January 2023 Discover CircRes

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.