model was injected with MG53 serine 255 to alanine mutant, the protein better protected the animals against myocardial infarction than injection with the wild type MG53, recipients of which had poor insulin sensitivity. Based on these findings, the authors suggest MG53 serine 255 alanine mutant could be developed into a heart protective drug, for use in diabetic and non-diabetic patients alike.
Nicole Purcell: Today, Dr Aaron Phillips and Dr Kevin O'Gallagher from University of Calgary are with me to discuss their study, the Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans in our December 2 issue of Circulation Research. Thank you for joining me today.
Kevin O'Gallagher: Hello, my name's Dr Kevin O'Gallagher. I'm a British Heart Foundation clinician scientist and interventional cardiologist at Kings College London and Kings College Hospital NHS Foundation Trust.
Aaron Phillips: Hello, my name's Dr Aaron Phillips. I'm an associate professor in physiology, pharmacology, cardiac sciences, biomedical engineering and clinical neurosciences at the University of Calgary in the Hotchkiss Brain Institute and Libin Cardiovascular Institute. I am also the director of the Restore Network, which is a large platform at the University of Calgary spanning all these groups, developing new tools and techniques for translational research into neurological conditions.
Nicole Purcell: There are a lot of authors involved in this study. While all could not join us, I appreciate you taking the time to discuss your findings today. Your paper deals with looking at neurovascular control in humans. Two primary regulatory pathways are neurovascular coupling, or NVC, and dynamic cerebral autoregulation. Dr Phillips, can you explain what NVC to our audience, and what does dysregulation lead to?
Aaron Phillips: Yeah, thanks Nicole and I'm happy to be here. Thank you for the invitation. NVC, or neurovascular coupling, we've been studying it for about 15 years. At its fundamental level, it's kind of this elegant interplay between neurons, which unfortunately have very limited capacity for substrate storage. The brain has very limited substrate storage capacity, and so neurons need to very rapidly match their metabolic activity to the blood flow that's being delivered to them, and that needs to happen locally, for areas of the brain that have greater metabolic needs as opposed to other areas.
What happens, in terms of dysregulation or conditions that are associated with dysregulation, it's an interesting story because we still really need to understand the mechanisms fully, in order to suss out what clinical conditions should have dysfunction of this unit. We know that certain conditions, such as vascular cognitive impairment, even spinal cord injury, we've done some work in stroke patients, it seems to be dysfunctional in all of these conditions, but understanding exactly why it's dysfunctional, we're still establishing that.
Nicole Purcell: Great. You were talking about how it's the connection or interplay between blood flow, so we're talking about altered blood pressure seems to play a key role in neurovascular coupling. So, for those listeners not familiar with this field, can you explain how nitric oxide synthase and its isoforms, how this relates to NVC?
Aaron Phillips: Well, nitric oxide synthase is an enzyme that produces nitric oxide that's expressed primarily in neurons. Nitric oxide is a powerful vasodilator. It actually works on quite a rapid time course. So, we surmised, we suspected, and there were some preclinical work before our human study, that neuronal sources of nitric oxide, being that nitric oxide is a potent vasodilator, we thought that would be likely to be mediating a large part of the neurovascular coupling response.
Nicole Purcell: Great. So, Dr O'Gallagher, based on that, what was your main objective or hypothesis of this study, and how is your study novel from those that have already just suggested, looked at NOS regulation for cerebral blood flow?
Kevin O'Gallagher: Thanks very much for the invite to talk. I mean, we hypothesized that nNOS would have a role in regulating neurovascular coupling. I think the novelty of our study is that although people have been interested in NOS and its regulation of cerebral vascular and cardiovascular blood flow, it's only relatively recently that there has become an agent available that will specifically inhibit nNOS, and therefore give us an idea of what it is doing, rather than previous inhibitors which just inhibit all of the three NOS isoforms. It was really that the development of the agent was what allowed us to do this study. I think it was really through that, that makes this an interesting finding that nNOS does play a role in neurovascular coupling, and really pushes the field forward ever so slightly.
Nicole Purcell: Great. So, as you pointed out, this is a specific nNOS inhibitor, which is known as SMTC. It's a synthetic L-Arginine analog, right? That's really what sets your study apart. Can you tell us a little bit the audience, whether that be you, Dr Phillips or Dr O'Gallagher, about what your study was and what did you find, and how did an ambition of using this SMTC to inhibit nNOS affect systemic hemodynamic changes and NVC?
Aaron Phillips: Yeah, I think both of us can probably speak to this interchangeably and add in different elements of the experiment. This is kind of a summary of the study, I guess. In advance of this, adding on what Kevin had just said in terms of the novelty of the study and the importance, we had done a lot of work previous to this paper where we were one of the groups that helped establish neurovascular coupling as a measure that could be tested in humans. This involved kind of understanding metabolism of the eye, how that's coupled to the visual cortex, and how to measure blood flow on a high temporal resolution in the visual cortex in response to visual input. That's why we used very well standardized perturbations involving tracking an eye, tracking a dot on a screen at a known one rate and a known one amplitude of movement, while also measuring the hyperemic response in the posterior brain.
Then we kind of went on and developed some new measures, developed some software that we're now proud is used in a few different labs around the world, that kind of automatically takes that input of repetitive eyes opening and closing and that hyperemic response, and it breaks it down into a single wave form. A single hyperemic response is superimposed of 10, 15, 20 cycles of those eyes open and eyes closed, and then when we superimpose all the wave forms together, we can generate different metrics from that hyperemic response that correspond to different elements.
One of the ways where software can, I guess dice out the hyperemic response, is by timing. We can look at very specific unique time windows over that 30 seconds of eyes open, and we can also look at the slope of the response, as well as we recently did some dimensionality reduction techniques and looked at specific computed measures of that hyperemic response. We published that a few years ago. Those were some of the tools that enabled this study, along with a fantastically unique drug that really could isolate that neuron expression of NOS and the capacity of nNOS to mediate neurovascular coupling.
Kevin O'Gallagher: Obviously, we're going to use a systemic infusion of SMTC, the study drug, and we've used that before and shown it to be safe. But because a systemic infusion of SMTC through peripheral and systemic nNOS inhibition does cause an increase in systemic vascular resistance, and therefore an increase in mean arterial pressure of around about 7 mm of mercury, in addition to a cline placebo control condition, we also felt the need to have a pressure control condition. For that, we used phenylephrine to match the rise in mean arterial pressure that we anticipated we'd see with SMTC. We ended up with 12 healthy volunteers who attended on three separate visits, and so we had a party randomized double blinded intervention study where we measured the neurovascular coupling metrics, both before and after an infusion of one of the three conditions on each particular visit.
Aaron Phillips: I just wanted to add into that, we had found previously that mean arterial pressure does have an effect on the hyperemic response. This was actually classically found by 1960s by Harper and Glass in a dog study, but we've repeated that in humans and kind of found that the ability of the brain to kind of... It's reserve for further vasodilation is dependent on pressure. As you drop it, neurovascular coupling will go away, and as you increase it, neurovascular coupling will increase partially, so it's important to standardize the mean arterial pressure levels. I always liken it to your water pressure in your house. You can't turn on a faucet with a given pressure unless you have that in the system upstream. That was a really important aspect of the study.
Nicole Purcell: That was quite unique for your study, too. Not a lot of people have control for pressure.
Aaron Phillips: Correct.
Kevin O'Gallagher: I think it reflects the challenges of these healthy volunteer studies where you're trying to look at one particular part of the cardiovascular system, because as a cardiologist, if we were doing a study like this, looking at cardiovascular regulation, we would put a catheter into the coronary arteries in patients who had come for angiograms, and we'd give a local infusion of SMTC, as we've done in studies before. But with healthy volunteers, and ethically it really demanded a systemic infusion, so it was a really nice workaround to have that pressure control condition.
Nicole Purcell: So, can you tell us a little bit about what your findings were?
Kevin O'Gallagher: I think testament to the study design and the rigorous methodology that we employed, we did find with the resting steady state hemodynamics that SMTC condition performed as we would expect, and as we've seen in prior studies where we've given a systemic dose in that compared to both placebo and pressure control conditions, SMTC decreased cardiac output, and it decreased stroke volume, and also increased systemic vascular resistance, so very much as expected the resting hemodynamic conditions.
Aaron Phillips: Yeah, thanks. Just adding onto that, moving on into some of the cerebral vascular measures. So again, we were measuring posterior cerebral artery velocity, blood velocity and specific responsiveness that it has to a visual stimuli. Between conditions, we didn't see a change in resting posterior cerebral artery velocity, so that was consistent between the conditions. Where we saw most of our change actually was in this very early period, the first five seconds of what we're going to call the hyperemic response, or the first five seconds of the neurovascular coupling response. That's where we saw our primary effect. We didn't see an effect in almost any of the neurovascular coupling measures that we generated in the actual sustained period after that initial rise, so that's where we saw our key inhibition with nNOS inhibition. What permitted that was the phenylephrine control group, again, allowing us to really look at apples and apples, not apples and oranges.
Nicole Purcell: Great. So that early transient change that you saw, that as you said, hyperemic response, what therapeutic implications does this have for the field?
Kevin O'Gallagher: Well, certainly there are conditions in which nNOS dysfunction, nNOS may be implicated, we mentioned a couple in the paper, some neurodegenerative diseases. But also, I think the field is now open for any vascular mediated headache syndrome, such as migraine, to investigate the potential role of nNOS from that angle. Then we haven't touched on already, but as well as dysfunctional, so decreased nNOS activity, there's also some conditions in which there's dysregulation or abnormally increased nNOS function. Again, we've highlighted this kind of study methodology is a tool that could be used to investigate those types of conditions.
Aaron Phillips: These are all terrific points, and I think there's a lot of conditions where neurovascular coupling is impaired, and it's worth exploring them and understanding the specific role where nNOS might be a part of it. I also think there's a lot of interesting basic science surrounding this, in terms of the mechanisms. What was really interesting in this study, which is still kind of wracking my brain, is why didn't more of the neurovascular coupling response go away? This is a highly selective inhibitor for what was potentially thought by some groups to be a large mediator, this response. It was a relatively small inhibitory effect, and isolated to a small part of the neurovascular coupling response, just that early phase. So, still lots of work to do to kind of dice out the other pathways. They're probably highly redundant. This is such a critical mechanism in the central nervous system. Getting at it and humans is going to be tricky, but we're excited about the future and exploring some of those other avenues on the mechanistic cascade.
Nicole Purcell: Based on the fact that you just had 12 healthy individuals, what do you see as some of the limitations of your study going forward, thinking about what you did?
Kevin O'Gallagher: I think you've just hit on a key limitation. It was a small number of volunteers. They were all healthy, so we can't extrapolate these findings to conditions such as hypertension, where we know from other studies that cardiovascular responses, nNOS responses are impaired Also, this was a noninvasive study. We looked at the blood flow through Doppler, but we don't really know the effect of SMTC on cerebral artery diameter or other markers like that, so I think those are important limitations to mention.
Nicole Purcell: I know I didn't ask this, and I know it was mentioned in the paper, but for our audience, and it was a small sample size, but did you see any sex differences between your male and female cohort?
Kevin O'Gallagher: No. We did analyze for that and there were no sex differences. But again, it's an important limitation in that we didn't control for things like phase of the menstrual cycle. And again, with those limitations, all the results should be interpreted with those in mind.
Nicole Purcell: Were there any challenges to the study that you found?
Kevin O'Gallagher: I work in London in the UK, where we performed this study related protocols, and Professor Phillips from University of Calgary, his team flew over to perform the studies. I think there was a real organizational challenge because we had a relatively small time window in which to get all of the volunteers and their three study visits done. But I think it's testament to just how well Professor Phillips runs his team, and how fantastic a team they are in working together that all of those challenges were minimized and everything. It ran fairly smoothly, and certainly, the data was connected back in early 2020. I think we all retrospectively breathed a sigh of relief when the Covid pandemic started and we realized that had we had to reschedule another set of visits, we would've then knocked the study back a couple of years. So yeah, there were organizational challenges, but it was an absolute pleasure to work with Professor Phillips's and his team in this.
Aaron Phillips: To add to that, I mean, it's not really related to necessarily the challenges, but I was going to list kind of the exact same thing. In the background. Kevin, and Professor Shaw, and Dr Gallagher were a tour de force on organizing quite a complicated study that involves some invasive protocols and unique experimental drug infusion. Getting all of that ethically approved, and organized, and structured, that was probably one of the biggest challenges of pulling this study off.
Nicole Purcell: Great. It was a very nice study. So lastly, what future studies are needed or have come out of this work that you'd like to tell us about?
Aaron Phillips: Mechanistically, I would still like to explore why nNOS inhibition doesn't seem to affect the sustained elevation in blood flow. This maybe means going back to some of the astrocyte mediated mechanisms, and understanding knocking out, knocking in, exploring some of those. I'd also like to continue to study the neurovascular cupping response itself in clinical conditions. This may be a tool for helping to characterize the severity of a given neurovascular condition over time, and kind of validating this outcome measure as potentially a clinical tool and further expanding its research application.
Kevin O'Gallagher: I would just add to that, that I tend to come to all of these things from a cardiologist light, and there are some conditions in cardiology where the microvascular is involved, and so the interest is then to see whether there's a linkage between the dysfunctional coronary microvascular responses with then cerebral microvascular responses. So again, I think there's plenty of future work to be done in that sphere.
Nicole Purcell: Well, I want to thank you so much for joining me today, Dr Kevin O'Gallagher and Dr Aaron Phillips, for discussing your exciting findings with me today, and I look forward to seeing your future work. Thank you.
Aaron Phillips: Thank you so much.
Kevin O'Gallagher: Thank you so much.
Nicole Purcell: That's it for highlights from the December 2 issue of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Drs Aaron Phillips and Kevin O'Gallagher. This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles provided by Ruth Williams.
I am your host, Dr Nicole Purcell, filling in for Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more, visit ahajournals.org.
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This month on Episode 42 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the October 28 and November 11th issues of Circulation Research. This episode also features an interview with Dr Miguel Lopez-Ramirez and undergraduate student Bliss Nelson from University of California San Diego about their study, Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformations.
Article highlights:
Jia, et al. Prohibitin2 Maintains VSMC Contractile Phenotype
Rammah, et al. PPARg and Non-Canonical NOTCH Signaling in the OFT
Wang, et al. Histone Lactylation in Myocardial Infarction
Katsuki, et al. PCSK9 Promotes Vein Graft Lesion Development
Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today, I'm going to be highlighting articles from our October 28th and our November 11th issues of Circ Res. I'm also going to have a chat with Dr Miguel Lopez-Ramirez and undergraduate student Bliss Nelson, about their study, Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformations. But, before I get into the interviews, here are a few article highlights.
Cindy St. Hilaire: The first article is from our October 28th issue, and the title is, PHB2 Maintains the Contractile Phenotype of Smooth Muscle Cells by Counteracting PKM Splicing. The corresponding author is Wei Kong, and the first authors are Yiting Jia and Chengfeng Mao, and they are all from Peking University. Insults to blood vessels, whether in the form of atherosclerosis, physical injury, or inflammation, can trigger vascular smooth muscle cells to transition from a contractile state to a proliferative and migratory one. Accompanying this conversion is a switch in the cells' metabolism from the mitochondria to glycolysis. But what controls this switch? To investigate, this group compared the transcriptomes of contractile and proliferative smooth muscle cells.
Among the differentially expressed genes, more than 1800 were reciprocally up and down regulated. Of those, six were associated with glucose metabolism, including one called Prohibitin-2, or PHB2, which the team showed localized to the artery wall. In cultured smooth muscle cells, suppression of PHB2 reduced expression of several contractile genes. While in rat arteries, injury caused a decrease in production of PHB2 itself, and of contractile markers.
Furthermore, expression of PHB2 in proliferative smooth muscle cells could revert these cells to a contractile phenotype. Further experiments revealed PHB2 controlled the splicing of the metabolic enzyme to up-regulate the phenotypic switch. Regardless of mechanism, the results suggest that boosting PHB2 might be a way to reduce adverse smooth muscle cell overgrowth and conditions such as atherosclerosis and restenosis.
Cindy St. Hilaire: The second article I'm going to highlight is also from our October 28th issue, and the first authors are Mayassa Rammah and Magali Theveniau-Ruissy. And the corresponding authors are Francesca Rochais and Robert Kelly. And they are all from Marseille University. Abnormal development of the heart's outflow track, which ultimately forms the bases of the aorta and the pulmonary artery, accounts for more than 30% of all human congenital heart defects. To gain a better understanding of outflow tract development, and thus the origins of such defects, this group investigated the role of transcription factors thought to be involved in specifying the superior outflow tract, or SOFT, which gives rise to the subaortic myocardium, and the inferior outflow tract, which gives rise to the subpulmonary myocardium. Transcription factor S1 is over-expressed in superior outflow tract cells and the transcription factors, TBX1 and PPAR gamma, are expressed in inferior outflow tract cells.
And now this group has shown that TBX1 drives PPAR gamma expression in the inferior outflow tract, while Hess-1 surpasses PPAR gamma expression in the superior outflow tract. Indeed, in mouse embryos lacking TBX1, PPAR gamma expression was absent in the outflow tract. While in mouse embryos lacking Hess-1, PPAR gamma expression was increased and PPAR gamma positive cells were more widespread in the outflow tract.
The team also identified that signaling kinase DLK is an upstream activator of Hess-1 and a suppressor of PPAR gamma. In further detailing the molecular interplay regulating outflow tract patterning, the work will shed light on congenital heart disease etiologies, and inform potential interventions for future therapies.
Cindy St. Hilaire: The third article I want to highlight is from our November 11th issue of Circulation Research, and the title is Histone Lactylation Boosts Reparative Gene Activation Post Myocardial Infarction. The first author is Jinjin Wang and the corresponding author is Maomao Zhang, and they're from Harbin Medical University. Lactylation of histones is a recently discovered epigenetic modification that regulates gene expression in a variety of biological processes. In inflammation, for example, a significant increase in histone lactylation is responsible for switching on reparative genes and macrophages when pro-inflammatory processes give way to pro-resolvin ones.
The role of histone lactylation in inflammation resolution has been shown in a variety of pathologies, but has not been examined in myocardial infarction. Wang and colleagues have now done just that. They isolated monocytes from the bone marrow and the circulation of mice at various time points after induced myocardial infarctions, and examined the cells' gene expression patterns. Within a day of myocardial infarction, monocytes from both bone marrow and the blood had begun upregulating genes involved in inflammation resolution. And, concordant with this, histone lactylation was dramatically increased in the cells, specifically at genes involved in repair processes.
The team went on to show that injection of sodium lactate into mice boosted monocyte histone lactylation and improved heart function after myocardial infarction, findings that suggest further studies of lactylation's pro-resolving benefits are warranted.
Cindy St. Hilaire: The last article I want to highlight is titled, PCSK9 Promotes Macrophage Activation via LDL Receptor Independent Mechanisms. The first authors are Shunsuke Katsuki and Prabhash Kumar Jha, and the corresponding author is Masanori Aikawa, and they are from Brigham and Women's Hospital in Harvard. Statins are the go-to drug for lowering cholesterol in atherosclerosis patients. But the more recently approved PCSK9 inhibitors also lower cholesterol and can be used to augment or replace statins in patients where these drugs are insufficient.
PCSK9 is an enzyme that circulates in the blood and destroys the LDL receptor, thereby impeding the removal of bad cholesterol. The enzyme also appears to promote inflammation, thus potentially contributing to atherosclerosis in two ways. This group now confirms that PCSK9 does indeed promote pro-inflammatory macrophage activation and lesion development, and does so independent of its actions on the LDL receptor.
The team assessed PCSK9-induced lesions in animals with saphenous vein grafts, which are commonly used in bypass surgery but are prone to lesion regrowth. They found that LDL receptor lacking graft containing mice had greater graft macrophage accumulation and lesion development when PCSK9 activity was boosted than when it was not. The animal's macrophages also had higher levels of the pro-inflammatory factor expression. Together, this work shows that PCSK9 inhibitors provide a double punch against atherosclerosis and might be effective drugs for preventing the all too common failure of saphenous vein grafts.
Cindy St. Hilaire: So, today with me I have Dr Miguel Lopez-Ramirez and undergraduate student Bliss Nelson from the University of California in San Diego, and we're going to talk about their study, Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformation Disease, and this article is in our November 11th issue of Circulation Research. Thank you both so much for joining me today. Before we talk about the science, want to just maybe tell me a little bit about yourselves?
Bliss Nelson: My name is Bliss Nelson. I'm a member of Miguel Lopez-Ramirez's lab here at UC San Diego at the School of Medicine. I'm an undergraduate student here at UC San Diego. I'm actually a transfer student. I went to a community college here in California and I got involved in research after I transferred.
Cindy St. Hilaire: What's your major?
Bliss Nelson: I'm a cognitive science major.
Cindy St. Hilaire: Excellent. You might be the first undergrad on the podcast, which is exciting.
Bliss Nelson: Wow. What an honor. Thank so much.
Cindy St. Hilaire: And Miguel, how about you?
Miguel Lopez-Ramirez: Yes, thank you. Well, first thank you very much for the opportunity to present our work through this media. It's very exciting for us. My name is Miguel Alejandro Lopez-Ramirez, and I'm an assistant professor in the Department of Medicine and Pharmacology here at UCSD.
Cindy St. Hilaire: Wonderful. I loved your paper, because, well, first, I don't think I've talked about cerebral cavernous malformations. So what are CCMs, and why are they so bad?
Bliss Nelson: Cerebral cavernous malformations, or CCMs for short, are common neurovascular lesions caused by a loss of function mutation in one of three genes. These genes are KRIT1, or CCM1, CCM2 and PDCD10, or CCM3, and generally regarded as an endothelial cell autonomous disease found in the central nervous system, so the brain and the spinal cord.
The relevance of CCMs is that it affects about one in every 200 children and adults,
and this causes a lifelong risk of chronic and acute hemorrhaging. CCMs can be
quiescent or dynamic lesions. If they are dynamic, they can enlarge, regress, or
behave progressively, producing repetitive hemorrhaging and exacerbations of the
disease.
Other side effects of the disease could be chronic bleedings, focal neurological deficits, headaches, epileptic seizures and, in some cases, death. There's no pharmacological treatment for CCMs. There's only one type of option some patients may have, which would be to have surgery to cut out the lesions. But of course this depends on where the lesion or lesions are in the central nervous system, if that's even an option. So sometimes there's no option these patients have, there's no treatment, which is what propels our lab to towards finding a pharmacological treatment or uncovering some of the mechanisms behind that.
Cindy St. Hilaire: Do people who have CCM know that they have them or sometimes it not detected? And when it is detected, what are the symptoms?
Bliss Nelson: Sometimes patients who have them may not show any symptoms either ever in their lifetime or until a certain point, so really the only way to find out if you were to have them is if you went to go get a brain scan, if you went to go see a doctor, or if you started having symptoms. But also, one of the issues with CCMs is that they're very hard to diagnose, and in the medical community there's a lack of knowledge for CCMs, so sometimes you may not get directed to the right specialist in time, or even ever, and be diagnosed.
Miguel Lopez-Ramirez: I will just add a little bit. It is fabulous, what you're doing. I think this is very, very good. But yes, that's why they're considered rare disease, because it's not obvious disease, so sometimes most of the patient, they go asymptomatic even when they have one lesions, but there's still no answers of why patients that are asymptomatics can become symptomatics. And there is a lot in neuro study, this study that we will start mentioning a little bit more in detail. We try to explain these transitions from silent or, quiescent, lesion, into a more active lesion that gives the disability to the patient.
Some of the symptoms, it can start even with headaches, or, in some cases, they have more neurological deficits that could be like weakness in the arms or loss of vision. In many cases also problems with the speech or balance. So it depends where the lesion is present, in the brain or in the spinal cord, the symptoms that the patient will experience. And some of the most, I will say, severe symptoms is the hemorrhagic stroke and the vascular thrombosis and seizure that the patients can present. Those would be the most significant symptoms that the patient will experience.
Cindy St. Hilaire: What have been some limitations in the study of CCMs? What have been limitations in trying to figure out what's going on here?
Bliss Nelson: The limitations to the disease is that, well, one, the propensity for lesions, or the disease, to come about, isn't known, so a lot of the labs that work on it, just going down to the basic building blocks of what's even happening in the disease is a major problem, because until that's well established, it's really hard to go over to the pharmacological side of treating the disease or helping patients with the disease, without knowing what's going on at the molecular level.
Cindy St. Hilaire: You just mentioned molecular level. Maybe let's take a step back. What's actually going on at the cellular level in CCMs? What are the major cell types that are not happy, that shift and become unhappy cells? Which are the key players?
Bliss Nelson: That's a great question and a great part of this paper. So when we're talking about the neuroinflammation in the disease, our paper, we're reporting the interactions between the endothelium, the astrocytes, leukocytes, microglia and neutrophils, and we've actually coined this term as the CaLM interaction.
Cindy St. Hilaire: Great name, by the way.
Bliss Nelson: Thank you. All props to Miguel. And if you look at our paper, in figure seven we actually have a great graphic that's showing this interaction in play, showing the different components happening and the different cell types involved in the CaLM interaction that's happening within or around the CCM lesions.
Cindy St. Hilaire: What does a astrocyte normally do? I think our podcast listening base is definitely well versed in probably endothelial and smooth muscle cell and pericyte, but not many of us, not going to lie, including me, really know what a astrocyte does. So what does that cell do and why do we care about its interaction with the endothelium?
Miguel Lopez-Ramirez: Well, the astrocytes play a very important role. Actually, there are more astrocytes than any other cells in the central nervous system, so that can tell you how important they are. Obviously play a very important role maintaining the neurological synapses, maintaining also the hemostasis of the central nervous system by supporting not only the neurons during the neural communication, but also by supporting the blood vessels of the brain.
All this is telling us that also another important role is the inflammation, or the response to damage. So in this case, what also this study proposed, is that new signature for these reactive astrocytes during cerebral malformation disease. So understanding better how the vasculature with malformations can activate the astrocytes, and how the astrocytes can contribute back to these developing of malformations. It will teach us a lot of how new therapeutic targets can be implemented for the disease.
This is part of this work, and now we extend it to see how it can also contribute to the communication with immune cells as Bliss already mentioned.
Cindy St. Hilaire: Is it a fair analogy to say that a astrocyte is more similar to a pericyte in the periphery? Is that accurate?
Miguel Lopez-Ramirez: No, actually there are pericytes in the central nervous system as well. They have
different roles. The pericyte is still a neuron cell that give the shape, plays a role in the contractility and maintains the integrity of the vessels, while the astrocyte is more like part of the immune system, but also part of the supporting of growth factors or maintaining if something leaks out of the vasculature to be able to capture that.
Cindy St. Hilaire: You used a handful of really interesting mouse models to conduct this study. Can you tell us a little bit about, I guess, the base model for CCM and then some of the unique tools that you used to study the cells specifically?
Bliss Nelson: Yeah, of course. I do a lot of the animal work in the lab. I'd love to tell you about the mouse model. So to this study we use the animal model with CCM3 mutation. We use this one because it is the most aggressive form of CCM and it really gives us a wide range of options to study the disease super intricately. We use tamoxifen-regulated Cre recombinase under the control of brain endothelial specific promoter, driving the silencing of the gene CCM3, which we call the PDCD10 betco animal, as you can see in our manuscript. To this, the animal without the Cre system, that does not develop any lesions, that we use as a control, we call the PDCD10 plox. And these animals are injected with the tamoxifen postnatally day one, and then for brain collection to investigate, wcollected at different stages. So we do P15, which we call the acute stage, P50, which we term the progressive stage, and then P80, which is the chronocytes stage. And after enough brain collections, we use them for histology, gene expression, RNA analysis, flow cytometry, and different imaging to help us further look into CCMs.
Cindy St. Hilaire: How similar is a murine CCM to a human CCM? Is there really good overlap or are there some differences?
Miguel Lopez-Ramirez: Yes. So, actually, that's a very good question, and that's part of the work that we are doing. This model definitely has advantages in which the lesions of the vascular formations are in an adult and juvenile animals, which represent an advantage for the field in which now we will be able to test pharmacological therapies in a more meaningful, way where we can test different doses, different, again, approaches. But definitely, I mean, I think I cannot say that it's only one perfect model for to mimic the human disease. It's the complementary of multiple models that give us certain advantages in another, so the integration of this knowledge is what will help us to understand better the disease.
Cindy St. Hilaire: That's great. I now want to hear a little bit about your findings, because they're really cool. So you took two approaches to study this, and the first was looking at the astrocytes and how they become these, what you're calling reactive astrocytes, and then you look specifically at the brain endothelium. So could you maybe just summarize those two big findings for us?
Miguel Lopez-Ramirez: Yeah, so, basically by doing these studies we use trangenic animal in this case that they give us the visibility to obtain the transcripts in the astrocytes. And basically this is very important because we don't need to isolate the cells, we don't need to manipulate anything, we just took all the ribosomes that were basically capturing the mRNAs and we profile those RNAs that are specifically expressed in the astrocytes.
By doing this, we actually went into looking at in depth the transcripts that were altered in the animals that developed the disease, in this case the cerebral cavernous malformation disease, and what we look at is multiple genes that were changing. Many of them were already described in our previous work, which were associated with hypoxia and angiogenesis. But what we found in this work is that now there were a lot of genes associated with inflammation and coagulation actually, which were not identified before.
What we notice is that now these astrocytes, during the initial phase of the vascular malformation, may play a more important role in angiogenesis or the degradation of the vessels. Later during the stage of the malformation, they play a more important role in the thrombosis, in the inflammation, and recruitment of leukocyte
That was a great advantage in this work by using this approach and looking in detail, these astrocytes. Also, we identified there were very important signature in these astrocytes that we refer as a reactive astrocytes with neuroinflammatory properties. In the same animals, basically, not in the same animal, but in the same basically the experimental approach, we isolated brain vasculature. And by doing the same, we actually identified not only the astrocyte but also the endothelium was quite a different pattern that we were not seeing before. And this pattern was also associated with inflammation, hypoxia and coagulation pathways.
That lead us to go into more detail of what was relevant in this vascular malformations. And one additional part that in the paper this is novel and very impactful, is that we identify inflammasome as a one important component, and particularly in those lesions that are multi-cavernous.
Now we have two different approaches. One, we see this temporality in which the lesions forms different patterns in which the initial phase maybe is more aneugenic, but as they become more progressive in chronocytes, inflammation and hypoxy pathways are more relevant for the recruitment of the inflammatory cells and also the precipitation of immunothrombosis.
But also what we notice is that inflammasome in endothelial and in the leukocytes may play an important role in the multi-cavernous formation, and that's something that we are looking in more detail, if therapeutics or also interventions in these pathways could ameliorate the transition of phases between single lesions into a more aggressive lesions.
Cindy St. Hilaire: That's kind of one of the follow up questions I was thinking about too is, from looking at the data that you have, obviously to get a CCM, there's a physical issue in the vessel, right? It's not formed properly. Does that form influence the activation of the astrocyte, and then the astrocytes, I guess, secrete inflammatory factors, target more inflammation in the vessel? Or is there something coming from the CCM initially that's then activating the astrocyte? It's kind of a chicken and the egg question, but do you have a sense of secondary to the malformation, what is the initial trigger?
Miguel Lopez-Ramirez: The malformations in our model, and this is important in our model, definitely start by producing changes in the brain endothelial. And as you mention it, these endothelium start secreting molecules that actually directly affect the neighboring cells.
One of the first neighboring cells that at least we have identified to be affected is the astrocytes, but clearly could be also pericytes or other cells that are in the neurovascular unit or form part of the neurovascular unit. But what we have seen now is that this interaction gets extended into more robust interactions that what you were referring as the CaLM interactions.
Definitely I think during the vascular malformations maybe is the discommunication that we identify already few of those very strong iteration that is part of the follow up manuscript that we have. But also it could be the blood brain barrier breakdown and other changes in the endothelium could also trigger the activation of the astrocytes and brain cells.
Cindy St. Hilaire: What does your data suggest about potential future therapies of CCM? I know you have a really intriguing statement or data that showed targeting NF-kappa B isn't likely going to be a good therapeutic strategy. So maybe tell us just a little bit about that, but also, what does that imply, perhaps, of what a therapeutic strategy could be?
Bliss Nelson: Originally we did think that the inhibition of NF-kappa B would cause an improvement potentially downstream of the CCMs. And unexpectedly, to our surprise, the partial or total loss of the brain endothelial NF-kappa B activity in the chronic model of the mice, it didn't prevent or cause any improvement in the lesion genesis or neuroinflammation, but instead it resulted in a trend to increase the number of lesions and immunothrombosis, suggesting that the inhibition of it is actually worsening the disease and shouldn't be used as a target for therapeutical approaches.
Miguel Lopez-Ramirez: Yes, particularly that's also part of the work that we have ongoing in which NF-kappa B may also play a role in preventing the further increase of inflammation. So that is something that it can also be very important. And this is very particular for certain cell types. It's very little known what the NF-kappa B actually is doing in the brain endothelial during malformations or inflammation per se. So now it's telling us that this is something that we have to consider for the future.
Also, our future therapeutics of what we propose are two main therapeutic targets. One is the harmful hypoxia pathway, which involves activation, again, of the population pathway inflammation, but also the inflammasomes. So these two venues are part of our ongoing work in trying to see if we have a way to target with a more safe and basically efficient way this inflammation.
However, knowing the mechanisms of how these neuroinflammation take place is what is the key for understanding the disease. And maybe even that inflammatory and inflammatory compounds may not be the direct therapeutic approach, but by understanding these mechanisms, we may come with new approaches that will help for safe and effective therapies.
Cindy St. Hilaire: What was the most challenging part of this study? I'm going to guess it has something to do with the mice, but in terms of collecting the data or figure out what's going on, what was the most challenging?
Bliss Nelson: To this, I'd like to say that I think our team is very strong. We work very well together, so I think even the most challenging part of completing this paper wasn't so challenging because we have a really strong support system among ourselves, with Miguel as a great mentor. And then there's also two postdocs in the lab who are also first authors that contributed a lot to it.
Cindy St. Hilaire: Great. Well, I just want to commend both of you on an amazing, beautiful story. I loved a lot of the imaging in it, really well done, very technically challenging, I think, pulling out these specific sets of cells and investigating what's happening in them. Really well done study. And Bliss, as an undergraduate student, quite an impressive amount of work. And I congratulate both you and your team on such a wonderful story.
Bliss Nelson: Thank you very much.
Miguel Lopez-Ramirez: Thank you for Bliss and also Elios and Edo and Katrine, who all contributed
enormously to the completion of this project.
Cindy St. Hilaire: It always takes a team.
Miguel Lopez-Ramirez: Yes.
Cindy St. Hilaire: Great. Well, thank you so much, and I can't wait to see what's next for this story.
Cindy St. Hilaire: That's it for the highlights from October 28th and November 11th issues of Circulation Research. Thank you so much for listening. Please check out the Circ Res Facebook page and follow us on Twitter and Instagram with the handle @circres and #discovercircres. Thank you to our guests, Dr Miguel Lopez-Ramirez and Bliss Nelson. This podcast is produced by Ashara Retniyaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for our highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahagenerals.org.
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This month on Episode 41 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the September 30 and October 14 issues of Circulation Research. This episode also features an interview with Dr Kory Lavine and Dr Chieh-Yu Lin from Washington University St. Louis, to discuss their study, Transcriptional and Immune Landscape of Cardiac Sarcoidosis.
Article highlights:
Tian, et al. EV-Mediated Heart Brain Communication in CHF
Wleklinski, et al. Impaired Dynamic SR Ca Buffering Causes AD-CPVT2
Masson, et al. Orai1 Inhibition as a Treatment for PAH
Li, et al. F. Prausnitzii Ameliorates Chronic Kidney Disease
Cindy St. Hilaire: Hi, and welcome to Discover Circ Res, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cynthia St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to highlight articles from our September 30th and October 14th issues of Circulation Research.
I'm also going to have a chat with Dr Kory Lavine and Dr Chieh-Yu Lin from Washington University St. Louis, and we're going to discuss their study Transcriptional and Immune Landscape of Cardiac Sarcoidosis. But before I get to the interview, I'm going to highlight a few articles.
Cindy St. Hilaire: The first article I'm going to share is Extracellular Vesicles Regulate Sympathoexcitation by Nrf2 in Heart Failure. The first author of this study is Changhai Tian, and the corresponding author is Irving Zucker, and they are at University of Nebraska. After a myocardial infarction, increased oxidative stress in the heart can contribute to adverse cardiac remodeling, and ultimately, heart failure. Nrf2 is a master activator of antioxidant genes, suggesting a protective role, but studies in rats have shown its expression to be suppressed after MI, likely due to upregulation of Nrf2-targeting microRNAs. These microRNAs can also be packaged into vesicles and released from stressed heart cells.
Now, this group has shown that rats and humans with chronic heart failure have an abundance of these microRNA-containing EVs in their blood. In the rats with chronic heart failure, these extracellular vesicles were found to be taken up by neurons of the rostral ventrolateral medulla, RVLM, wherein the microRNA suppressed Nrf2 expression. The RVLM is a brain region that controls the sympathetic nervous system, and in the presence of EVs, it is ramped up by sympathetic excitation. Because such elevated sympathetic activity can induce the fight or flight response, including increased heart rate and blood pressure, this would likely worsen heart failure progression. The team, however, found that inhibiting microRNAs in the extracellular vesicles prevented Nrf2 suppression in the RVLM and sympathetic activation, suggesting the pathway could be targeted therapeutically.
Cindy St. Hilaire: The next article I want to highlight is titled, Impaired Dynamic Sarcoplasmic Reticulum Calcium Buffering in Autosomal Dominant CPVT2. The first author of this study is Matthew Wleklinski, and the corresponding author is Bjӧrn Knollmann, and they are at Vanderbilt University.
Exercise or emotional stress can prompt the release of catecholamine hormones, which induce a fast heart rate, increased blood pressure, and other features of the fight or flight response. For people with catecholaminergic polymorphic ventricular tachycardia, or CPVT, physical activity or stress can cause potentially lethal arrhythmias. Mutations of calsequestrin-2, or CASQ2, which is a sarcoplasmic reticulum calcium-binding protein, is a major cause of CPVT, and can be recessive or dominant in nature.
For many recessive mutations, disease occurs due to loss of CASQ2 protein. This group investigated a dominant lysine to arginine mutation in this protein, and found by contrast, protein levels remain normal. In mice carrying the mutation, not only was the level of CASQ2 comparable to that in control animals, but so, too, was the protein's subcellular localization. The mutation instead interfered with CASQ2's calcium binding or buffering capability within the sarcoplasmic reticulum. The result was that upon catecholamine injection or exercise, the unbound calcium released prematurely from the sarcoplasmic reticulum, triggering spontaneous cell contractions. In uncovering this novel molecular etiology of CPVT, the work provides a basis for studying the consequences of other dominant CASQ2 mutations.
Cindy St. Hilaire: The next article I want to highlight is from our October 14th issue of Circulation Research, and the title of the article is ORAI1 Inhibitors as Potential Treatments for Pulmonary Arterial Hypertension. The first author is Bastien Masson, and the corresponding author is Fabrice Antigny, and they're from Inserm in France. In pulmonary arterial hypertension, the arteries of the lungs become progressively obstructed, making it harder for the heart to pump blood through them, ultimately leading to right ventricular hypertrophy and heart failure. A contributing factor in the molecular pathology of pulmonary arterial hypertension is abnormal calcium handling within the pulmonary artery smooth muscle cells. Indeed, excess calcium signaling causes these cells to proliferate, migrate, and become resistant to apoptotic death, thus leading to narrowing of the vessel.
This group now identified the calcium channel ORAI1 as a major culprit behind this excess signaling. Samples of lung tissue from pulmonary arterial hypertension patients and a pulmonary arterial hypertension rat model had significantly upregulated expression of this channel compared with controls. And in patient pulmonary arterial smooth muscle cells, the high ORAI1 levels resulted in heightened calcium influx, heightened proliferation, heightened migration and reduced apoptosis. Inhibition of ORAI1 reversed these effects. Furthermore, in pulmonary hypertension model rats, ORAI1 inhibition reduced right ventricle systolic pressure and attenuated right ventricle hypertrophy when compared with untreated controls. This study indicates that ORAI1 inhibitors could be a new potential target for treating this incurable condition.
Cindy St. Hilaire: The last article I want to share is titled Faecalibacterium Prausnitzii Attenuates CKD via Butyrate-Renal GPR43 Axis. The first author of this study is Hong-Bao Li, and the corresponding author is Tao Yang, and they are from the University of Toledo.
Progressive renal inflammation and fibrosis accompanied by hypertension are hallmarks of chronic kidney disease, which is an incurable condition affecting a significant chunk of the world's population. Studies indicate that chronic kidney disease is linked to gut dysbiosis. Specifically, depletion of lactobacillus bifidobacterium and faecalibacterium, prompting investigations into the use of probiotics. While supplements including lactobacillus and bifidobacterium have shown little effectiveness in chronic kidney disease, supplementations with F. prausnitzii have not been investigated.
Now, this group has shown in a mouse model of chronic kidney disease that oral administration of F. prausnitzii has beneficial effects on renal function, reducing renal fibrosis and inflammation. This bacterial supplementation also produced the short chain fatty acid butyrate, which was found to be at unusually low levels in the blood samples from the CKD model mice and from chronic kidney disease patients. Oral supplementation with this bacterium boosted butyrate levels in the mice, and in fact, oral administration of butyrate itself mimicked the effects of the bacteria. These findings suggest that supplementation with F. prausnitzii or, indeed, butyrate could be worth investigating as a treatment for chronic kidney disease.
Cindy St. Hilaire: Today I have with me Dr Kory Lavine and Dr Chieh-Yu Lin from Washington University St. Louis, and we're going to talk about their paper, Transcriptional and Immune Landscape of Cardiac Sarcoidosis. This is in our September 30th issue of Circulation Research. Welcome, and thank you for taking the time to speak with me today.
Chieh-Yu Lin: Thank you for inviting us. It's a great honor to be here today.
Kory Lavine: Thank you.
Cindy St. Hilaire: Really great paper, ton of data, and hopefully, we can pick some of it apart. But before we get into it, I actually want to just talk about sarcoidosis generally. I know it's a systemic inflammatory disease that has this kind of aggregation of immune cells as its culprit, and it can happen in a bunch of different organs. It's mostly in the lung, but it's also, like you're studying, in the heart. Can you just give us a little bit of background? What is sarcoidosis, and how common is cardiac sarcoidosis?
Chieh-Yu Lin: Well, this is actually a great question, and I'll try to answer it. You actually capture one of the most important kind of features for sarcoidosis. It happens in all kind of organ system, mostly commonly in lung, in lymph nodes, but also in heart, spleen, even in brain, or even orbit, like eyes. It's really a truly multisystemic disease that has been characterized by this aggregate of macrophages, or myeloid cells, with scattered multinucleated giant cells, as the name implies, have multiple nuclear big, chunky, cells that form an aggregate. That's kind of like a pathognomonic feature for sarcoidosis, whether it's happening in lung, in the heart. When any organ system, a lot of studies has been done, but as of now, a very clear pathogenesis or mechanism has been, I would say, still pretty elusive, or still remain quite unclear, despite all the great effort has been made in this field.
The other thing is that a lot of the studies actually focusing on pulmonary sarcoidosis for good reasons. Actually, that's one of the most common manifestations. For cardiac sarcoidosis, although it's only effect in probably, I would say depends on the data, 20% to 30% of the outpatient that with sarcoidosis, with or without lung involvement. It's actually carry a very significant clinical implications as of matter that the presentation of cardiac sarcoidosis can be devastating and sometimes actually fatal. Some of the study actually show that cardiac sarcoidosis actually higher, up to 80%, just because the first presentation's actually, unfortunately, sudden cardiac death. That's why Kory and I, we teamed up. I'm a cardiothoracic pathologist, so in my clinical practice I see specimens and samples from human body, from patient suffer from sarcoidosis, both in lung, lymph node, and heart. Kory is an outstanding heart failure, heart transplant cardiologist, see the other end, which is the patient care. This disease, specifically in heart, its presentation and its pathogens in heart, really attracts our attention.
Cindy St. Hilaire: Do we know any or some of the potential causes? Why it would start, maybe in a different patient population, but also in the heart versus the lung? Do we know anything about that process?
Kory Lavine: We know nothing about it. Sarcoid has no known etiology. There's been thoughts in the past that it may be driven by infection, the typical pathogens or autoimmune ideologies, but really, there's little data out there to support those possibilities. Right now, the field's wide open. The other challenge is we don't really have a good way to treat this disease, so a lot of the therapies available are things like steroids, which can have some effect on the disease but carry a lot of risk of complications. The other agents that we sometimes use to lower the doses of steroids, things like methotrexate and azathioprine, are only modestly effective.
These are really the motivation for Chieh-Yu and myself to pursue this. We don't really know what causes the disease, and we don't really have very good treatments. We really wanted to take the first step, that's to study the real disease, and understand what are the pathologic cell types that are present within the granuloma, which is these aggregation of immune cells that Chieh-Yu was speaking about.
Cindy St. Hilaire: What is actually happening at the beginning of this disease? These granulomas form, and then what is the pathological progression in the heart? What goes on there?
Chieh-Yu Lin: This is actually another great question that I will say there's not much that has been discovered because, especially in human tissue, every time we have a sample, it's actually a kind of time point. We cannot do a longitudinal study. But in general speaking, very little is known about how it's initiated because it will need to accumulate to a certain disease burden for this to have a clinical symptom sign and be manifested, and then being clinically studied. We do know that in both heart and lung after treatment of progressions, it's usually in, a general speaking, going through a phase from a more proliferative means that it's creating more granulomas, more inflammatory cell aggregate, to a more fibrotic phase. Means that sometimes you actually see the granuloma start to disappear or dissipate, and then showing this kind of dense collagen and fibrosis. That has been commonly documented in both lung and heart sarcoidosis.
The other things is that very difficult to study this disease that we do not have a great animal model, so we cannot use animal model to try to approximate or really study the disease pathogenesis. There are several animal models they try to use microbacteria or infectious agents, and these infectious agents can create morphologically similar granuloma, per se, but just like in human body. For instance, patients suffer from TB in their lung, biopsy will show this. But clinically, these are two very distinct disease entities, even though they look alike. Even in the heart, one of the conditions that we study in our paper is giant cell myocarditis, as the name implying having multinucleated giant cells granuloma. It looks really alike under microscopy for pathologists like me, but their clinical course in response to treatment is drastically different. This type of barriers and in the current limitations of our study tool makes, as Kory just said, this is really a wide open. We just know so little despite all the effort.
Cindy St. Hilaire: Yeah. I'm guessing based on this granuloma information, to start with, the obvious question you went after is going after the immune cell populations that possibly contribute to sarcoidosis. To do this, because you have the human tissue, you went for single cell transcriptional profiling, which is a great use of the technology. But what biological sources did you use, and how did you go about choosing patient? Because the great thing about single cell is you can do just that, you can look at however many thousands of cells in one patient. But how do you make sure or check that that is broadly seen versus just a co-founding observation in that patient?
Kory Lavine: We use explanted hearts and heart tissue from patients that underwent either heart transplantation or implementation of LVADs. It's a pretty big hunk of myocardium, and we're lucky to work with outstanding pathologists both at WashU, JU, as well as our collaborators at Duke. Between the two institutions, we're able to pull together a collection of tissues where we knew there were granulomas within that piece of tissue we analyzed. You bring up an important challenge. You need to make sure the disease and cause of the disease is present in the tissue that you're analyzing, otherwise you'll not come up with the data that really is informative.
Chieh-Yu Lin: Kory beautifully answered the question, but I just wanted to add one little thing, and that's also why we use various different modalities. Some of them is more inside you, like the NanoString Technologies' spatial transcriptomic. You can visualize and confirm that we are studying the phenomenon that has been described for sarcoidosis, and then using multichannel immunofluorescence to validate our sequencing data, to complement such limitations of certain technology.
Cindy St. Hilaire: Especially, I feel like with this diseased tissue that it's such a large tissue, there's so much information, it's really hard to dig in and figure out where the signal is. This was a wonderful paper for kind of highlighting, integrating all these new technologies with also just classical staining. Makes for great pictures as well. How does this cellular landscape of cardiac sarcoidosis compare to a normal heart? What'd you find?
Chieh-Yu Lin: This is a great question. Compared to normal heart, we have been talking about this accumulation of macrophages with scattered multinucleated giant cells. For the similar landscape, first and foremost, you do not see those type of accumulations in brain microscopy or by myeloid markers in the heart. Although, indeed, in even normal heart tissue we have rest and macrophages. It just doesn't form such morphological alterations. But then we dive deep into it, and then we found that from a different cell type perspective, we realized that the granuloma is composed by several different type of inflammatory cells, with most of the T cells and NKT cells kind of adding periphery. The myeloid cells, including the multinucleated giant cells also, are kind of in the center of the granuloma of the sarcoidosis. Then, we further dive in and realize that there are at least six different subtype of myeloid cells that is contributing to the formation of this very eye-catching distinctive granular malformations, and to just never feel first off and foremost, of course, is those multinucleated giant cells that is really distinct, even on the line microscopy] routine change stand.
And then we have a typical monocyte that's more like a precursor being recently recruited to the heart, and we finally sent the other four different type of myeloid cell that carry different markers, and then improving the resident macrophages. Especially for me as a pathologist, I'm using my eye and looking at stand every day, is actually these six type of cells, myeloid cells, actually form a very beautiful special kind of distribution with the connections or special arrangement with all different type, kind of like multinucleated giant cell in the middle, flanked by HLA-DR positive epithelioid macrophages, kind of scatter, and then with dendritic cells and a typical monocyte at the peripheral, and then resident macrophage kind of like in the mix of the seas of granuloma information. All these are distinct from normal heart tissues that does carry a certain amount of macrophages, but just don't form this orchestrated architectural distinct structure that's composed of this very complicated landscape.
Cindy St. Hilaire: Those images, I think it was figure six, it's just gorgeous to look at, the model you made. One of the questions I was thinking is there must be a significance between these cells that are on the periphery and those that are in the center of this granuloma. Do you have an idea or can we speculate as to are some more cause and some more consequence of the granuloma? Were you able to capture any more information about maybe the initiating steps of these from your study?
Kory Lavine: That's a great question, and a question the field has had for a long time. Now, we know there's different populations of cells. The single cell data allows us to understand what are the transcriptional differences and distinctions between them to gain some insights. One thing that we do know from the field is that disease activity correlates with mTOR activity within these granulomas. We took advantage of phospho-S6 kinase staining as a downstream marker of mTOR activity, and Ki-67 is a marker of self proliferation.
Which of these populations within the granuloma might be most active with respect to mTOR and respect to proliferation? If you ask most people in the field, they would jump up and say, "It's the giant cell in the middle." We found that that's not actually the case at all. It's the macrophages that surround the giant cell, the ones that are HLA-DR positive, the epithelioid macrophages, and the ones that are SYLT-3 positive that are scattered around them. That's really interesting and could make a lot of sense, and leads to hypothesis that perhaps activation mTOR signaling within certain parts of the granuloma might be sufficient to set up the rest of the architecture. That's something that we can explore in animal models, and are doing so to try to create a cause and effect relationship.
Cindy St. Hilaire: Yeah, and I was actually thinking about this, too, in relation to kind of the resident macrophages versus infiltrating macrophages or even just infiltrating immune cells. Do you know the original source of the cells that make up the granuloma? Is it mostly resident immune, or are they recruited in?
Kory Lavine: We can make predictions from the single cell data where you can use trajectory analysis to make strong predictions about what the origin of different populations might be. What those analyses predicted is that the giant cells and the cells that surround the giant cells, the HLA-DR positive and SYLT-3 positive macrophages, come from monocytes. That's the prediction, and, of course, resident macrophages do not. However, that prediction has to be tested, and that's the beauty and importance of developing animal models. The wonderful thing today is we now have genetic tools to do that. We can ask that question.
Cindy St. Hilaire: I don't know. Maybe you don't want to spoil the lead of the next paper, but what kind of mouse model are you thinking about trying?
Kory Lavine: Yeah. First of all, let me talk about the tools that are available, because they're published in Circulation Research, of course. We have a nice tool to specifically mark, track and delete in tissue resident macrophages using a CX3CR1 ERT pre-mouse, and taking advantage of the concept that tissue macrophages don't turn over from monocytes and turn over from themselves. We can give tamoxifen to label all monocytes macrophages in Dcs with that CRE, and then wait a period of time where only the resident macrophages remain labeled. We can use that trick to modulate mTOR signaling as a first step, and ask whether mTOR signaling is required in that population. We've now developed a new genetic tool to do the same thing in just recruited macrophages.
Cindy St. Hilaire: What was the most challenging aspect of this study? There's a lot of moving parts. I'm sure probably the data analysis alone is challenging, but what would you say is the most challenging?
Kory Lavine: I think you alluded to this early on, but the most challenging thing is collecting the right tissues to analyze, and that's not a small feat or a small effort here. All the technologies are a lot of fun, and everything works so well today compared to many years ago when we trained, so it's an exciting time to do science. The most challenging and time-consuming component was assembling a group of tissues that we could do single-cell sequencing on between our group and our colleagues at Duke, and then creating validation cohorts that we did across several different institutions, including our own as well as Stanford. That team effort in building that team is the most important, challenging, and honestly, enjoyable part of this.
Chieh-Yu Lin: I cannot agree more what Kory just said. I think that that's the challenging and the fun part, and that we're very fortunate to really have a great team to tackle this questions in multiple from multiple institute. I just want to add one more thing that, particularly for me as a cardiopathologist, one of the hardest things is I've known how to look or diagnose sarcoidosis for years, but seeing the data emerging that is so complicated and then beyond my reliable eyes in understanding, it's kind of mentally very challenging but very fun to really open and broaden the vision. It's not just how it looks like just giant cells in macrophages.
Cindy St. Hilaire: What do you think about in terms of diagnostics or even potential therapies? How do you think this data that you have now can be leveraged towards those objectives, whether it's screening for new cell types that are really key to this granuloma formation versus therapeutically targeting them?
Kory Lavine: This study opens new doors, and right now, diagnosis of sarcoids islimited by trying to biopsy, which, in the heart, is limited by sample bias. You certainly can biopsy the wrong area because you don't know whether a granuloma is in the area or not. We do do some cardiac and other imaging studies like FDG-PET scans, which are helpful but are not perfect, and each of them has their individual limitations. One of the beauties of our study is it identifies new markers of macrophage populations that live within the granuloma, many of which are unique to this disease.
That suggests that there's maybe an opportunity to develop imaging tracers that can identify those populations more specifically than our current PET imaging studies do, which rely simply on glucose uptake. It also opens up the possibility that we may able to take blood samples and identify some of these cell types within the blood, and have more simple testing for our patients. I think in terms of therapy, you alluded to it earlier, these concepts about mTOR signaling, that could be a new therapeutic avenue that needs to be rigorously explored in preclinical models. We're lucky already to have very good mTOR inhibitors available in clinical practice today.
Cindy St. Hilaire: Obviously, opening new doors is amazing because it's more information, but often a good study leads to even more questions to be asked. What question, or maybe what questions, are you guys going to go after next?
Chieh-Yu Lin: Well, that list is very long, and then that's actually the exciting thing about doing this research. There's no bad questions, in some sense. All the way from diagnosis, management, monitoring, therapeutic, how we predict where the patient can respond, that's the whole clinical side. Even the basic science side, we still haven't really answered the question, although our data suggests where that multinucleated giant cells coming from. It's very eye catching. How do they form, even though our data suggests it's from the recruited macrophages. But that's still a long way from the recruited macrophage, monocyte to that gigantic bag of nuclei in the very fluffy cytoplasm.
And then, how the granuloma, as we discussed earlier in this discussion, really initially from a relatively normal background myocardium to form this disease process. There are just so many questions that we can ask. There are, of course, several fronts that we would like to focus on. Kory already nicely listed some of them. First and foremost is actually to establish animal model to enable us to do more details in mechanistic studies, because human tissue, as good as it is, it's kind of like a snapshot, just one time point, and it really limits our ability to test our hypothesis. Animal model, certainly, is one of the major directions that we are going forward, but also the other side, like more clinical science also to develop novel noninvasive methodologies to diagnose and to hopefully monitor this patient population in a better way.
Cindy St. Hilaire: Well, it's beautiful work. I was actually reading this paper this weekend at a brunch place just next door to my house, and the guy sitting next to me happened to see over my shoulder the title and said that his father had passed away from it. This is hopefully going to help lots of people in the future, and really help to make the models that we need to ask, "What's happening in this disease?" Thank you so much for taking the time to speak with me, and congratulations on what seems to be a landmark study in understanding what's going on in this disease.
Chieh-Yu Lin: Thank you so much. It's a pleasure.
Cindy St. Hilaire: That's it for our highlights from the September 30th and October 14th issues of Circulation Research. Thank you so much for listening. Please check out the Circ Res Facebook page, and follow us on Twitter and Instagram with the handle @CircRes, and hashtag Discover Circ Res. Thank you so much to our guests, Dr Kory Lavine and Dr Chieh-Yu Lin from Washington University St. Louis. This podcast is produced by Ashara Retniyaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles was provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover Circ Res, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors of the American Heart Association. For more information, please visit ahajournals.org.
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This month on Episode 40 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the September 2 and September 16 issues of the journal. This episode also features an interview with Dr Jun Yoshioka, and Dr Yoshinobu Nakayama, from the City University of New York, about their study, Interaction of ARRDC-4 with GLUT1 Mediates Metabolic Stress in the Ischemic Heart.
Article highlights:
Jin, et al. Gut Dysbiosis Promotes Preeclampsia
Mengozzi, et al. SIRT1 in Human Microvascular Dysfunction
Hu, et al. Racial Differences in Metabolomic Profiles and CHD
Garcia-Gonzales, et al. IRF7 Mediates Autoinflammation in Absence of ADAR1
Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to be highlighting some articles from September 2nd, and September 16th issues of CircRes. And I'm also going to have a conversation with Dr Jun Yoshioka, and Dr Yoshinobu Nakayama, from the City University of New York, about their study, Interaction of ARRDC-4 with GLUT1 Mediates Metabolic Stress in the Ischemic Heart. But, before I get to the interview, I'm going to highlight a few articles.
The first article is from our September 2nd issue, and it's titled, Gut Dysbiosis Promotes Preeclampsia by Regulating Macrophages, and Trophoblasts. The first author is Jiajia Jin, and the corresponding author is Qunye Zhang from the Chinese National Health Commission.
Preeclampsia is a late-stage pregnancy complication that can be fatal to the mother, and the baby. It's characterized by high blood pressure, and protein in the urine. The cause is unknown, but evidence suggests the involvement of inflammation, and impaired placental blood supply. Because gut dysbiosis can influence blood pressure, and inflammation has been observed in preeclamptic patients, Jin and colleagues examined this link more closely. They found that women with preeclampsia had altered gut microbiome. Specifically, a reduction in a species of bacteria that produced short-chain fatty acids, and lower short-chain fatty acid levels in their feces, in their serum, and in their placentas. And preeclamptic women had lower short-chain fatty acid levels in their feces, in their serum, and in their placentas compared with women without preeclampsia.
They found that fecal transfers from the preeclampsia women to rats with a form of the condition exacerbated the animals' preeclampsia symptoms, while fecal transfers from control humans alleviated the symptoms. Furthermore, giving rats an oral dose of short-chain fatty acids or short-chain fatty acid producing bacteria decreased the animals' blood pressure, reduced placental inflammation, and improved placental function. This work suggests that short-chain fatty acids, and gut microbiomes could be a diagnostic marker for preeclampsia. And microbial manipulations may even alleviate the condition.
The second article I want to share is also from our September 2nd issue, and it's titled, Targeting SIRT1 Rescues Age and Obesity-Induced Microvascular Dysfunction in Ex Vivo Human Vessels. And this study was led by Alessandro Mengozzi from University of Pisa.
With age, the endothelial lining of blood vessels can lose its ability to control vasodilation, causing the vessel to narrow and reduce blood flow. This decline in endothelial function has been associated with age related decrease in the levels of the enzyme, SIRT1. And artificially elevating SIRT1 in old mice improves animals' endothelial function. Obesity, which accelerates endothelial dysfunction, is also linked to low SIRT1 levels.
In light of these SIRT1 findings, Mengozzi, and colleagues examined whether increasing the enzyme's activity could improve the function of human blood vessels. The team collected subcutaneous microvessels from 27 young, and 28 old donors. And both age groups included obese, and non-obese individuals. SIRT1 levels in the tissue were, as expected, negatively correlated with age and obesity, and positively correlated with baseline endothelium dependent vasodilatory function. Importantly, incubating tissue samples from older, and obese individuals with a SIRT1 agonist, restored the vessel’s vasodilatory functions. This restoration involved a SIRT1 induced boost to mitochondrial function, suggesting that maintaining SIRT1 or its metabolic effect might be a strategy for preserving vascular health in aging, and in obesity.
The third article I want to share is from our September 16th issue. And this one is titled, Differences in Metabolomic Profiles Between Black And White Women and Risk of Coronary Heart Disease. The first author is Jie Hu, and the corresponding author is Kathryn Rexrode, and they're from Brigham and Women's Hospital, and Harvard University.
In the US, coronary heart disease, and coronary heart disease-related morbidity, and mortality is more prevalent among black women than white women. While racial differences in coronary heart disease risk factors, and socioeconomic status have been blamed, this group argues that these differences alone cannot fully explain the disparity. Metabolomic variation, independent of race, has been linked to coronary heart disease risk. Furthermore, because a person's metabolome is influenced by genetics, diet, lifestyle, environment and more, the authors say that it reflects accumulation of many cultural, and biological factors that may differ by race.
This group posited that if racial metabolomic differences are found to exist, then they might partially account for differences in coronary heart disease risk. This study utilized plasma samples from nearly 2000 black women, and more than 4500 white women from several different cohorts. The team identified a racial difference metabolomic pattern, or RDMP, consisting of 52 metabolites that were significantly different between black, and white women. This RDMP was strongly linked to coronary heart disease risk, independent of race, and known coronary heart disease risk factors. Thus, in addition to socioeconomic factors, such as access to healthcare, this study shows that racial metabolomic differences may underlie the coronary heart disease risk disparity.
The last article I want to share is also from our September 16th issue, and it is titled, ADAR1 Prevents Autoinflammatory Processes in The Heart Mediated by IRF7. The first author is Claudia Garcia-Gonzalez, and the corresponding author is Thomas Braun, and they are from Max Planck University.
It's essential for a cell to distinguish their own RNA from the RNA of an invading virus to avoid triggering immune responses inappropriately. To that end, each cell makes modifications, and edits its own RNA to mark it as self. One type of edit made to certain RNAs is the conversion of adenosines to inosines. And this is carried out by adenosine deaminase acting on RNA1 or ADAR1 protein. Complete loss of this enzyme causes strong innate immune auto reactivity, and is lethal to mice before birth. Interestingly, the effects of ADAR1 loss in specific tissues is thought to vary. And the effect in heart cells in particular has not been examined.
This study, which focused on the heart, discovered that mice lacking ADAR1 activity specifically in cardiomyocytes, exhibit autoinflammatory myocarditis that led to cardiomyopathy. However, the immune reaction was not as potent as in other cells lacking ADAR1. Cardiomyocytes did not exhibit the sort of upsurge in inflammatory cytokines, and apoptotic factors seen in other cells lacking ADAR1. And the animals themselves did not succumb to heart failure until 30 weeks of age. The author suggests that this milder reaction may ensure the heart resists apoptosis, and inflammatory damage because, unlike some other organs, it cannot readily replace cells.
Cindy St. Hilaire: Today I have with me, Dr Jun Yoshioka, and Dr Yoshinobu Nakayama, and they're from City University of New York. And today we're going to talk about their paper, Interaction of ARRDC4 With GLUT1 Mediates Metabolic Stress in The Ischemic Heart. And this is in our September 2nd issue of Circulation Research. So, thank you both so much for joining me today.
Jun Yoshioka: Thank you for having us. We are very excited to be here.
Cindy St. Hilaire: It's a great publication, and also had some really great pictures in it. So, I'm really excited to discuss it. So, this paper really kind of focuses on ischemia, and the remodeling in the heart that happens after an ischemic event. And for anyone who's not familiar, ischemia is a condition where blood flow, and thus oxygen, is restricted to a particular part of the body. And in the heart, this restriction often occurs after myocardial infarctions, also called heart attacks. And so, cardiomyocytes, they require a lot of energy for contraction, and kind of their basic functions. And in response to this lack of oxygen, cardiomyocytes switch their energy production substrate. And so, I'm wondering if before we start talking about your paper, you can just talk about the metabolic switch that happens in a cardiac myocyte in the healthy state versus in the ischemic state.
Jun Yoshioka: Sure. As you just said, that the heart never stops beating throughout the life. And it's one of the most energy demanding organs in the body. So, under normal conditions, cardiac ATP is mainly derived from fatty acid oxidation, and glucose metabolism contributes a little bit less in adult cardiomyocytes. However, under stress conditions such as ischemia, glucose uptake will become more critical when oxidative metabolism is interrupted by a lack of oxygen. That is because glycolysis is a primary anaerobic source of energy. We believe this metabolic adaptation is essential to preserve high energy phosphates and protect cardiomyocytes from lethal injuries. The concept of shifting the energy type of stress preference toward glucose, as you just said, has been actually long proposed as an effective therapy against MI. For example, GIK glucose insulin petition is classic.
Now, let me explain how glucose uptake is regulated. Glucose uptake is facilitated by multiple isophones of glucose transporters in cardiomyocytes. Mainly group one and group four, and the minor, with a minor contribution of more recently characterized STLT1. In this study, we were particularly interested in group one because group one is a basal glucose transporter.
Dr Ronglih Liao, and Dr Rong Tian's groups reported nearly two decades ago that the cardiac over-expression of group one prevents development of heart failure, and ischemic damage in mice. Since they are remarkable discoveries, the precise mechanism has not yet been investigated enough, at least to me. Especially how acute ischemic stress regulates group one function in cardiomyocytes. We felt that this mechanism is important because there is a potential to identify new strategies around group one, to reduce myocardiac ischemic damage. That is why we started this project hoping to review a new mechanism by which a protein family, called alpha-arrestins, controls cardiac metabolism under both normal, and diseased conditions.
Cindy St. Hilaire: That is a perfect segue for my next question, actually, which is, you were focusing on this arrestin-fold protein, arrestin domain-containing protein four or ARRDC4. So, what is this family of proteins? What are arrestin-fold proteins? And before your study, what was known about a ARCCD4, and its relationship to metabolism, and I guess specifically cardiomyocyte metabolism?
Jun Yoshioka: So, the arrestin mediated regulation of steroid signaling is actually common in cardiomyocytes. Especially beta, not the alpha, beta-arrestins have been well characterized as an adapter protein for beta-adrenergic receptors. Beta-arrestins combine to activate beta-adrenergic receptors on the plasma membrane, promote their endosomal recycling, and cause desensitization of beta-adrenergic signaling. Over the past decade, however, this family, the arrestin family, has been extended to include a new class of alpha-arrestins. But unlike beta-arrestins, the physiological functions of alpha-arrestins remain largely unclear based in mammalian cells. Humans, and mice have six members of alpha-arrestins including Txnip, thioredoxin interacting protein called Txnip, and five others named alpha domain-containing protein ARRDC1 2, 3, 4 and 5. Among them Txnip is the best studied alpha-arrestin. And Txnip is pretty much the only one shown to play a role in cardiac physiology.
Txnip was initially thought to connect alternative stress and metabolism. However, it is now known that the Txnip serves as an adapter protein for the endocytosis of group one, and group four to mediate acute suppression of glucose influx to cells. In fact, our group has previously shown that the Txnip knockout mice have an enhanced glucose uptake into the peripheral tissues, as well as into the heart. Now, in this study, our leading player is ARRDC4. The arrestin-domains of ARRDC4 have 42% amino acid sequence similarities to Txnip. This means that the structurally speaking ARRDC4 is a brother to Txnip. So, usually the functions of arrestins are expected to be related to their conserved arrestin-domains. So, we were wondering whether two brothers, Txnip, and ARRDC4, may share the same ability to inhibit the glucose transport. That was a starting point where we initiated this project.
Cindy St. Hilaire: That's great. And so, this link between ARRDC4, and the cardiac expression of gluten one and gluten four, I guess, mostly gluten one related to your paper, that really wasn't known. You went about this question kind of based on protein homology. Is that correct?
Jun Yoshioka: That is right.
Cindy St. Hilaire: And so, ARRDC4 can modulate glucose levels in the cell by binding, and if I understand it right, kind of helping that internalization process of glute one. Which makes sense. You know, when you have glucose come into the cell, you don't want too much. So, the kind of endogenous mechanism is to shut it off, and this ARRDC4 helps do that. But you also found that this adapter protein impacts cellular stress, and the cellular stress response. So, I was wondering if you could share a little bit more about that because I thought that was quite interesting. It's not just the metabolic impact of regulating glucose. There's also this cellular stress response.
Jun Yoshioka: Right? So, Txnip is known to induce oxidative stress. But about the ARRDC4, we found that ARRDC4 actually does not induce oxidative stress. Instead, we found that it reproducibly causes ER, stress rather than oxidative stress. So, let Yoshinobu talk about the ER stress part. Yoshinobu, can you talk about how you found the ER stress story?
Yoshinobu Nakayama: So, then let's talk about the, yeah, ER stress caused by ARRDC4. The ER stress caused by ARRDC4, year one was the biggest challenge in this study, because it's a little bit difficult to how we found a link of the glucose metabolism to the effect of the ARRDC4, only our stress. And at the other point of the project, we noticed that a ARRDC4 causes ER stress reproducibly, but we did not know how. So, both group one, and ARRDC4 are membrane proteins mainly localized near the plasma membrane. Then how does ARRDC4 regulate the biological process inside in the plasma radical? So, we then hypothesize that ARRDC4 induces intercellular glucose depravation by blocking cellular glucose uptake, and then interferes with protein glycosylation, thereby disturbing the ER apparatus. That makes sense because inhibition of group one trafficking by ARRDC4 was involved in the unfolded protein response in ischemic cardiomyocytes.
Cindy St. Hilaire: So how difficult was that to figure out? How long did that take you?
Yoshinobu Nakayama: How long? Yeah. Is this the question?
Cindy St. Hilaire: It's always a hard question.
Yoshinobu Nakayama: I think it's not several weeks. Maybe the monthly, months project. Yeah.
Cindy St. Hilaire: Okay. It's always fun when, you know, you're focusing on one angle, and then all of a sudden you realize, oh, there's this whole other thing going on. So, I thought it was a really elegant tie-in between the metabolism, but also just the cellular stress levels. It was really nice.
So, you created a full body knockout of ARRDC4 in the mouse, and you did all the proper kind of phenotyping. And at baseline everything's normal, except there's a little bit of changes in the blood glucose levels. But I also noticed when you looked at the expression of ARRDC4 in different tissues, it was very high in the lungs, and also in the intestines. And so, I know your study didn't focus on those tissues, but I was wondering if you could possibly speculate what ARRDC4 is doing in those tissues? Is it something similar? Do those cells under stress have any particular metabolic switching that's similar?
Jun Yoshioka: Well, actually we don't have any complete answer for that question, because like you said, we didn't focus on lung, and other tissues. But I could say that actually the brother of ARRDC4, Txnip, is also highly expressed in lung, and bronchus, and in those organs. So, it's interesting because, which means that, the molecule is very oxygen sensitive, I will say. Both brothers. But that's all we know for now. But that's a very great point. And then we are excited to, you know.
Cindy St. Hilaire. Yeah.
Jun Yoshioka: Move on to the other tissues.
Cindy St. Hilaire: I was thinking about it just because I've actually recently reviewed some papers on pulmonary hypertension. So, when I saw that expression, that was the first thing I thought of was, oh, they should put these mice in a sugen/hypoxia model, and see what happens.
Jun Yoshioka: Right?
Cindy St. Hilaire: So, there's an idea for you, Yoshinobu. A K-99 grant or something. And also, because it's a full body knockout, even when you're looking at the heart, obviously the cardiomyocytes are really the most metabolically active cell, but cardiac fibroblasts are also a major component of the heart tissue. And so, do you know, is the, I guess, effects or the protectiveness of the ARRDC4 knockout heart, is it mostly because of the role in the cardiomyocytes or is there a role for it also in the fibroblast?
Yoshinobu Nakayama: Yeah, that's a very great question. Yeah. So, although we use the systemic knockout mice in the study, we believe that the beneficial effect of ARRDC4 deficiency is cardiac, autonomous. But this is because cardioprotection was demonstrated in the isolated heart experiments. But, you know, root is still uniformly expressing all cell types within the heart.
To address this, we have tested the specific effects of ARRDC4 on cardiac fibroblasts, and inflammatory cells. ARRDC4 knockout hearts had a twofold increase in myocardial glucose uptake over wild-type hearts during insulin-free perfusion. However, an increase in glucose uptake in isolated cardiac fibroblast or inflammatory cells was relatively mild, with about 1.2 fold increase over wild-type cells.
Thus we conclude that cardiomyocytes are the measure contributed to the cardiac metabolic shift. And then the mechanism within cardiomyocytes should play the major role in cardioprotection.
Jun Yoshioka: I might, at one point, because, you know, the fibroblasts, they don't need to beat, right?
Cindy St. Hilaire: Right.
Jun Yoshioka: The inflammasome cells. They don't need to beat neither. So, they don't need that much energy. So, the cardiomyocytes energy metabolism is very important. So, that's why this mechanism is kind of more important in cardiomyocytes than other cell types.
Cindy St. Hilaire: Yeah. And I think, you know, your phenotyping of the mice at baseline show that there's really no effect in a cell that's not under stress. So, it's really, really nice finding. Yeah.
This article, I should say, is featured on the cover of the September 2nd Circulation Research issue. And it's got this really nice 3D modeling of the binding of ARRDC4 to glute one. And I was reading the paper, and the methods said, you use some AI for that. So, I'm sure other people have heard, too, AI in protein modeling is important. But AI in art, right? There's that new DALL-E 2 program. So how are you able to do this? How did that work?
Jun Yoshioka: So, our study used is called AlphaFold, which applies the artificial intelligence-based deep learning method. AlphaFold, nowadays, everybody really is interested in AlphaFold. AlphaFold uses structural, and genetic data to come up with a model of what the protein of interest should look like. So, that is also how we got the protein structure, ARRDC4. We think that the ability of AlphaFold to precisely predict the protein structure from amino acid sequence would be a huge benefit to life sciences, including of course, cardiovascular science research, because of high cost, and technical difficulties in experimental methods.
It's very useful if you can computationally predict the complex from individual structures of ARRDC4. And group one, which is actually structure of group one, is available in a protein data bank. But ARRDC4, it was not available. That's why we used AlphaFold.
And then we use the docking algorithm called Hdoc. So, based on these AI analysis, we could successfully identify specific residues in a C terminal arrestin domain as an international interface, that regulates group one function. So, we believe this AI method will pretty much accelerate efforts to understand the protein, protein interactions. And we believe that will enable more advanced drug discovery, for example, in very near future.
Cindy St. Hilaire: Yeah, it's really great. I started thinking about it in terms of some of the things I'm studying. So yeah, it was really nice.
Jun Yoshioka: Try next time.
Cindy St. Hilaire: Yeah, I will, I will. Actually, I went to the website, and was playing with it before I got on the call with you. So, how do you think your findings can be leveraged towards informing clinical decision making or even developing therapeutics?
Jun Yoshioka: So, let me talk about what needs to be done. There are more things we must do.
Cindy St. Hilaire: Always. Yeah.
Jun Yoshioka: One of the most clinically relevant questions is whether ARRDC4 inhibition actually can mitigate development of post MI heart failure, and reduce mortality in the chronic phase, not the acute phase. Because in this paper we just did the seven day post MI, which is kind of like acute to subacute phase. But you never know what's going to happen in the chronic phase, right? And that is actually not so simple to answer because there are so many issues that you should consider. For example, Dr E. Dale Abel's lab has reported previously that cardiomyacites, specific group one, knockout in mice does not really accelerate the transition from compensated hypotrophy to heart failure. Also, the same group has shown that the overexpression group one does not actually prevent LV dysfunction in the mouse model of pressure overload. So, it is possible that ARRDC knockout can be, do much, or even harmful to LV remodeling in a chronic phase because chronic phase, it's not, it's getting hypoxy conditions, right?
Cindy St. Hilaire: Yeah. So, it really might be something, I guess, personalized medicine is not the phrase I'm looking for. But I guess temporarily modulated, it would be something maybe we can figure out in an acute phase versus.
Jun Yoshioka: Chronic phase.
Cindy St. Hilaire: Yeah. Yeah.
Jun Yoshioka: This makes sense. Because, you know, high capacity of ATP synthesis, by oxidating metabolism, could be important for chronic heart failure. So, it's selecting substrates. Energy substrates is no longer, you know, that issue. So, I'm not sure I'm answering your question, but this is the point that we consider to move on to the next.
Cindy St. Hilaire: Well, that's great. And I think that was my next question, really. What is next? Are you really going to try to pinpoint where you could possibly target?
Jun Yoshioka: Right. So, the first point we have to figure out about chronic phase, and another point we are interested in, is what's going on at the level of mitochondria. Does ARRDC4 knockout hearts have a different activity of electron transport chain or glycolytic enzymes within mitochondria?
Cindy St. Hilaire: Or even mitochondrial fission infusion because it's, you know, it's a machinery.
Jun Yoshioka: Yeah. And how about the other essential pathways in glucose metabolism such as mTOR, AMPK and HEF1, and so on. So, all these must be determined to help understand the more precise role of ARRDC4 in cardiac metabolism, we believe.
Cindy St. Hilaire: It's a wonderful study, and now we have even more questions to ask using your great model. Congratulations again.
Yoshinobu Nakayama: Thank you so much.
Cindy St. Hilaire: Dr Yoshioka, and Dr Nakayama.
Jun Yoshioka: Thank you.
Cindy St. Hilaire: A wonderful paper, and congrats on getting the cover, and thank you so much for joining me today.
Jun Yoshioka: Thanks well so much for having us.
Yoshinobu Nakayama: Thank you.
Cindy St. Hilaire: That's it for the highlights from our September 2nd, and our September 16th issues of Circulation Research. Thank you so much for listening. Please check out our CircRes Facebook page, and follow us on Twitter, and Instagram with the handle @circres, and hashtag discovercircres. Thank you to our guests, Dr Jun Yoshioka, and Dr Yoshinobu Nakayama.
This podcast is produced by Ishara Ratnayaka, edited by Melissa Stonerm, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on the go source for the most exciting discoveries in basic cardiovascular research.
This program is copyright of the American Heart Association 2022. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.
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