CCN4 regulates vascular smooth muscle cell migration and proliferation

The migration and proliferation of vascular smooth muscle cells (VSMCs) are essential elements during the development of atherosclerosis and restenosis. An increasing number of studies have reported that extracellular matrix (ECM) proteins, including the CCN protein family, play a significant role in VSMC migration and proliferation. CCN4 is a member of the CCN protein family, which controls cell development and survival in multiple systems of the body. Here, we sought to determine whether CCN4 is involved in VSMC migration and proliferation. We examined the effect of CCN4 using rat cultured VSMCs. In cultured VSMCs, CCN4 stimulated the adhesion and migration of VSMCs in a dose-dependent manner, and this effect was blocked by an antibody for integrin α5β1. CCN4 expression was enhanced by the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). Furthermore, knockdown of CCN4 by siRNA significantly inhibited the VSMC proliferation. CCN4 also could up-regulate the expression level of marker proteins of the VSMCs phenotype. Taken together, these results suggest that CCN4 is involved in the migration and proliferation of VSMCs. Inhibition of CCN4 may provide a promising strategy for the prevention of restenosis after vascular interventions.     

Selective expression of an endogenous inhibitor of FAK regulates proliferation and migration of vascular smooth muscle cells

Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538-540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [(3)H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals.     

Syndecan-4 is required for thrombin-induced migration and proliferation in human vascular smooth muscle cells

Thrombin is a mitogen and chemoattractant for vascular smooth muscle cells (SMCs) and may contribute to vascular lesion formation. We have previously shown that human SMCs, when stimulated with thrombin, release basic fibroblast growth factor (bFGF), causing phosphorylation of FGF receptor-1 (FGFR-1). Treatment with bFGF-neutralizing antibodies (anti-bFGF) or heparin inhibits thrombin-induced DNA synthesis. We concluded that thrombin may stimulate entry into the cell cycle via bFGF release and FGFR-1 activation. In the present study, we demonstrate a requirement for not only FGFR-1 but also syndecan-4, a transmembrane heparan-sulfate proteoglycan. Inhibition of syndecan-4 expression using small interfering RNA (siRNA) resulted in reduced DNA synthesis by human SMCs after stimulation with thrombin (10 nmol/liter). Anti-bFGF antibody, which inhibits DNA synthesis in control cells, had no inhibitory effect when syndecan-4 expression was reduced by siRNA. Thrombin- or bFGF-induced SMC migration, determined in Boyden chamber assays, was reduced in cells treated with syndecan-4 or FGFR-1 siRNA or by anti-bFGF. Thrombin induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in a biphasic pattern. Although thrombin-mediated ERK phosphorylation at 5 min was not affected by syndecan-4 or FGFR-1 siRNA, ERK phosphorylation at later time points was reduced. We conclude that thrombin-released bFGF binds to syndecan-4 and FGFR-1, which is required for thrombin-induced mitogenesis and migration.     

PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells

Human vascular smooth muscle cell proliferation and migration contribute to vascular remodeling in pulmonary hypertension and atherosclerosis. The precise mechanisms that regulate structural remodeling of the vessel wall remain unknown. This study tests the hypothesis that phosphatidylinositol 3-kinase (PI3K) activation is both necessary and sufficient to mediate human pulmonary vascular smooth muscle (PVSM) cell proliferation and migration. Microinjection of human PVSM cells with a dominant-negative class IA PI3K inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis by 65% (P < 0.001; chi(2) analysis) compared with cells microinjected with control plasmid, whereas microinjection of cells with a constitutively active class IA PI3K (p110*-CA) was sufficient to induce DNA synthesis (mitotic index of p110*-CA-microinjected cells was 15% vs. 3% in control cells; P < 0.01). Transfection of PVSM cells with p110*-CA was also sufficient to promote human PVSM cell migration. In parallel experiments, stimulation of human PVSM cells with PDGF induced PI3K-dependent activation of Akt, p70 S6 kinase, and ribosomal protein S6 but not mitogen-activated protein kinase. PDGF-induced proliferation and migration was inhibited by LY-294002. These results demonstrate that PI3K signaling is both necessary and sufficient to mediate human PVSM cell proliferation and migration and suggest that the activation of PI3K may play an important role in vascular remodeling.     

Clusterin regulates vascular smooth muscle cell nodule formation and migration

Vascular smooth muscle cells (VSMC) are the principal cellular component of the blood vessel wall where they exist in a differentiated state to maintain vascular tone. However, VSMC are not terminally differentiated and can be induced to dediffentiate, proliferate, and migrate. In fact, smooth muscle cell migration from the vascular wall into the lumen of the vessel is a central feature of occlusive vascular pathologies including atherosclerosis and intimal hyperplasia. In vitro, in the presence of an extracellular matrix, cultured vascular smooth muscle cells can migrate and invade the underlying gelatinous matrix, form multicellular nodular aggregations, and secrete the glycoprotein clusterin. Nodular cultures appear to mimic some of the properties of differentiated VSMC, in vivo. Here, to test the hypothesis that clusterin functions to modulate the formation of VSMC nodules and to facilitate cell migration a clusterin negative VSMC clone, SM-CLU13AS (Moulson and Millis, 1999, J Cell Physiol 180:355), was transiently transfected with plasmid pRcCMVCLU that contains the full-length porcine clusterin cDNA sequence under control of the CMV promoter. The transiently transfected VSMC culture expressed and secreted clusterin and formed nodules. To determine if clusterin regulates VSMC migration we used modified Boyden chamber assays. Clusterin, at 10 microg/ml, clearly promotes VSMC migration. In addition, a 15 amino acid synthetic peptide, representing amino acids 118-132 [KQTCMKFYARVCRSG] of the mature clusterin polypeptide, inhibits VSMC attachment to gelatinous substrate. Finally, clusterin appears to have a role in regulating endogenous clusterin expression in the clusterin negative clone. These results clearly establish that clusterin has functional role in VSMC nodule formation and support the conclusion that clusterin is a critical component of smooth muscle cell phenotypic modulation.     

Monocyte-expressed urokinase regulates human vascular smooth muscle cell migration in a coculture model

Interactions of vascular smooth muscle cells (VSMC) with monocytes recruited to the arterial wall at a site of injury, with resultant modulation of VSMC growth and migration, are central to the development of vascular intimal thickening. Urokinase-type plasminogen activator (uPA) expressed by monocytes is a potent chemotactic factor for VSMC and might serve for the acceleration of vascular remodeling. In this report, we demonstrate that coculture of human VSMC with freshly isolated peripheral blood-derived human monocytes results in significant VSMC migration that increases during the coculture period. Accordingly, VSMC adhesion was inhibited with similar kinetics. VSMC proliferation, however, was not affected and remained at the same basal level during the whole period of coculture. The increase of VSMC migration in coculture was equivalent to the uPA-induced migration of monocultured VSMC and was blocked by addition into coculture of soluble uPAR (suPAR). Analysis of uPA and uPAR expression in cocultured cells demonstrated that monocytes are a major source of uPA, whose expression increases in coculture five-fold, whereas VSMC display an increased expression of cell surface-associated uPAR. These findings indicate that upregulated uPA production by monocytes following vascular injury acts most likely as an endogenous activator of VSMC migration contributing to the remodeling of vessel walls.     

Oxygen regulates mitogen-stimulated proliferation of fetal human airway smooth muscle cells

Increased airway smooth muscle (ASM) content is characteristic of infants with chronic lung disease of prematurity/bronchopulmonary dysplasia. Oxygen therapy, reactive oxygen species (ROS), and immature antioxidant defenses are major risk factors in chronic lung disease of prematurity/bronchopulmonary dysplasia, but their interrelationship is unclear. The direct effects of raised Po2 and modulation of ROS were examined on proliferation of cultured fetal human ASM cells. A bell-shaped relationship was found between Po2 and DNA synthesis induced by fetal bovine serum, platelet-derived growth factor, and basic fibroblastic growth factor, with peak responses occurring at 10-kPa Po2. Changes in DNA synthesis by Po2 did not occur in the absence of mitogen. ROS generation, estimated by dichlorodihydrofluorescein oxidation, was increased by mitogens but was unaffected by nonmitogens (bradykinin, histamine). There was an inverse relationship between ROS generation and Po2, and mitogen-induced ROS generation was substantially potentiated as the Po2 fell. H2O2 mimicked the effect of Po2 on fetal bovine serum-stimulated proliferation, whereas treatment with antioxidants (GSH, N-acetylcysteine) reduced it. These data demonstrate that increases in Po2 above levels found in utero modulate proliferation of fetal ASM cells but only in the presence of growth factors. They also strongly suggest that, under these conditions, proliferation is mediated in part by generation of ROS.     

CREG inhibits migration of human vascular smooth muscle cells by mediating IGF-II endocytosis

We previously determined that the cellular repressor of E1A-stimulated genes, (CREG) plays a role in the maintenance of the mature phenotype of vascular smooth muscle cells (SMCs). This study aimed to identify the role of CREG in modulating the migration of SMCs. Recombinant virus-mediated CREG expression inhibited the cellular migration of cultured SMCs associated with down-regulated activity of matrix metalloproteinase-9 (MMP-9). In contrast, CREG knockdown via the retroviral transfer of short hairpin RNAs promoted cellular migration. Enzyme-linked immunosorbent assay and endocytosis analysis revealed that CREG knockdown attenuated the internalization and increased secretion of insulin-like growth factor (IGF)-II. Western blot analysis demonstrated that both phosphoinositide 3-kinase (PI3K) and phosphatase Akt were enhanced in CREG knockdown SMCs. Furthermore, the effect of CREG knockdown on SMC migration was abrogated in a dose-dependent manner by the addition of either IGF-II neutralizing antibody or the PI3K inhibitor, LY294002. These results indicate that the CREG knockdown-mediated increase in IGF-II secretion promoted cellular migration in SMCs via the PI3K/Akt signal pathway. Additionally, blockage of IGF-II binding to the mannose-6-phosphate/IGF-II receptor (M6P/IGF2R) by IGF2R antibody or recombinant IGF2R fragment attenuated the endocytosis of IGF-II in cells overexpressing CREG. This indicates that M6P/IGF2R is involved in the regulation of CREG-mediated IGF-II endocytosis. In summary, these data demonstrate for the first time that CREG plays a critical role in the inhibition of SMC migration, as well as maintaining SMCs in a mature phenotype. These results may provide a new therapeutic target for vascular disease associated with neointimal hyperplasia.     

Protocatechuic aldehyde inhibits migration and proliferation of vascular smooth muscle cells and intravascular thrombosis

TAR DNA-binding protein of 43 kDa (TDP-43) is the major component of the intracellular inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we show that both monoclonal (60019-2-Ig) and polyclonal (10782-2-AP) anti-TDP-43 antibodies recognize amino acids 203-209 of human TDP-43. The monoclonal antibody labeled human TDP-43 by recognizing Glu204, Asp205 and Arg208, but failed to react with mouse TDP-43. The antibodies stained the abnormally phosphorylated C-terminal fragments of 24-26 kDa in addition to normal TDP-43 in ALS and FTLD brains. Immunoblot analysis after protease treatment demonstrated that the epitope of the antibodies (residues 203-209) constitutes part of the protease-resistant domain of TDP-43 aggregates which determine a common characteristic of the pathological TDP-43 in both ALS and FTLD-TDP. The antibodies and methods used in this study will be useful for the characterization of abnormal TDP-43 in human materials, as well as in vitro and animal models for TDP-43 proteinopathies.     

Heparin interactions with cultured human vascular endothelial and smooth muscle cells: incidence on vascular smooth muscle cell proliferation

The binding, internalization, and metabolism of [3H]-heparin by human umbilical vein endothelial cells (HUVEC) and human umbilical arterial smooth muscle cells (HUASMC) have been characterized using size-exclusion HPLC. Incubation of HUVEC with [3H]-heparin demonstrated selective binding of high-molecular-weight (MW) components (MW = 21 kd), which was followed by rapid, temperature-dependent internalization. Over the next 3 hours, this internalized [3H]-heparin was degraded to low-MW fragments (MW = 0.9 kd). Primary cultures of HUASMC selectively bound extremely high-MW components (MW = 40 kd) and also smaller components whose MW (0.9 kd) corresponded to that of the heparin metabolite(s) formed by HUVEC. Subcultured HUASMC bound only the 40-kd components. Internalization of heparin by smooth muscle cells (SMC) was significantly slower than that determined for HUVEC, and even after 4 hours there was no evidence of the heparin being metabolized. However, when incubating primary rabbit aortic SMC with purified low-MW heparin fragment(s) produced in culture by HUVEC, a significantly lower proliferative response of these cells (IC50 = 18.4 micrograms/ml) was obtained. Virtually no effect was observed with subcultured SMC in the range of the tested concentrations (0-20 micrograms/ml). These fragments were 10- to 15-fold more effective in inhibiting primary SMC growth than was standard heparin. Furthermore, heparin fractions in the same range of molecular weights, purified either after nitrous acid or heparinase depolymerization of standard heparin, showed no activity on primary SMC growth, thus indicating a high degree of selectivity of the heparin metabolite(s) produced by HUVEC in culture.     

LncRNA LEF1-AS1 regulates the migration and proliferation of vascular smooth muscle cells by targeting miR-544a/PTEN axis

Long noncoding RNAs (lncRNAs) play important roles in endothelium development. A lncRNA, LEF1-AS1, is recently emerging as a potent mediator of the proliferation and migration of a number of cells, including smooth muscle cells. However, the effects of LEF1-AS1 in atherosclerosis remains largely unknown. Specimens from patients with coronary artery atherosclerosis were collected. The quantitative real-time polymerase chain reaction was used to analyze levels of LEF1-AS1 and microRNA-544a (miR-544a). Western blot analysis was used to assess PTEN, P-Akt, and T-Akt protein expression. Proliferation, migration, and invasion of cells were analyzed by cell counting kit-8 assay, scratch wound assay, and transwell assay, respectively. The interaction between LEF1-AS1, miR-544a, and PTEN was probed using bioinformatical analysis and dual-luciferase assay. In plasma and tissue of patients with coronary artery atherosclerosis, LEF1-AS1 was upregulated and miR-544a was downregulated. A negative correlation was found between LEF1-AS1 and miR-544a. miR-544a overexpression reversed the inhibition of LEF1-AS1 in smooth muscle cell proliferation and invasion, which were mediated through the PTEN pathway. LEF1-AS1 regulates smooth muscle cell proliferation and migration through the miR-544a/PTEN axis, indicating that LEF1-AS1 may be a potential therapeutic target in atherosclerosis.     

Nucleolin regulates the proliferation of vascular smooth muscle cells in atherosclerotic via Aurora B

Vascular smooth muscle cells (VSMCs) play a significant role in atherosclerosis. As a multifunctional protein, nucleolin (NCL) is involved in many important physiological and pathological processes. In this study, we aimed to investigate the role of nucleolin in VSMCs proliferation and cell cycle. The expression of nucleolin increased in VSMCs of mice with aortas advanced plaques. With the left common carotid-artery ligation-injury model, immunofluorescence staining revealed that nucleolin and Ki67 expression increased in VSMCs in mice left carotid artery compared with right carotid artery after surgery. POVPC or ox-LDL up-regulated nucleolin mRNA and protein expression in a dose- and time-dependent manner in HAVSMCs. POVPC (5μg/ml) or ox-LDL (50μg/ml) promoted the proliferation of HAVSMCs. Nucleolin ablation relieved the pro-proliferation role of VSMCs. The cell cycle assay and cell ability results showing that POVPC or ox-LDL increased the proliferation, but nucleolin ablation inhibited the proliferation of HAVSMCs. And nucleolin ablation can prevent DNA replication at S phase and induce cell cycle arrest in S phase. The bioinformatics database predicts protein-protein interactions with nucleolin and aurora B. Nucleolin overexpression and ablation affected the expression of aurora B. These findings indicate for the first time that nucleolin actively involved the proliferation of VSMCs via aurora B.     

miR‐145 inhibits the proliferation and migration of vascular smooth muscle cells by regulating autophagy

miR‐145, the most abundant miRNA in the vascular smooth muscle cells (VSMCs), regulates VSMC function in intimal hyperplasia. It has been reported that autophagy participates in the regulation of proliferation and migration of VSMCs. However, the effect of miR‐145 on autophagy and related mechanism in the proliferation and migration of VSMCs remains unclear. Therefore, we aimed to determine the effect of miR‐145 on autophagy and the mechanism in VSMCs. Cell autophagy was determined by transmission electron microscope, mRFP‐GFP‐LC3 assay and Western blotting. A recombinant lentivirus containing miR‐145 was used to construct VSMCs with miR‐145 overexpression. We found that miR‐145 expression was decreased, and autophagy was increased in the carotid arteries of C57BL/6J mice with intimal hyperplasia and TGF‐β1‐stimulated VSMCs. Furthermore, miR‐145 overexpression inhibited cell autophagy, whereas miR‐145 inhibition promoted autophagy in TGF‐β1‐stimulated VSMCs. Meanwhile, miR‐145 inhibited the proliferation and migration of VSMCs. More importantly, our study showed that autophagy inhibition augmented the inhibitory effect of miR‐145 on the proliferation and migration of VSMCs. In addition, we found that the sirtuins are not direct targets of miR‐145 in the proliferation and migration of VSMCs. These results suggest that miR‐145 inhibits the proliferation and migration of VSMCs by suppressing the activation of autophagy.     

Oleic acid and adipokines synergize in inducing proliferation and inflammatory signalling in human vascular smooth muscle cells

In the context of obesity, perivascular fat produces various adipokines and releases free fatty acids, which may induce inflammation and proliferation in the vascular wall. In this study we investigated how adipokines, oleic acid (OA) and the combined treatment regulate human vascular smooth muscle cell (hVSMC) proliferation and migration and the underlying signalling pathways. Adipocyte‐conditioned media (CM) generated from human adipocytes induces a prominent proliferation and migration of hVSMC. Autocrine action of adiponectin totally abolishes CM‐induced proliferation. Furthermore, OA but not palmitic acid induces proliferation of hVSMC. CM itself does not contain fatty acids, but CM in combination with OA markedly enhances proliferation of hVSMC in a synergistic way. Both the nuclear factor (NF)‐κB and the mammalian target of rapamycin (mTOR) pathway were synergistically activated under these conditions and found to be essential for hVSMC proliferation. Expression of iNOS and production of nitric oxide was only enhanced by combined treatment inducing a marked release of VEGF. Combination of OA and VEGF induces an additive increase of hVSMC proliferation. We could show that the combination of CM and OA led to a synergistic proliferation of hVSMC. Expression of iNOS and production of nitric oxide were only enhanced under these conditions and were paralleled by a marked release of VEGF. These results suggest that the combined elevated release of fatty acids and adipokines by adipose tissue in obesity might be critically related to hVSMC dysfunction, vascular inflammation and the development of atherosclerosis.     

Inhibition of PTEN expression and activity by angiotensin II induces proliferation and migration of vascular smooth muscle cells

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor and has been suggested recently to be involved in the regulation of cardiovascular diseases. The molecular mechanisms of this regulation are however poorly understood. This study shows that down regulation of PTEN expression and activity by angiotensin II (Ang II) increased proliferation and migration of vascular smooth muscle cells (VSMCs). The presence of Ang II induced rapid PTEN phosphorylation and oxidation in accordance with increased AKT and FAK phosphorylation. The Ang II‐mediated VSMC proliferation and migration was inhibited when cellular PTEN expression was increased by AT1 inhibitor losartan, PPARγ agonist rosiglitazone, NF‐κB inhibitor BAY 11‐7082. Over expression of PTEN in VSMCs by adenovirus transduction also resulted in inhibition of cell proliferation and migration in response to Ang II. These results suggest that PTEN down‐regulation is involved in proliferation and migration of VSMCs induced by Ang II. This provides insight into the molecular regulation of PTEN in vascular smooth muscle cells and suggests that targeting the action of PTEN may represent an effective therapeutic approach for the treatment of cardiovascular diseases. J. Cell. Biochem. 114: 174–182, 2012. © 2012 Wiley Periodicals, Inc.     

Inhibition of proliferation and apoptosis of vascular smooth muscle cells by ghrelin

To evaluate the possible role of ghrelin in the development of atherosclerosis, its effects on tumor necrosis factor (TNF)-alpha-induced proliferation and apoptosis of vascular smooth muscle cells (VSMCs) were investigated. Rat VSMCs were pretreated with different concentrations of ghrelin and then with TNF-alpha. VSMC proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry method. Apoptosis was detected using propidium iodide and Annexin-V labeling method. Exogenous ghrelin (10-1000 ng/ml) significantly inhibited TNF-alpha-induced proliferation of VSMCs in a concentration-dependent manner. Treatment with 1000 ng/ml ghrelin was most effective at inhibiting VSMC proliferation rate and the expression of proliferating cell nuclear antigen. However, treatment with des-acyl ghrelin affected neither proliferation nor PCNA expression. In contrast, TNF-alpha-induced apoptosis of VSMCs was inhibited by both ghrelin and des-acyl ghrelin in concentration-dependent manners, with maximal inhibition observed for both compounds at 1000 ng/ml. Taken together, our results suggested that ghrelin inhibited both the proliferation and apoptosis of rat VSMCs. Furthermore, the former effect is probably mediated by the growth hormone secretagogue receptor type 1a receptor, while the latter effect may be mediated through other receptors.