Xinbo Zhang

Ten-Eleven Translocation-2 (TET2) Is a Master Regulator of Smooth Muscle Cell Plasticity

Background— Smooth muscle cells (SMCs) are remarkably plastic. Their reversible differentiation is required for growth and wound healing but also contributes to pathologies such as atherosclerosis and restenosis. Although key regulators of the SMC phenotype, including myocardin (MYOCD) and KLF4, have been identified, a unifying epigenetic mechanism that confers reversible SMC differentiation has not been reported. Methods and Results— Using human SMCs, human arterial tissue, and mouse models, we report that SMC plasticity is governed by the DNA-modifying enzyme ten-eleven translocation-2 (TET2). TET2 and its product, 5-hydroxymethylcytosine (5-hmC), are enriched in contractile SMCs but reduced in dedifferentiated SMCs. TET2 knockdown inhibits expression of key procontractile genes, including MYOCD and SRF, with concomitant transcriptional upregulation of KLF4. TET2 knockdown prevents rapamycin-induced SMC differentiation, whereas TET2 overexpression is sufficient to induce a contractile phenotype. TET2 overexpression also induces SMC gene expression in fibroblasts. Chromatin immunoprecipitation demonstrates that TET2 coordinately regulates phenotypic modulation through opposing effects on chromatin accessibility at the promoters of procontractile versus dedifferentiation-associated genes. Notably, we find that TET2 binds and 5-hmC is enriched in CArG-rich regions of active SMC contractile promoters (MYOCD, SRF, and MYH11). Loss of TET2 and 5-hmC positively correlates with the degree of injury in murine models of vascular injury and human atherosclerotic disease. Importantly, localized TET2 knockdown exacerbates injury response, and local TET2 overexpression restores the 5-hmC epigenetic landscape and contractile gene expression and greatly attenuates intimal hyperplasia in vivo. Conclusions— We identify TET2 as a novel and necessary master epigenetic regulator of SMC differentiation.

Macrophage deficiency of miR‐21 promotes apoptosis, plaque necrosis, and vascular inflammation during atherogenesis

Atherosclerosis, the major cause of cardiovascular disease, is a chronic inflammatory disease characterized by the accumulation of lipids and inflammatory cells in the artery wall. Aberrant expression of microRNAs has been implicated in the pathophysiological processes underlying the progression of atherosclerosis. Here, we define the contribution of miR‐21 in hematopoietic cells during atherogenesis. Interestingly, we found that miR‐21 is the most abundant miRNA in macrophages and its absence results in accelerated atherosclerosis, plaque necrosis, and vascular inflammation. miR‐21 expression influences foam cell formation, sensitivity to ER‐stress‐induced apoptosis, and phagocytic clearance capacity. Mechanistically, we discovered that the absence of miR‐21 in macrophages increases the expression of the miR‐21 target gene, MKK3, promoting the induction of p38‐CHOP and JNK signaling. Both pathways enhance macrophage apoptosis and promote the post‐translational degradation of ABCG1, a transporter that regulates cholesterol efflux in macrophages. Altogether, these findings reveal a major role for hematopoietic miR‐21 in atherogenesis.

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

Endothelium derived nitric oxide synthase negatively regulates the PDGF-survivin pathway during flow-dependent vascular remodeling.

Chronic alterations in blood flow initiate structural changes in vessel lumen caliber to normalize shear stress. The loss of endothelial derived nitric oxide synthase (eNOS) in mice promotes abnormal flow dependent vascular remodeling, thus uncoupling mechanotransduction from adaptive vascular remodeling. However, the mechanisms of how the loss of eNOS promotes abnormal remodeling are not known. Here we show that abnormal flow-dependent remodeling in eNOS knockout mice (eNOS (−/−)) is associated with activation of the platelet derived growth factor (PDGF) signaling pathway leading to the induction of the inhibitor of apoptosis, survivin. Interfering with PDGF signaling or survivin function corrects the abnormal remodeling seen in eNOS (−/−) mice. Moreover, nitric oxide (NO) negatively regulates PDGF driven survivin expression and cellular proliferation in cultured vascular smooth muscle cells. Collectively, our data suggests that eNOS negatively regulates the PDGF-survivin axis to maintain proportional flow-dependent luminal remodeling and vascular quiescence.

Posttranscriptional regulation of lipid metabolism by non-coding RNAs and RNA binding proteins

Alterations in lipoprotein metabolism enhance the risk of cardiometabolic disorders including type-2 diabetes and atherosclerosis, the leading cause of death in Western societies. While the transcriptional regulation of lipid metabolism has been well characterized, recent studies have uncovered the importance of microRNAs (miRNAs), long-non-coding RNAs (lncRNAs) and RNA binding proteins (RBP) in regulating the expression of lipid-related genes at the posttranscriptional level. Work from several groups has identified a number of miRNAs, including miR-33, miR-122 and miR-148a, that play a prominent role in controlling cholesterol homeostasis and lipoprotein metabolism. Importantly, dysregulation of miRNA expression has been associated with dyslipidemia, suggesting that manipulating the expression of these miRNAs could be a useful therapeutic approach to ameliorate cardiovascular disease (CVD). The role of lncRNAs in regulating lipid metabolism has recently emerged and several groups have demonstrated their regulation of lipoprotein metabolism. However, given the high abundance of lncRNAs and the poor-genetic conservation between species, much work will be needed to elucidate the specific role of lncRNAs in controlling lipoprotein metabolism. In this review article, we summarize recent findings in the field and highlight the specific contribution of lncRNAs and RBPs in regulating lipid metabolism.

Endothelial Glucocorticoid Receptor Suppresses Atherogenesis

Objective— The purpose of this study was to determine the role of the endothelial glucocorticoid receptor in the pathogenesis of atherosclerosis. Approach and Results— Control mice and mice lacking the endothelial glucocorticoid receptor were bred onto an Apoe knockout background and subjected to high-fat diet feeding for 12 weeks. Assessment of body weight and total cholesterol and triglycerides before and after the diet revealed no differences between the 2 groups of mice. However, mice lacking the endothelial glucocorticoid receptor developed more severe atherosclerotic lesions in the aorta, brachiocephalic artery, and aortic sinus, as well as a heightened inflammatory milieu as evidenced by increased macrophage recruitment in the lesions. Conclusions— These data suggest that the endothelial glucocorticoid receptor is important for tonic inhibition of inflammation and limitation of atherosclerosis progression in this model.

Endothelial Glucocorticoid Receptor Suppresses Atherogenesis—Brief Report

Objective— The purpose of this study was to determine the role of the endothelial glucocorticoid receptor in the pathogenesis of atherosclerosis. Approach and Results— Control mice and mice lacking the endothelial glucocorticoid receptor were bred onto an Apoe knockout background and subjected to high-fat diet feeding for 12 weeks. Assessment of body weight and total cholesterol and triglycerides before and after the diet revealed no differences between the 2 groups of mice. However, mice lacking the endothelial glucocorticoid receptor developed more severe atherosclerotic lesions in the aorta, brachiocephalic artery, and aortic sinus, as well as a heightened inflammatory milieu as evidenced by increased macrophage recruitment in the lesions. Conclusions— These data suggest that the endothelial glucocorticoid receptor is important for tonic inhibition of inflammation and limitation of atherosclerosis progression in this model.

Absence of ANGPTL4 in adipose tissue improves glucose tolerance and attenuates atherogenesis

Alterations in ectopic lipid deposition and circulating lipids are major risk factors for developing cardiometabolic diseases. Angiopoietin-like protein 4 (ANGPTL4), a protein that inhibits lipoprotein lipase (LPL), controls fatty acid (FA) uptake in adipose and oxidative tissues and regulates circulating triacylglycerol-rich (TAG-rich) lipoproteins. Unfortunately, global depletion of ANGPTL4 results in severe metabolic abnormalities, inflammation, and fibrosis when mice are fed a high-fat diet (HFD), limiting our understanding of the contribution of ANGPTL4 in metabolic disorders. Here, we demonstrate that genetic ablation of ANGPTL4 in adipose tissue (AT) results in enhanced LPL activity, rapid clearance of circulating TAGs, increased AT lipolysis and FA oxidation, and decreased FA synthesis in AT. Most importantly, we found that absence of ANGPTL4 in AT prevents excessive ectopic lipid deposition in the liver and muscle, reducing novel PKC (nPKC) membrane translocation and enhancing insulin signaling. As a result, we observed a remarkable improvement in glucose tolerance in short-term HFD-fed AT-specific Angptl4-KO mice. Finally, lack of ANGPTL4 in AT enhances the clearance of proatherogenic lipoproteins, attenuates inflammation, and reduces atherosclerosis. Together, these findings uncovered an essential role of AT ANGPTL4 in regulating peripheral lipid deposition, influencing whole-body lipid and glucose metabolism and the progression of atherosclerosis.

miR-33 Regulation of Adaptive Fibrotic Response in Cardiac Remodeling

Despite substantial improvements in prevention, diagnosis, and therapeutic strategies, cardiovascular diseases remain the leading cause of mortality and morbidity worldwide. Cardiac fibrosis, a critical hallmark of maladaptive hypertrophy and heart failure (HF), is characterized by the excessive and uncontrolled accumulation of extracellular matrix (ECM) by cardiac fibroblasts (CFs) in the interstitial and perivascular space.1 CFs, primarily of embryonic epicardial and endothelial origins,2 are the predominant noncardiomyocyte population, accounting for 20% of total cell population in the adult murine heart.3 In addition to their traditional functions in regulating ECM synthesis and metabolism and controlling cardiac fibrosis, ECM remodeling, scar formation, and tissue repair, CFs serve as functionally pluralistic cells involved in inflammatory responses, cardiomyocyte survival, and vasculogenesis.4,5 Article, see p 835 In the adult heart, CFs are present in a quiescent state with low proliferative capacity and are responsible for ECM homeostasis, providing a structural scaffold for cardiomyocytes. CFs also express gap junction protein connexin-43, which mediates electric coupling of cardiomyocytes and CFs.6 The adult mammalian heart has limited regenerative capacity after pathological injury. The repair of wounds in the heart consists of the removal of dead cardiomyocytes and replacement of myocytes loss by a collagen-based fibrotic scar to preserve myocardial integrity and cardiac function. The importance of CFs in myocardial remodeling and wound healing has been extensively studied and involves all of the 3 phases of the healing process: acute inflammatory response, proliferation, and late scar maturation. After an acute myocardial injury, CFs release proinflammatory cytokines, which promote their own proliferation in a feed-forward loop and trigger differentiation into a myofibroblast phenotype. This hyperproliferative cell type secretes high levels of proinflammatory and profibrotic factors and ECM proteins. This adaptive fibrosis can maintain structural integrity and prevent the injured heart from dysfunction and rupture; …

MiR-33 regulation of stretch-induced intimal hyperplasia in vein grafts

Vein graft bypass surgery has become the most commonly performed revascularization technique in patients with coronary artery disease, the leading cause of mortality and morbidity worldwide. Vein grafts adapt to the new arterial environment, and the structural vascular remodelling and intimal thickening in the vein graft wall is the main cause of restenosis after vascular reconstruction. Intimal accumulation of smooth muscle cells (SMCs) contributes to the thickening and narrowing of the vessel lumen through pro-inflammatory cytokine-induced cell migration and local cell proliferation. Proliferation of SMCs is a crucial event in the pathogenesis of intimal hyperplasia, which is thought to be an important determinant of successful vein graft adaptation. Although the disease process has been described, the underlying mechanisms are still unclear. Work over the last decade has uncovered prominent roles for noncoding RNAs in several cardiovascular disorders including the failure of vein graft bypass. MicroRNAs (miRNAs) are highly conserved small non-coding RNA molecules involved in the regulation of gene expression at the post-transcriptional level. Huang and co-workers identified microRNA-33 (miR-33) as a major regulator of SMC proliferation and neointimal hyperplasia in vein grafts. Huang and colleagues found that miR-33 expression was markedly attenuated in grafted veins. The authors observed an inverse correlation between miR-33 levels and increased intimal thickening and SMC proliferation. To determine whether miR-33 directly controls SMC proliferation, the authors performed a series of elegant studies, including BrdU incorporation and CCK-8 assays. They found that miR-33 overexpression markedly inhibited SMC proliferation. By contrast, antagonism miR-33 enhances SMC proliferation. These findings are consistent with previous reports establishing miR-33 as an important regulator of cell proliferation and cell cycle progression. To dissect the molecular mechanisms by which miR-33 controls SMCs proliferation, Huang et al. analysed miR-33-predicted targets mRNAs using a number of computational algorithms. They identified bone morphogenetic protein 3 (BMP3) as a novel miR-33 target-gene. BMP3 is a member of the transforming growth factor beta (TGF-b) superfamily and promotes mesenchymal stem cell proliferation though the TGF-b/Activin signalling pathway. The authors found significantly upregulated BMP3 expression in grafted veins, while miR-33 showed an opposite regulation. They also utilized gainand lossof function approaches to demonstrate that exogenous BMP3 accelerated venous SMC proliferation, whereas knock-down of BMP3 exhibited the opposite effect. Most importantly, exogenous BMP3 abolished the inhibitory effects of miR-33 on SMC proliferation. Further studies showed that the phosphorylation of SMAD2 and SMAD5, two molecules downstream of BMP3, were regulated by miR-33 in a BMP3-dependent manner. Together, these observations support the hypothesis that miR-33 protects SMCs proliferation and neointimal hyperplasia by repressing BMP3. The authors also analysed the function of miR-33 in venous SMC proliferation in response to mechanical cyclic stretch, the predominant mechanical force influencing SMCs structural organization, function, and gene expression. Consistent with the in vivo vein graft model, cyclic stretch decreased the expression of miR-33 accompanied by elevated BMP3 expression and increased phosphorylation of SMAD2 and SMAD5 in vitro. By treating the SMCs with miR-33 mimics or BMP3 specific siRNA, the authors further validated the important role of miR-33 and BMP3 on venous SMC proliferation in response to cyclic stretch. Notably, injection of agomiR-33 attenuated neointimal formation and repressed cell proliferation in grafted veins by regulating BMP3 expression and phosphorylation of SMAD2 and SMAD5. As expected, BMP3 overexpression using lentivirus negated the effects of agomiR-33 on intimal thickening occurring in the vein grafts, suggesting the effects of miR-33 on venous SMC proliferation and neointimal hyperplasia are dependent on BMP3 expression. It is worth noting that each microRNA can regulate multiple target mRNAs and each target mRNA can also be regulated by multiple microRNAs. Previously, miR-33 has been demonstrated to play an important role in the regulation of cell proliferation and cell cycle progression by targeting cyclin-dependent kinase 6 (CDK6), cyclin D1, and p53, by which control hepatocyte proliferation, replicative senescence of mouse embryonic fibroblasts and haematopoietic stem cell self-renewal. CDK6 is a D-cyclin-activated kinase involved in driving the cell cycle through interactions with cyclins D1, D2, and D3 in G1 phase of the cell cycle, while p53 induces G1 arrest in the cell cycle by regulating p21 expression. Whether these target genes are involved in miR-33dependent regulation of mechanical stretch-induced proliferation of