Promotes Vascular Smooth Muscle Cells Proliferation Research Articles

Serum and glucocorticoid-regulated kinase 1 promotes vascular smooth muscle cell proliferation via regulation of β-catenin dynamics

In response to arterial intimal injury vascular smooth muscle cells (VSMCs) within the vessel wall proliferate upon exposure to growth factors, accumulate, and form a neointima that can occlude the vessel lumen. Serum and glucocorticoid inducible kinase 1 (SGK1) is a growth factor-responsive kinase; however its role in VSMC proliferation is not fully understood. Here, we examined growth factor-dependent regulation of SGK1 and defined a molecular role for SGK1 in stimulation of VSMC proliferation. We found that stimulation of VSMCs with the pro-proliferative growth factor, platelet-derived growth factor BB (PDGF) significantly increased SGK1 mRNA, protein, and kinase activity in aortic VSMCs in vitro. To test the hypothesis that activation of SGK1 activity promotes VSMC proliferation, we examined the effects of stable expression of constitutively active (S422D) and kinase-defective (S422A) mutants of SGK1 on VSMC growth. We found that activation of SGK1 increased, whereas interference of SGK1 signaling inhibited VSMC growth in vitro. Consistent with these findings, expression of the S422D mutant augmented both basal and PDGF-induced BrdU uptake in VSMCs. Conversely, PDGF-induced BrdU uptake was attenuated in VSMCs expressing S422A. Furthermore, we determined that activated SGK1 enhanced basal and PDGF-dependent G1→S cell cycle transition, whereas dominant-negative SGK1 abrogated G1→S cell cycle transition under similar conditions. Downstream signaling by active SGK1 induced basal and PDGF-induced phosphorylation of glycogen synthase kinase 3β, an effect which was attenuated when SGK1 activity was blocked by expression of the kinase-defective mutant, S422A. We also found that transfection of S422D enhanced β-catenin-nuclear localization and activation of the TOP/Flash and cyclin D1 transcriptional reporters. These effects were significantly blunted in VSMCs transfected with the S422A mutant. Our results provide compelling evidence of a role for SGK1 in stimulation of arterial VSMC growth via regulation of β-catenin dynamics and implicate SGK1 in the progression of intimal narrowing following arterial injury. Hence, the findings presented here point to inhibition of SGK1 activity as a novel therapeutic approach for the treatment of occlusive vascular diseases.

Abstract 12: Basigin Promotes Vascular Smooth Muscle Proliferation and Pulmonary Hypertension

Background: Cyclophilin A (CyPA) is secreted from vascular smooth muscle cells (VSMC) by oxidative stress and promotes VSMC proliferation. However, the role of CyPA and its extracellular receptor Basigin (Bsg) in the pathogenesis of pulmonary hypertension (PH) remains to be elucidated. In this study, we tested our hypothesis that CyPA/Bsg signaling promotes the development of PH. Methods and Results: In the pulmonary arteries of PH patients, immunostaining revealed strong expression of CyPA and Bsg (n=5). In cultured human pulmonary arterial VSMC, hypoxia (O 2 2%) significantly increased CyPA secretion and Bsg expression compared with normoxic condition (O 2 21%) (n=4 per group). To determine the role of CyPA/Bsg signaling in PH development, CyPA +/- and Bsg +/- mice were exposed to hypoxia (O 2 10%) for 4 weeks. The pulmonary arteries (PA) of CyPA +/- and Bsg +/- mice exposed to normoxia did not differ in morphology compared with their littermate controls. In contrast, CyPA +/- (n=12) and Bsg +/- mice (n=15) exposed to hypoxia revealed significantly reduced right ventricular systolic pressure (RVSP), PA remodeling and RV hypertrophy compared with their littermate controls (all P<0.01). Importantly, after transplantation of bone marrow, the severity of PH was still exacerbated in Bsg +/+ recipient mice compared with Bsg +/- recipient mice regardless of the source of bone marrow (Bsg +/+ or Bsg +/- ), suggesting the crucial role of Bsg in PA. To further evaluate the role of Bsg, we harvested VSMC from Bsg +/+ and Bsg +/− mice. VSMC proliferation was significantly reduced in Bsg +/− compared with Bsg +/+ in response to 2% FBS, suggesting the crucial role of Bsg in VSMC proliferation. Mechanistic studies demonstrated that Bsg +/- VSMC revealed reduced expression of oxidative stress genes, less secretion of cytokines/chemokines and growth factors. Finally, in the clinical study, plasma CyPA levels in PH patients were increased in accordance with the severity of pulmonary vascular resistance (P<0.001). Furthermore, event-free curve revealed that high plasma CyPA levels predicted poor outcome in PH patients (death or lung transplantation, P<0.001). Conclusions: These results indicate the crucial role of extracellular CyPA and vascular Bsg in the pathogenesis of PH.

TSH stimulates the proliferation of vascular smooth muscle cells

Subclinical hypothyroidism (SCH) was reported to be associated with atherosclerosis (AS) in recent studies. Thyroid hormone levels are normal in patients with SCH, but the levels of thyroid-stimulating hormone (TSH) are increased. Thyroid-stimulating hormone receptor (TSHR) in extra-thyroidal tissues plays a pathophysiological role in these conditions. Our previous results demonstrated that TSHR was functional in hepatocytes and revealed elevated total cholesterol levels in the serum, which were an independent risk factor for AS. TSHR is expressed in vascular smooth muscle cells (VSMCs), and VSMC proliferation plays an important role in the development of AS. Cell proliferation was measured by the MTT assay. Intracellular cyclic AMP (cAMP) was measured using a cAMP ELISA kit. Cells were analyzed using a flow cytometer to determine the cell-cycle phase of each cell. For the purpose of detecting cyclin A and cyclin D, immunohistochemical staining and western blots were performed. Real-time PCR was used to assess the VSMC phenotypes. TSH increased cell progression into the G2/M phases and induced VSMC proliferation; thus, functional TSHR was present on VSMCs. Furthermore, the expression of cyclin D1 and cyclin A was increased. In addition, the results indicated that VSMCs undergo a phenotypic transformation from a contractile state to a synthetic state after treatment with different concentrations of TSH. Elevated TSH can promote VSMC proliferation through the cAMP-dependent pathway.

Oxidized High-Density Lipoprotein Induces the Proliferation and Migration of Vascular Smooth Muscle Cells by Promoting the Production of ROS

As the major atheroprotective particle in plasma, high-density lipoprotein(HDL) is oxidized during atherosclerotic processes. Oxidized HDL(ox-HDL) may lose its cardioprotective properties and develop a proinflammatory and proatherogenic phenotype. The proliferation and migration of vascular smooth muscle cells(VSMCs) play a crucial role in atherogenesis. However, the influence of ox-HDL on VSMC proliferation and migration remains poorly understood. VSMCs were treated with native HDL(N-HDL) or ox-HDL at varying concentrations for different time intervals and used in several analyses. The degree of cell proliferation was assayed using CCK-8 kits. The level of cell migration was determined using a Transwell chamber and scratch-wound assay. The presence of intracellular reactive oxygen species(ROS) was detected based on ROS-mediated 2',7'-dichlorofluorescein fluorescence. The activation of NADPH oxidase was measured in terms of the Rac1 activity and NADP(＋)/NADPH ratio. Compared to N-HDL, ox-HDL significantly promoted VSMC proliferation and migration in a dose-dependent manner. In addition, ox-HDL remarkably activated NADPH oxidase and enhanced ROS generation in the VSMCs. Diphenyleneiodonium chloride, an inhibitor of NADPH oxidase, and N-acetylcysteine, a ROS scavenger, efficiently inhibited the ROS production triggered by ox-HDL and subsequently blocked the proliferating and migrating effects of ox-HDL in the VSMCs. Ox-HDL significantly induces VSMC proliferation and migration by promoting NADPH oxidase activation and ROS production. Furthermore, the inhibition of NADPH oxidase and ROS generation blocks the proliferation and migration of VSMCs induced by ox-HDL. These proliferating and migrating effects of ox-HDL are closely related to its proinflammatory and proatherogenic roles.

Crosstalk between TGF-β/Smad3 and BMP/BMPR2 signaling pathways via miR-17–92 cluster in carotid artery restenosis

In the recent decades, carotid angioplasty and stenting (CAS) has been developed into a credible option for the patients with carotid stenosis. However, restenosis remains a severe and unsolved issue after CAS treatment. Restenosis is characterized by neointimal hyperplasia, which is partially caused by vascular smooth muscle cells (VSMC) proliferation. However, the molecular mechanism involved in the restenosis is still unclear. In this study, we demonstrated a functional crosstalk between two TGF-β superfamily signaling pathway members, Smad3 and BMPR2, in VSMC proliferation. Smad3 plays an important role in the TGF-β/Smad3 signaling pathway, and is significantly upregulated in the carotid artery with restenosis to promote VSMC proliferation. In contrast, BMP receptor II (BMPR2), an inhibitor of VSMC proliferation is downregulated in carotid restenosis. We further found that BMPR2 downregulation is mediated by miR-17-92 cluster, which is transcriptionally regulated by Smad3. Thus, Smad3 upregulation and Smad3/miR-17-92 cluster-dependent BMPR2 downregulation are likely to promote VSMC proliferation and restenosis. Taken together, our results may provide novel clues for early diagnosis of carotid restenosis and developing new therapeutic strategy.

Reactive Oxygen Species Promote Vascular Smooth Muscle Cell Proliferation

Do reactive oxygen species play a role in the pathogenesis of cardiovascular diseases? In 1992, Dr Berk and his group published an article that began to answer this question. During the following years, they performed a series of research projects that established the role of reactive oxygen species in vascular smooth muscle cell proliferation and cardiovascular diseases. It is now widely recognized that production of intracellular reactive oxygen species (ROS) is substantially involved in the pathogenesis of cardiovascular diseases, in part by promoting vascular smooth muscle cell (VSMC) proliferation. However, 20 years ago, it was elusive why VSMCs proliferate in response to arterial injury. Thus, Dr Berk et al hypothesized that ROS generated during arterial injury could be a common mechanism involved. Using xanthine/xanthine oxidase to generate ROS, they were able to demonstrate that ROS stimulate VSMC proliferation in vitro and that H2O2 was primarily responsible for xanthine/xanthine oxidase–induced VSMC DNA synthesis.1 This Circulation Research article was the first milestone article that directly demonstrated the role of ROS in VSMC proliferation, triggering the studies that followed targeting the role of ROS in the pathogenesis of cardiovascular diseases. Until now, this article has been referenced by more than 450 articles. Three years later, they further reported that both O2− and H2O2 stimulate VSMC growth but only O2− rapidly activates MAP kinase, suggesting that additional signal events are required for the mitogenic effects of H2O2.2 Based on these reports, it has been elucidated that the excess amount of ROS (oxidative stress) promotes VSMC proliferation and potentially develops cardiovascular diseases. In the subsequent 5 years, they focused on the interesting finding that ROS stimulates ERK1/2 in a biphasic manner in VSMCs. Finally, they demonstrated that one explanation …

B cell translocation gene, a direct target of miR-142-5p, inhibits vascular smooth muscle cell proliferation by down-regulating cell cycle progression

Vascular smooth muscle cell (VSMC) proliferation plays a key role in neointimal hyperplasia and restenosis. Here we report the role of the microRNA miR-142-5p and its downstream target genes on the proliferation of cultured VSMCs. miR-142-5p promoted VSMC proliferation by down-regulating B cell translocation gene 3 (BTG3). We found that BTG3 inhibited the expression of cell cycle regulatory genes and cell growth. As shown by luciferase reporter assay, miR-142-5p bound directly to the 3′-untranslated region of BTG3. Overexpression of miR-142-5p induced expression of cell cycle regulatory genes. Thus, BTG3, a novel, direct target of miR-142-5p, negatively regulates VSMC proliferation.

Abstract 537: Platelet Derived Growth Factor Induced Vascular Smooth Muscle Cell Proliferation is Associated with Enhanced Glut1 Expression/glycosylation and Glucose Uptake

Objective The role of glucose transporters (GLUTs) in the phenotypic regulation of vascular smooth muscle cells (VSMCs) has not been fully characterized. The present study has examined the effects of PDGF ( vs insulin) on GLUT isoform expression/localization, GLUT1 glycosylation, glucose uptake and ATP formation during VSMC proliferation. Methods Human aortic VSMCs were exposed to PDGF (30 ng/ml) or insulin (1 μM) for different time intervals. GLUT isoform expression was determined by qPCR and immunoblot analysis. GLUT isoform distribution/localization was determined by subcellular fractionation studies and immunocytochemical analysis. Glucose utilization and ATP formation were determined by glucose oxidation assay and luminescent ATP detection assay, respectively. The role of GLUT1 in VSMCs was determined by GLUT1 siRNA knockdown studies. Results Exposure of VSMCs to PDGF resulted in significant increases in glucose uptake and cell proliferation, compared with insulin. In addition, PDGF and insulin enhanced the mRNA expression levels of total GLUT isoforms (GLUT1 through 14) to ~4317/10 5 B2M transcripts and ~8,174 transcripts/10 5 B2M transcripts, respectively, compared with control (~3259 transcripts/10 5 B2M transcripts). In particular, PDGF and insulin enhanced GLUT1 mRNA to 1711 ± 38/10 5 B2M transcripts and 3387 ± 58 transcripts/10 5 B2M transcripts, respectively. Immunoblot analysis using whole cell homogenates showed a significant increase in GLUT1 protein expression in PDGF or insulin-treated VSMCs. Importantly, subcellular localization studies using PDGF-treated VSMCs revealed enhanced GLUT1 protein expression and its glycosylation in the plasma membrane. Furthermore, PDGF enhanced glucose metabolism as revealed by increased glucose utilization and ATP formation. Down regulation of GLUT1 using target-specific siRNA significantly attenuated PDGF-induced glucose uptake. Conclusions Enhanced expression and glycosylation of plasma membrane-localized GLUT1 isoform plays a major role in promoting VSMC proliferation, which may contribute to accelerated atherosclerosis.

Rho-associated protein kinase isoforms stimulate proliferation of vascular smooth muscle cells through ERK and induction of cyclin D1 and PCNA

Abnormal proliferation and migration of vascular smooth muscle cells (VSMC) plays an important role in vascular diseases. The Rho-associated protein kinase (ROCK) signaling pathway is now well recognized for its role in VSMC migration and proliferation. Recently, a number of studies revealed that different isoforms of ROCK have distinct functions in VSMCs. We have reported that ROCK1, rather than ROCK2, induces platelet-derived growth factor (PDGF)-BB-stimulated migration of VSMCs. In the current study, we aimed to investigate the roles of ROCK1/2 in PDGF-induced rat aorta VSMC proliferation by manipulating ROCK gene expression. The results revealed that knock-down of both ROCK1 and ROCK2 by siRNA technology decreased PDGF-BB-generated VSMC proliferation by inhibiting the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1. In addition, up-regulation of ROCK1 expression through transfection, further increased the proliferation of VSMCs induced by PDGF-BB. The ERK inhibitor U0126 reduced the proliferation and expression of PCNA and cyclinD1, and ROCK1 and ROCK2 siRNA decreased the level of ERK in the nucleus. These results demonstrated that ROCK1 and ROCK2 could promote VSMC proliferation through ERK nuclear translocation, regulating the expression of PCNA and cyclin D1 protein.

Involvement of IL-1 and Oncostatin M in Acanthosis Associated With Hypertensive Leg Ulcer

Hypertensive leg ulcer (HLU) is an inflammatory disease characterized by intense pain, alteration of vascularization, and skin necrosis. The optimal treatment relies on surgical removal of necrotic tissues covered by a split-skin graft. We studied the histomorphology of the lesions and investigated the involvement of inflammatory cells and cytokines to further define the physiopathology of HLU. We report epidermis acanthosis and a preferential occlusion of the precapillary arterioles with infiltration of neutrophils, macrophages, and T lymphocytes in the dermis. OSM, IL-1β, and IL-6 were overexpressed in the ulcer, whereas the Th17-derived cytokines were not. In vitro, the addition of IL-1β and OSM promoted acanthosis and destructuring of reconstructed epidermis. Exogenous IL-1β and OSM synergistically induced epidermal acanthosis in mice. These data show that OSM and IL-1β are not only a biological characteristic signature of HLU, but these cytokines reflect a specific inflammatory state, directly involved in the pathogenesis. We suggest that anti-cytokine biotherapies could be an alternative strategy to surgery to treat HLU.

Endothelial cells on the proliferation and expression of intercellular adhesion molecule 1 and interleukin 8 of vascular smooth muscle cells

The aim of this study was to investigate the influence of activated endothelial cells on the proliferation and secretion of vascular smooth muscle cells (VSMCs). Cultured lung microvascular endothelial cells were treated with or without tumor necrosis factor alpha (TNF-α; 10 ng/mL) for 6 h, and the supernatant was collected and filtered. The supernatant with TNF-α was called fluid A, and that without TNF-α was called fluid B. VSMCs were cultured and divided into 3 groups with different media as follows: activated medium [fluid A and Dulbecco's modified Eagle medium (DMEM); activated group], inactivated medium (fluid B and DMEM; inactivated group), and DMEM only (control group) for 24 h. Intercellular adhesion molecule 1 (ICAM-1), interleukin (IL)-8, and IL-6 levels in the supernatant of VSMCs were measured with enzyme-linked immunosorbent assay 0 and 24 h after grouping. The proliferation of VSMCs was detected with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ICAM-1 and IL-8 increased above baseline values in the 3 groups; the maximum increase occurred in activated medium. The optical densities in MTT assay of the activated, inactivated, and control groups was 1.35 ± 0.11, 1.01 ± 0.09, and 0.29 ± 0.01, respectively, which correlated positively with the initial IL-6 level in the supernatant of the VSMCs (r = 0.63, P < 0.05). TNF-α-activated endothelial cells promote VSMC proliferation and secretion of ICAM-1 and IL-8 by elevating IL-6 release.

EFFECTS OF SUPPRESSOR OF CYTOKINE SIGNALLING-1 ON PROLIFERATION INDUCED BY OXIDATIVE LOW-DENSITY LIPOPROTEIN IN VASCULAR SMOOTH MUSCLE CELLS

ObjectivesExcessive proliferation vascular smooth muscle cells(VSMC) can promote the development of atherosclerosis.And, in the development of atherosclerosis, oxidative low-density lipoprotein (oxLDL) enhance excessive proliferation of VSMC.As studies have showed, down-regulation...

EFFECTS OF XERODERMA PIGMENTOSUM B GENE ON PROLIFERATION AND APOPTOSIS CONDUCTED BY INTERLEUKIN-6 IN VASCULAR SMOOTH MUSCLE CELLS

ObjectivesExcessive proliferation vascular smooth muscle cells (VSMC) can promote the development of atherosclerosis. And, in the development of atherosclerosis, interleukin-6 (IL-6) enhance excessive proliferation of VSMC. As studies have showed,...

EFFECTS OF XERODERMA PIGMENTOSUM D GENE ON PROLIFERATION INDUCED BY INTERLEUKIN-6 IN VASCULAR SMOOTH MUSCLE CELLS

ObjectivesExcessive proliferation vascular smooth muscle cells (VSMC) can promote the development of atherosclerosis. And, in the development of atherosclerosis, interleukin-6 (IL-6) enhance excessive proliferation of VSMC.As studies have showed, up-regulation...

DiGeorge Syndrome Critical Region 8 (DGCR8) Protein-mediated microRNA Biogenesis Is Essential for Vascular Smooth Muscle Cell Development in Mice

DiGeorge Critical Region 8 (DGCR8) is a double-stranded RNA-binding protein that interacts with Drosha and facilitates microRNA (miRNA) maturation. However, the role of DGCR8 in vascular smooth muscle cells (VSMCs) is not well understood. To investigate whether DGCR8 contributes to miRNA maturation in VSMCs, we generated DGCR8 conditional knockout (cKO) mice by crossing VSMC-specific Cre mice (SM22-Cre) with DGCR8(loxp/loxp) mice. We found that loss of DGCR8 in VSMCs resulted in extensive liver hemorrhage and embryonic mortality between embryonic days (E) 12.5 and E13.5. DGCR8 cKO embryos displayed dilated blood vessels and disarrayed vascular architecture. Blood vessels were absent in the yolk sac of DGCR8 KOs after E12.5. Disruption of DGCR8 in VSMCs reduced VSMC proliferation and promoted apoptosis in vitro and in vivo. In DGCR8 cKO embryos and knockout VSMCs, differentiation marker genes, including αSMA, SM22, and CNN1, were significantly down-regulated, and the survival pathways of ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated. Knockout of DGCR8 in VSMCs has led to down-regulation of the miR-17/92 and miR-143/145 clusters. We further demonstrated that the miR-17/92 cluster promotes VSMC proliferation and enhances VSMC marker gene expression, which may contribute to the defects of DGCR8 cKO mutants. Our results indicate that the DGCR8 gene is required for vascular development through the regulation of VSMC proliferation, apoptosis, and differentiation.

Abstract 32: EBP50 and NF-κB: A Feed-Forward Regulation of Vascular Inflammation

Inflammation plays a fundamental role in the development of cardiovascular diseases such as atherosclerosis and restenosis. The vascular response to inflammation in these diseases requires communication between multiple cell types such as vascular smooth muscle cells (VSMC) and macrophages. We have identified a scaffolding protein, Ezrin Binding Protein 50 (EBP50, also known as NHERF-1), which is expressed at very low levels in normal vessels but is up-regulated following vascular injury. EBP50 promotes VSMC proliferation and neointima formation after wire injury in mouse femoral arteries. In the current study, we hypothesized that EBP50 functions as a central mediator linking macrophage activation and the response of vessels to inflammation. Similar to the observations in vessels, treatment of primary VSMC and macrophages with LPS or TNFα increased EBP50 expression. This increase was dependent on NF-κB signaling because pharmacological inhibition of NF-κB and expression of a dominant negative IκBα abolished the induction of EBP50. Interestingly, LPS-induced activation of NF-κB (determined by phosphorylation of IKKα/β, degradation of IκBα, and p65 phosphorylation) was impaired in EBP50 null VSMC and macrophages. Moreover, nuclear translocation of the p65 subunit of NF-κB was also reduced in EBP50 -/- VSMC. Consistent with the role of EBP50 on NF-κB, LPS- and TNFα-induced macrophage activation (determined by the expression of the inflammatory markers iNOS, TNFα, and IL-1β) was reduced in EBP50 -/- macrophages and mice. Similarly, the expression of iNOS, VCAM-1, and ICAM-1 in VSMC was decreased in EBP50 -/- VSMC and mice. Collectively, these observations indicate that EBP50 and NF-κB participate in a positive feedback loop leading to increased macrophage activation and enhanced response of VSMC to inflammation, suggesting its involvement in vascular remodeling.

MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation

Mitogen activated protein kinase (MAPK) phosphorylation of connexin 43 (Cx43) is associated with proliferation of vascular smooth muscle cells (VSMC) in atherosclerosis. We aimed to investigate whether MAPK phosphorylation of Cx43 directly regulates VSMC proliferation. Using in vivo models of VSMC proliferation e.g. carotid treatments with platelet‐derived growth factor–BB (PDGF), we identified that MAPK phosphorylated Cx43 interacts with the cell cycle control proteins cyclin E and CDK2. To confirm this in vitro we isolated primary Cx43−/− VSMC and transfected these with constructs for Cx43 containing null phosphorylation (alanine) or phospho‐mimetic (aspartate) mutations of the MAPK serines. Co‐immunoprecipitation and proliferation studies in transfected cells combined with analytical size exclusion studies of purified Cx43 C‐terminus and cyclin E proteins demonstrated that MAPK phosphorylation of Cx43 is critical for its binding with cyclin E and for VSMC proliferation in vitro. Finally, using a novel knock‐in mouse containing Cx43 MAPK alanine mutations (Cx43‐MK4A), we showed that the null‐phosphorylation mutation disrupts interactions between Cx43 and cyclin E and VSMC proliferation in vivo. We conclude that MAPK phosphorylated Cx43 is a novel interacting partner of cyclin E and is required to promote VSMC proliferation.

Nucleotide-Binding Oligomerization Domain Protein 2 Deficiency Enhances Neointimal Formation in Response to Vascular Injury

Nucleotide-binding oligomerization domain protein 2 (NOD2) stimulates diverse inflammatory responses resulting in differential cellular phenotypes. To identify the role of NOD2 in vascular arterial obstructive diseases, we investigated the expression and pathophysiological role of NOD2 in a vascular injury model of neointimal hyperplasia. We first analyzed for neointimal hyperplasia following femoral artery injury in NOD2(+/+) and NOD2(-/-) mice. NOD2(-/-) mice showed a 2.86-fold increase in neointimal formation that was mainly composed of smooth muscle (SM) α-actin positive cells. NOD2 was expressed in vascular smooth muscle cells (VSMCs) and NOD2(-/-) VSMCs showed increased cell proliferation in response to mitogenic stimuli, platelet-derived growth factor-BB (PDGF-BB), or fetal bovine serum, compared with NOD2(+/+) VSMCs. Furthermore, NOD2 deficiency markedly promoted VSMCs migration in response to PDGF-BB, and this increased cell migration was attenuated by a phosphatidylinositol 3-kinase inhibitor. However, protein kinase C and c-Jun N-terminal kinase inhibitors exerted negligible effects. Moreover, muramyl dipeptide-stimulated NOD2 prevented PDGF-BB-induced VSMCs migration. Functional NOD2 was found to be expressed in VSMCs, and NOD2 deficiency promoted VSMCs proliferation, migration, and neointimal formation after vascular injury. These results provide evidence for the involvement of NOD2 in vascular homeostasis and tissue injury, serving as a potential molecular target in the modulation of arteriosclerotic vascular disease.

Vitisin B, a resveratrol tetramer, inhibits migration through inhibition of PDGF signaling and enhancement of cell adhesiveness in cultured vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) play an important role in normal vessel formation and in the development and progression of cardiovascular diseases. Grape plants contain resveratrol monomer and oligomers and drinking of wine made from grape has been linked to “French Paradox”. In this study we evaluated the effect of vitisin B, a resveratrol tetramer, on VSMC behaviors. Vitisin B inhibited basal and PDGF-induced VSMC migration. Strikingly, it did not inhibit VSMC proliferation but inversely enhanced cell cycle progression and proliferation. Among the tested resveratrol oligomers, vitisin B showed an excellent inhibitory activity and selectivity on PDGF signaling. The anti-migratory effect by vitisin B was due to direct inhibition on PDGF signaling but was independent of interference with PDGF binding to VSMCs. Moreover, the enhanced VSMC adhesiveness to matrix contributed to the anti-migratory effect by vitisin B. Fluorescence microscopy revealed an enhanced reorganization of actin cytoskeleton and redistribution of activated focal adhesion proteins from cytosol to the peripheral edge of the cell membrane. This was confirmed by the observation that enhanced adhesiveness was repressed by the Src inhibitor. Finally, among the effects elicited by vitisin B, only the inhibitory effect toward basal migration was partially through estrogen receptor activation. We have demonstrated here that a resveratrol tetramer exhibited dual but opposite actions on VSMCs, one is to inhibit VSMC migration and the other is to promote VSMC proliferation. The anti-migratory effect was through a potent inhibition on PDGF signaling and novel enhancement on cell adhesion.

MiR-125b Regulates Calcification of Vascular Smooth Muscle Cells

Vascular calcification is a prominent feature of atherosclerosis and is closely linked to osteoporosis. Cellular differentiation is regulated by various microRNAs (miRs), including miR-125b, which is known to be involved in osteoblast differentiation. However, no specific miR has been defined that modulates vascular calcification. Herein, we assessed the impact of miR-125b in osteogenic transformation of vascular smooth muscle cells. Osteogenic transdifferentiation of human coronary artery smooth muscle cells was induced by osteogenic medium and enhanced the formation of mineralized matrix, resulting in a significantly higher mineral deposition after 21 days. Increased expression of miR-125b was time-dependent in human coronary artery smooth muscle cells and diminished during osteogenic transdifferentiation. At day 21, miR-125b was significantly reduced (-42%) compared with that in the untreated control. The expression of miR-processing enzymes, RNase III endonucleases DICER1 and DROSHA, was also decreased. Furthermore, inhibition of endogenous miR-125b promoted osteogenic transdifferentiation, as measured by increased alkaline phosphatase activity and matrix mineralization. Expression analysis revealed the osteoblast transcription factor SP7 (osterix) as a target of miR-125b. In vivo, miR-125b was decreased in calcified aortas of apolipoprotein E knockout mice. In conclusion, our results suggest that miR-125b is involved in vascular calcification in vitro and in vivo, at least partially by targeting SP7. Evaluating the role of miRs in arterial calcification in vivo may have important therapeutic implications.