Kathleen A. Martin - US grants

simian immunodeficiency virus; virus receptors; receptor binding; biological signal transduction; protein structure function; mitogen activated protein kinase; G protein; calcium flux; chemokine; HIV envelope protein gp120; tissue /cell culture; molecular cloning;

DESCRIPTION (provided by applicant): Drug-eluting stents have revolutionized revascularization of coronary artery lesions by largely preventing restenosis. Importantly, emerging long term safety data suggests that rapamycin-eluting stents pose an elevated risk of late thrombosis, likely due to incomplete healing of the endothelium. An ideal stent drug would, therefore, selectively inhibit proliferation and promote differentiation of vascular smooth muscle cells (VSMC), without inhibiting re-endothelialization. In our studies of the molecular mechanisms underlying VSMC phenotypic modulation, a process necessary for angiogenesis, atherosclerosis, and restenosis, we have discovered that the mTOR inhibitor rapamycin promotes VSMC differentiation by inducing a new program of gene expression, including contractile proteins. We have found that rapamycin inhibition of the mTOR effector S6K1, and the resulting activation of Akt2 is necessary for this effect. Surprisingly, Akt1 inhibited VSMC differentiation. We have also made the exciting discovery that rapamycin activates a VSMC transcription factor, GATA-6, and that this factor is necessary for rapamycin-induced differentiation. This transcription factor is specific to VSMC and not found in endothelial cells. We find that rapamycin also induces expression of the master regulatory transcriptional coactivator myocardin that promotes VSMC differentiation. The mTOR pathway is well known to regulate protein synthesis. Notably, we identify regulation of VSMC-specific transcription as a novel function for this pathway. We hypothesize that rapamycin regulation of transcription factors via Akt2 is a critical mechanism by which this drug inhibits proliferation and promotes differentiation in VSMC. We aim to understand the mechanisms by which rapamycin regulates prodifferentiation transcription factors in VSMC. (1) In addressing this hypothesis, we aim to determine how rapamycin activation of Akt2 regulates GATA-6. We hypothesize that rapamycin induces phosphorylation of GATA-6 that leads to its nuclear translocation and activation. We will use siRNA, VSMC from Akt1 or Akt2 knockout mice, and GATA-6 phosphorylation site mutants to determine which kinase phosphorylates GATA-6, and how phosphorylation influences GATA-6 activity. We hypothesize that a phospho-mimetic mutant GATA-6 may be constitutively active, and therefore a potential VSMC-specific prodifferentiation therapeutic. (2) We aim to use siRNA methods to determine the role of myocardin in rapamycin-induced expression. We will determine whether and how rapamycin promotes myocardin expression and/or activity using DNA binding, chromatin immunoprecipitation, and promoter reporter methods. (3) We will determine whether Akt2 deletion exacerbates, and Akt1 deletion diminishes intimal hyperplasia, and whether the therapeutic response to rapamycin requires Akt2, using an injury model in wild type, Akt1 or Akt2 knockout mice. We propose that understanding these downstream targets of rapamycin and the molecular mechanisms by which they are regulated will provide key targets for development of improved stent therapeutics. PUBLIC HEALTH RELEVANCE Cardiovascular disease is a major cause of morbidity and mortality in the western world. While stents coated with the drug rapamycin have greatly reduced the risk of restenosis (re-blockage of the vessel) after coronary artery angioplasty, recent findings have revealed that they confer a small but significant risk of heart attack or death. This project aims to understand the molecular mechanisms underlying the beneficial anti-restenotic response of vascular smooth muscle cells to rapamycin, as this knowledge may allow us to tailor future therapeutics to inhibit these cells specifically, avoiding detrimental side effects on other cell types that can cause the dangerous complications.

DESCRIPTION (provided by applicant): Regulation of VSMC phenotype remains a key unanswered question in vascular smooth muscle cell (VSMC) biology. VSMC retain a remarkable plasticity to de-differentiate and re-enter the cell cycle allowing for growth and healing. However, such plasticity can also contribute to severe vascular pathologies, including restenosis, graft failure, atherosclerosis, and transplant vasculopathy. Remarkably, despite intense study, the process regulating plasticity is largely unknown with few therapies successfully targeting this process. With the growing numbers of patients suffering from vascular disease the discovery of novel targets is urgently warranted. While rapamycin analogs are efficacious drug-eluting stent agents, the risk of late-stent thrombosis and subsequent need for long term antiplatelet therapy still complicates their use. Our previous studies have revealed that the mTORC1 inhibitor, rapamycin, promotes VSMC differentiation, revealing cell type-specific transcription as a novel function of the mTORC1 pathway. Moreover, we implicated feedback activation of Akt2 as critical for rapamycin-induced differentiation. We have discovered distinct roles for Akt1 and Akt2 in the response to vascular injury. We have also identified TET2 and LMO7 as novel mTORC1-regulated proteins that modulate VSMC phenotype. Based upon these exciting Preliminary Results we will address the overall hypothesis that the mTORC1 pathway governs VSMC phenotype and response to injury through epigenetic (TET2) and transcriptional (LMO7) regulation, and that Akt isoforms play distinct roles in the pathology of restenosis and therapeutic response to rapamycin. In Specific Aim 1, we will determine the role of TET2 in VSMC phenotypic modulation and how it is regulated by rapamycin. In Specific Aim 2, we will determine the role of LMO7 in rapamycin- induced VSMC differentiation in vitro and in response to injury in vivo. In Specific Aim 3, we will determine the differential roles of Akt1 and Akt2 in injury response in vascular tissues. If our goals are achieved, we will have identified key regulatory elements in VSMC plasticity. Understanding the critical mechanisms by which mTORC1 regulates VSMC phenotype will lead to improved cardiovascular therapeutics.

DESCRIPTION (provided by applicant): Vascular smooth muscle cells (VSMC) exhibit a remarkable plasticity that allows for reversible differentiation and de-differentiation. This propety is distinct from skeletal or cardiac myocytes, which undergo terminal differentiation. Smooth muscle plasticity is important in normal development, growth, and wound healing, but also contributes to pathologies including atherosclerosis, intimal hyperplasia, hypertension, and transplant arteriosclerosis. Because VSMC exhibit this unique plasticity, we hypothesized that regulatory mechanisms found in other types of stem cells might also be involved in VSMC phenotypic modulation. Indeed, our preliminary data demonstrate that de-differentiated VSMC express multiple stem cell genes, including Oct4, Nanog, and KLF4. As the TET (ten-eleven translocation) family of chromatin-modifying proteins has recently been implicated as essential regulators of pluripotency in embryonic and hematopoietic stem cells, we investigated the roles of TET proteins in VSMC. Our preliminary data reveal that TET2 is expressed at high levels in VSMC, and is induced by differentiating agents but repressed by PDGF-BB. We find that TET2 is necessary and sufficient for SMC differentiation, where TET2 associates with contractile gene promoters and increases accessibility, but represses chromatin at stem cell gene promoters. We further found that TET2 is expressed at high levels in normal vessels, but markedly downregulated in disease states, including human atherosclerosis and intimal hyperplasia in mice. Importantly, TET2 overexpression rescues intimal hyperplasia post-arterial injury. We hypothesize that TET2 is a novel epigenetic master regulator of VSMC phenotype. We believe we have uncovered a fundamental new mechanism that is the basis for smooth muscle plasticity. As such, this work has important implications for multiple vascular diseases. In the following specific aims, we propose to address mechanisms of action of TET2, and determine the role of TET2 in vascular disease models. In Specific Aim 1, we will use cutting edge methods to determine the epigenetic mechanisms by which TET2 regulates methylation and gene expression at contractile and stem cell promoters. In Specific Aim 2, we will use mouse models to determine the role of TET2 in the pathogenesis of cardiovascular diseases, including atherosclerosis, intimal hyperplasia, and graft arteriosclerosis. Because we have found that epigenetic regulation by TET2 lies at the apex of VSMC phenotypic modulation, controlling other known mediators such as myocardin, SRF, and KLF4, we propose that understanding the mechanisms of TET2 action at the cellular and disease level will generate new therapeutic approaches for treatment and prevention of cardiovascular disease.

DESCRIPTION (provided by applicant): Regulation of VSMC phenotype remains a key unanswered question in vascular smooth muscle cell (VSMC) biology. VSMC retain a remarkable plasticity to de-differentiate and re-enter the cell cycle allowing for growth and healing. However, such plasticity can also contribute to severe vascular pathologies, including restenosis, graft failure, atherosclerosis, and transplant vasculopathy. Remarkably, despite intense study, the process regulating VSMC plasticity is largely unknown with few therapies successfully targeting this process. With the growing numbers of patients suffering from vascular disease the discovery of novel targets is urgently warranted. We have made the exciting discovery that de-differentiated VSMC express genes associated with stem cell pluripotency, including Sox2, Oct4, Nanog, and KLF4. We propose that these stem cell-associated genes account for the unique plasticity of mature VSMC. Recent groundbreaking studies have identified the TET (ten-eleven translocations) family of chromatin modifying proteins as key mediators of pluripotency in embryonic and hematopoietic stem cells. Our Preliminary Results implicate TET2 as an epigenetic master regulator of VSMC phenotype. Importantly, we find that TET2 inhibits expression of stem cell-associated genes and classic markers of the de-differentiated phenotype. We previously discovered that the mTORC1 inhibitor, rapamycin, promotes VSMC differentiation. We now find that rapamycin regulates TET2 expression. Remarkably, we find that TET2 also regulates miRNAs that can modulate both differentiation-specific and stem cell-associated gene expression. We hypothesize that TET2 is a master regulator of VSMC phenotype through its coordinated regulation of the promoters of contractile and stem cell-associated genes, as well as of miRNAs. In Specific Aim 1, we will determine the role of stem cell-associated genes in VSMC phenotype. In Specific Aim 2, we will determine the role of TET2-regulated miRNAs in VSMC phenotype. In Specific Aim 3, we will determine whether targeting TET2 or stem cell-associated genes has therapeutic utility in in vivo models of intimal hyperplasia. If our goals are achieved, we will have identified a nove mechanism underlying VSMC plasticity. Understanding the critical mechanisms by which mTORC1 regulates VSMC phenotype will lead to improved cardiovascular therapeutics.

Novel insights into intimal hyperplasia in cardiac allograft vasculopathy Biotechnical advances in surgical and percutaneous interventions have greatly improved cardiovascular disease therapies. However, restenosis arising from uncontrolled vascular smooth muscle cell (SMC) proliferation and migration leading to occlusive intimal hyperplasia, remains a major unresolved hurdle. SMC possess a unique ability to alter their phenotype in response to environmental stimuli, which allows for vascular healing and growth. However, this SMC plasticity also contributes to cardiovascular pathologies, including intimal hyperplasia following revascularization procedures. A particularly resistant and deadly form of intimal hyperplasia occurs in cardiac allograft vasculopathy (CAV) where chronic immune injury mediated by IFN? promotes diffuse, and often severe, SMC intimal hyperplasia throughout the vessels of the grafted organ, leading to ischemic organ failure. A better understanding of this SMC response is urgently warranted to identify potential targets for therapy for CAV. mTORC1 inhibitors have shown promise for CAV but are limited by adverse effects. By understanding the molecular targets downstream of mTORC1, we may be able to recapitulate the benefits of mTORC1 inhibition in SMC while preventing systemic complications. The recent discovery of the clonal origin of some SMC lesions, including in atherosclerosis, has shifted paradigms in how we view vascular disease. Indeed, such ?pioneering? cells that give rise to clonal lesions may be involved in the early pathogenesis of neointima in CAV. Moreover, epigenetics may play a major role in this process, but we have limited understanding of how epigenetics influence CAV. We have recently identified TET2 as a master epigenetic regulator of SMC phenotype that is induced by the mTORC1 inhibitor rapamycin. TET2 is repressed in intimal hyperplasia post-injury and in atherosclerotic lesions (Circulation 2013). We now demonstrate that TET2 is downregulated in SMC in human CAV, in mouse allograft models, and by IFN? in cultured SMC. In the absence of a therapeutic method to overexpress TET2 throughout the coronary vasculature, we propose that miRNAs that repress TET2 expression, such as miR29 and others, could be targeted for CAV therapy. To identify novel mechanisms and therapeutic targets, we have established a mouse heterotopic heart transplant model of CAV. We hypothesize that epigenetic (chromatin and miRNAs) and transcriptional changes alter SMC gene expression, promoting intimal hyperplasia in the coronary arteries of transplanted hearts. Using biotechnological advances, we have developed a coordinated, complementary, non-overlapping 3-pronged approach toward furthering our understanding of and developing new treatments for CAV that includes: 1) clonality and initiating events, 2) epigenomics and transcriptomics, and 3) miRNA-based therapies. We have recruited an outstanding internationally recognized team of surgeon-scientists, pathologists, vascular biologists, epigenetics/bioinformatics and miRNA experts to address these goals.

Mature vascular smooth muscle cells (SMC) retain the ability to reversibly dedifferentiate and dramatically alter their phenotype in response to environmental cues. This plasticity allows for vascular repair and growth, but also contributes to cardiovascular pathologies including atherosclerosis, intimal hyperplasia, aneurysm, transplant arteriosclerosis, and others. Little is known about the epigenetic control of SMC phenotypic switching. We have recently identified TET2 as a novel master epigenetic regulator of SMC phenotype that promotes SMC differentiation and is downregulated in diseased vessels. TET2 promotes expression of key transcriptional drivers of SMC differentiation including myocardin and SRF while simultaneously inhibiting expression of KLF4, a transcription factor associated with dedifferentiation in SMC and pluripotency in stem cells. In addition to its known function of generating the epigenetic mark 5 hydroxymethylcytosine (5hmC), we have determined that TET2 also influences histone methylation, indicating that TET2 is involved in global chromatin remodeling in SMC. Others have shown association between TET2 and HDACs in hematopoietic cells. Our new preliminary data indicate a physical and functional association between TET2 and histone acetyltransferases (HATs). HATs acetylate histones in enhancer regions to promote cell type-specific gene expression. We have made the surprising observation that the HATs p300 and CBP, often considered to be interchangeable, have opposing roles in regulating SMC gene expression. We find that p300 is required to induce SMC differentiation in culture, while CBP is required for de-differentiation. Notably, these HATs also oppositely influence 5hmC at contractile promoters in SMC, and we detect a differentiation-dependent association between p300 and TET2. We hypothesize that p300 and CBP regulate distinct enhancers at contractile- and synthetic phenotype-specific genes, respectively, and are critical factors in SMC phenotypic switching. We further propose that TET2/p300 interactions may coordinately regulate chromatin conformation. The overall goal of this proposal is to identify the mechanisms by which p300, CBP, and TET coordinately regulate SMC phenotypic plasticity. We will employ state-of-the art molecular biology approaches, animal models, and advanced genomics techniques to address the central hypothesis that p300 and CBP regulate opposing programs of gene expression by acetylating distinct cis regulatory elements, and that HATs and TET2 work in concert to remodel chromatin during SMC phenotype modulation. Aim 1 will determine the mechanistic roles of p300 and CBP in SMC phenotypic modulation. Aim 2 will aim to define the opposing roles of p300 and CBP on vascular injury response in vivo. Aim 3 will determine how enhancers are regulated by p300, CBP, and TET2 using deletion approaches in vitro and in vivo. Collectively, these studies will lead to new understanding of SMC phenotypic modulation, which has potential for generating new preventive and therapeutic strategies for cardiovascular diseases.

Role of LMO7 in atherosclerosis Atherosclerosis is a major cause of cardiovascular disease morbidity and mortality, including myocardial infarction (MI), stroke, and peripheral vascular disease. As plaque rupture is a key factor in atherothrombotic events, understanding the determinants of plaque stability is critical. The underlying molecular mechanisms are poorly understood, but thin cap fibroatheromas, characterized by inflammation, matrix metalloprotease (MMP) activity, large necrotic cores, and thin fibrous caps, are considered more vulnerable to rupture. Vascular smooth muscle cells (SMC) play a critical role in plaque stabilization by forming the fibrous cap that covers the lipid-laden plaque and necrotic core. Recent studies have revealed the paradigm-changing findings that SMC comprise a greater portion of the plaque interior than previously appreciated by transdifferentiating to phenotypes that lack SMC markers, and that investment of the plaque by SMC-derived cells appears to be atheroprotective. Thus, SMC play a central role in regulating both plaque size and stability. Multiple lines of evidence support a protective role for TGF? signaling in plaques. We have recently identified the protein LIM Domain Only 7 (LMO7) as a key negative feedback regulator of TGF? signaling in SMC that promotes wound healing resolution (Xie et al, Circulation, 2019). Mice with global or inducible smooth muscle-specific knockout of LMO7 (SM-LMO7-/-) exhibit enhanced TGF? signaling and extracellular matrix (ECM) synthesis compared to controls following vascular injury. We find that LMO7 represses the TGF? pathway at multiple levels. In new studies, we demonstrate that SM-LMO7-/- mice develop plaques of similar size but with features of increased stability compared to controls in the ApoE-/- high fat diet (HFD) model. The SM- LMO7-/- plaques have reduced necrotic core size, decreased CD68+ cells, increased ACTA2 and collagen staining, and thicker fibrous caps. Preliminary lineage tracing data in these mice reveals that SM-LMO7-/- increases the number of transdifferentiated SMC-derived cells in lesions, a phenotype that may be protective. Preliminary data in human carotid specimens reveals that LMO7 mRNA expression is increased 5.6X in plaque vs normal artery, and is enriched in ruptured vs non-ruptured lesions. We hypothesize that LMO7 loss of function in SMC promotes more stable plaques in mice and humans. In Aim 1, we will determine the role of SMC LMO7 in plaque composition and gene expression using comprehensive staining and single cell RNA-sequencing analyses. In Aim 2, we will dissect underlying mechanisms, and in Aim 3, we directly test the role of SMC LMO7 in lesion stability by assessing plaque rupture in mice, as well as LMO7 expression and localization in human ruptured vs stable lesions. These studies will provide insights into the pathophysiology of atherosclerotic plaque remodeling with potential therapeutic implications.