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July 2019 Discover CircRes

Discover CircRes

Release Date: 07/18/2019

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Discover CircRes

  This month on the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from recent issues of Circulation Research and talks with Denisa Wagner and Nicoletta Sorvillo about their article on how PAD4 in blood promotes VWF strings and thrombosis. Article highlights: Goodyer et al: ScRNA-seq of the Cardiac Conduction System   Xiong et al: Chemotaxis Mediated Second Heart Field Deployment   Ranchoux et al: Pulmonary Hypertension and Metabolic Syndrome   Rühl et al. Thrombin/APC Response in FVL and FII 20210G>A   Mahmoud et al. LncRNA...

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July 2019 Discover CircRes show art July 2019 Discover CircRes

Discover CircRes

This month on the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from recent issues of Circulation Research and talks with Steve Lim and James M. Murphy about their article on nuclear FAK regulation of smooth muscle cell proliferation. Article highlights: Li et al: Histone Turnover in Adult Heart Kurosawa et al: Celastramycin Ameliorates Pulmonary Hypertension Urban et al: NOS3 Gene Polymorphism and Coronary Heart Disease   Transcript Cindy...

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Discover CircRes

Cindy S.H.:                         Hi. Welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal Circulation Research. I'm your host, Cindy St. Hilaire, and my goal is to bring you highlights of articles published in the Circ Research Journal as well as have in-depth conversations with senior scientists and the junior trainees who have led the most exciting discoveries in our current issues. Today is our premier episode, so I want to take...

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This month on the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from recent issues of Circulation Research and talks with Steve Lim and James M. Murphy about their article on nuclear FAK regulation of smooth muscle cell proliferation.

Article highlights:

Li et al: Histone Turnover in Adult Heart

Kurosawa et al: Celastramycin Ameliorates Pulmonary Hypertension

Urban et al: NOS3 Gene Polymorphism and Coronary Heart Disease

 
Transcript

Cindy S.:                               Hi, welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal, Circulation Research. I'm your host Cindy St. Hilaire, and I'm an assistant professor of medicine and bioengineering at the University of Pittsburgh. My goal as host of this podcast is to share with you highlights from recent articles published in the July 5th and July 19th issues of the Circulation Research Journal. We'll also have an in-depth conversation with Drs. Steve Limb and James Murphy, from the University of South Alabama College of Medicine, who are the lead authors in one of the exciting discoveries presented in the July 5th issue.

Cindy S.:                               The first article I want to share with you is titled, Replication-Independent Histone Turnover Underlines the Epigenetic Homeostasis in the Adult Heart. The co-first authors are Yumei Li, Shanshan Ai, Xianhong Yu, and the corresponding author is Aibin He. This research was conducted at the Institute of Molecular Medicine Beijing Key Laboratory of Cardiometabolic Molecular Medicine and the Peking-Tsinghua Center for Life Sciences. Both of which are part of the Peking University in Beijing, China.

Cindy S.:                               In the nucleus of cells, DNA is packaged into a structure called chromatin. Chromatin can reside in an open state that is permissive to gene transcription, or closed state where transcription is inhibited. The core units of chromatin are called nucleosomes. A nucleosome consists of DNA that is wrapped around proteins called histones. It's the position of these nucleosomes that determines whether the chromatin allows for DNA transcription or not. There is a large body of research that is focused on understanding the epigenetic processes that promote or repress transcription. Most of this research focuses on the processes that read, write, and erase covalent histone modifications. But, histones are proteins, and proteins, as we all know, have finite half-lives.

Cindy S.:                               Far less research has been conducted to understand the dynamics of histone assembly and disassembly on specific regions of DNA. In this study, the authors took a novel approach of using a GFP-tagged histone H2B protein to track in vivo the rate at which nucleosomes are replaced in cardiac chromatin, and to what extent this rate varies across the genome of those cells. This is particularly interesting, and a particularly good cell type to study, as cardiomyocytes rarely divide or proliferate in the adult heart. What they found was intriguing. Nucleosome recycling is not even across the epigenome of cardiac cells. Instead, gene promoters, enhancer, and other regulatory regions that are known to promote gene transcription all exhibited a higher histone turnover rate than regions of the epigenome that are not occupied by these permissive remarks.

Cindy S.:                               Further, they found greater histone turnover at loci for cardiac specific transcription factors as compared to loci for pluripotency transcription factors. This implies preferential access to these regions. Digging further into the mechanism, they discovered that the repressive chromatin regulator, EED, promoted this histone turnover. The epigenetic signature is what helps to define the identity and function of a fully differentiated cell. This study suggests that loss of histone turnover may promote loss of the proper epigenetic signature of a fully differentiated cell. These exciting findings suggest replication independent histone turnover is a requirement in maintaining both epigenetic and functional homeostasis in the adult heart. From this, one may hypothesize that perhaps aberrations in histone turnover contribute to age related diseases in the cardiac tissue, as well as possibly other tissues.

Cindy S.:                               The next article I'd like to highlight is titled, Identification of Celastramycin as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension-High-throughput Screening of 5,562 Compounds. The first author is Ryo Kurosawa, and the corresponding author is Hiroaki Shimokawa, both from the department of cardiovascular medicine at Tohoku University Graduate School of Medicine in Sendai, Japan. This article is focusing on the disease pulmonary arterial hypertension.

Cindy S.:                               Pulmonary arterial hypertension, or PAH, is a disease that stems from the increased proliferation of arterial smooth muscle cells in the lungs. This proliferation leads to a progressive occlusion of the pulmonary arteries. This occlusion also causes increased pressure in the right heart ventricle. That can lead to heart failure, and ultimately death. Basal dilatory drugs are currently used as therapy in PAH, as they help to open the blood vessels, which can alleviate some of the symptoms. However, these drugs do not target the underlying cause of the symptoms, which is the hyperproliferation of the smooth muscle cell.

Cindy S.:                               To identify novel compounds that inhibit smooth muscle cell proliferation, Kurosawa and colleagues used a high-throughput approach. They isolated cells from patients with pulmonary arterial hypertension and used these cells in a high-throughput approach to test 5,562 novel molecules on their ability to inhibit the proliferation of these cells. This unbiased approach yielded several potential compounds that potentially reduced smooth muscle cell proliferation from these patients, and also had very minimal deleterious effects on healthy control smooth muscle cells. From there, the team tinkered with the structure of the drug Celastramycin to try to increase its efficacy, and with that tinkering they found in vitro, that their new molecule could reduce both the inflammatory signal that helps to drive the proliferation of the smooth muscle cells, as well as reactive oxygen species, which helps to drive the inflammatory signaling.

Cindy S.:                               Moving forward to in vivo studies, the team found that their new treatment also reduced right ventricle systolic pressure and hypertrophy in three different rodent models of pulmonary arterial hypertension. This treatment improved exercise capacity in one of the models. Together, these exciting results indicate that Celastramycin could be developed as a potential therapy for pulmonary arterial hypertension.

Cindy S.:                               The last paper we're going to talk about before switching to our interview with Drs. Steve Limb and James Murphy, is a paper titled, 15-Deoxy-Δ12,14-Prostaglandin J2 Reinforces the Anti-Inflammatory Capacity of Endothelial Cells With a Genetically Determined Nitric Oxide Deficit. The co-first authors are Ivelina Urban, Martin Turinsky, Sviatlana Gehrmann, and the corresponding author is Marcus Hecker, all from the department of cardiovascular physiology at Heidelberg University in Heidelberg, Germany.

Cindy S.:                               Nitric oxide is a vasodilatory and anti-inflammatory molecule, and thus, beneficial to cardiovascular health. Homozygosity of a single nucleotide polymorphism, or SNP, is a gene nitric oxide synthase results in reduced ability of endothelial cells to produce nitric oxide, specifically in response to fluid share stress. Decreased bioavailability of nitric oxide in the vessel wall helps to promote atherosclerosis. The SNP that we're referring to in this paper is called T-786C, where TT homozygosity is considered the control, or healthy genotype, and CC homozygosity is the disease associated. CC homozygosity of this SNP is predictive of atherosclerotic related diseases, and consequently, individuals with CC homozygosity have an increased risk for coronary heart disease.

Cindy S.:                               Now, despite this detrimental evidence, homozygous patients do not develop atherosclerosis at an accelerated rate. This suggests that there's a compensatory mechanism at play. To identify how CC homozygous cells compensate for reduced nitric oxide synthase activity, the authors utilized human umbilical vein endothelial cells, that are also called huvecs, that harbored either the TT or the CC version of this SNP. They also used these in combination with a monocytic cell line.

Cindy S.:                               Urban and colleagues found that under fluid share stress conditions, human endothelial cells homozygous with for the CC variant, had increased production of an anti-inflammatory prostaglandin called 15d-PGJ2. Signaling, via this prostaglandin, helps to compensate in part for the reduced endo production. This prostaglandin suppressed monocyte activation by reducing expression of pro-inflammatory genes such as aisle 1 beta, and decreased monocyte transmigration through endothelial cells. The team also found that patients with coronary heart disease were more likely to have the CC homozygous variant than age match controls. Thus, not only did they identify a partial compensatory mechanism, the authors suggest that 15d-PGJ2 could be a useful biomarker for the diagnosis of coronary heart disease.

Cindy S.:                               So that's it for the highlights of the July issues of Circulation Research. Thank you very much to Ruth Williams, who writes the In This Issue copy for the journal, as well as the editorial team at the journal and at the podcast.

Cindy S.:                               Okay, so now we're going to talk to our team of first author and last author. Today's paper that we're talking about is Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-Mediated Cyclin D1 Transcription. The first authors of this papers are Kyuho Jeong, Jung-Hyun Kim, and James M. Murphy, and the corresponding author is Steve Lim. Today, we're going to be speaking with James and Steve about this paper. So thank you, both of you, for joining us today.

Steve Lim:                           Thanks for having us today.

Cindy S.:                               Great. Congratulations on your beautiful paper. I was wondering if maybe we could just start by both of you introducing yourselves, telling us your current position, and maybe about how you came into this field.

Steve Lim:                           Hi, Cindy. We appreciate the opportunity to discuss our paper. I'm Steve Lim, an associate professor in the department of biochemistry and molecular biology and medicine at the University of South Alabama. I received my PhD from University of Alabama at Birmingham. I did my post-doctoral training studying the law of FAK in cancer biology at UC San Diego Moores Cancer Center. In 2012 I started my own lab here at South Alabama, where I decided to focus on vascular biology using some pharmacy data I generated at the end of my post-doctoral study.

James Murphy:                 I'm James Murphy, I'm a post-doctoral fellow in Dr. Lim's lab at the University of South Alabama. My path to science was a little different than most. I got an undergraduate and graduate degree in mathematics before I joined the PhD program here at South Alabama. Due to a family history of cardiovascular related deaths, I decided to join Dr. Lim's lab due to his interest in studying vascular disease to find new therapeutic targets.

Cindy S.:                               Interesting, a math major.

James Murphy:                 Yeah.

Cindy S.:                               Has that been able to help with any of your basic science studies?

James Murphy:                 I'm pretty good at doing concentrations.

Cindy S.:                               You're the expert in lab math.

James Murphy:                 Yeah. I think the logic skills and critical thinking skills that I picked up in math really help out here in science.

Cindy S.:                               Oh, I bet, that's wonderful. You're the dream PhD student who can hit the ground running with M1V1 equals M2V2. Great. Well, thank you so much. I really liked this paper because I love the mechanosensing and how does a cell read what's outside, and how does that message get brought to the inside. Really, that's what you're finding in this paper, specifically looking at how FAK is mediating transcriptional regulation. Maybe you can start by just telling us, what was your overarching question when you started this study?

Steve Lim:                           Sure. It is very well-known fact that promotes cell proliferation and migration through interior receptors and gross factor receptor signaling. Both of which are key components in the smooth muscle cell hyperplasia. So naturally, we asked ourselves a simple question, "Is FAK activity important for smooth muscle cell proliferation, and leading into hyperplasia?"

Cindy S.:                               So when you say FAK activity I think one thing that's interesting in your paper is, FAK really has kind of two different functions, and one is the kinase function. A kinase is when it can phosphorylate another protein, so it itself is an enzyme. But, then it has another function, so can you maybe tell us about those different functions of FAK?

Steve Lim:                           Right. So FAK can function as a kinase, as well as a kind of independent scaffold, which can recruit different proteins. In the paper, we specifically described a kinase independent function as a nuclear function, nuclear FAK function.

Cindy S.:                               Interesting. So what premise, or what gaps and knowledge were present before your study, that you were trying to address?

Steve Lim:                           Actually, a study showed that the knocking off FAK in the smooth muscle cells prevented neointimal hyperplasia. As just you asked question, FAK has two different functions. Since FAK has both kind of dependent and independent [inaudible 00:14:48], this study lets the unanswered question, which of these two different functions of FAK plays a larger role in dealing with hyperplasia. We aimed to inhibit FAK activity to distinguish between FAK kind of dependent and independent roles in dealing with hyperplasia.

Cindy S.:                               Interesting. How exactly were you able to do that? How could you take and dissect apart the two different functions of this protein?

Steve Lim:                           We started off with a small pile of experiment to test if a small molecule FAK inhibitor could block neointimal hyperplasia, and we were very surprised at the degree to FAK inhibition actually prevented neointimal hyperplasia following vascular injury.

Cindy S.:                               Yeah, and that's in figure one. I was looking at that, it's quite striking.

Steve Lim:                           Actually, to distinguish these two different functions we generated new genetic FAK–Kinase-Dead mouse model in conjunction with a FAK inhibitor model, and that would allow us to study a lot of FAK activity in smooth muscle cells.

Cindy S.:                               Great. James, could you tell us about the mouse model that you developed for this study, and the specific mutations that you created and what you were allowed to test with those models.

James Murphy:                 So the FAK–Kinase-Dead knock-in model was actually generated during Dr. Lim's post-doctoral studies.

Cindy S.:                               Is that the exciting data?

James Murphy:                 The mutation is just a simple lysine to arginine mutation of amino acid 454. What they found was that, actually, homozygous kinase-dead embryos was lethal. So you need FAK activity to actually develop a full grown organism. We kind of had to cross a hetero wild-type kinase-dead mouse with a phlox FAK mouse, which eventually, if you cross with tissue-specific Cres, what you end up with is a phlox wild-type or a phlox kinase-dead mouse. Then, when you treat Tamoxifen in your Cre mouse, then you delete one copy of wild-type FAK and you're left with either a single copy of wild-type FAK, or a single copy of kinase-dead FAK.

Cindy S.:                               Very nice. So for your study, you used, if I recall correctly, the myosin-11, Tamoxifen-inducible Cre model. Can you maybe talk about why you chose that model and why not the SM22 Cre or a non-inducible model? What was your strategy?

James Murphy:                 As I mentioned, FAK activity is important for embryo genesis, so we thought we had to use an inducible model, so as to make sure we had an adult mouse at the time of the experiment. We originally actually had the SMA Cre model, however, some grant reviewers had told us that we should kind of shift to the more myosin-11 mouse to be more specific to the vascular. One downside to that, as we mentioned in the paper, is that that's actually only on the Y chromosome, so you can only use male mice.

Cindy S.:                               Yes. But, at least it's in only the smooth muscle cells. Is that kind of the pros and cons of that model?

James Murphy:                 Yes, and the MYH-11 Cre is kind of the most accepted model when you're doing smooth muscle studies.

Cindy S.:                               Great. So can both of you go over some of the key findings of your paper? If we're going to say this in a tweet, what would we say?

James Murphy:                 In a tweet. So I think, as we talked about, FAK can go to the nucleus. It's kind of constantly shuttling between the nucleus and the cytoplasm, at least what we've been able to observe in vitro. However, kind of a its localization in vivo still kind of was up in the air at the time. However, our immunostaining data actually rebuild that healthy uninjured arteries primarily showed FAK was in the nucleus. Suggesting that FAK was inactive, and maybe somehow suppressing smooth muscle cell proliferation by staying in the nucleus. But, after wire injury, FAK not only increased its activation, but also shifted to be primarily within the cytoplasm, and eventually we showed that that increase of GABA4 protein stability leading to proliferation.

Cindy S.:                               Very interesting. That's great. So what was the hardest part of this whole study?

James Murphy:                 Dr. Lim did the preliminary FAK inhibitor studies, but he had people when he started his own lab, he had to teach us how to do the wire injury. At first, learning gets kind of technical, you have to get used to using the microscope.

Cindy S.:                               Could you describe the wire injury model for us?

James Murphy:                 Yes. What you do is you anesthetize the mouse and you actually locate the femoral artery, and you want to kind of reveal the muscular branch. What you do is you add suture proximal and distal to the muscular branch of the femoral artery to stop blood flow. Then, you're going to cut a small incision in the muscular branch, and you insert a small wire through the branch up into the femoral artery towards the iliac branch. What this does is denude the endothelial layer and kind of causes an extension of the artery, damaging the smooth muscle layer. Once you remove it and suture off the muscular branch, then after a couple weeks you start to see hyperplasia.

Cindy S.:                               Interesting. So what does this model clinically?

James Murphy:                 This model kind of mimics angioplasty procedures that one may have if they have an occluded artery. There's multiple angioplasty procedures. There's a physical dislodging and opening of the artery. Then, there's some other methods such as using a stent to keep it open.

Cindy S.:                               Great. Very interesting. What do you think would happen in maybe, I don't know, an LDLR knockout that was crossed with your FAK kinase deficient mutant? What do you think would happen in an athero model?

James Murphy:                 We're actually-

Cindy S.:                               Or is that the next paper? We don't have to talk about it if it's the next paper.

James Murphy:                 We're actually currently testing that right now.

Cindy S.:                               Oh, okay.

James Murphy:                 So that's kind of our next step is to test this in atherosclerotic models to see what happens.

Cindy S.:                               So, what might this mean for potential therapeutic target? How could we leverage this data to possibly translate it to the clinical setting, even if it's far off? What might we want to do moving forward?

Steve Lim:                           Speaking of translational potential, currently most of the treatment options for narrow vessels rely on thrombolytic stents, that provides local delivery of anti-proliferative drugs. However, DES comes with several disadvantages, including location, work, size of these affected vessels. In fact, inhibitors are under cancer clinical development, have never been used in the vascular diseases. Our study, I think, at least to show the potential for using this type of FAK inhibitors in treating hyperplasia, which was not possible before.

Cindy S.:                               That's interesting. So essentially, there's already potential, therapy's already available that would just have to be tested in this new ... in this new vascular realm, essentially.

Steve Lim:                           Yeah. I was thinking about effication of these type of drugs. I think it could be, as you said, PAH could be one of the targets, because they're not really useful drugs available now. In the future, what we actually, we started already, but it's known, these moments of proliferation plays key role in the arthrosclerosis progression. Studies targeting neointimal hyperplasia and atherosclerosis, it's not existing. I think in the future probably, we would like to test whether in fact inhibition and the smooth muscle cells reduce its atherosclerity in animal models, and hopefully in humans.

Cindy S.:                               Yeah, yeah, hopefully in humans, always. Yeah, and in those mouse models, there's always interesting studies where you can block things from the beginning. But, I think one of the beautiful things about the mouse model that you created, the fact that it's Tamoxifen inducible, you could essentially let that atherosclerotic plaque build up for a bit and then knock it out and see if it can reverse it. So the model you created is a really wonderful tool to use for a whole bunch of studies. So congratulations.

Steve Lim:                           Thank you.

Cindy S.:                               Yeah, I thought the most interesting aspect of this paper was really the fact that it could link this FAK protein, this integrin signal mediating protein to the transcription factor GABA4. So could you possibly tell us a little bit about that interaction, and exactly what GAB is doing in the smooth muscle cell?

Steve Lim:                           I actually think that the identifying GABA4 factor actually was one of the difficulties, because normal cells do not express GABA4, that's what is known. I think it's because, based on our finding, the smooth muscle cells in vivo, you could package more predominantly localized in vivo, the nuclear FAK is predominant. So that nuclear FAK finds GABA4 and reduces ability through the process on degradation. But, actually, not changing ... Nuclear factor is not changed. GABA4 mRNA are the expression, so GABA4 is always expressed in smooth muscle cells. But, we never see in healthy, or very freshly isolated smooth muscle cells. We never see Gaba4. That was the most difficult part actually.

Cindy S.:                               So the mRNA is always there, it's just never making it to a protein that accumulates in any measurable quantity.

Steve Lim:                           So you become a protein, but FAK, nuclear FAK kills all GABA4 in the nucleus.

Cindy S.:                               That's the proteasome mediated degradation?

Steve Lim:                           Right. Then, GABA4 actually promotes cycling D1 transcription. So no GABA4, no cycling the new one, and smooth muscle cells do not cycle.

Cindy S.:                               Interesting. So can you maybe close the loop and tell us essentially what's in figure nine, like this. Could you talk us through that?

Steve Lim:                           It summarizes in figure nine, I think it would be best, we can put two different situations. In healthy R3, FAK is in the nucleus, and GABA4 is reduced, cycling D1 is not expressed, and smooth muscle cells become high acid. They don't proliferate. But, in injured, actually, FAK localization is it's the vaso injury promotes FAK localization, vaso injury shifts FAK nuclear localization to cytoplasm. Actually, FAK is activated. Now, GABA4, that increases cycling T1 expression. So that causes intimal hyperplasia. That could be a kind of summary.

Cindy S.:                               No, that's perfect. Congratulations on a very nice paper. I thoroughly enjoyed reading it, and I enjoyed even more speaking with the two of you. So thank you very much.

Steve Lim:                           Well, thank you so much.

Cindy S.:                               Thank you for listening. I'm your host Cindy St. Hilaire, and this is Discover CircRes, your source for the most up to date and exciting discoveries in basic cardiovascular research.