Novel Paracrine Functions of Smooth Muscle Cells in Supporting Endothelial Regeneration Following Arterial Injury.
ABSTRACT: RATIONALE:Regeneration of denuded or injured endothelium is an important component of vascular injury response. Cell-cell communication between endothelial cells and smooth muscle cells (SMCs) plays a critical role not only in vascular homeostasis but also in disease. We have previously demonstrated that PKC? (protein kinase C-delta) regulates multiple components of vascular injury response including apoptosis of SMCs and production of chemokines, thus is an attractive candidate for a role in SMC-endothelial cells communication. OBJECTIVE:To test whether PKC?-mediated paracrine functions of SMCs influence reendothelialization in rodent models of arterial injury. METHODS AND RESULTS:Femoral artery wire injury was performed in SMC-conditional Prkcd knockout mice, and carotid angioplasty was conducted in rats receiving transient Prkcd knockdown or overexpression. SMC-specific knockout of Prkcd impaired reendothelialization, reflected by a smaller Evans blue-excluding area in the knockout compared with the wild-type controls. A similar impediment to reendothelialization was observed in rats with SMC-specific knockdown of Prkcd. In contrast, SMC-specific gene transfer of Prkcd accelerated reendothelialization. In vitro, medium conditioned by AdPKC?-infected SMCs increased endothelial wound closure without affecting their proliferation. A polymerase chain reaction-based array analysis identified Cxcl1 and Cxcl7 among others as PKC?-mediated chemokines produced by SMCs. Mechanistically, we postulated that PKC? regulates Cxcl7 expression through STAT3 (signal transducer and activator of transcription 3) as knockdown of STAT3 abolished Cxcl7 expression. The role of CXCL7 in SMC-endothelial cells communication was demonstrated by blocking CXCL7 or its receptor CXCR2, both significantly inhibited endothelial wound closure. Furthermore, insertion of a Cxcl7 cDNA in the lentiviral vector that carries a Prkcd shRNA overcame the adverse effects of Prkcd knockdown on reendothelialization. CONCLUSIONS:SMCs promote reendothelialization in a PKC?-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of endothelial cells from uninjured endothelium.
Project description:The LIM-only protein FHL2, also known as DRAL or SLIM3, has a function in fine-tuning multiple physiological processes. FHL2 is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells and conflicting data have been reported on the regulatory function of FHL2 in SMC phenotype transition. At present the function of FHL2 in SMCs in vascular injury is unknown. Therefore, we studied the role of FHL2 in SMC-rich lesion formation. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice showed accelerated lesion formation with enhanced Ki67 expression compared with wild-type (WT)-mice. Consistent with these findings, cultured SMCs from FHL2-KO mice showed increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. Overexpression of FHL2 in SMCs inhibited CyclinD1 expression and CyclinD1-knockdown blocked the enhanced proliferation of FHL2-KO SMCs. We also observed increased CyclinD1 promoter activity in FHL2-KO SMCs, which was reduced upon ERK1/2 inhibition. Furthermore, FHL2-KO SMCs showed enhanced migration compared with WT SMCs. In conclusion, FHL2 deficiency in mice results in exacerbated SMC-rich lesion formation involving increased proliferation and migration of SMCs via enhanced activation of the ERK1/2-CyclinD1 signaling pathway.
Project description:Vascular endothelial cells (ECs) at arterial branches and curvatures experience disturbed blood flow and induce a quiescent-to-activated phenotypic transition of the adjacent smooth muscle cells (SMCs) and a subsequent smooth muscle hyperplasia. However, the mechanism underlying the flow pattern-specific initiation of EC-to-SMC signaling remains elusive. Our previous study demonstrated that endothelial microRNA-126-3p (miR-126-3p) acts as a key intercellular molecule to increase turnover of the recipient SMCs, and that its release is reduced by atheroprotective laminar shear (12 dynes/cm2) to ECs. Here we provide evidence that atherogenic oscillatory shear (0.5 ± 4 dynes/cm2), but not atheroprotective pulsatile shear (12 ± 4 dynes/cm2), increases the endothelial secretion of nonmembrane-bound miR-126-3p and other microRNAs (miRNAs) via the activation of SNAREs, vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Knockdown of VAMP3 and SNAP23 reduces endothelial secretion of miR-126-3p and miR-200a-3p, as well as the proliferation, migration, and suppression of contractile markers in SMCs caused by EC-coculture. Pharmacological intervention of mammalian target of rapamycin complex 1 in ECs blocks endothelial secretion and EC-to-SMC transfer of miR-126-3p through transcriptional inhibition of VAMP3 and SNAP23. Systemic inhibition of VAMP3 and SNAP23 by rapamycin or periadventitial application of the endocytosis inhibitor dynasore ameliorates the disturbed flow-induced neointimal formation, whereas intraluminal overexpression of SNAP23 aggravates it. Our findings demonstrate the flow-pattern-specificity of SNARE activation and its contribution to the miRNA-mediated EC-SMC communication.
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:Active interactions between endothelial cells and smooth muscle cells (SMCs) are critical to maintaining the SMC phenotype. Exosomes play an important role in intercellular communication. However, little is known about the mechanisms that regulate endothelial cells and SMCs crosstalk. We aimed to determine the mechanisms underlying the regulation of the SMC phenotype by human umbilical vein endothelial cells (HUVECs) through exosomes. We found that HUVECs overexpressing miR-206 upregulated contractile marker (?-SMA, Smoothelin and Calponin) mRNA expression in SMCs. We also found that the expression of miR-206 by HUVECs reduced exosome production by regulating ADP-Ribosylation Factor 6 (ARF6) and sodium/calcium exchanger 1 (NCX1). Using real-time PCR and western blot analysis, we showed that HUVEC-derived exosomes decreased the expression of contractile phenotype marker genes (?-SMA, Smoothelin and Calponin) in SMCs. Furthermore, a reduction of the miR-26a-containing exosomes secreted from HUVECs affects the SMC phenotype. We propose a novel mechanism in which miR-206 expression in HUVECs maintains the contractile phenotype of SMCs by suppressing exosome secretion from HUVECs, particularly miR-26a in exosomes, through targeting ARF6 and NCX1.
Project description:In resistance arteries, endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). Endothelial nitric oxide synthase (eNOS) is present in the luminal side of the EC (apical EC) and the basal side of the EC (MEJ). To test if these eNOS pools acted in sync or separately, we co-cultured ECs and SMCs, then stimulated SMCs with phenylephrine (PE). Adrenergic activation causes inositol [1,4,5] triphosphate (IP3) to move from SMC to EC through gap junctions at the MEJ. PE increases MEJ eNOS phosphorylation (eNOS-P) at S1177, but not in EC. Conversely, we used bradykinin (BK) to increase EC calcium; this increased EC eNOS-P but did not affect MEJ eNOS-P. Inhibiting gap junctions abrogated the MEJ eNOS-P after PE, but had no effect on BK eNOS-P. Differential lipid composition between apical EC and MEJ may account for the compartmentalized eNOS-P response. Indeed, DAG and phosphatidylserine are both enriched in MEJ. These lipids are cofactors for PKC activity, which was significantly increased at the MEJ after PE. Because PKC activity also relies on endoplasmic reticulum (ER) calcium release, we used thapsigargin and xestospongin C, BAPTA, and PKC inhibitors, which caused significant decreases in MEJ eNOS-P after PE. Functionally, BK inhibited leukocyte adhesion and PE caused an increase in SMC cGMP. We hypothesize that local lipid composition of the MEJ primes PKC and eNOS-P for stimulation by PE, allowing for compartmentalized function of eNOS in the blood vessel wall.
Project description:RATIONALE:Vascular smooth muscle cell (SMC) migration is an important pathological process in several vascular occlusive diseases, including atherosclerosis and restenosis, both of which are accelerated by diabetes mellitus. OBJECTIVE:To determine the mechanisms of abnormal vascular SMC migration in type 2 diabetes, the obese Zucker rat (ZO), a model of obesity and insulin resistance, was studied. METHODS AND RESULTS:In culture, ZO aortic SMCs showed a significant increase in Nox4 mRNA and protein levels compared with the control lean Zucker rat (ZL). The sarco-/endoplasmic reticulum Ca(2+) ATPase (SERCA) nitrotyrosine-294,295 and cysteine-674 (C674)-SO(3)H were increased in ZO SMCs, indicating oxidant stress. Unlike ZL SMC, nitric oxide (NO) failed to inhibit serum-induced SMC migration in ZO. Transfection of Nox4 small interference RNA or overexpression of SERCA2b wild type, but not C674S mutant SERCA, restored the response to NO. Knockdown of Nox4 also decreased SERCA oxidation in ZO SMCs. In addition, transforming growth factor-?1 via Smad2 was necessary and sufficient to upregulate Nox4, oxidize SERCA, and block the antimigratory action of NO in ZO SMCs. Corresponding to the results in cultured SMCs, immunohistochemistry confirmed that Nox4 and SERCA C674-SO(3)H were significantly increased in ZO aorta. After common carotid artery injury, knockdown of Nox4 by adenoviral Nox4 short hairpin RNA decreased Nox4 and SERCA C674-SO(3)H staining and significantly decreased injury-induced neointima. CONCLUSION:These studies indicate that the upregulation of Nox4 by transforming growth factor-?1 in ZO SMCs is responsible for the impaired response to NO by a mechanism involving the oxidation of SERCA C674. Knockdown of Nox4 inhibits oxidation of SERCA, as well as neointima formation, after ZO common carotid artery injury.
Project description:Apoptosis of smooth muscle cells (SMCs) is a prominent pathological characteristic of abdominal aortic aneurysm (AAA). We have previously shown that SMC apoptosis stimulates proinflammatory signaling in a mouse model of AAA. Here, we test whether protein kinase C-? (PKC?), an apoptotic mediator, participates in the pathogenesis of AAA by regulating apoptosis and proinflammatory signals.Mouse experimental AAA is induced by perivascular administration of CaCl(2). Mice deficient in PKC? exhibit a profound reduction in aneurysmal expansion, SMC apoptosis, and transmural inflammation as compared with wild-type littermates. Delivery of PKC? to the aortic wall of PKC?(-/-) mice restores aneurysm, whereas overexpression of a dominant negative PKC? mutant in the aorta of wild-type mice attenuates aneurysm. In vitro, PKC?(-/-) aortic SMCs exhibit significantly impaired monocyte chemoattractant protein-1 production. Ectopic administration of recombinant monocyte chemoattractant protein-1 to the arterial wall of PKC?(-/-) mice restores inflammatory response and aneurysm development.PKC? is an important signaling mediator for SMC apoptosis and inflammation in a mouse model of AAA. By stimulating monocyte chemoattractant protein-1 expression in aortic SMCs, upregulated PKC? exacerbates the inflammatory process, in turn perpetuating elastin degradation and aneurysmal dilatation. Inhibition of PKC? may serve as a potential therapeutic strategy for AAA.
Project description:UNLABELLED:Vascular smooth muscle cells (SMCs) from distinct anatomic locations derive from different embryonic origins. Here we investigated the respective potential of different embryonic origin-specific SMCs derived from human embryonic stem cells (hESCs) to support endothelial network formation in vitro. SMCs of three distinct embryological origins were derived from an mStrawberry-expressing hESC line and were cocultured with green fluorescent protein-expressing human umbilical vein endothelial cells (HUVECs) to investigate the effects of distinct SMC subtypes on endothelial network formation. Quantitative analysis demonstrated that lateral mesoderm (LM)-derived SMCs best supported HUVEC network complexity and survival in three-dimensional coculture in Matrigel. The effects of the LM-derived SMCs on HUVECs were at least in part paracrine in nature. A TaqMan array was performed to identify the possible mediators responsible for the differential effects of the SMC lineages, and a microarray was used to determine lineage-specific angiogenesis gene signatures. Midkine (MDK) was identified as one important mediator for the enhanced vasculogenic potency of LM-derived SMCs. The functional effects of MDK on endothelial network formation were then determined by small interfering RNA-mediated knockdown in SMCs, which resulted in impaired network complexity and survival of LM-derived SMC cocultures. The present study is the first to show that SMCs from distinct embryonic origins differ in their ability to support HUVEC network formation. LM-derived SMCs best supported endothelial cell network complexity and survival in vitro, in part through increased expression of MDK. A lineage-specific approach might be beneficial for vascular tissue engineering and therapeutic revascularization. SIGNIFICANCE:Mural cells are essential for the stabilization and maturation of new endothelial cell networks. However, relatively little is known of the effect of the developmental origins of mural cells on their signaling to endothelial cells and how this affects vessel development. The present study demonstrated that human smooth muscle cells (SMCs) from distinct embryonic origins differ in their ability to support endothelial network formation. Lateral mesoderm-derived SMCs best support endothelial cell network complexity and survival in vitro, in part through increased expression of midkine. A lineage-specific approach might be beneficial for vascular tissue engineering and therapeutic revascularization.
Project description:Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels--smooth muscle and endothelial cells--and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.
Project description:Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) are in close contact with blood vessels. SMC phenotypes can be altered during pathological vascular remodeling. However, how SMC phenotypes affect EC properties remains largely unknown. In this study, we found that PDGF-BB-induced synthetic SMCs suppressed EC proliferation and migration while exhibiting increased expression of anti-angiogenic factors, such as endostatin, and decreased pro-angiogenic factors, including CXC motif ligand 1 (CXCL1). Cyclopentenyl cytosine (CPEC), a CTP synthase inhibitor that has been reported previously to inhibit SMC proliferation and injury-induced neointima formation, induced SMC redifferentiation. Interestingly, CPEC-conditioned SMC culture medium promoted EC proliferation and migration because of an increase in CXCL1 along with decreased endostatin production in SMCs. Addition of recombinant endostatin protein or blockade of CXCL1 with a neutralizing antibody suppressed the EC proliferation and migration induced by CPEC-conditioned SMC medium. Mechanistically, CPEC functions as a cytosine derivate to stimulate adenosine receptors A1 and A2a, which further activate downstream cAMP and Akt signaling, leading to the phosphorylation of cAMP response element binding protein and, consequently, SMC redifferentiation. These data provided proof of a novel concept that synthetic SMC exhibits an anti-angiogenic SMC phenotype, whereas contractile SMC shows a pro-angiogenic phenotype. CPEC appears to be a potent stimulator for switching the anti-angiogenic SMC phenotype to the pro-angiogenic phenotype, which may be essential for CPEC to accelerate re-endothelialization for vascular repair during injury-induced vascular wall remodeling.