October 2020 Discover CircRes

Oct 15, 2020

This month on Episode 17 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the September 25 and October 9 issues of Circulation Research. This episode features an in-depth conversation with Drs David Dichek, Sina Gharib and Tomáš Vaisar regarding their study titled Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture.

Article highlights:
Cai, et al. Single Cell RNA-Seq in Arteriosclerosis
Schuhmann, et al. CD84 in Ischemic Stroke
VanOudenhove, et al. Gene Regulatory Dynamics of Developing Human Heart
Nie, et al. Periostin is a Target to Treat PH

Dr Cindy St. Hilaire: Hi, 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 four articles selected from the late September and early October issues of CircRes. I'll also be interviewing Drs David Dichek, Sina Gharib, and Tomáš Vaisar regarding their study titled Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture.
The first article I want to share is titled Single Cell RNA Sequencing of Allograft Cells in Transplant Arteriosclerosis. The first author is Jingjing Cai and the corresponding author is Qingbo Xu from Zhejiang university in Hangzhou, China. Arteriosclerosis is a major contributor to organ transplant failure. The thickening and stiffening of arteries within the grafts results in diminished blood flow supplies and diminished organ function. While it is well understood that atherosclerosis is an inflammatory disease, the details of the cellular and molecular players on this transplant-specific pathology are lacking. Now, Cai and colleagues used single cell RNA sequencing to identify a consensus of cells and cytokines in sclerotic transplanted aortas in mice. Two weeks after transplant, the grafted vessels exhibited signs of arteriosclerosis and by four weeks, this remodeling had worsened significantly.
Analyzing the RNA transcripts of over 12,000 individual cells isolated at both two and four weeks, the team discovered that the number of T-cells was greatly increased throughout the process. An early abundance of macrophages gave way to a later wave of B cells and there was also evidence of the development of tertiary lymphoid tissue. They further found that chemokine CCL121 was up regulated after transplant, both the mRNA in the tissues, as well as the protein levels in the animal's blood. The authors then went on to show that blocking CCL121 or its attracting partner, CXC3, significantly delayed arteriosclerosis in the grafted vessel. Hence, this work not only defines the cellular and molecular drivers of arteriosclerosis in grafted vessels, but highlights potential molecular targets for future therapeutic interventions.
The second article I want to share is titled CD84 Links T-cell and Platelet Activity in Cerebral Thrombo-inflammation in Acute Stroke. The first author is Michael Schuhmann and the corresponding author is David Stegner, and the work was completed at University of Würzburg in Germany. Ischemic stroke is caused by the occlusion of cerebral blood vessels and it is a leading cause of death and disability worldwide. Despite treatments to degrade or remove clots such as mechanical thrombectomy, infarct size itself can continue to grow even when blood perfusion is re-established. Thrombo-inflammatory processes are thought to mediate this worsening injury, with both T-cells and activated platelets playing a role. Because both T-cells and activated platelets express CD84, which is a self-binding adhesion molecule involved in lymphocyte activation, this team tested the hypothesis that CD84 might mediate stroke inflammatory processes.
They went on to show that mice lacking CD84 have smaller infarct sizes with reduced T-cell inflammation after stroke than wild-type animals. Furthermore, mice that specifically lack CD84 in either T-cells or platelets also experienced smaller infarcts. The team went on to show that CD84 promotes T cell migration in vitro. And then in patients with stroke, high expression of CD84 in platelets was associated with poor outcomes. Together, these results suggest that activated CD84-secreting platelets encourage inflammatory T cell migration to the infarct site. And that blocking CD84 activity could be a novel therapeutic strategy for minimizing inflammatory injury after stroke.
The third article I want to share is titled Epigenomic and Transcriptomic Dynamics During Human Heart Organogenesis. The first author is Jennifer VanOudenhove and the corresponding author is Justin Cotney. And they're from the University of Connecticut. Congenital heart defects, or CHDs, are common birth abnormalities and while some genes have been linked to congenital heart defects, the majority, close to 60%, have unknown etiologies. It's thought that multiple genetic and environmental factors contribute to congenital heart defects. One of which could be variations in both cis and trans regulatory regions of the genome. To find such heart specific regulatory regions, this team examined heart tissue from human embryos obtained four to eight weeks after conception.
They performed chromatin immunoprecipitation experiments to scour the heart genomes for histone modifications associated with increased or decreased gene transcription. They also performed transcriptome analysis to see whether the genomic regions identified by chip corresponded with the activity status of nearby genes. In total, the team found more than 12,000 previously unknown enhancers that were enriched for binding sites for heart specific transcription factors, some of which included GATA, MEF2 and Nkx. These binding sites tended to be close to genes activated in the heart. Many of the regions also contain sequence variations that have been associated with atrial fibrillation. These newly identified sites are potential congenital heart defect candidate loci and the authors have now made their data readily available so that other investigators may study it.
The last article I want to share with you before we switch to our interview is titled Periostin: a Potential Therapeutic Target for Pulmonary Hypertension? The first author is Xiaowei Nie from the Shenzhen Third People's Hospital and the corresponding authors are Jingyu Chen and Jin-Song Bian from the Wuxi People's Hospital and the National University of Singapore, respectively. Pulmonary hypertension, or PH for short, is a life-threatening disease where an excess in the proliferation of vascular smooth muscle cells and the deposition of extracellular matrix thickens the walls of the lung vasculature, which leads to an increase in pulmonary blood pressure and ultimately contributes to right heart failure. Vasodilatory medications can be used to treat the symptoms of the disease. However, these medications do not prevent or reverse the underlying pathogenic remodeling. This study now suggests that drugs targeting the secreted extracellular matrix protein, periostin, might be a potential therapeutic strategy for the treatment of pulmonary hypertension.
Periostin is an abundant protein in the lung arteries of pulmonary hypertension patients. And it is thought to be involved in cell adhesion and wound healing mechanisms, such as the proliferation and the migration of smooth muscle cells. The team confirmed increased production of periostin in patient lungs, and also found the same to be true for mice with an induced model of pulmonary hypertension. They also showed that genetic deletion of periostin attenuated pulmonary hypertension in mice, while suppression of periostin via RNA inhibition could even reverse pathological vessel thickening and the subsequent right ventricle hypertrophy. The team went on to identify factors HIF-1a and TrkB as factors that mediate periostin's effects in cultured arterial cells. And they suggest that blocking either of these factors or by blocking periostin itself could be a novel strategy for the treatment of pulmonary hypertension patients.
Drs Tomáš Vaisar, Sina Gharib, and David Dichek from the University of Washington in Seattle, Washington are here with me today and we're going to discuss their recent study titled Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture. Thank you all so much for joining me today and congratulations on this beautiful and interesting study. So this is an atherosclerotic study, but unlike many in the field, it's really looking at the end stage event called plaque rupture. So for those listeners who are unfamiliar with the term, plaque rupture is when an atherosclerotic plaque degrades and its contents are exposed to the circulation, which can then induce a clotting event and lead to all sorts of adverse pathologies, myocardial infarction, transient ischemic events, stroke. So I'm wondering if we if maybe you can give us a little bit of background about what's known and what really is unknown at least before your study regarding plaque rupture.

Dr David Dichek: So the pathogenesis of acute myocardial infarction and stroke was really unknown for many years. And the idea that it was due to acute thrombosis was really confirmed by a study probably 30 years ago, that did angioscopy in the coronary arteries, proximal to a myocardial infarction, and visualized actual clot so that the clot was confirmed to be associated with the acute event. At that point, the question became why would a coronary artery form a clot? And that led to identification of, or histologic studies that identified ruptured caps of atherosclerotic plaques, exposure of the blood to the thrombogenic contents of the lesion, and a thrombus.
However, it was not known what the initiating event was in rupture of the plaque cap. And there were a lot of hypotheses and a lot of nice work, but it does still remain an unknown. A significant amount of focus has been devoted to the possibility that proteolysis is the initiating event. And that was sort of the takeoff point from our study because we had developed a mouse model where proteolysis clearly was associated with rupture of plaque caps. And we decided we wanted to get more into the biochemistry of what was going on and go beyond the histology. So that was really what led up to our study.

Dr Cindy St. Hilaire: It's really interesting. So, mice are really good and obviously really useful, very well-known model systems to study atherosclerosis and particularly the initial drivers and maybe the mechanisms of the disease pathogenesis, but like many models systems, they're not perfect. So I'm wondering if you could discuss the limitations of murine model systems and specifically for this study, how you were able to overcome some of those limitations.

Dr David Dichek: So the limitations of mouse models of plaque rupture are that essentially none of them duplicate the histology of human plaque rupture, particularly the thrombus that occurs on top of the plaque rupture. So there are various mouse models where caps are disrupted, but there's not acute thrombosis. It has been argued in the vascular community as to whether these models are authentic models of plaque rupture, because they don't have the superimposed thrombosis. And the counter argument is well, mice aren't people, they have different hemostatic and coagulation factors that may be differentially regulated. The hemodynamics of small mouse arteries is different from human mouse arteries. And the fact that you don't get a thrombus doesn't necessarily mean that you're not modeling the process that would cause it.
So we really accepted that argument as being valid and felt that the occurrence of frank plaque rupture, and that was in our Circulation paper in 2010, in these lesions in the mice, really validated it as an authentic model of cap disruption. And so I agree it's arguable, whether this is an authentic model. But we actually took that issue head on by saying, well, is the biochemistry of the ruptured plaque similar to the biochemistry of a ruptured human plaque? And that if there were similarities that we would gain more confidence in our model being an authentic model of plaque rupture and that it matched not only the histology, but also the biochemistry.

Dr Cindy St. Hilaire: One of the main tools you used, you used shotgun proteomics, which I think is just a great name for it. And also a algorithmic learning tool or analysis tool called proteomaps. I was wondering if you could give us a little bit of background about the proteomic aanalysis involved, what does that entail? Especially, when you're comparing a teeny tiny mouse plaque to a larger human plaque and then how that analysis was done?

Dr Tomáš Vaisar: So the shotgun proteomics term was coined by John Yates, but way back in the early 1990s. And it's essentially the way how you enumerate the proteins and in more recent forms, you even quantify the abundance of the proteins in a very complex mixture. So shotgun proteomics essentially takes a protein sample. And in this case it was an extract of the tissue and uses protease, trypsin typically, to cut the proteins down to peptides, which are relatively small and relatively well
behaved compared to intact proteins. And then using tandem mass spectrometry combined with liquid chromatography separation, basically aims to sequence every single of those peptides or majority of the peptides. And then based from the identification of the sequence of individual peptides piece back together like a jigsaw puzzle, what was the protein present in the original mixture?

Dr Cindy St. Hilaire: That's so interesting. I know it's been around for a while. I'm always impressed by it.

Dr Tomáš Vaisar: And then the other approach we use it's called Proteomap developed by Ben Cravatt, collaborator on this paper at La Jolla. And that approach uses basically the shotgun approach but as a first step uses gel electrophoresis, SDS PAGE electrophoresis to fractionate the very complex mixture to size segments.

Dr Tomáš Vaisar: So you run your complex mixture on a gel, you slice it by size and then run shotgun proteomics experiment on each of those slices after gel digestion. And then Ben developed a set of tools where you identify the proteins and their abundance in each of the bands based on the size and the way it's applied to formation or mapping of proteolytic events is based on the idea of that intact protein will show up at the molecular weight of the intact protein but if it was cleaved by a protease, it will also show up at the molecular weight, which is smaller corresponding to the fragments formed by proteolysis. Then you use set of bioinformatics tools to piece this all together and generate the Proteomaps.

Dr Cindy St. Hilaire: You pick it apart, throw it in the machine and then put it back together. That's so cool. It's so amazing.
So what were the main findings that you were able to pull out of your comparisons? And I think you had three main groups, if I understood it right? There's the transgenic mouse that has the plaques that don't rupture, and then there's the atherosclerotic mouse that had the transgenic bone marrow, and then you had the human. Can you tell us a little bit about the different groups you compared and then what ultimately you found?

Dr David Dichek: Sure. Yes. You are absolutely right. We had what we refer to as the straight transgenic mice that are either transgenic for macrophage overexpressed uPA or not. We also had older mice who had advanced atherosclerosis and receive bone marrow transplants from mice that either had uPA overexpression in bone marrow or not and then we had the human plaque. So those were the three groups.
So what we found was that looking at the proteome of those three groups, we were able to find some common biological processes, and this was really Sina's work, but taking the proteomics data and analyzing it with sophisticated bioinformatics tools. We looked not only at the overlap in specific proteins among the models, but the overlap in biological processes, because it may be in different species that there are different proteins, different actors carrying out the same roles. And that's been described in other systems as well.
So we were able to identify not only common biological processes, but surprisingly, we were able to identify decreases in specific category of proteins, basement membrane proteins that were common to two of the models, the straight transgenic and the human model and loss of these proteins certainly has a plausible role in precipitating plaque rupture. So I think one aspect of the analysis that's worthy of note is that we initially thought we would observe more profound changes in the bone marrow transplant mice because they had more advanced atherosclerosis. And in fact, we found fewer changes than in the straight transgenic mice, but thinking about it after letting the data talk to us, rather than trying to impose our own on the data-

Dr Cindy St. Hilaire: Always a good idea.

Dr David Dichek: ... was that the straight transgenic mice were telling us we've been overexpressing urokinase for 20 weeks since we were conceived, and the bone marrow transplant mice had it for only eight weeks. And indeed they had far less loss of basement membrane proteins and far fewer changes in their plaques than the mice that had expressed it for a longer time. And so when one placed the three groups in a chronology of exposure to protease activity with the bone marrow transplant mice, being the shortest exposure than the straight transgenic mice, and then the humans who've had decades of exposure, it really tells you a nice chronological story about the biological processes leading to plaque rupture. And I think that's a generally applicable lesson and can be applied to other problems in cardiovascular biology. And that is when you have a biological process for which you can't get human tissue until after it's occurred because you can't go in and biopsy.

Dr Cindy St. Hilaire: I have that problem with valve calcifications that you can't take your valve out early.

Dr David Dichek: If you can get a mouse model that duplicates the pathology, then you have access to the steps leading up to the event. And that's what we tried to construct in this study. And really it was really Sina’s analyses that allowed us to make those connections.

Dr Sina Gharib: Yeah. Of course, David was kind of the mastermind behind the design of the experiments on the developing of genetic models and Tomáš is a renowned expert in proteomics analysis. And I kind of joined more on the bioinformatics component of this study, tried to put some of the large data that was being generated together. And as David and Tomáš mentioned, of course atherosclerosis is a very complex disease with many, many components. And then of course the mouse model doesn't quite capture all the different pathophysiological events that happen. So one of the aims of this study was to try to integrate and merge the findings from these model without coming a priori with a bias or a pathway or a candidate gene, we decided to do a relatively unbiased shotgun proteomics approach, we actually do for everything.
So the challenge then was how to put it all together. And as Tomáš mentioned, there are statistical tools to try to identify a relative abundance of proteins. But, a few things that pure biologists often don't have to encounter is, you're not one or two different proteins, you're looking at thousands of proteins. So there are issues, statistical issues, such as multiple comparisons. If you looked for changes, you're going to find changes just by random chance. So a lot of statistical adjustments had to be made to ensure that those were adjusted for. This are also
pathways and processes that were coming out of these results. There's many different pathways that were interrogated. And again, statistically, you want to adjust for the fact that many of those could have been there by random chance.
So there's a fair amount of statistical methods that need to be applied for this data. We also did somewhat more sophisticated pathway analysis where we develop networks based on the differential expressed proteins between the ruptured and unruptured plaques to identify connection among these proteins and identify hubs which are highly connected nodes that could potentially drive the biology of a network. So other types of kind of deeper statistical analysis was done, which are maybe more hypothesis-generating because we actually did not follow up on some of these candidates, but I think they really do provide a map or framework to then pursue more mechanistic experiments to see what happens if we knocked out this highly connected node at the plaque rupture site to see if we can either stabilize or manipulate the biology as plaque rupture.

Dr Cindy St. Hilaire: Yeah. I mean, that's really the strength of these unbiased approaches is you can come up with so many more novel targets and pathways that might be contributing. So they're just really great. So one thing I found really interesting, you mentioned that you saw a clear distinction in the proteome and I think it was specifically talking about the human samples because they were large enough to see ruptured area versus non ruptured area, but you really saw a distinct difference in the proteomes of the ruptured area of the plaque versus the non ruptured area of the plaque. And obviously the models you were using are overexpressing a protease. So of course there's a role for proteolysis in this process, which you've now firmly established, but I'm wondering if there are other processes that might also erode the basement membrane. And did you pull up anything that might suggest of other things that are happening or even are there other hypothesis out there that we could test with an approach like yours?

Dr David Dichek: I think the pathways that came up have probably all been implicated previously. We have processes like inflammation and complement activation, immune response, thrombosis. That's a post-hoc event. I think what was most unexpected was the decrease in the abundance to basement membrane proteins rather than collagens. So collagen has become the sort of signature protein of stable and unstable plaques and used as a surrogate, people do Picrosirius red staining. It's easy to detect with a histochemical stain, you don't even need an antibody. And surprisingly, we found very few differences in collagens and actually no differences in the type 1 or type 3 collagen, which are thought to be the primary stabilizers of the plaque cap. They weren't significantly different in between ruptured and stable areas of the same plaque. So that was certainly a big surprise.

Dr Cindy St. Hilaire: Yeah. Because that would indicate that it's not necessary... We always say thinning of the cap, which obviously we know that there's remodeling, it can get thinner. But you kind of found that the contents were the same, but it's the basement really that was eroded.

Dr David Dichek: Yeah. The basement membrane proteins were lost. It used to be said in physiology that if you discovered something new, you should just go to the German literature and go back 30 years and it had already been described. And so, looking back in the literature, there are actually the work of Jean-Baptiste Michelle in France and a scientist in Finland, Petri Kovanen, have actually focused on the potential role of basement membrane in unstable atherosclerosis many years ago, but it was kind of buried in the collagen hypothesis. And I think it needs resurrection.

Dr Cindy St. Hilaire: Well, I think this paper has done that so well done there. That's great.

Dr Tomáš Vaisar: Would be worthwhile to know that of course, the way you prepare the samples may affect what exactly you're seeing. But we've done very careful characterization of the sample preparation of the extraction procedure to focus, to enrich the exosomal matrix proteins because of this collagen hypothesis. And even with that, we basically saw no difference.

Dr David Dichek: Yeah. I think that's an excellent point. If we hadn't found collagen in our extracts, we would not be able to conclude a lot about it. And how you do the extraction, how you process the samples here can really influence what you find. We call it unbiased, but there are technical biases that enter, especially in sample preparation, but our extraction process really was able to extract collagens as well as elastin, which is really infamous for being a-

Dr Cindy St. Hilaire: Difficult.

Dr David Dichek: ... I really think we were getting a good sampling of the matrix here.

Dr Cindy St. Hilaire: I don’t know iif there is an answer for this question, but it's something I'm always thinking about. We always talk about athero being so prevalent because there's no kind of evolutionarily the way to tamp it down, it happens later in life. But can you think of any advantage that the vasculature would have in eroding the basement membrane or altering proteases in a response? I was just trying to think, is this just harnessing a wound healing process that's gone awry or could this ever be protective at all in any way?

Dr David Dichek: Well, I think you hit the nail on the head, at least according to my bias. It's a healing response gone awry and that you can really draw out the pathways, basement membrane digestion release of chemotactic peptides as part of the inflammatory response, attraction of more inflammatory cells and then a potential healing response that unfortunately results in digestion of the matrix, which has a morbid or fatal consequence rather than physiologic remodeling. And you're right, that's not selected against. It's selected for, in settings in earlier life infections, for example, perhaps neoplasia, but it's not selected against in late life because people are post reproductive.

Dr Cindy St. Hilaire: So what's next for these studies? What questions are you going to attack next with either these models or with some of your proteomic findings?

Dr David Dichek: Well, we were just talking about that recently, Tomáš and I, and I think we'd like to look at... For one study, we're interested in doing, plaques that are high risk based on MRI imaging, which is really very well developed here at the University of Washington. And many of those patients have endarterectomies and they don't have ruptured plaques. So they are in a high risk group. So they undergo a endarterectomy for that. Not because they've had a plaque rupture and those plaques might be particularly instructive because they're pre event and won't have the healing response to thrombotic response. And it would be really interesting to see if our studies were confirmed. So that's one direction we're going in.

Dr Cindy St. Hilaire: That would be amazing. Luckily, you have access to a whole bunch of human tissue for those kinds of really high impact studies.

Dr Sina Gharib: I just wanted to point out that one of the advantages of doing proteomics and being part of the scientific community is that we made all this data available in the manuscript for other researchers to access and confirm. So, really probably the best way to procced with this is to have other investigators replicate our findings and expand on it. So I just want to bring that up because all of that data that was generated has been included within the supplements of this manuscripts and it's accessible to any scientist who wants to pursue further.

Dr David Dichek: Yeah, I would add one other direction we'd like to go is we still like to know what the substrates are. We think their disappearance based on their abundance is due to proteolysis. But boy, would it be exciting if we could detect fragments. We were unable to do that in the study, probably because they were lost either in vivo or in processing. Technical advancements in that area, and maybe Tomáš can speak to that, might enable us to actually find more direct evidence of proteolysis.

Dr Tomáš Vaisar: Yeah, I mean, to start with, it's really hard to determine physiological substrates of proteases. There's a huge amount of literature identifying proteolytic substrates in vitro, but the physiological substrates are really extremely hard to determine, and especially physiologic in vivo confirming that because in vitro, in a tube, you can mix whatever you want and you modify the ratio of proteins to protease substrate, and you can cleave almost everything with anything. It's a little exaggeration, but it's close. While the physiology substrates in the really complex milia of tissues is extremely hard. And so there has been several approaches developed and one of them is the Proteomaps. The other one is an approach called TAILS developed by Chris Overall at UBC that uses the idea of formation of the neo termini and then tagging the neo termini. So that in the actual sample, you can specifically detect these neo termini formed. But even with that approach, it's really hard to determine what are actual physiological substrates. And on top of that, what are the cleavage sites of the proteases?

Dr Cindy St. Hilaire: And I guess the third being, if those substrates are cleaved, are they circulating and can we detect them in a blood sample? That would be, I guess, the gold standard. Well, thank you all so much for joining me today. Congratulations on this really very cool study. Being into human and translational work, I always love mouse studies that bring in lots of human samples. So congratulations on that. And I look forward to your future publications on this.

Dr Tomáš Vaisar: Thanks a lot.

Dr David Dichek: Thanks.

Dr Cindy St. Hilaire: That's it for the highlights from the late September and early October issues of Circulation Research. Thank you so much 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 David Dichek, Sina Gharib, and Tomáš Vaisar. This podcast is produced by Rebecca McTavish and Ishara Ratnayake, 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, your on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research.