June 2022 Discover Circ Res

This month on Episode 37 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the May 27th issue of the journal and also provides an overview of the Compendium on Basic Models of Cardiovascular Disease featured in the June 10 issue of Circulation Research. This episode also features an interview between Dr Nikki Purcell, Circulation Research Social Media Editor and Associate Professor at Huntington Medical Research Institute and Dr Mark Feinberg, Dr Rulin Zhuang, and Dr Jingshu Chen from Brigham and Women's Hospital in Harvard Medical School to discuss their study, Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Access in CD4+ T-Cells.   Article highlights:   Liang, et al. Tenascin-X Inhibits EndMT and Atherosclerosis   Jin, et al. Lineage Tracing of Pericardial Cavity Macrophages   Rosenzweig, et al. Basic Models of Cardiovascular 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 be highlighting articles from our May 27th and June 10th issues of Circulation Research. Dr Nikki Purcell, an Associate Professor from the Huntington Medical Research Institute and my colleague on the CircRes Editorial Board, is going to interview Dr Mark Feinberg, Dr Rulin Zhuang and Dr Jingshu Chen from Brigham and Women's Hospital in Harvard Medical School and they're going to discuss their study, Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Access in CD4+ T-Cells.   Cindy St. Hilaire:        The first article I'm going to highlight is titled Tenascin-X Mediates Flow Induced Suppression of EndMT and Atherosclerosis. The first author is Guozheng Liang and the corresponding author is Stefan Offermanns and they're from the Max Planck Institute. Atherosclerotic plaques in arteries arise when blood flow is reduced or turbulent. These are commonly regions where the vessels are curved or branched. Disturbed flow in these regions can prompt the local endothelial cells to undergo a process called e-to-mesenchymal transition or EndMT, which in turn contributes to atherosclerotic plaque formation. Exactly why turbulent, but not laminar flow prompts EndMT is not known. Using in vitro and in vivo experiments, this group discovered a critical EndMT suppressor protein expressed at high levels in endothelial cells exposed to lamina flow, but not exposed to turbulent flow. Mice that lack the protein, which is called Tenascin-X or TN-X, exhibited signs of EndMT and inflammation throughout their aorta and they were more prone to atherosclerosis. TN-X binds and blocks the function of the cytokine TGF beta, which is a potent driver of EndMT. Inhibiting the activity of TGF beta, whether with an anti TGF beta antibody or by deletion of its receptors, prevented the EndMT promoting effects of TN-X loss. Together, the results suggest that bolstering or mimicking the effects of TN-X may be a novel strategy for preventing atherosclerosis progression.   Cindy St. Hilaire:        The second article I want to share is titled, Genetic Lineage Tracing of Pericardial Cavity Macrophages in the Injured Heart. The first author is Hengwei Jin and the corresponding author is Bin Zhou and they're from the Chinese Academy of Science. If the heart suffers an infarction, monocytes and macrophages travel to the injury site via the bloodstream and locally from within the heart itself. A recent Immunity paper claims that macrophages in the surrounding pericardial cavity also can infiltrate the heart to aid in its repair. New work from Jin and colleagues, however, does not support these findings. The discrepancies seem in part to be related to the way that pericardial cavity macrophages were tracked. In the Immunity study, pericardial cavity macrophages were tracked by labeling with fluorescent beads or transplantation of trackable pericardial cavity macrophages into the recipient mice. Both of these strategies showed the cells entering the myocardium after infarction.   Cindy St. Hilaire:        However, in the Circ Research study, mice were engineered to have trackable endogenous pericardial cavity macrophages. Using these animals, the team found that while pericardial cavity macrophages did migrate to the surface of the heart, they did not significantly penetrate the tissue. Further discrepancies between the studies were apparent in loss of function experiments. Where the initial study found pericardial cavity macrophage loss led to increased myocardial fibrosis and left ventricle stiffness, the new study did not. Because myocardial infiltration of pericardial cavity macrophages represents a paradigm shift in heart injury research, the authors say its existence requires rigorous testing and for now at least, it remains debatable.   Cindy St. Hilaire:        The June 10th issue of Circ Research is our Compendium on Basic ModelsoOf Cardiovascular Diseases. The articles in this compendium are Illuminating the Mechanisms Underlying Sex Differences in Cardiovascular Disease by Carrie Weiss and Karen Rue, Animal Models to Study Cardiac Arrhythmias by Daniel Blackwell and Bjorn Knollmann, Animal Models of Exercise from Rodents to Pythons by Margaret Hastings, Anthony Rosenzweig and colleagues, Animal Models of Atherosclerosis Supportive Notes and Tricks of the Trade by Anton Gastera and Goran Hanson and colleagues, Heart Failure with Preserved Ejection Fraction, Heterogeneous Syndrome, Diverse Preclinical Models by Jason Roh and colleagues, Large and Small Animals of Heart Failure with Reduced Ejection Fraction by Patrick Pilz, Ronglih Liao and colleagues, CRISPR Modeling and Correction of Cardiovascular Disease by Ning Liu and Eric Olson, Animal Models of Cardiovascular Complications of Pregnancy by Zoltan Arany, Denise Hilfiker-Kleiner and S Ananth Karumanchi, Animal Models of Dysregulated Cardiac Metabolism by Heiko Bugger, Nicole Burn and Dale Abel, Biomedical Imaging and Experimental Models of Cardiovascular Disease by Marielle Scherrer-Crosbie and David Sosnovik, Zebrafish Models of Cardiac Disease from Fortuitous Mutants to Precision Medicine by Juan Gonzalez-Rosa and Cellular and Engineered Organoid Cardiovascular Models by Dilip Thomas and Joe Wu and colleagues.   Nikki Purcell:              Hi, I'm Dr Nicole Purcell, Associate Professor in the Cardiovascular Division at Huntington Medical Research Institute and today Dr Mark Feinberg, Dr Rulin Zhuang and Dr Jingshu Chen from Brigham and Women's Hospital in Harvard Medical School are with me to discuss their study, Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Access in CD4 T-Cells in our May 27th issue of Circulation Research. Thank you for joining me today.   Mark Feinberg:          Thanks for having us. We're glad to be here.   Rulin Zhuang:             Thank you.   Nikki Purcell:              There were a lot of authors involved in this study, and while all could not join us today, I appreciate you taking the time to discuss your findings. So, your paper is dealing with vascular disease, often associated with elevated blood pressure or hypertension. A hallmark of advanced vascular diseases fibrosis is in the heart. When we talk about fibrosis, most investigators would think of interstitial fibrosis, but your paper focuses on perivascular. So Dr Feinberg, what is perivascular fibrosis and what led you to focus in hypertension?   Mark Feinberg:          Thanks, it's a great question. So perivascular fibrosis is characterized by an increased accumulation of connective tissue around blood vessels. There are many cell types that contribute to this process. Fibroblasts, obviously, that produce a lot of extracellular matrix and a wide range of collagens, but other cell types, muscle cells, which are sensitive to humoral factors, Ang-2, endothelial and other cytokines and growth factors, and more recently getting more attention, our immune cells, including infiltrated T-cells, which are important mediators of crosstalk between fibroblasts and extra matrix that actively modulate this fibrotic response. Clinically, perivascular fibrosis is a hallmark of several age related conditions that we see in our patients, hypertension, diabetes, chronic kidney disease, really all are involved with extensive extracellular remodeling. Many of our patients with elderly patients with hypertension have left ventricular hypertrophy, stiff heart and pure diastolic dysfunction, as well as arterial stiffness, which can contribute to a range of diseases from heart failure, MI, stroke and organ damage and including kidney disease.   Mark Feinberg:           So we actually started off wondering if there are any key transcription factors that may be involved in CD-4 T cell effector functions, given potential role of CD-4 T cells in this hypertensive response, and that perhaps may underlie the development of blood pressure and organ injury. With that idea, if you can understand a signaling pathway, perhaps it might impact the development of blood pressure, cardiovascular modeling, particularly with interstitial fibrosis and end organ injury. To be honest, we did not expect to find a factor that regulated perivascular fibrosis and end organ damage, but had no effect on blood pressure or interstitial fibrosis. This was a real surprise in the subject of the paper.   Nikki Purcell:              Like you said, the importance of T cells and perivascular fibrosis falling hypertension with Ang 2 infusion has recently been demonstrated, but you know, you guys were really focusing on what was the mechanism, trying to understand that. So, Dr Feinberg, can you elaborate on why you chose to focus on the CD-4+ T cells and hypertension in particular, how it came about the transcription factor, Kruppel like factor 10 or KLF 10?   Mark Feinberg:          Yeah. Great question. So we over accumulating studies many years now that CD-4 T cells play a particular important role, mediating hypertension, and a variety of preclinical models. For example, studies from David Harrison's or Steven Crowley's groups perform some classical experiments using immunocompromised mice. These are either RAG1 or SCID mice, which as, you know, have defective T or B cells. What's really interesting about those seminal papers was one that Ang2 mediated increase in blood pressure and the associated cardiac and kidney injury in these mice was severely blocked and two only when there was adoptive transfer of CD-4 T cells did that restore these deleterious effects in response to Ang2, indicating really for the first time that CD-4 T cells are key mediators of blood pressure and organ injury, and predominantly they focus on interstitial fibrosis and remodeling. However, the factors that mediate the CD-4 T cell effect on end organ damage or blood pressure really have been poorly defined over the years.   Mark Feinberg:          And so work from our group and others have identified over several years a transcription factor called KLF10 or Kruppel like factor 10 belongs to a family about 17 total Kruppel like factors. This one is expressed highly in CD-4 T cell subsets, both factors and T regulatory cells and work from our group and actually those are others have, have shown that KLF10 can regulate T cell factors. They're more hyperactivated and also the T regulatory subsets don't exhibit what we call immunosuppressive or anti-inflammatory properties. We've shown this now in the context of athero, we've developed CD-4 specific health and knockout mice, and when placed on a high fat diet, those mice developed obesity and insulin resistance. However, the role for KLF10 in CD-4 T cells and hypertension really was unknown and really came into this thinking that this was going to play a role in development of blood pressure and interstitial fibrosis. So it was, again, a real surprise for us.   Nikki Purcell:              I'm glad you mentioned that. Dr Zhuang, given the role of the CD-4T+  cells in controlling hypertension, you would've expected the blood pressure to be increased in your CDF4KLF10 knockout mice, but surprisingly, there was no difference. Can you tell us why you think this happened?   Rulin Zhuang:             Yeah, that's true. When we found severe cardiovascular fibrosis and vascular remodeling in TKO mice, after Ang2 infusion, the first potential explanation comes to us that there should be a difference in blood pressure, but we didn't find any difference in after 28 days and even 42 days after Ang2 treatment. So previous literatures did indicate that hypertension and is related cardiovascular injury could result from other forms of mediators, which is independent of blood pressure. Which suggests that blood pressure alone perhaps is not sufficient to predict end organ damage in hypertension. You know, it is because of the lack of difference in blood pressure that allow us to explore another molecule insights into perivascular fibrosis.   Nikki Purcell:              So Dr Chen, you found that the CD-4 positive KLF10 knockout mice had perivascular fibrosis in multiple organs, both the aorta, hearts and kidneys following Ang2 infusion. How did you identify that IL-9 was mediating these effects on perivascular fibrosis?   Jingshu Chen:             Actually after we found the phenotype in Ang2 treated TKO mice, we start to find some possible mediators involved in these multiple organ perivascular fibrosis. Firstly, we detect the expression of angiotensin to receptors in CD-4 positive T Cells, but no difference was observed between TKO and our Cre mice. KLF10 also have another name, which is TIEG1, TGFbeta Inducible Early Gene-1. So next we checked the TGFbeta signaling. But we didn't find any difference in the rational level of TGFbeta one to three between Cre and knockout mice in the CD-4+ T cells that made it. Also, we didn't find any impact of calcium in TGFbeta signaling in vivo supported by our different RNA seek dataset for some pathway analysis. We performed calcium flux profiling from our Ang2 treated TKO and Cre mice. Although we do find some cytokines, slightly changed TKO mice to treatment, IL-9 nine was the only one significantly increase dramatically in both male and female TKO miceIL-9 nine was reported in the regulation of the immune responses and played a pro fibrotic role in lung fibrosis and liver fibrosis. We'll assume that perhaps IL-9 contributes to perivascular fibrosis. We gave the recombinant IL-9, our control mice, which have a less perivascular fibrosis after Ang2 treatment. After we giving them recombinant IL-9, we do found more perivascular fibrosis, which is efficiently phenocopy what we observed in our transgenic mice. Also, our further study found that calcium could bind into IL-9 promoter and interact with HTAC-1 to inhibit IL-9 activation. That is where we make a conclusion that we consider that calcium deficient C4 positive release more IL-9 that introduce perivascular fibrosis.   Mark Feinberg:          I might add that, the discovery of this phenotype was almost missed. And I think it was interesting when Rulin described some of the initial H and E from the hearts of these mice and remember him saying there wasn't a lot of interstitial fibrosis, but there was lots of thickening outside the blood vessel wall. And what was interesting is that several of the aortas didn't show this, but it turned out those aortas Rulin had actually stripped for different reasons. And then when he repeated it without stripping and looking at all the organs, aorta, heart, kidney, you could tell easily who was the knockout, just blank, looking at the H and E slide. And he searched very hard for interstitial fibrosis. I actually had a colleague, Rick Mitchell who's a cardiac pathologist at the Brigham, review these slides in a blinded manner who, who verified that there was a lot of perivascular fibrosis in multiple organs, but no clear difference in interstitial fibrosis. That made us really excited about our new pathway to explore.   Nikki Purcell:              Nice. That leads you nice that you were talking about the strip because Dr Zhuang and  Chen, you had through your RNA seek data found that calcium signaling were dominantly upregulated in those nonstripped aorta. That's the perivascular adventitia tissue wasn't removed in those, in the CD-4 + KLF10 knockout mice after Ang2 treatment. To further investigate calcium signal involved in the fibroblast and myophytes differentiation, you had gone and nicely isolated primary fibroblasts from these blood vessels. And that can be quite tricky. So can you tell us about what method was used to purify these fibroblasts for this study?   Jingshu Chen:             Yeah, it's actually very interesting progress for isolating those because when we search literatures, there's actually no well-established method to isolate from the aortas. We actually go from the aorta digestion to make sure we have a very good viability of the digestive cells. And then after that, Dr Zhuang and I kept discussing at lab, like why we just use the BES to isolate it's use the antibody, find the BES to isolate the fibroblasts. We tried a lot of methods and then finally we find optimize the protocol by use of magnetic BES these, but also to remove a lot of other cell types and to make sure we get the good purity of the fibroblasts. It's a very nice protocol and we actually published in the Atherosclerosis journal. It is already online. So I hope it can benefit the field. Anyone who can use it.   Nikki Purcell:              Yeah. It's these beautiful pictures that you've got from that isolation in the paper. You used both the cells as well as the tissue for several RNA Seq overlapping data sets in this manuscript. You used those stripped and non-stripped aortas from the KD-4 + KLF10 knockout Cre mice exposed to Ang2. But then as you talked about Dr Zhuang, you also use from a group of Ang2 to treated mice that received that anti IL-9 monoclonal antibody. What are some of the main findings that you found from these dataset that you'd like others to know?   Jingshu Chen:             Followed by the discussion, we are having the non-stripped and stripped aortas at very first, we sent off on the stripped aortas for sequencing, and we don't find a lot of genes are regulated actually only 200, 300 genes were regulated. It comes to the question actually, the perivascular fibrosis happened in adventitia. We sent out again for the nonstripped aorta, which has a perivascular fibrosis areas included. And luckily, we find like thousands of genes were regulated and a lot of the genes by perform the gene oncology pathway analysis, we found, calcium pathways and fibrosis are very enriched for the top 10 significant pathways.   Nikki Purcell:              Nice. And so once you found that you were very interested and you were able to isolate these primary fibroblasts from your KLF10 knockout mice, you then performed single cell RNA sequencing on these. Were there specific fibroblasts populations of interest that were driving this perivascular fibrosis that you found?   Jingshu Chen:             Yes. The single cell RNA Seq we perform as we digest the whole aorta, including the adventitia tissues and then make a cell suspension center for Seq. And the beauty and the privilege of the single cell is you actually can cluster different cell types. And then you can extract only the fibrous part from the whole aortic cells. And because we perform the single cell, including 50,000 cells, it's a very good population for us to look into. And by extract only the fibroblast clusters, we actually reclustered and find, there's actually nine clusters within this fibroblast clusters and followed by that we actually perform a very interesting analysis called RNA velocity. It is actually measuring or calculating the splices and unspliced mRNAs. And then by using this analysis can predict the potential future directions of this, the cells. And by utilizing that analysis, we actually found actually fibroblast original type can gradually progress to a more fibrosis or a mild fibrous type by looking at the splices and unspliced ratio. And which is the picture we showed in the manuscript.   Mark Feinberg:          I might add that I was really impressed with the single cell seq. When you look at the vascular fibrosis, it looks like chaos. And the single cell seq provides a fair amount of order that I had not anticipated. We would be able to appreciate what I mean by that is Jingshu was able to characterize these nine subgroups and with the velocity analysis, prioritize them in terms of who are the major drivers. And this, for example, the subgroup eight was highly expressed in this collagen eight, A1 for example, that we think is a pathological fibroblasts whereas the canonical fibroblasts markers were in this earlier fibroblast subgroup one, and there's many other fibroblast cellular identities in between, including those that have endothelial mesenchymal markers and many others. We've learned quite a bit from it. And I have to say for others, it might be interested it's worth the investment, because it provides clarity.   Nikki Purcell:              Thank you. So because of the heterogeneity, was there any sex differences between your male and female knockout mice on hypertension and this perivascular fibrosis that you saw? Mark Feinberg:          Phenotypically we did not notice any difference. The knockout females do not similar to the males they don't have increased blood pressure. They still develop accelerated perivascular fibrosis and many of the IL-9 signaling pathway. We didn't just because of cost issues, I think did not send out for the female single cell seek just because the phenotype was similar. But it's a great question. We, we probably should just to verify things.   Nikki Purcell:              So what are the translational implications of your findings and how might you use this for treating vascular fibrotic diseases?   Mark Feinberg:          We hope this study highlights the importance that hypertension is clearly a heterogeneous disease and multiple signaling pathways are likely responsible separately for different phenotypic manifestations of an organ disease. In particular, we show that perivascular fibrosis and interstitial fibrosis are distinct and are controlled by the former, by the CD-4, KLF10, IL-9 signaling pathway. And this may be leveraged for therapeutic benefit someday in patients. While early, these preclinical studies, we hope will inform the potential feasibility of considering IL-9 neutralizing antibodies, for example, in a proof of concept study in patients with uncontrolled or what we call refractory hypertension or chronic kidney disease that have a lot of perivascular fibrosis and that maybe that would improve or benefit the end organ damage that unfortunately occurs in those patients.   Nikki Purcell:              What's on the horizon in the lab, what future studies are needed or have come from this work that you'd like to tell us about?   Mark Feinberg:          There's actually a lot to do. We have some preliminary data that suggests that these CD-4 T cells probably there's a lot of crosstalk, not just with fibroblasts, but between muscle cells and endothelial cells. We show in the supplement of this paper that there's impairment of some muscle cell dependent, vaso relaxation and contraction in myograph studies. We have some preliminary data to suggest that endothelial dependent vaso relaxations also ongoing. The big question is how does KLF10 be an IL-9, maybe in a parakin manner impact the progression of disease in the intima. Where we actually spend most of the time in the lab thinking about the inner most lining of the blood vessel wall. This was a real treat to think about it from outside in. That's a real major focus in the lab is trying to understand the signaling pathway and maybe there's some crosstalk with IL-9 and other factors that control KLF10.   Jingshu Chen:             For me, it's because we have seen this heterogeneities of the aortic cells using single cell. It's actually a bunch of, for us to do, like we can utilize the single cell technology to see how IL-9 is affecting the aortic walls for example, the fibroblasts, and also we can be more like to do a cell-cell talk like how endothelial cells, fibroblasts or other immune of key cells, especially like KLF10 is knocked out in T cells, how the cells talk and how we can observe this by using the data mining technique.   Rulin Zhuang:             Actually in our study we've showed in the last figure is about the visualization of the IL-9 and we do found there it's reversed the angio to induce the fibrovascular fibrosis. The actually previous paper showed IL-9 can treat liver and heart and lung fibrosis. We assume that is probably IL-9 is a kind of profibrotic cytokine and can be evaluated further in the future to seeing other fibrosis like heart, interstitial maybe, I don't know, but it's a good way to detect.   Nikki Purcell:              Great. Thank you so much for joining me today, Dr Feinberg, Zhuang and Chen, and for discussing your exciting findings and I look forward to seeing your future work. Thank you.   Mark Feinberg:          Thanks so much for having us.   Cindy St. Hilaire:        That's it for highlights from the May 27th and June 10th 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 guest host Dr Nikki Purcell and our interviewees, Dr Mark Feinberg, Dr Rulin Zhuang and Dr Jingshu Chen. 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, 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.