Jul 15, 2021
This month on Episode 26 of Discover CircRes, host Cindy St. Hilaire highlights four original research articles featured in the June 25th and July 9th issues of Circulation Research. This episode also features an in-depth conversation with Dr Hirofumi Watanabe, Dr Ariel Gomez, and Dr Maria Luisa Sequeira-Lopez from the University of Virginia about their study, The Renin Cell Baroreceptor, A Nuclear Mechanotransducer Central for Homeostasis.
Article highlights:
Mesirca, et al. Electrical Remodeling of the AV Node in Athletes
Yang, et al. Macrophage-Mediated Inflammation in COVID-19 Heart
Örd, et al. Functional Fine-Mapping of CAD/MI GWAS Variants
Akhter, et al. EC-S1PR1 Activity Directs Vascular Repair
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'll be highlighting the
articles presented in our June 25th and July 9th issues of
Circulation Research. I'm also going to speak with Dr Hirofumi
Watanabe, Dr Ariel Gomez and Dr Maria Luisa Sequeira-Lopez from the
University of Virginia about their study, The Renin Cell
Baroreceptor, A Nuclear Mechanotransducer Central for
Homeostasis.
Cindy St. Hilaire: The first article I want to share comes from the June 25th issue of Circ Res and is titled Intrinsic Electrical Remodeling Underlies Atrial Ventricular Block in Athletes. The first authors are Pietro Mesirca, Shu Nakao, Sarah Dalgas Nissen, and the corresponding author is Alicia D'Souza. And they're from the University of Manchester in the UK.
Cindy St. Hilaire: Endurance training has cardiovascular benefits, but when taken to extremes, it can elicit heart problems such as atrial ventricular block or AV block. AV block is the impaired conduction through the AV node. In fact, some endurance athletes require pacemakers later in life due to AV block. One hypothesis for this conundrum is that the problem stems from disruptions in the autonomic nervous system. This study shows that in fact, the intrinsic electrophysiology of the heart is to blame. They used trained race horses, as well as mice, subjected to endurance swimming as models for human endurance athletes. Electrocardiograms on the animals showed that just like human athletes, the race horses and the swim-trained mice exhibited signs of AV node dysfunction that is not seen in sedentary controls.
Cindy St. Hilaire: Because the dysfunction also persisted when the autonomic nervous system was blocked, the team examined molecular changes within the heart itself. They found that ion channels, HCN4 and Cav1.2, were less abundant in the AV nodes of trained animals than those of the controls. The team went on to identify two microRNAs regulating HCN4 and Cav1.2 production and showed that suppression of these microRNAs restored normal heart electrophysiology in the mice. If the result holds true for humans, this could pave the way for novel treatments for AV block.
Cindy St. Hilaire: The second article I want to share is titled An Immuno-Cardiac Model for Macrophage-Mediated Inflammation in COVID-19 Hearts. The first authors are Liuliu Yang, Yuling Han, Fabrice Jafre, Benjamin Nilson-Payant and Yaron Bram. And the corresponding author is Shuibing Chen. And they're from Cornell University Medical Center.
Cindy St. Hilaire: COVID-19 is primarily a respiratory disease, but cardiac complications are common and appear to be linked with worsening outcomes. Post-mortem examinations of COVID-19 patients’ hearts have revealed abnormally high numbers of macrophages, suggesting that these cells have a role in the heart pathology. To investigate this possibility, this group co-cultured macrophages and cardiomyocytes, both which were derived from human induced pluripotent stem cells and infected the cultures with SARS-CoV-2 virus. Upon infection, both cell types increased their rates of apoptosis. However, the number of cardiomyocytes succumbing to the cell death process was far higher than that of macrophages. When cardiomyocytes were infected with the virus in the absence of macrophages, their rate of apoptosis dropped.
Cindy St. Hilaire: The team showed that macrophages produced large amounts of the inflammatory cytokines, IL-6 and TNF, in response to the virus and that trading the cardiomyocytes directly with the cytokines could similarly induce apoptosis. Blocking IL-6 and TNF alpha signaling prevented the macrophage-driven cardiomyocyte death. The team then identified two FDA approved drugs, ranolazine and tofacitinib, that prevented the virus-induced cardiomyocyte death in vitro and suggest that these drugs now be investigated in larger animal models.
Cindy St. Hilaire: The next article I want to share is titled Single-Cell Epigenomics and Functional Fine-Mapping of Atherosclerosis GWAS Loci. The first author is Tiit Ord, and the corresponding author is Minna Kaikkonen, from the University of Eastern Finland.
Cindy St. Hilaire: Genome-wide association studies, or GWAS studies, have identified hundreds of genetic loci associated with coronary artery disease and myocardial infarction. And many of these genes likely play a role in atherosclerotic development. However, most of these loci are located in non-coding intergenic regions of the genome. Thus, their functional effects on atherosclerosis development are not clear. Non-coding regions of the genome may contain gene regulatory elements, including cell type specific enhancers. And because such enhancer elements often have open chromatin structures, this team profiled the chromatin accessibility of single cells in human atherosclerotic plaques.
Cindy St. Hilaire: They found that many cell type-specific assessable regions overlapped with both transcription factor binding motifs, as well as GWAS-identified coronary artery disease loci. Using an algorithm called Cicero, the team was able to predict likely genes under the control of these accessible intergenic regions. They found that in more than 30 cases, they were able to confirm these intergenic regions control gene expression in in vitro assays. This work highlights the power of chromatin accessibility mapping for homing in on GWAS loci with transcriptional effects, and for identifying the likely genes they regulate.
Cindy St. Hilaire: The last article I want to share is titled Programming to S1PR1+ Endothelial Cells Promote Restoration of Vascular Integrity. The first author is Mohammed Zahid Akhter, and the corresponding author is Dolly Mehta, and they're from the University of Illinois College of Medicine.
Cindy St. Hilaire: Endothelial cells line the lumen of our blood vessels, forming a barrier that regulates the transport of nutrients, fluids and circulating cells to and from tissues. The lipid signaling molecule, sphingosine-1-phosphate, or S1P, and its receptor, S1PR1, promote endothelial barrier integrity. But how S1P and S1PR1 signaling might restore barrier function to inflammation-induced leaky vessels is unclear.
Cindy St. Hilaire: Using mice with fluorescently tagged S1PR1, this group showed that when mice are given a dose of the bacterial endotoxin, LPS, which induces lung inflammation, there's a dramatic boost in the proportion of growing lung endothelial cells. This boost in S1PR1+ endothelial cells is due to their increase in proliferation.
Cindy St. Hilaire: The authors go on to show that this proliferation is accompanied by increased production of the transcription factors involved in S1P synthesis and secretion. When they transplanted S1PR1+ cells into mice whose endothelial cells lacked the receptor, they could rescue the leaky blood vessels. By detailing the cells and molecular players responsible for vessel recovery after inflammation, this work may inform repair boosting therapies for chronic inflammatory conditions.
Cindy St. Hilaire: So today with me, I have Dr Hirofumi Watanabe, Dr Ariel Gomez and Dr Maria Luisa Sequeira-Lopez, from the University of Virginia. And they are all with me to discuss their study, The Renin Cell Baroreceptor, a Nuclear Mechanotransducer Central for Homeostasis. And this article is in our July 9th issue of Circulation Research. So thank you all for joining me today. I think we're spanning 13 time zones, so I appreciate you all making the effort.
Maria Luisa Sequeira-Lopez: It's our pleasure. Thank you.
Ariel Gomez: Thank you.
Hirofumi Watanabe: Thank you.
Cindy St. Hilaire: I won't lie, the Renin-Angiotensin-Aldosterone System is quite complex, so we're not going to try to break it all down here, but it is essential for the regulation of fluid balance and blood pressure in the body. Without it, things go quite awry. And your study is focusing on the kidney cell that produces renin in response to the minute changes in the blood pressure and the composition and the volume of the extracellular fluid in the body. So I'm wondering if, before we jump into the study, if you can give us a bit of background about these renin-producing cells and what is known about the renal pressure sensing system?
Maria Luisa Sequeira-Lopez: So in the adult mammalian kidney, renin cells are located at the tip of the afferent arterioles at the entrance to the glomeruli. So that's why they are called juxtaglomerular cells. They synthesize and release renin. This is then, as you mentioned, the rate-limiting enzyme for the renin-angiotensin system that controls blood pressure and fluid-electrolyte homeostasis. However, during early embryonic development, as demonstrated many years ago, renin cells are widely distributed along the renal arterial tree and inside the glomerulus and the interstitium. And with maturation they differentiated to vascular smooth muscle cells and they end up being located in the juxtaglomerular area.
Maria Luisa Sequeira-Lopez: But in response to a homeostatic challenge, such as hypertension, dehydration, hemorrhage, there is an increase in the number of renin-expressing cells along the renal arterial tree, resembling the embryonic counter. And this occurs mostly by re-expression of renin from vascular smooth muscle cells that descended from originally renin-expressing cells. And when the challenge passes, then they stop expressing renin and become vascular smooth muscle cells again. So renin cells are extremely plastic and they can switch back and forth from an endocrine to a contractile phenotype.
Cindy St. Hilaire: I'm really glad you mentioned the vascular smooth muscle cell angle because I actually have a question about that later on. But before I get to that question, one of the things that I love reading in studies is when a current paper references much older work that often has a really intricate or insightful observation. And in your paper you cited, I believe it was in 1957, was the first real hypothesis that there is an existence of this pressure sensing mechanism in the kidney, what we're calling this baroreceptor. Yet, that was a long time ago and the identity has really been elusive. So I was wondering why has it just been so difficult to really pin down this baroreceptor and how this pressure and fluid sensing works in these cells?
Ariel Gomez: So it was elusive, as you said. The reason is the researchers didn't have the tools to actually study it. It really requires an evolution, conceptual evolution, and scientific evolution, as well as technical development. And so we were fortunate over time, over the years. We developed ways to mark the cells endogenously with the appropriate fluorescent markers, genetically engineer, then develop models that allowed to drop the blood pressure in a consistent manner, and so forth. And we could follow the lineage of these cells and study them as they move back and forth from their phenotypes. So I think it was a matter of even Dr Tovian, who is the person that you mentioned, Lou Tovian, who I actually met a long time ago. So he even postulated that maybe it was a stretch mechanism, and that's one of the great contributions of Hirofumi who figure out how to stretch the cells using different ways of doing that.
Cindy St. Hilaire: So in your quest to identify this baroreceptor, you use several murine models. A surgical tool, but also several genetic tools. And I was wondering if you could share a little bit about that initial surgical model, that aortic constriction and maybe the pros and cons about that method?
Hirofumi Watanabe: And so we established surgical model of in mice. We created inductation between the roots of the right and left renal arteries. By the surgery, and our right kidney receives high pathogen pressure, and the left kidney receives low pathogen pressure. And this surgery model resulted in a marked difference in the expression of renin in each kidney. And by RT2 PCR and in situ hybridization, renin was decreased in the right kidneys and increased in the left kidneys.
Cindy St. Hilaire: Excellent. So it's a really powerful model because you can use the same mouse to look at the same...
Ariel Gomez: Right. So the beauty of that is that, Hirofumi, by doing that, he got rid of any genetic variation between the mice. Because you are doing the high and low pressure in the same mouse.
Maria Luisa Sequeira-Lopez: And another question I can think that we have said was when, if you calculate the number of cells that increase in one kidney and decreases in the other one, if you add them, it ends up being the number of cells in a non-aortic coarctation mouse. So it looks like-
Cindy St. Hilaire: It's a literal seesaw. That's beautiful. At least the math works out in your favor in the end. That's great.
Maria Luisa Sequeira-Lopez: And that's something that Luis Tovian didn't see, because what he did is he increased the perfusion pressure in an isolated kidney and what he observed was less granulation. So it was an indirect method to find less renin in those kidneys. But with a low pressure, he didn't observe an increase in renin, or increase in granulation. What we know that really happens.
Cindy St. Hilaire: So you mentioned smooth muscle cells in the beginning of our discussion and my training has been in smooth muscle cells, vascular smooth muscle cells, mostly though focused on the aorta, especially in mice. A lot of times we just say smooth muscle cells, but people are really talking about the aortic smooth muscle cells in the mice. And in humans, in the coronaries. But we use the mouse aortic smooth muscle cells as the model, which you can obviously see when you frame it out like that, some issues. And one of the things we talk about at least in athero is the cell plasticity and this phenotype switching from the contractile quiescent state to one that's associated with disease processes.
Cindy St. Hilaire: And we've really evolved on what we've known about that. It used to be just about the migration and proliferation. Now it's about the actual phenotypic switching into different kinds of cells. Macrophage-like cells, for one. And yours really was the first to bring to my eyes that there's probably many more regarding that. So could you maybe expand a little bit on these renal smooth muscle cells or renin-like cells maybe, and what's happening in that disease process? And do we know the point at which it can switch and make renin and go back versus switches and doesn't return? Is that part of the disease process?
Ariel Gomez: We describe the plasticity of the smooth muscle cells from the renal arterioles long time ago. I mean, I think, I would say that even at the beginning of my career. And at that time people didn't use that term so much, plasticity. We didn't know how to call it because it was a switch back and forth from a smooth muscle contractile phenotype to endocrine without, at the moment, without causing disease. And the cells were able to come back to be smooth muscle cells. But the period of the stimulation was only a week or so. So during that time, the cells can go back and forth. And now we know that they do that. But if you create a persistent stimulation, and this is another paper that we are working with Hirofumi and Maria Luisa, if you create a knockout renin or knockout of angiotensin receptors or so forth, the stimulation doesn't stop because there is no angiotensin.
Ariel Gomez: And so under those conditions, the cells reach a point in which they become very aggressive, almost embryonic-like. They are constantly stimulated. They are attempting to reestablish the phenotype and in doing so, they create these concentric vascular hypertrophy. And I don't know whether we are going to send the paper to Circulation Research or to where, but we are still writing it. After that, we don't know whether they can come back because they are so seriously sick. And we know that they are responsible for this, but this is another paper.
Maria Luisa Sequeira-Lopez: Another thing that I wanted to add is that these cells have been extremely difficult to study. Ariel has been developing many, many tools that allow him to dissect them and cover many secrets of the cells. But if you... First because they are very, very few in the kidney. And there were no markers to isolate them. And if you put them in culture, now that we can have them live with a person marker, they stop expressing renin and making renin within 24-48 hours. So it's difficult to study. So that's why Hirofumi [inaudible 00:19:21] how the system works. Stimulating them with cyclic AMP, they go back like renin. If not, they differentiate into vascular smooth muscle cells. It looks like that's their default pathway. So they need to sense that there is a need for renin to increase the blood pressure and electrolyte homeostasis. So that's one of the characteristics of the cells. But if you stimulate constantly, as Ariel said, then they may be hard to… They cannot come back.
Cindy St. Hilaire: It's over the tipping point a bit.
Maria Luisa Sequeira-Lopez: Yes.
Cindy St. Hilaire: In your discussion you mentioned another study from your group that kind of took more of a developmental angle. And you mentioned that you had identified unique chromatin structures of renin-producing cells, and you also identified what are called super enhancers that help dictate the differentiation of these running progenitor cells into renin producing cells. And then in your mechanical stimuli experiments, you mentioned identifying similar chromatin signatures. And I was wondering what this might suggest in regards to the disease pathogenesis. And I guess I'm thinking about it in terms of in many diseased states, we see this activation of developmental programs that either are not stopped or just go on and are even higher expressed than in developmental programs. And is that you think is happening in these renin cells? A developmental program gone awry?
Ariel Gomez: Yeah, definitely. I definitely think so. I think we all, the three of us think that way. Yeah. I think it's an exaggeration of a developmental program. One thing that we didn't mention and why the vessels get so sick is because during development, these cells contribute to the formation of the vasculature. And so when they regress so much trying to make renin... And they make it. I mean, they go from 20,000 units to 2 million of renin, right? And they never stop. But when they regress so much, they regressed on embryonic stage and they think that they need to make more blood vessels to actually increase the flow and the oxygenation of the tissue. But in doing so, they create more pathology. So maybe, Hirofumi, I don't know if you're going to ask him, but one of those super enhancers is the Lamin A/C gene. And he has studied that in this Circulation Research paper that we are talking about.
Maria Luisa Sequeira-Lopez: I just wanted to add that they also make lots of angiogenic factors to make the vessels.
Cindy St. Hilaire: Got it. So developmentally, they're activating more production of renin but they're also producing these pro angiogenic cytokines and really driving that…
Ariel Gomez: BGF. They produce a type of BGF or angiopoietins.
Cindy St. Hilaire: Interesting.
Ariel Gomez: Yeah. And things like that.
Cindy St. Hilaire: I really liked reading about this magnetic bead experiment that you used as the mechanical stimuli. Frankly, I saw the picture and I brought it to my lab and said, "Guys, figure out how to do this." Can you explain a little bit about it? It seemed really nice, really elegant and very tuneable. So I'm excited. I'm sure many more people are excited to hear about it.
Hirofumi Watanabe: So we applied coated magnetic beads to the cultured ring cells. Then we placed a magnet above the cells so we can pull the cells by magnetic force.
Cindy St. Hilaire: How strong is the magnet that it doesn't just rip everything up?
Hirofumi Watanabe: Yeah. We cannot observe the shapes of the cells, but yeah, I hope it's just stretch.
Cindy St. Hilaire: Yeah. Well, it certainly elicited an effect. So, in terms of future translational potential, what do you think about these findings that suggest either potential future therapies or even targets that we can use to develop therapies? Is there a future therapeutic angle to these really interesting biomechanical findings?
Ariel Gomez: Discovering or knowing the structure of these pressure sensing mechanism, I think we'll eventually have many applications because it will be applicable to hypertension, of course. And maybe we can begin to think... Not yet because it's really a fundamental discovery, it's not yet at that stage. But eventually the information can be used to start thinking about treatments that are addressing those particular structures that are involved from the beta one, integrating all the way to the nucleus. And little by little people started developing epigenetic therapies, right? And we are testing some of these compounds in our lower authority. Not with this model, with other models. But I think eventually we will be able to do what was the dream. It was really a dream years ago, was to do molecular therapy, right? And so a small compound development will play an important role. And eventually driving the molecules to the exact place in the genome is... So it would be not only patient-oriented, personalized medicine, but local specific. That should be the goal of medicine in the future. I won't be there when we get there.
Cindy St. Hilaire: I don't know. CRISPR is moving things rather fast, so that's great.
Ariel Gomez: Oh, yeah. You're right. You're right. You're right about that. Okay.
Cindy St. Hilaire: So what's next in this project? What do you think is the next low hanging fruit? Now that you've identified this baroreceptor or maybe a component of a larger baroreceptor family, what do you think is the next most important question?
Maria Luisa Sequeira-Lopez: We want to know what is in-between. And the bigger one integrating and the Lamin A/C. And also, we want to see how fast this reacts. So we'll be doing experiments with the constriction for just a few hours, and harvest both kidneys and we will try to do single cell RNA-seq and a from those vials.
Hirofumi Watanabe: I think we want to study how Lamin A/C regulates renin expression in renin cells, so chromatic modification initiated by changes in particle pressure more.
Ariel Gomez: And I think the... What I've been now pushing a little bit is to remember that there is another cell in there that is in between the pressure and the JG cells. And that is the endothelium cell. Right? And so, they are communicating with one another. So we are going to engage some... In fact, it's already happening. A member of the lab is already working with the same model that Hirofumi used, looking at endothelial cells label also using aninterfering promoter linked to a fluorescent protein. So we want to know what happens to the endothelial cells, because they are receiving the brunt of the pressure. And we don't know how they sense. We described the mechanosensing capability of the JG cells, the renin cells, but the whole system is probably a lot more complex than what we think.
Cindy St. Hilaire: I think that's the lesson of renin angiotensin signaling. It's always more complex.
Ariel Gomez: Yeah. Exactly.
Cindy St. Hilaire: Well, thank you all so much for joining me today. This is a beautiful study, very elegant. And I liked the new kind of in vitro models with this bead system. And congratulations on a whole lot of work. The amount of mice was probably a lot. I look forward to your future studies and learning what's happening at this endothelial renin cell junction.
Maria Luisa Sequeira-Lopez: Thank you. And we feel honored that you chose us.
Ariel Gomez: Yeah. Well, so I want to thank you for interviewing us. But I want to say that Hirofumi spent three years in the lab and he did a magnificent amount of work.
Cindy St. Hilaire: Wow. Yeah. I would have guessed a lot longer.
Ariel Gomez: Yeah. So he did a lot of work. And I'm very, very proud of what he has accomplished.
Maria Luisa Sequeira-Lopez: Yes. And I would like to add also that we were very lucky to have an expert in integrins, Dr DeSimone, who is the chair of Cell Biology at UVA and when we went and told him that we thought that this could be part of a mechanism sensing receptor, he started collaborating with us and opened his lab for us and trained Hirofumi with some experiments. It was really highly collaborative.
Cindy St. Hilaire: That's it for the highlights from our June 25th and July 19th 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 Hirofumi Watanabe, Dr Ariel Gomez, and Dr Maria Luisa Sequeira-Lopez.
Cindy St. Hilaire: This podcast was produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles was 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, 2021. 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.