Histone Acetyltransferases p300 and CBP Coordinate Distinct Chromatin Remodeling Programs in Vascular Smooth Muscle Plasticity.

BACKGROUND: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methylcytosine dioxygenase TET2 acting as a master regulator of smooth muscle cell phenotype. The histone acetyl-transferases p300 and CREB-binding protein (CBP) are highly homologous and often considered to be interchangeable, and their roles in smooth muscle cell phenotypic regulation are not known. METHODS: We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (inducible knockout [iKO]; p300iKO or CBPiKO), and in samples of human intimal hyperplasia. RESULTS: P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in vessel remodeling after vascular injury. Medial p300 expression and activity were repressed by injury, but CBP and histone acetylation were induced in neointima. Knockdown experiments revealed opposing effects of p300 and CBP in the VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300iKO mice exhibited severe intimal hyperplasia after arterial injury compared with controls, whereas CBPiKO mice were entirely protected. In normal aorta, p300iKO reduced, but CBPiKO enhanced, contractile protein expression and contractility compared with controls. Mechanistically, we found that these histone acetyl-transferases oppositely regulate histone acetylation, DNA hydroxymethylation, and PolII (RNA polymerase II) binding to promoters of differentiation-specific contractile genes. Our data indicate that p300 and TET2 function together, because p300 was required for TET2-dependent hydroxymethylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 coimmunoprecipitated with p300, and this interaction was enhanced by rapamycin but repressed by platelet-derived growth factor (PDGF) treatment, with p300 promoting TET2 protein stability. CBP did not associate with TET2, but instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile protein promoters. Furthermore, CBP inhibited TET2 mRNA levels. Immunostaining of cardiac allograft vasculopathy samples revealed that p300 expression is repressed but CBP is induced in human intimal hyperplasia. CONCLUSIONS: This work reveals that p300 and CBP serve nonredundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific histone acetyl-transferases may hold therapeutic promise for cardiovascular diseases.

Targeting smooth muscle cell phenotypic switching in vascular disease.

OBJECTIVE: The phenotypic plasticity of vascular smooth muscle cells (VSMCs) is central to vessel growth and remodeling, but also contributes to cardiovascular pathologies. New technologies including fate mapping, single cell transcriptomics, and genetic and pharmacologic inhibitors have provided fundamental new insights into the biology of VSMC. The goal of this review is to summarize the mechanisms underlying VSMC phenotypic modulation and how these might be targeted for therapeutic benefit. METHODS: We summarize findings from extensive literature searches to highlight recent discoveries in the mechanisms underlying VSMC phenotypic switching with particular relevance to intimal hyperplasia. PubMed was searched for publications between January 2001 and December 2020. Search terms included VSMCs, restenosis, intimal hyperplasia, phenotypic switching or modulation, and drug-eluting stents. We sought to highlight druggable pathways as well as recent landmark studies in phenotypic modulation. RESULTS: Lineage tracing methods have determined that a small number of mature VSMCs dedifferentiate to give rise to oligoclonal lesions in intimal hyperplasia and atherosclerosis. In atherosclerosis and aneurysm, single cell transcriptomics reveal a striking diversity of phenotypes that can arise from these VSMCs. Mechanistic studies continue to identify new pathways that influence VSMC phenotypic plasticity. We review the mechanisms by which the current drug-eluting stent agents prevent restenosis and note remaining challenges in peripheral and diabetic revascularization for which new approaches would be beneficial. We summarize findings on new epigenetic (DNA methylation/TET methylcytosine dioxygenase 2, histone deacetylation, bromodomain proteins), transcriptional (Hippo/Yes-associated protein, peroxisome proliferator-activity receptor-gamma, Notch), and ß3-integrin-mediated mechanisms that influence VSMC phenotypic modulation. Pharmacologic and genetic targeting of these pathways with agents including ascorbic acid, histone deacetylase or bromodomain inhibitors, thiazolidinediones, and integrin inhibitors suggests potential therapeutic value in the setting of intimal hyperplasia. CONCLUSIONS: Understanding the molecular mechanisms that underlie the remarkable plasticity of VSMCs may lead to novel approaches to treat and prevent cardiovascular disease and restenosis.

TET2 Protects Against Vascular Smooth Muscle Cell Apoptosis and Intimal Thickening in Transplant Vasculopathy.

BACKGROUND: Coronary allograft vasculopathy (CAV) is a devastating sequela of heart transplant in which arterial intimal thickening limits coronary blood flow. There are currently no targeted therapies to prevent or reduce this pathology that leads to transplant failure. Vascular smooth muscle cell (VSMC) phenotypic plasticity is critical in CAV neointima formation. TET2 (TET methylcytosine dioxygenase 2) is an important epigenetic regulator of VSMC phenotype, but the role of TET2 in the progression of CAV is unknown. METHODS: We assessed TET2 expression and activity in human CAV and renal transplant samples. We also used the sex-mismatched murine aortic graft model of graft arteriopathy (GA) in wild-type and inducible smooth muscle-specific Tet2 knockout mice; and in vitro studies in murine and human VSMCs using knockdown, overexpression, and transcriptomic approaches to assess the role of TET2 in VSMC responses to IFNγ (interferon γ), a cytokine elaborated by T cells that drives CAV progression. RESULTS: In the present study, we found that TET2 expression and activity are negatively regulated in human CAV and renal transplant samples and in the murine aortic graft model of GA. IFNγ was sufficient to repress TET2 and induce an activated VSMC phenotype in vitro. TET2 depletion mimicked the effects of IFNγ, and TET2 overexpression rescued IFNγ-induced dedifferentiation. VSMC-specific TET2 depletion in aortic grafts, and in the femoral wire restenosis model, resulted in increased VSMC apoptosis and medial thinning. In GA, this apoptosis was tightly correlated with proliferation. In vitro, TET2-deficient VSMCs undergo apoptosis more readily in response to IFNγ and expressed a signature of increased susceptibility to extrinsic apoptotic signaling. Enhancing TET2 enzymatic activity with high-dose ascorbic acid rescued the effect of GA-induced VSMC apoptosis and intimal thickening in a TET2-dependent manner. CONCLUSIONS: TET2 is repressed in CAV and GA, likely mediated by IFNγ. TET2 serves to protect VSMCs from apoptosis in the context of transplant vasculopathy or IFNγ stimulation. Promoting TET2 activity in vivo with systemic ascorbic acid reduces VSMC apoptosis and intimal thickening. These data suggest that promoting TET2 activity in CAV may be an effective strategy for limiting CAV progression.

Reduced Platelet miR-223 Induction in Kawasaki Disease Leads to Severe Coronary Artery Pathology Through a miR-223/PDGFRß Vascular Smooth Muscle Cell Axis.

RATIONALE: Kawasaki disease (KD) is an acute vasculitis of early childhood that can result in permanent coronary artery structural damage. The cause for this arterial vulnerability in up to 15% of patients with KD is unknown. Vascular smooth muscle cell dedifferentiation play a key role in the pathophysiology of medial damage and aneurysm formation, recognized arterial pathology in KD. Platelet hyperreactivity is also a hallmark of KD. We recently demonstrated that uptake of platelets and platelet-derived miRNAs influences vascular smooth muscle cell phenotype in vivo. OBJECTIVE: We set out to explore whether platelet/vascular smooth muscle cell (VSMC) interactions contribute to coronary pathology in KD. METHODS AND RESULTS: We prospectively recruited and studied 242 patients with KD, 75 of whom had documented coronary artery pathology. Genome-wide miRNA sequencing and droplet digital PCR demonstrated that patient with KD platelets have significant induction of miR-223 compared with healthy controls (HCs). Platelet-derived miR-223 has recently been shown to promote vascular smooth muscle quiescence and resolution of wound healing after vessel injury. Paradoxically, patients with KD with the most severe coronary pathology (giant coronary artery aneurysms) exhibited a lack of miR-223 induction. Hyperactive platelets isolated from patients with KD are readily taken up by VSMCs, delivering functional miR-223 into the VSMCs promoting VSMC differentiation via downregulation of PDGFRß (platelet-derived growth factor receptor ß). The lack of miR-223 induction in patients with severe coronary pathology leads to persistent VSMC dedifferentiation. In a mouse model of KD (Lactobacillus casei cell wall extract injection), miR-223 knockout mice exhibited increased medial thickening, loss of contractile VSMCs in the media, and fragmentation of medial elastic fibers compared with WT mice, which demonstrated significant miR-223 induction upon Lactobacillus casei cell wall extract challenge. The excessive arterial damage in the miR-223 knockout could be rescued by adoptive transfer of platelet, administration of miR-223 mimics, or the PDGFRß inhibitor imatinib mesylate. Interestingly, miR-223 levels progressively increase with age, with the lowest levels found in <5-year-old. This provides a basis for coronary pathology susceptibility in this very young cohort. CONCLUSIONS: Platelet-derived miR-223 (through PDGFRß inhibition) promotes VSMC differentiation and resolution of KD induced vascular injury. Lack of miR-223 induction leads to severe coronary pathology characterized by VSMC dedifferentiation and medial damage. Detection of platelet-derived miR-223 in patients with KD (at the time of diagnosis) may identify patients at greatest risk of coronary artery pathology. Moreover, targeting platelet miR-223 or VSMC PDGFRß represents potential therapeutic strategies to alleviate coronary pathology in KD. Graphic Abstract: A graphic abstract is available for this article.

Platelet-derived miR-223 promotes a phenotypic switch in arterial injury repair.

Upon arterial injury, endothelial denudation leads to platelet activation and delivery of multiple agents (e.g., TXA2, PDGF), promoting VSMC dedifferentiation and proliferation (intimal hyperplasia) during injury repair. The process of resolution of vessel injury repair, and prevention of excessive repair (switching VSMCs back to a differentiated quiescent state), is poorly understood. We now report that internalization of APs by VSMCs promotes resolution of arterial injury by switching on VSMC quiescence. Ex vivo and in vivo studies using lineage tracing reporter mice (PF4-cre × mT/mG) demonstrated uptake of GFP-labeled platelets (mG) by mTomato red-labeled VSMCs (mT) upon arterial wire injury. Genome-wide miRNA sequencing of VSMCs cocultured with APs identified significant increases in platelet-derived miR-223. miR-223 appears to directly target PDGFRß (in VSMCs), reversing the injury-induced dedifferentiation. Upon arterial injury, platelet miR-223-KO mice exhibited increased intimal hyperplasia, whereas miR-223 mimics reduced intimal hyperplasia. Diabetic mice with reduced expression of miR-223 exhibited enhanced VSMC dedifferentiation and proliferation and increased intimal hyperplasia. Our results suggest that horizontal transfer of platelet-derived miRNAs into VSMCs provides a novel mechanism for regulating VSMC phenotypic switching. Platelets thus play a dual role in vascular injury repair, initiating an immediate repair process and, concurrently, a delayed process to prevent excessive repair.

LMO7 Is a Negative Feedback Regulator of Transforming Growth Factor ß Signaling and Fibrosis.

BACKGROUND: Vascular smooth muscle cells (SMCs) synthesize extracellular matrix (ECM) that contributes to tissue remodeling after revascularization interventions. The cytokine transforming growth factor ß (TGF-ß) is induced on tissue injury and regulates tissue remodeling and wound healing, but dysregulated signaling results in excess ECM deposition and fibrosis. The LIM (Lin11, Isl-1 & Mec-3) domain protein LIM domain only 7 (LMO7) is a TGF-ß1 target gene in hepatoma cells, but its role in vascular physiology and fibrosis is unknown. METHODS: We use carotid ligation and femoral artery denudation models in mice with global or inducible smooth muscle-specific deletion of LMO7, and knockout, knockdown, overexpression, and mutagenesis approaches in mouse and human SMC, and human arteriovenous fistula and cardiac allograft vasculopathy samples to assess the role of LMO7 in neointima and fibrosis. RESULTS: We demonstrate that LMO7 is induced postinjury and by TGF-ß in SMC in vitro. Global or SMC-specific LMO7 deletion enhanced neointimal formation, TGF-ß signaling, ECM deposition, and proliferation in vascular injury models. LMO7 loss of function in human and mouse SMC enhanced ECM protein expression at baseline and after TGF-ß treatment. TGF-ß neutralization or receptor antagonism prevented the exacerbated neointimal formation and ECM synthesis conferred by loss of LMO7. Notably, loss of LMO7 coordinately amplified TGF-ß signaling by inducing expression of Tgfb1 mRNA, TGF-ß protein, αv and ß3 integrins that promote activation of latent TGF-ß, and downstream effectors SMAD3 phosphorylation and connective tissue growth factor. Mechanistically, the LMO7 LIM domain interacts with activator protein 1 transcription factor subunits c-FOS and c-JUN and promotes their ubiquitination and degradation, disrupting activator protein 1-dependent TGF-ß autoinduction. Importantly, preliminary studies suggest that LMO7 is upregulated in human intimal hyperplastic arteriovenous fistula and cardiac allograft vasculopathy samples, and inversely correlates with SMAD3 phosphorylation in cardiac allograft vasculopathy. CONCLUSIONS: LMO7 is induced by TGF-ß and serves to limit vascular fibrotic responses through negative feedback regulation of the TGF-ß pathway. This mechanism has important implications for intimal hyperplasia, wound healing, and fibrotic diseases.

Opposing Actions of AKT (Protein Kinase B) Isoforms in Vascular Smooth Muscle Injury and Therapeutic Response.

OBJECTIVE: Drug-eluting stent delivery of mTORC1 (mechanistic target of rapamycin complex 1) inhibitors is highly effective in preventing intimal hyperplasia after coronary revascularization, but adverse effects limit their use for systemic vascular disease. Understanding the mechanism of action may lead to new treatment strategies. We have shown that rapamycin promotes vascular smooth muscle cell differentiation in an AKT2-dependent manner in vitro. Here, we investigate the roles of AKT (protein kinase B) isoforms in intimal hyperplasia. APPROACH AND RESULTS: We found that germ-line-specific or smooth muscle-specific deletion of Akt2 resulted in more severe intimal hyperplasia compared with control mice after arterial denudation injury. Conversely, smooth muscle-specific Akt1 knockout prevented intimal hyperplasia, whereas germ-line Akt1 deletion caused severe thrombosis. Notably, rapamycin prevented intimal hyperplasia in wild-type mice but had no therapeutic benefit in Akt2 knockouts. We identified opposing roles for AKT1 and AKT2 isoforms in smooth muscle cell proliferation, migration, differentiation, and rapamycin response in vitro. Mechanistically, rapamycin induced MYOCD (myocardin) mRNA expression. This was mediated by AKT2 phosphorylation and nuclear exclusion of FOXO4 (forkhead box O4), inhibiting its binding to the MYOCD promoter. CONCLUSIONS: Our data reveal opposing roles for AKT isoforms in smooth muscle cell remodeling. AKT2 is required for rapamycin's therapeutic inhibition of intimal hyperplasia, likely mediated in part through AKT2-specific regulation of MYOCD via FOXO4. Because AKT2 signaling is impaired in diabetes mellitus, this work has important implications for rapamycin therapy, particularly in diabetic patients.

Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice.

Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of Tet2-mutant cells in atherosclerosis-prone, low-density lipoprotein receptor-deficient (Ldlr-/-) mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome-mediated interleukin-1ß secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic TET2 mutations in blood cells play a causal role in atherosclerosis.

Activation of the retinoid X receptor modulates angiotensin II-induced smooth muscle gene expression and inflammation in vascular smooth muscle cells.

The retinoid X receptor (RXR) partners with numerous nuclear receptors, such as the peroxisome proliferator activated receptor (PPAR) family, liver X receptors (LXRs), and farnesoid X receptor (FXR). Although each heterodimer can be activated by specific ligands, a subset of these receptors, defined as permissive nuclear receptors, can also be activated by RXR agonists known as rexinoids. Many individual RXR heterodimers have beneficial effects in vascular smooth muscle cells (SMCs). Because rexinoids can potently activate multiple RXR pathways, we hypothesized that treating SMCs with rexinoids would more effectively reverse the pathophysiologic effects of angiotensin II than an individual heterodimer agonist. Cultured rat aortic SMCs were pretreated with either an RXR agonist (bexarotene or 9-cis retinoic acid) or vehicle (dimethylsulfoxide) for 24 hours before stimulation with angiotensin II. Compared with dimethylsulfoxide, bexarotene blocked angiotensin II-induced SM contractile gene induction (calponin and smooth muscle-α-actin) and protein synthesis ([(3)H]leucine incorporation). Bexarotene also decreased angiotensin II-mediated inflammation, as measured by decreased expression of monocyte chemoattractant protein-1 (MCP-1). Activation of p38 mitogen-activated protein (MAP) kinase but not extracellular signal-related kinase (ERK) or protein kinase B (Akt) was also blunted by bexarotene. We compared bexarotene to five agonists of nuclear receptors (PPARα, PPARγ, PPARδ, LXR, and FXR). Bexarotene had a greater effect on calponin reduction, MCP-1 inhibition, and p38 MAP kinase inhibition than any individual agonist. PPARγ knockout cells demonstrated blunted responses to bexarotene, indicating that PPARγ is necessary for the effects of bexarotene. These data demonstrate that RXR is a potent modulator of angiotensin II-mediated responses in the vasculature, partially through inhibition of p38.

Pharmacologic uncoupling of angiogenesis and inflammation during initiation of pathological corneal neovascularization.

Pathological neovascularization occurs when a balance of pro- and anti-angiogenic factors is disrupted, accompanied by an amplifying inflammatory cascade. However, the interdependence of these responses and the mechanism triggering the initial angiogenic switch have remained unclear. We present data from an epithelial debridement model of corneal neovascularization describing an initial 3-day period when a substantial component of neovascular growth occurs. Administration of selective inhibitors shows that this initial growth requires signaling through VEGFR-2 (vascular endothelial growth factor receptor-2), independent of the accompanying inflammatory response. Instead, increased VEGF production is found prominently in repair epithelial cells and is increased prior to recruitment of neutrophil/granulocytes and macrophage/monocytes. Consequently, early granulocyte and monocyte depletion has little effect on corneal neovascularization outgrowth. These data indicate that it is possible to pharmacologically uncouple these mechanisms during early injury-driven neovascularization in the cornea and suggest that initial tissue responses are coordinated by repair epithelial cells.

SDF-1α induction in mature smooth muscle cells by inactivation of PTEN is a critical mediator of exacerbated injury-induced neointima formation.

OBJECTIVE: PTEN inactivation selectively in smooth muscle cells (SMC) initiates multiple downstream events driving neointima formation, including SMC cytokine/chemokine production, in particular stromal cell-derived factor-1α (SDF-1α). We investigated the effects of SDF-1α on resident SMC and bone marrow-derived cells and in mediating neointima formation. METHODS AND RESULTS: Inducible, SMC-specific PTEN knockout mice (PTEN iKO) were bred to floxed-stop ROSA26-ß-galactosidase (ßGal) mice to fate-map mature SMC in response to injury; mice received wild-type green fluorescent protein-labeled bone marrow to track recruitment. Following wire-induced femoral artery injury, ßGal(+) SMC accumulated in the intima and adventitia. Compared with wild-type, PTEN iKO mice exhibited massive neointima formation, increased replicating intimal and medial ßGal(+)SMC, and enhanced vascular recruitment of bone marrow cells following injury. Inhibiting SDF-1α blocked these events and reversed enhanced neointima formation observed in PTEN iKO mice. Most recruited green fluorescent protein(+) cells stained positive for macrophage markers but not SMC markers. SMC-macrophage interactions resulted in a persistent SMC inflammatory phenotype that was dependent on SMC PTEN and SDF-1α expression. CONCLUSION: Resident SMC play a multifaceted role in neointima formation by contributing the majority of neointimal cells, regulating recruitment of inflammatory cells, and contributing to adventitial remodeling. The SMC PTEN-SDF-1α axis is a critical regulator of these events.