Long noncoding RNA H19 promotes vascular remodeling by sponging let-7a to upregulate the expression of cyclin D1

Vascular remodeling is mainly caused by excessive proliferation of vascular smooth muscle cells (VSMCs). Noncoding RNAs (ncRNAs) have emerged as important regulators in diverse pathological processes. Previous work has shown the functions and mechanisms of long noncoding RNA H19 (LncRNA H19) on VSMCs. As long noncoding RNAs (lncRNAs) are complex in their mechanisms of action, the aim of the study is to identify if there are any other molecular mechanisms of LncRNA H19 on VSMCs. In vivo studies demonstrated that cyclin D1 was overexpressed in neointima of balloon-injured artery. In vitro studies identified that the overexpression of LncRNA H19 promoted VSMCs proliferation and cyclin D1 upregulation. On the contrary, cellular proliferation and expression of cyclin D1 were inhibited in VSMCs after infection with let-7a. Furthermore, luciferase reporter assays and RNA pull-down assays were used to explore the regulatory mechanism, we found that LncRNA H19 functioned as a competing endogenous RNA (ceRNA) by sponging let-7a to promote the expression of the target gene cyclin D1. In conclusion, LncRNA H19 positively regulated cyclin D1 expression through directly binding to let-7a in VSMCs. Our findings provide new insight into the mechanism of LncRNA H19 in VSMCs proliferation and vascular remodeling, and further indicate the implications of LncRNA H19 in the diagnosis and treatment of vascular proliferative diseases.     

Characterization of transcriptional and posttranscriptional properties of native and cultured phenotypically modulated vascular smooth muscle cells

Various in vitro models are used for studying phenotypic modulation of vascular smooth muscle cells (VSMCs) and the established culture of vascular smooth muscle cells (cVSMCs) is most often used for this purpose. On the other hand, vascular interstitial cells (VICs) are native phenotypically modulated VSMCs present in blood vessels under normal physiological conditions. The aim of this work has been to compare the difference in expression of a number of VSMC-specific markers, which are commonly used for the characterisation of phenotypic modulation of VSMCs, between freshly dispersed VSMCs, VICs and cVSMCs from rat abdominal aorta. Our experiments show that VICs are present in the rat aorta and express markers of VSMCs. Both VICs and cVSMCs display the presence of sparse individual stress fibres enriched in alpha smooth muscle actin (αSM-actin), whereas in VSMCs, this protein is more densely packed. Compared with contractile VSMCs, both VICs and cVSMCs display decreased expression of VSMC-specific markers such as smoothelin, myosin light chain kinase and SM22α; however, the expression of two major cytoskeletal and contractile proteins (smooth muscle myosin heavy chain and αSM-actin) was downregulated in cVSMCs but not in VICs compared with contractile VSMCs. These results suggest different mechanisms for the phenotypic modulation of cVSMCs and VICs. VICs might therefore represent a novel convenient model for studying molecular mechanisms that govern the phenotypic modulation of VSMCs.     

Lin28a up‐regulation is associated with the formation of restenosis via promoting proliferation and migration of vascular smooth muscle cells

To explore the potential role of Lin28a in the development of restenosis after percutaneous transluminal angioplasty, double‐balloon injury surgery and mono‐balloon injury surgery were used to establish restenosis and atherosclerosis models, respectively, so as to better distinguish restenosis from atherosclerotic lesions. Immunohistochemical analysis revealed that significantly higher expression of Lin28a was observed in the iliac arteries of restenosis plaques than that of atherosclerosis plaques. Immunofluorescence studies showed the colocalization of Lin28a with α‐smooth muscle actin in restenosis plaques, rather than in atherosclerosis plaques, which suggested that Lin28a might be related to the unique behaviour of vascular smooth muscle cells (VSMCs) in restenosis. To further confirm above hypothesis, Lin28a expression was up‐regulated by transfection of Lenti‐Lin28a and inhibited by Lenti‐Lin28a‐shRNA transfection in cultured VSMCs, and then the proliferation and migration capability of VSMCs were detected by EdU and Transwell assays, respectively. Results showed that the proliferation and migration of VSMCs were significantly increased in accordance with the up‐regulation of Lin28a expression, while above behaviours of VSMCs were significantly suppressed after inhibiting the expression of Lin28a. In conclusion, the up‐regulation of Lin28a exerts its modulatory effect on VSMCs’ proliferation and migration, which may play a critical role in contributing to pathological formation of restenosis.     

Differences in proliferation rate between CADASIL and control vascular smooth muscle cells are related to increased TGFβ expression

Cerebral autosomal‐dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial fatal progressive degenerative disorder. One of the pathological hallmarks of CADASIL is a dramatic reduction of vascular smooth muscle cells (VSMCs) in cerebral arteries. Using VSMCs from the vasculature of the human umbilical cord, placenta and cerebrum of CADASIL patients, we found that CADASIL VSMCs had a lower proliferation rate compared to control VSMCs. Exposure of control VSMCs and endothelial cells (ECs) to media derived from CADASIL VSMCs lowered the proliferation rate of all cells examined. By quantitative RT‐PCR analysis, we observed increased Transforming growth factor‐β (TGFβ) gene expression in CADASIL VSMCs. Adding TGFβ‐neutralizing antibody restored the proliferation rate of CADASIL VSMCs. We assessed proliferation differences in the presence or absence of TGFβ‐neutralizing antibody in ECs co‐cultured with VSMCs. ECs co‐cultured with CADASIL VSMCs exhibited a lower proliferation rate than those co‐cultured with control VSMCs, and neutralization of TGFβ normalized the proliferation rate of ECs co‐cultured with CADASIL VSMCs. We suggest that increased TGFβ expression in CADASIL VSMCs is involved in the reduced VSMC proliferation in CADASIL and may play a role in situ in altered proliferation of neighbouring cells in the vasculature.     

Effects of serotonin on expression of the LDL receptor family member LR11 and 7-ketocholesterol-induced apoptosis in human vascular smooth muscle cells

Serotonin (5-HT) is a known mitogen for vascular smooth muscle cells (VSMCs). The dedifferentiation and proliferation/apoptosis of VSMCs in the arterial intima represent one of the atherosclerotic changes. LR11, a member of low-density lipoprotein receptor family, may contribute to the proliferation of VSMCs in neointimal hyperplasia. We conducted an in vitro study to investigate whether 5-HT is involved in LR11 expression in human VSMCs and apoptosis of VSMCs induced by 7-ketocholesterol (7KCHO), an oxysterol that destabilizes plaque. 5-HT enhanced the proliferation of VSMCs, and this effect was abolished by sarpogrelate, a selective 5-HT2A receptor antagonist. Sarpogrelate also inhibited the 5-HT-enhanced LR11 mRNA expression in VSMCs. Furthermore, 5-HT suppressed the 7KCHO-induced apoptosis of VSMCs via caspase-3/7-dependent pathway.     

Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation

Although much physiology in resistance vessels has been attributed to the cytoplasmic connection between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), little is known of the protein expression between the two cell types. In an attempt to identify the proteins between ECs and VSMCs, mouse cremaster arterioles were stained with phalloidin-Alexa 594 and viewed on a confocal microscope that resolved "actin bridges" within the internal elastic lamina between ECs and VSMCs. To determine the incidence of protein, the pixel intensity from the antibodies on actin bridges were compared with the pixel intensity from antibodies within ECs or VSMCs. N-cadherin, desmin, connexin (Cx)40, and Cx43 and phosphorylated Cx43 at serine-368 were identified on actin bridges, but NG2, CD31, and Cx45 were not evident. Cx37 expression was more variable than the other connexins examined. Using this method on rat mesentery, we confirm the previously published predominance of Cx37 and Cx40 at the myoendothelial junction that was determined using electron microscopy. We conclude that this new method represents an important screening mechanism in which to rapidly test for protein expression between ECs and VSMCs and possibly a first-step in quantifying protein expression at the myoendothelial junction.     

The effects of myocyte enhancer factor 2A gene on the proliferation, migration and phenotype of vascular smooth muscle cells

The genetic basis for the phenotypic switching of vascular smooth muscle cells (VSMCs) is unclear in atherosclerosis. Recent studies showed that the 21‐base pair deletion mutation (Δ21) in myocyte enhancer factor 2A (MEF2A) gene could be an inherited marker for coronary artery disease. MEF2A mutation may affect the phenotypic switching of VSMCs. Human aortic VSMCs were used. Four groups of VSMCs transfected with green fluorescent protein plasmid (control group), MEF2A wild‐type (WT) plasmid (WT group), MEF2A Δ21 plasmid (Δ21 group) or MEF2A siRNA (siRNA group) were studied. The proliferation of VSMCs was determined by methylthiazolyldiphenyl‐tetrazolium bromide, and the migration of VSMCs was measured by Millicell chamber. The protein expressions of MEF2A, smooth muscle α‐actin, SM22α, osteopontin and p38 mitogen‐activated protein kinase signaling pathway were detected by Western blotting. MEF2A protein expression was knockdown by siRNA transfection. MEF2A protein was overexpressed in WT and Δ21 groups. Δ21 and siRNA groups obviously showed more proliferation (methylthiazolyldiphenyl‐tetrazolium bromide, 0.63 vs 0.66 vs 0.31, P < 0.01) and migration (52.6 vs 58.0 vs 21.2, P < 0.01) of VSMCs as compared with the WT group. In addition, the transfection of Δ21 and siRNA could induce the down‐regulation of smooth muscle α‐actin and SM22α (P < 0.01) and the up‐regulation of osteopontin (P < 0.01) in VSMCs. The phosphorylated p38 signaling pathway expression was significantly enhanced in the Δ21 and siRNA groups as compared with that of the WT group (P < 0.01). These results suggest that MEF2A dominant negative mutation and RNA silence could induce the phenotypic switching of VSMCs, leading to its increased proliferation and migration, and p38 mitogen‐activated protein kinase signaling pathway may participate in it. Copyright © 2011 John Wiley & Sons, Ltd.     

Regulation of alpha7-integrin expression in vascular smooth muscle by injury-induced atherosclerosis

Injury of vascular smooth muscle cells (VSMCs) by allylamine (AAM) leads to phenotypic changes associated with atherogenic progression including increased proliferation, migration, and alterations in cell adhesion. In the present study, the relationship between AAM-induced vascular injury and expression of the alpha(7)-integrin subunit was investigated. The alpha(7)-mRNA and protein expression were examined using real-time RT-PCR, fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and immunoblotting. In cultured VSMCs from aortas of AAM-treated rats (70 mg/kg for 20 days), alpha(7)-mRNA levels were increased more than twofold compared with control cells. No change was seen in beta(1)-integrin expression. FACS analysis revealed increased cell surface expression of alpha(7)-protein (25 +/- 9%; *P < 0.05). AAM treatment of naive VSMCs enhanced alpha(7)-mRNA expression (2.4 +/- 0.7-fold, mean +/- SE; *P < 0.05). The increased alpha(7)-mRNA expression was attenuated by the amine oxidase inhibitor semicarbazide and the antioxidant pyrrolidine dithiocarbamate, which confirms a role for oxidative stress in modulating alpha(7)-expression. In vivo alpha(7)-mRNA and protein expression were enhanced in the aortas of AAM-treated rats. In addition, increased alpha(7)-integrin expression facilitated AAM VSMC adhesion to laminin more efficiently compared with control (51 +/- 2%; *P < 0.05). Chemical injury induced by AAM significantly enhances alpha(7)-integrin expression in VSMCs. These findings implicate for the first time the expression of alpha(7)-integrin during the response of VSMCs to vascular injury.     

A new approach to study fibroblast migration

This paper presents a new approach to study cell migration. Human tendon fibroblasts were plated on silicone membranes coated with 10 microg/ml ProNectin-F. The silicone surfaces were micro-fabricated with parallel microgrooves, with 10 microm ridge and groove width, and 3 microm groove depth. Fibroblasts grown in the microgrooves had an elongated shape and oriented along the microgroove direction. They also moved along the same direction instead of "random walk" when cells migrate on smooth culture surfaces. In response to TGF-beta1 (5 ng/ml) treatment, these fibroblasts on the microgrooved surfaces were differentiated into myofibroblasts, as judged by an elevated expression of alpha-smooth muscle actin (alpha-SMA), a specific marker for myofibroblasts. Moreover, these myofibroblasts were found to be approximately 30% less motile compared to that of untreated fibroblasts. Thus, use of microgrooved surface may be an effective approach to detect difference in cell motility because cell migration on the microgrooved surface is one dimensional and hence easier to be quantified than two-dimensional random movement on conventional smooth culture surfaces.     

Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells

Phorbol ester induces actin cytoskeleton rearrangements in cultured vascular smooth muscle cells. Calponin and SM22 alpha are major components of differentiated smooth muscle and potential regulators of actin cytoskeleton interactions. Here we show that actin fibers decorated with h1 CaP remain stable, whereas SM22 alpha-decorated actin bundles undergo rapid reorganization into podosomes within 30 min of PDBu exposure. Ectopic expression of GFP alpha-actinin had no effect on the stability of the actin cytoskeleton and alpha-actinin was transported rapidly into PDBu-induced podosomes. Our results demonstrate the involvement of CaP and SM22 alpha in coordinating the balance between stabilization and dynamics of the actin cytoskeleton in mammalian smooth muscle. We provide evidence for the existence of two functionally distinct actin filament populations and introduce a molecular mechanism for the stabilization of the actin cytoskeleton by the unique actin-binding interface formed by calponin family-specific CLIK23 repeats.     

Loss of FoxO3a prevents aortic aneurysm formation through maintenance of VSMC homeostasis

Vascular smooth muscle cell (VSMC) phenotypic switching plays a critical role in the formation of abdominal aortic aneurysms (AAAs). FoxO3a is a key suppressor of VSMC homeostasis. We found that in human and animal AAA tissues, FoxO3a was upregulated, SM22α and α-smooth muscle actin (α-SMA) proteins were downregulated and synthetic phenotypic markers were upregulated, indicating that VSMC phenotypic switching occurred in these diseased tissues. In addition, in cultured VSMCs, significant enhancement of FoxO3a expression was found during angiotensin II (Ang II)-induced VSMC phenotypic switching. In vivo, FoxO3a overexpression in C57BL/6J mice treated with Ang II increased the formation of AAAs, whereas FoxO3a knockdown exerted an inhibitory effect on AAA formation in ApoE^−/− mice infused with Ang II. Mechanistically, FoxO3a overexpression significantly inhibited the expression of differentiated smooth muscle cell (SMC) markers, activated autophagy, the essential repressor of VSMC homeostasis, and promoted AAA formation. Our study revealed that FoxO3a promotes VSMC phenotypic switching to accelerate AAA formation through the P62/LC3BII autophagy signaling pathway and that therapeutic approaches that decrease FoxO3a expression may prevent AAA formation.Subject terms: Cell biology, Diseases     

Selective expression of the proprotein convertases furin, pc5, and pc7 in proliferating vascular smooth muscle cells of the rat aorta in vitro.

The aim of this study was to investigate whether transformation of quiescent vascular smooth muscle cells (VSMCs) into proliferating secretory cells is accompanied by an expression of processing enzymes that activate de novo-synthesized growth factors. Three enzymes belonging to the family of the kexin/subtilisin-like mammalian proprotein convertases (PCs), furin, PC5, and PC7, were found to be upregulated after balloon denudation in vivo. To determine their importance in these cell processes, we investigated their gene regulation using a short-term organ culture system. After incubation of rat aorta for 4 and 24 hr in serum-free medium, we demonstrated a significant induction of VSMC proliferation. The affected subset of VSMCs, positive for alpha-smooth muscle actin, also expressed proliferating cell nuclear antigen (PCNA). Our results revealed a parallel upregulation of furin, PC5, and PC7 in PCNA-immunolabeled cells. As a substrate model for comparison with PCs we used nerve growth factor (NGF). NGF is known to be activated by PCs. As shown by Northern blotting analysis, NGF mRNA concentration was significantly increased in cultured explants. NGF was released into the culture medium. In conclusion, both PCs and NGF are coordinately modulated on induction of VSMC proliferation.     

Expression and Preliminary Functional Profiling of the let-7 Family during Porcine Ovary Follicle Atresia

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.     

Effects of cyclic stretch on the molecular regulation of myocardin in rat aortic vascular smooth muscle cells

Background

The expression of myocardin, a cardiac-restricted gene, increases during environmental stress. How mechanical stretch affects the regulation of myocardin in vascular smooth muscle cells (VSMCs) is not fully understood. We identify the mechanisms and pathways through which mechanical stretch induces myocardin expression in VSMCs.

Results

Rat VSMCs grown on a flexible membrane base were stretched to 20% of maximum elongation, at 60 cycles per min. An in vivo model of aorta-caval shunt in adult rats was also used to investigate myocardin expression. Cyclic stretch significantly increased myocardin and angiotensin II (AngII) expression after 18 and 6 h of stretch. Addition of extracellular signal-regulated kinases (ERK) pathway inhibitor (PD98059), ERK small interfering RNA (siRNA), and AngII receptor blocker (ARB; losartan) before stretch inhibited the expression of myocardin protein. Gel shift assay showed that myocardin-DNA binding activity increased after stretch. PD98059, ERK siRNA and ARB abolished the binding activity induced by stretch. Stretch increased while myocardin-mutant plasmid, PD98059, and ARB abolished the promoter activity. Protein synthesis by measuring [³H]proline incorporation into the cells increased after cyclic stretch, which represented hypertrophic change of VSMCs. An in vivo model of aorta-caval shunt also demonstrated increased myocardin protein expression in the aorta. Confocal microscopy showed increased VSMC size 24 h after cyclic stretch and VSMC hypertrophy after creation of aorta-caval shunt for 3 days.

Conclusions

Cyclic stretch enhanced myocardin expression mediated by AngII through the ERK pathway in cultured rat VSMCs. These findings suggest that myocardin plays a role in stretch-induced VSMC hypertrophy.     

Behavior of dermal fibroblasts on microdot arrays yields insight into wound healing mechanisms

The behavior of fibroblasts on patterned substrates was examined in order to elucidate the role of dermal structure in wound healing. Dermal fibroblasts were cultured on micro-patterned silicone elastomer substrates designed to enforce cell adhesion only to fibronectin microdots. The morphology, expression of α-smooth muscle actin (α-SMA), proliferation, apoptotic cells, and soluble collagen production of cells were measured. Cells grown on patterned substrates showed some signs of a scar-fibroblast phenotype such as: elongated pseudopodia, enhanced expression of alpha smooth muscle actin (α-SMA), and increased collagen/pre-collagen, in comparison to unpatterned controls. Cells also showed low proliferation rates and high apoptotic index. The results showed that the microdot arrays, acting as a grid of limited focal adhesion sites, could force cells to adopt constrained morphologies and limited adhesion areas, which affect the cytoskeleton, ultimately leading to expression of a scar-tissue fibroblast phenotype. This study provides insight into the regulatory mechanisms of micro-topology on cell behavior in wound healing.     

Mechanical stretch modulates the promoter activity of the profibrotic factor CCN2 through increased actin polymerization and NF-kappaB activation

The connective tissue growth factor known as CCN2 is an inducible, profibrotic molecule that becomes aberrantly expressed in mechanical overload-bearing tissues. In this study, we found that CCN2 gene expression is rapidly induced in cyclically stretched bladder smooth muscle cells (SMCs) in vitro and in the detrusor muscle of a mechanically overloaded bladder in a rat model of experimental urethral obstruction. The activity of CCN2 promoter constructs, transiently transfected into cultured SMCs, was increased (up to 6-fold) by continuous cyclic stretching. Molecular analyses of the CCN2 promoter by serial construct deletions, cis-element mutagenesis, and electrophoretic mobility shift assays revealed that a highly conserved NF-kappaB binding site located within the CCN2 proximal promoter region is responsible for the activation of the promoter by stretch. Chromatin immunoprecipitation assays showed that NF-kappaB binds to the endogenous CCN2 promoter in both stretched cells and mechanically overloaded bladder tissues. Furthermore, stretch-dependent CCN2 promoter activity was significantly reduced upon inhibition of either phosphatidylinositol 3-kinase, p38 stress-activated kinase, or RhoA GTPase and was completely abolished upon inhibition of actin polymerization. Concordantly, actin polymerization was increased in either mechanically stretched cells or overloaded bladder tissues. Incubation of cultured SMCs with a cell-penetrating peptide containing the N-terminal sequence, Ac-EEED, of smooth muscle alpha-actin, altered both actin cytoskeleton organization and stretch-mediated nuclear relocation of NF-kappaB, and subsequently, it reduced CCN2 promoter activity. Thus, mechanical stretch-induced changes in actin dynamics mediate NF-kappaB activation and induce CCN2 gene expression, which probably initiates the fibrotic reactions observed in mechanical overload-associated pathologies.