A Human Pluripotent Stem Cell-Based Screen for Smooth Muscle Cell Differentiation and Maturation Identifies Inhibitors of Intimal Hyperplasia.
ABSTRACT: Contractile to synthetic phenotypic switching of smooth muscle cells (SMCs) contributes to stenosis in vascular disease and vascular transplants. To generate more contractile SMCs, we performed a high-throughput differentiation screen using a MYH11-NLuc-tdTomato human embryonic stem cell reporter cell line. We identified RepSox as a factor that promotes differentiation of MYH11-positive cells by promoting NOTCH signaling. RepSox induces SMCs to exhibit a more contractile phenotype than SMCs generated using PDGF-BB and TGF-β1, two factors previously used for SMC differentiation but which also cause intimal hyperplasia. In addition, RepSox inhibited intimal hyperplasia caused by contractile to synthetic phenotypic switching of SMCs in a rat balloon injury model. Thus, in addition to providing more contractile SMCs that could prove useful for constructing artificial blood vessels, this study suggests a strategy for identifying drugs for inhibiting intimal hyperplasia that act by driving contractile differentiation rather than inhibiting proliferation non-specifically.
Project description:Intimal thickening is an early phase of atherosclerosis characterized by differentiation of plaque smooth muscle cells (SMCs) from a contractile to a synthetic phenotype. We used laser microdissection (LMD) plus real-time RT-PCR to quantify mRNAs for calponin-1 and smoothelin, markers of the contractile phenotype, and for serum response factor (SRF), a regulator of SMC differentiation, in intimal and medial SMCs of human coronary arteries with intimal thickening. RNA expression was also analyzed by ISH and protein expression was detected by IHC. LMD plus RT-PCR found similar levels of SRF mRNA in intimal and medial SMCs, while medial mRNA levels for calponin-1 and smoothelin were higher. ISH confirmed that smoothelin mRNA levels in media exceeded those in intima, whereas SRF mRNA levels were similar at both sites. For calponin-1 and smoothelin, protein levels mirrored respective mRNA levels. By contrast, more medial than intimal SRF protein was present. Our results indicate that intimal SMCs exhibit a largely synthetic phenotype, perhaps reflecting lower intimal levels of SRF protein; ISH and LMD plus real-time RT-PCR provide comparable results; as a valuable alternative to ISH, LMD plus RT-PCR allows parallel measurement of several transcripts; and tissue gene expression studies must measure both protein and mRNA levels.
Project description: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.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.We identify TET2 as a novel and necessary master epigenetic regulator of SMC differentiation.
Project description:Outcomes of cardiovascular procedures, such as angioplasty and stent or bypass grafting are limited by failure, predominantly caused by pathological smooth muscle cell (SMC) proliferation, known as intimal hyperplasia. Local delivery of a genetically engineered herpes simplex virus (HSV) is known to block vascular SMC proliferation while allowing for re-endothelialization. However, the mechanism this mutant virus uses to prevent SMC hyperplasia is unknown. The Ras signaling cascade is activated in SMCs undergoing hyperplasia leading to phosphorylation of the mitogen-activated protein kinase (MAPK). In this study we tested the hypothesis that MAPK kinase (MEK) activity is the molecular basis by which SMCs are susceptible to mutant HSV. We show that genetically engineered herpes simplex-1 viruses (HSV-1) can target proliferating SMCs. We demonstrate that the molecular basis of this HSV-1 anti-proliferative effect is MEK activation in SMCs. We demonstrate efficacy and practicality of the MEK-dependent HSV-1 for the treatment of intimal hyperplasia in a clinically relevant in vivo model. Important to this strategy is the ability to modulate the effects by controlling viral dose. These results propel genetically engineered HSV-1 therapy towards clinical evaluation in treatment of intimal hyperplasia.
Project description:To gain insights into mechanisms by which intimal hyperplasia interferes with the repair process by investigating expression and function of the catalytic telomerase reverse transcriptase (TERT) subunit after vascular injury.Functional telomerase is essential to the replicative longevity of vascular cells. We found that TERT was de novo activated in the intima of injured arteries, involving activation of the nuclear factor ?B pathway. Stimulation of the isolated intimal smooth muscle cell (SMC) by basic fibroblast growth factor or tumor necrosis factor ? resulted in increased TERT activity. This depends on the activation of c-Myc signaling because mutation of the E-box in the promoter or overexpression of mitotic arrest deficient 1 (MAD1), a c-Myc competitor, abrogated the transcriptional activity. Inhibition of nuclear factor ?B in both intimal SMCs and the injured artery attenuated TERT transcriptional activity through reduction of c-Myc expression. Pharmacological blockade of TERT led to SMC senescence. Finally, depletion of telomerase function in mice resulted in severe intimal SMC senescence after vascular injury.These results support a model in which vascular injury induces de novo expression of TERT in intimal SMCs via activation of nuclear factor ?B and upregulation of c-Myc. The resumed TERT activity is critical for intimal hyperplasia.
Project description:Despite rapid progress in surgical techniques, there is still a significant lack of surgery-supportive pharmacological treatments. The aim of this study was to test the hypothesis that ursolic acid (UA) may prevent intimal hyperplasia of venous bypass grafts.The hypothesis was tested by means of primary cell isolation and culture followed by real-time polymerase chain reaction, western blotting, fluorescence microscopy and fluorescence-activated cell sorting analyses, as well as an in vivo rat model for intimal hyperplasia of venous bypass grafts and immunohistochemistry and histochemistry.The local application of UA significantly inhibited intimal hyperplasia in vivo (intimal thickness control: 25 µm, UA group: 18 µM-8 weeks after surgery). The UA treatment of grafts significantly resulted in reduced endothelial vascular cell adhesion molecule-1 (VCAM-1) expression, reduced infiltration of the grafts vessel wall by CD45-positive cells and increased smooth muscle cell (SMC) death. In in vitro condition, it could be shown that UA inhibits VCAM-1 expression downstream of NF?B and is likely to interfere with VCAM-1 protein synthesis in endothelial cells. Quantification of cell death in vascular smooth muscle cells treated with UA indicated that UA is a potent inducer of SMC apoptosis.Our results suggest that UA-mediated inhibition of endothelial VCAM-1 expression reduces the infiltration of venous bypass grafts by CD45-positive cells and inhibits intimal hyperplasia. Apoptosis induction in SMCs may be another method in which UA reduces intimal thickening. UA may constitute a surgery-supportive pharmacon that reduces intimal hyperplasia of vein grafts.
Project description:We replicated the rat common carotid artery (CCA) intima hyperplasia model and found the expression of a circular RNA, circRNA_009723 (circDcbld1), was markedly increased in the CCA with intimal hyperplasia. In vitro, the suppression of circDcbld1 in rat vascular smooth muscle cells (VSMCs) led the increase of contractile smooth muscle cell markers and the decrease of cell migration. In vivo, the injection of chemically modified circDcbld1 small interfering RNA (siRNA) lessened the formation of neointima in rat CCA after balloon injury. Further experiments proved that circDcbld1, as a competing endogenous RNA, interacted with miR-145-3p and upregulated the level of neuropilin-1 (Nrp1), thereby regulating the migration of VSMCs. In this study, we demonstrated a new mechanism by which circular RNA promotes intimal hyperplasia. We deem that intervention in the circDcbld1-miR-145-3p/Nrp1 pathway might be a feasible approach to alleviate the post-injury intimal hyperplasia.
Project description:Calcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that Proteoglycan 4 (PRG4) is a key molecular signature of calcified plaques, expressed in smooth muscle cell (SMC) rich regions. Here, we aimed to unravel the PRG4 role in vascular remodeling and intimal calcification. <i>PRG4</i> expression in human carotid endarterectomies correlated with calcification assessed by preoperative computed tomographies. PRG4 localized to SMCs in early intimal thickening, while in advanced lesions it was found in the extracellular matrix, surrounding macro-calcifications. In experimental models, <i>Prg4</i> was upregulated in SMCs from partially ligated <i>ApoE<sup>-/-</sup></i> mice and rat carotid intimal hyperplasia, correlating with osteogenic markers and <i>TGF</i><i>b1</i>. Furthermore, PRG4 was enriched in cells positive for chondrogenic marker SOX9 and around plaque calcifications in <i>ApoE<sup>-/-</sup></i> mice on warfarin. In vitro, <i>PRG4</i> was induced in SMCs by IFNg, TGFb1 and calcifying medium, while SMC markers were repressed under calcifying conditions. Silencing experiments showed that <i>PRG4</i> expression was driven by transcription factors <i>SMAD3</i> and <i>SOX9.</i> Functionally, the addition of recombinant human PRG4 increased ectopic SMC calcification, while arresting cell migration and proliferation. Mechanistically, it suppressed endogenous <i>PRG4</i>, <i>SMAD3</i> and <i>SOX9</i>, and restored SMC markers' expression. PRG4 modulates SMC function and osteogenic phenotype during intimal remodeling and macro-calcification in response to TGFb1 signaling, SMAD3 and SOX9 activation. The effects of PRG4 on SMC phenotype and calcification suggest its role in atherosclerotic plaque stability, warranting further investigations.
Project description:Atherosclerotic-associated diseases are the leading cause of death in the United States. Despite recent progress, interventional treatments for atherosclerosis can be complicated by restenosis resulting from neo-intimal hyperplasia. We have previously demonstrated that TGF-? and its downstream signaling protein Smad3 ? 1) are up-regulated following vascular injury, 2) together drive smooth muscle cell (SMC) proliferation and migration and 3) enhance the development of intimal hyperplasia. In order to determine a mechanism through which TGF-?/Smad3 promote these effects, Affymetrix gene expression arrays were performed on primary rat SMCs infected with Smad3 and stimulated with TGF-? or infected with GFP alone. More than 200 genes were differentially expressed (>2.0 fold change, p<0.05) in TGF-?/Smad3 stimulated SMCs. We then performed GO term enrichment analysis using the DAVID bioinformatics database and found that TGF-?/Smad3 activated the expression of multiple genes related to either development or cell differentiation, several of which have been shown to be associated with multipotent stem or progenitor cells. Quantitative real-time PCR confirmed up-regulation of several developmental genes including FGF1, NGF, and Wnt11 (by 2.5, 6 and 7 fold, respectively) as well as stem/progenitor cell associated genes CD34 and CXCR4 (by 10 and 45 fold, respectively). In addition, up-regulation of these factors at protein levels were also confirmed by Western blotting, or by immunocytochemistry (performed for CXCR4 and NGF). Finally, TGF-?/Smad3 down regulated transcription of SMC contractile genes as well as protein production of smooth muscle alpha actin, calponin, and smooth muscle myosin heavy chain. These combined results suggest that TGF-?/Smad3 stimulation drives SMCs to a phenotypically altered state of de-differentiation through the up-regulation of developmental related genes.
Project description:RATIONALE:Mutations in myosin heavy chain (MYH11) cause autosomal dominant inheritance of thoracic aortic aneurysms and dissections. At the same time, rare, nonsynonymous variants in MYH11 that are predicted to disrupt protein function but do not cause inherited aortic disease are common in the general population and the vascular disease risk associated with these variants is unknown. OBJECTIVE:To determine the consequences of the recurrent MYH11 rare variant, R247C, through functional studies in vitro and analysis of a knock-in mouse model with this specific variant, including assessment of aortic contraction, response to vascular injury, and phenotype of primary aortic smooth muscle cells (SMCs). METHODS AND RESULTS:The steady state ATPase activity (actin-activated) and the rates of phosphate and ADP release were lower for the R247C mutant myosin than for the wild-type, as was the rate of actin filament sliding in an in vitro motility assay. Myh11(R247C/R247C) mice exhibited normal growth, reproduction, and aortic histology but decreased aortic contraction. In response to vascular injury, Myh11(R247C/R247C) mice showed significantly increased neointimal formation due to increased SMC proliferation when compared with the wild-type mice. Primary aortic SMCs explanted from the Myh11(R247C/R247C) mice were dedifferentiated compared with wild-type SMCs based on increased proliferation and reduced expression of SMC contractile proteins. The mutant SMCs also displayed altered focal adhesions and decreased Rho activation, associated with decreased nuclear localization of myocardin-related transcription factor-A. Exposure of the Myh11(R247C/R247C) SMCs to a Rho activator rescued the dedifferentiated phenotype of the SMCs. CONCLUSIONS:These results indicate that a rare variant in MYH11, R247C, alters myosin contractile function and SMC phenotype, leading to increased proliferation in vitro and in response to vascular injury.
Project description:Intimal hyperplasia (IH) is the primary cause of vein bypass graft failure. The smooth muscle cell (SMC) is a key element of IH as it phenotypically switches from a contractile to a synthetic state which can become pathological. R7020, which is an engineered strain of Herpes Simplex Virus-1, inhibits IH in animal models. Although it has many characteristics which make it a strong candidate for use as a prophylactic agent how it inhibits IH is not well understood. The objective of this study was to identify modes of action used by R7020 to function in blood vessels that may also contribute to its inhibition of IH. The cytopathic effect of R7020 on SMCs was determined in vitro and in a rabbit IH model. In vitro assays with R7020 infected SMCs were used to quantify the effect of dose on the release kinetics of the virus as well as the effects of R7020 on cell viability and the adhesion of peripheral blood mononuclear cells (PBMCs) to SMCs in the absence and presence of tumor necrosis factor alpha (TNF-?). The observed cytopathic effect, which included R7020 positive filopodia that extend from cell to cell and the formation of syncytia, suggests that R7020 remains cell associated after egress and spreads cell to cell instead of by diffusion through the extracellular fluid. This would allow the virus to rapidly infect vascular cells while evading the immune system. The directionality of the filopodia in vivo suggests that the virus preferentially travels from the media towards the intima targeting SMCs that would lead to IH. The formation of syncytia would inhibit SMC proliferation as incorporated cells are not able to multiply. It was also observed that R7020 induced the fusion of PBMCs with syncytia suggesting the virus may limit the effect of macrophages on IH. Furthermore, R7020 inhibited the proliferative effect of TNF-?, an inflammatory cytokine associated with increased IH. Thus, the results of this study suggest that R7020 inhibits IH through multiple mechanisms.