U nlike most mature cells, smooth muscle cells (SMCs) are remarkably plastic and can dedifferentiate in response to environmental cues, 1,2 adding a layer of complexity to the regulation of gene expression. Although several transcription factors have been identified, a global mechanism that coordinately regulates SMC phenotype has yet to be uncovered. How SMC genes become silenced and then reactivated is unknown and is an area of intense investigation. Recent demonstration that the ten-eleven-translocation (TET) family of proteins is involved in DNA demethylation [3][4][5] prompted us to evaluate the role of the TET proteins in the modulation of SMC phenotype. Editorial see p 2002 Clinical Perspective on p 2057The TET proteins (TET1-TET3) are a recently discovered family of DNA demethylases. TET proteins oxidize 5-methylcytosine (5-mC) to generate 5-hydroxymethylcytosine (5-hmC), frequently called the sixth DNA base, in mammalian cells. 4,5 Through the base excision repair pathway, 5-hmC is then converted to unmethylated cytosine, leading to DNA demethylation and gene activation. [6][7][8] Therefore, the 5-hmC modification and the TET enzymes have emerged as key activators of gene expression. Studies of TET proteins and 5-hmC function in embryonic stem cells (ESCs) demonstrate that they play a major role in maintaining cellular pluripotency through the regulation of lineage-specific genes. 4,[9][10][11] In contrast to this role in ESC pluripotency, the TET proteins (and their 5-hmC products) have an opposing role in adult stem cells and somatic tissues. TET2 mutations have been described in several types of hematopoietic disorders in which the loss of TET2 has been shown to promote hematopoietic stem cell self-renewal.12 TET2 and 5-hmC levels are increased during neurogenesis, 13 and more recently, loss of TET2 and 5-hmC was demonstrated to be a key epigenetic event associated with 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 regul...

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Background Platelet abnormalities are well-recognized complications of diabetes mellitus (DM). Mitochondria play a central role in platelet metabolism and activation. Mitochondrial dysfunction is evident in DM. The molecular pathway for hyperglycemia-induced mitochondrial dysfunction in DM platelets is unknown. Methods and Results Using both human and humanized mouse models, we report that hyperglycemia-induced aldose reductase (AR) activation, and subsequent reactive oxygen species (ROS) production, leads to increased p53 phosphorylation (Ser15), which promotes mitochondrial dysfunction, damage and rupture by sequestration of the anti-apoptotic protein Bcl-xL. In a glucose dose dependent manner, severe mitochondrial damage leads to loss of mitochondrial membrane potential and platelet apoptosis (cytochrome c release, caspase 3 activation and phosphatidylserine exposure). Although platelet hyperactivation, mitochondrial dysfunction, AR activation, ROS production and p53 phosphorylation are all induced by hyperglycemia, we demonstrate that platelet apoptosis and hyperactivation are two distinct states, dependent upon the severity of the hyperglycemia and mitochondrial damage. Combined, both lead to increased thrombus formation in a mouse blood stasis model. Conclusions AR contributes to diabetes-mediated mitochondrial dysfunction and damage through the activation of p53. The degree of mitochondrial dysfunction and damage determines whether hyperactivity (mild damage) or apoptosis (severe damage) will ensue. These signaling components provide novel therapeutic targets for DM thrombotic complications.

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Smooth muscle cells (SMC) are the major cell type in blood vessels. Their principle function in the body is to regulate blood flow and pressure through vessel wall contraction and relaxation. Unlike many other mature cell types in the adult body, SMC do not terminally differentiate but retain a remarkable plasticity. They have the unique ability to toggle between a differentiated and quiescent “contractile” state and a highly proliferative and migratory “synthetic” phenotype in response to environmental stresses. While there have been major advances in our understanding of SMC plasticity through the identification of growth factors and signals that can influence the SMC phenotype, how these regulate SMC plasticity remains unknown. To date, several key transcription factors and regulatory cis elements have been identified that play a role in modulating SMC state. The frontier in understanding the molecular mechanisms underlying SMC plasticity has now advanced to the level of epigenetics. This review will summarize the epigenetic regulation of SMC, highlighting the role of histone modification, DNA methylation, and our most recent identification of a DNA demethylation pathway in SMC that is pivotal in the regulation of the SMC phenotypic state. Many disorders are associated with smooth muscle dysfunction, including atherosclerosis, the major underlying cause of stroke and coronary heart disease, as well as transplant vasculopathy, aneurysm, asthma, hypertension, and cancer. An increased understanding of the major regulators of SMC plasticity will lead to the identification of novel target molecules that may, in turn, lead to novel drug discoveries for the treatment of these diseases.

BackgroundBone repair is dependent on the presence of osteocompetent progenitors that are able to differentiate and generate new bone. Muscle is found in close association with orthopaedic injury, however its capacity to make a cellular contribution to bone repair remains ambiguous. We hypothesized that myogenic cells of the MyoD-lineage are able to contribute to bone repair.MethodsWe employed a MyoD-Cre+:Z/AP+ conditional reporter mouse in which all cells of the MyoD-lineage are permanently labeled with a human alkaline phosphatase (hAP) reporter. We tracked the contribution of MyoD-lineage cells in mouse models of tibial bone healing.ResultsIn the absence of musculoskeletal trauma, MyoD-expressing cells are limited to skeletal muscle and the presence of reporter-positive cells in non-muscle tissues is negligible. In a closed tibial fracture model, there was no significant contribution of hAP+ cells to the healing callus. In contrast, open tibial fractures featuring periosteal stripping and muscle fenestration had up to 50% of hAP+ cells detected in the open fracture callus. At early stages of repair, many hAP+ cells exhibited a chondrocyte morphology, with lesser numbers of osteoblast-like hAP+ cells present at the later stages. Serial sections stained for hAP and type II and type I collagen showed that MyoD-lineage cells were surrounded by cartilaginous or bony matrix, suggestive of a functional role in the repair process. To exclude the prospect that osteoprogenitors spontaneously express MyoD during bone repair, we created a metaphyseal drill hole defect in the tibia. No hAP+ staining was observed in this model suggesting that the expression of MyoD is not a normal event for endogenous osteoprogenitors.ConclusionsThese data document for the first time that muscle cells can play a significant secondary role in bone repair and this knowledge may lead to important translational applications in orthopaedic surgery.Please see related article: http://www.biomedcentral.com/1741-7015/9/136

Cardiovascular diseases are a class of heart or blood vessels diseases, which are now considered to be the leading cause of death globally. A number of recent studies have reported key roles for inflammation in the progression of diseased vessels and systematic heart failure. In particular, endoplasmic reticulum (ER) stress, which is mechanistically implicated in inflammation and autophagy, has now been associated with pathophysiological states in the cardiovascular system. Autophagy has also been identified as an important process in the progression of multiple cardiovascular diseases such as in atherosclerosis plaque progression and ischemia and/or reperfusion. In light of the above, it has been proposed that a link between inflammation, autophagy, and ER stress may exist that contribute to diseases of the heart and its supporting vessels. This review highlights current knowledge on the cross talk between these three biological processes, and the potential of targeting this pathway as a therapeutic approach for cardiovascular disorders and its related diseases.

Background: Aberrant expression of circular RNA (CircRNA) contributes to human diseases. CircRNAs regulate gene expression by sequestering specific microRNAs (miRNAs). In this study, we investigated whether CircMAP3K5 could act as a competing endogenous miR-22-3p sponge and regulate neointimal hyperplasia. Methods: CircRNA profiling from genome-wide RNA sequencing data was compared between human coronary artery smooth muscle cells (HCASMCs) treated with or without PDGF. Expression levels of circular MAP3K5 (CircMAP3K5) was assessed in human coronary arteries from autopsies on patients with dilated cardiomyopathy (DCM) or coronary heart disease (CHD). The role of CircMAP3K5 in intimal hyperplasia was further investigated in mice with AAV9-mediated CircMAP3K5 transfection. SMC-specific Tet2 knockout mice and global miR-22-3p knockout mice were used to delineate the mechanism by which CircMAP3K5 attenuated neointimal hyperplasia using the femoral arterial wire injury model. Results: RNA sequencing demonstrated that treatment with PDGF-BB significantly reduced expression of CircMAP3K5 in HCASMCs. Wire-injured mouse femoral arteries and diseased arteries from CHD patients (where PDGF-BB is increased) confirmed in vivo downregulation of CircMAP3K5 associated with injury and disease. Lentivirus-mediated overexpression of CircMAP3K5 inhibited the proliferation of HCASMCs. In vivo AAV9-mediated transfection of CircMap3k5 specifically inhibited SMC proliferation in the wire-injured mouse arteries, resulting in reduced neointima formation. Using a luciferase reporter assay and RNA pull-down, CircMAP3K5 was found to sequester miR-22-3p, which in turn inhibited the expression of TET2. Both in vitro and in vivo results demonstrate that the loss of miR-22-3p recapitulated the anti-proliferative effect of CircMap3k5 on VSMCs. In SMC-specific Tet2 knockout mice, loss of Tet2 abolished the CircMap3k5-mediated anti-proliferative effect on VSMCs. Conclusions: We identify CircMAP3K5 as a master regulator of TET2-mediated VSMC differentiation. Targeting the CircMAP3K5/miR-22-3p/TET2 axis may provide a potential therapeutic strategy for diseases associated with intimal hyperplasia including restenosis and atherosclerosis.