Tissue inhibitor of metalloproteinases-4 suppresses vascular smooth muscle cell migration and induces cell apoptosis

In a previous study, we have demonstrated that overexpression of the tissue inhibitors of metalloproteinases-4 (TIMP-4) can inhibit the neointima formation in the rat carotid model. To define the functions of tissue inhibitor of metalloproteinases-4 (TIMP-4) in SMCs, we transduced human TIMP-4 cDNA into rat aortic SMCs by using adenoviral vector. Overexpression of TIMP-4 blocked the conversion of pro-MMP-2 to the active form and inhibited basic fibroblast growth factor-induced migration by 56.7% (p < 0.01). Overexpression of TIMP-4 markedly increased apoptotic cell death without changing their proliferation. Importantly, overexpression of human TIMP-4 in the wall of balloon-injured rat carotid artery also increased SMC apoptosis. The percentages of TUNEL-positive cells of total cells increased significantly in AdTIMP-4 infected group compared with AdGFP infected group. These findings demonstrate that TIMP-4 can inhibit SMCs migration and induce apoptosis in vitro and in vivo, which may generate new targets for prevention and treatment of vascular diseases.     

Alterations of adrenomedullin and its receptor system components in calcified vascular smooth muscle cells

Vascular calcification is a common finding in many cardiovascular diseases. Paracrine/autocrine changes in calcified vessels, and the secreted factors participate in and play an important role in the progress of calcification. Adrenomedullin (ADM) is a potent vasodilator peptide secreted by vascular smooth muscle cells (VSMCs) and vascular endothelial cells. Recently, receptor activity-modifying proteins (RAMPs) have been shown to transport calcitonin receptor-like receptor (CRLR) to the cell surface to present either as CGRP receptor or ADM receptor. In this work, we explored the production of ADM, alterations and significance of ADM mRNA and its receptor system components—CRLR and RAMPs mRNA in calcified VSMCs. Our results showed that calcium content, ⁴⁵Ca²⁺ uptake and alkaline phosphatases (ALPs) activity in calcified VSMCs were increased, respectively, compared with control VSMCs. Content of ADM in medium was increased by 99% (p<0.01). Furthermore, it was found that the levels of ADM, CRLR, RAMP2 and RAMP3 mRNA in calcified cells were elevated, respectively, compared with that of control. The elevated levels of CRLR, RAMP2 and RAMP3 mRNA were significant correlation with ADM mRNA (r=0.83, 0.92 and 0.93, respectively, all p's<0.01) in calcified VSMCs. The results show that calcified VSMCs generate an increased amount of ADM, up-regulate gene expressions of ADM and its receptor system components—CRLR, RAMP2 and RAMP3, suggesting an important role of ADM and its receptor system in the regulation of vascular calcification.     

Shear stress induces apoptosis in vascular smooth muscle cells via an autocrine Fas/FasL pathway

Differentiated melanocytic cells produce melanin, through several redox reactions including tyrosinase-catalyzed DOPA oxidation to DOPA quinone. We now developed a method based on DOPA oxidase in-gel detection and Sypro Ruby fluorometric normalization to investigate induction of specific DOPA oxidase isoforms in response to hydrogen peroxide-mediated stress, and to ask whether this is associated with p53-dependent adaptive responses. This report shows that hydrogen peroxide leads to comparable induction of 60 and 55 kDa DOPA oxidases in poorly pigmented B16 melanoma, in contrast to sole induction of a major 55 kDa DOPA oxidase in their highly pigmented counterparts. In the latter cells, this response also increases p53 concomitant with joint induction of p53-activated proteins like the cell-cycle inhibitor p21WAF1 and pro-apoptotic bax, with no comparable effect on expression of anti-apoptotic bcl-2. Together, these data suggest that response to hydrogen peroxide involves p53-mediated growth-restrictive signaling and unequal induction of specific DOPA oxidases in melanocytic cells with unequal basal pigmentation.     

Increased sensitivity of serotonin on the voltage-dependent K channels in mesenteric arterial smooth muscle cells of OLETF rats

This study examined the mechanisms of hypertension in diabetes. We investigated the effects of serotonin (5-HT) on voltage-dependent K⁺ (Kv) channel activity, vasoconstriction, 5-HT receptor expression levels, and the involvement of protein kinase C (PKC) in mesenteric arteries of Otsuka Long-Evans Tokushima fatty (OLETF) rats compared with Long-Evans Tokushima Otsuka (LETO) rats. Blood pressure, body weight, blood glucose level, and mesenteric arterial wall thickness were greater in OLETF rats. The 5-HT-induced vasoconstriction of mesenteric arteries was greater in OLETF rats than in LETO rats and inhibited by the 5-HT_2A inhibitor inhibitor, ketanserin. The Kv currents in mesenteric arterial smooth muscle cells (MASMCs), determined using a perforated patch clamp technique, was inhibited by 1 mM 4-AP (42.5 ± 4.1% vs. 63.5 ± 2.3% in LETO vs. OLETF rats at +40 mV), but was insensitive to 1 mM TEA and 100 nM iberiotoxin. The inhibition of Kv current by 1 μM 5-HT in MASMCs was greater in OLETF rats than in LETO rats (17.1 ± 2.2% vs. 33.2 ± 2.7% in LETO vs. OLETF rats at +40 mV), and the inhibition was prevented by treatment with the PKCα- and β- selective inhibitor, Gö6976. The expression level of 5-HT_2A, but not 5-HT_2B, receptor and the expression levels of total PKC, PKCβ, and PKCε, but not PKCα, were higher in the mesenteric arteries of OLETF rats compared with LETO rats. The enhanced expression of 5-HT_2A receptor together with PKCβ may promote mesenteric vasoconstriction and increase vascular resistance in OLETF rats.     

Physiological effects of androgens on human vascular endothelial and smooth muscle cells in culture

Androgenic hormones are associated with atherosclerotic cardiovascular disease, although the underlying cellular and molecular mechanisms remain unclear. This study examines the impact of androgens on the physiology of human vascular endothelial cells (EC) and smooth muscle cells (SMC) in culture. Cells were incubated with testosterone, dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) at various physiological concentrations (5-50 nM) in the present or absence of an androgen receptor (AR) blocker flutamide (100 nM). Cell growth and death, DNA and collagen synthesis, and gene protein expression were assessed. It was shown that: (1) DHEA protected EC from superoxide injury via AR-independent mechanisms; (2) testosterone induced DNA synthesis and growth in EC via an AR-independent manner with activation of ERK1/2 activity; (3) DHT inhibited DNA synthesis and growth in EC in an AR-dependent manner; (4) testosterone and DHT enhanced ERK1/2 activation and proliferation in SMC via AR-independent and -dependent pathways, respectively; and (5) these androgens did not significantly affect collagen synthesis in SMC. We conclude that androgens possess multiple effects on vascular cells via either AR-dependent or -independent mechanisms. Testosterone and DHEA may be “beneficial” in preventing atherosclerosis by improving EC growth and survival; in contrast, stimulation of VSMC proliferation by testosterone and DHT is potentially “harmful”. The relationship of these in vitro effects by androgens to in vivo vascular function and atherogenesis needs to be further clarified.     

Caveolin-1 sensitizes vascular smooth muscle cells to mildly oxidized LDL-induced apoptosis

Oxidized low-density lipoprotein (oxLDL)-induced apoptosis of vascular cells may participate to plaque instability and rupture. Caveolin-1 has emerged as an important regulator of several signal transduction pathways and processes that play a role in atherosclerosis. In this study we examined the potential role of caveolin-1 in the regulation of oxLDL-induced Ca²⁺ signaling and apoptosis in vascular smooth muscle cells (VSMC). Cells expressing caveolin-1 were more susceptible to oxLDL-induced apoptosis, and this was correlated with enhanced Ca²⁺ entry and pro-apoptotic events. Moreover, caveolin-1 silencing by small interfering RNA decreased the level of apoptotic cells after oxLDL treatment. These findings provide new insights about the potential role of caveolin-1 in the regulation of oxLDL-induced apoptosis in vascular cells and its contribution to the instability of the plaque.     

Papaverine induces apoptosis in vascular endothelial and smooth muscle cells

Papaverine is a vasodilator commonly used in the treatment of vasospasmic diseases such as cerebral spasm associated with subarachnoid hemorrhage, and in the prevention of spasm of coronary artery bypass graft by intraluminal and/or extraluminal administration. In this study, we examined whether papaverine in the range of concentrations used clinically causes apoptosis of vascular endothelial and smooth muscle cells. Apoptotic cells were identified by morphological changes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In porcine coronary endothelial cells (EC) and rat aortic smooth muscle cells (SMC), papaverine at the concentration of 10(-3) M induced membrane blebbing within 1 hour of incubation. Nuclear condensation and fragmentation were found after 24 hours of treatment. The number of apoptotic cells stained with the TUNEL method was significantly higher in the EC and the SMC after 24 hours of incubation with papaverine at the concentrations of 10(-4) and 10(-3) M than their respective controls. Acidified saline solution (pH 4.8, as control for 10(-3) M papaverine hydrochloride) did not cause apoptosis in these cells. These results showed that papaverine could damage endothelial and smooth muscle cells by inducing changes which are associated with events leading to apoptosis. Since integrity of endothelial cells is critical for normal vascular function, vascular administration of papaverine for clinical use, especially at high concentrations (> or = 10(-4) M), should be re-considered.     

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.     

Regulation of ERK5 by insulin and angiotensin-II in vascular smooth muscle cells

ERK5 is involved in proliferation of vascular smooth muscle cells (VSMC). The proliferative actions of insulin and angiotensin-II (A-II) in VSMC are mediated in part by ERK1/2. We hypothesized that insulin and A-II also regulate ERK5 activity in VSMC. Acute treatment (<60min) with insulin or A-II increased phosphorylation of ERK1/2 at 15min and ERK5 at 5min. Chronic treatment (< or = 8h) with insulin increased ERK1/2 phosphorylation by 4h and ERK5 by 8h. A-II-stimulated phosphorylation of ERK1/2 by 8h and ERK5 by 4h. The EC(50) for insulin treatment effecting ERK1/2 and ERK5 phosphorylation was 1.5 and 0.1nM, whereas the EC(50) for A-II was 2nM, each. Insulin plus A-II induced an additive effect only on ERK5 phosphorylation. Inhibition of insulin- and A-II-stimulated phosphorylation of ERK5 and ERK1/2 by PD98059 and Wortmannin exhibited differential and time-dependent effects. Taken together, these data indicate that insulin and A-II regulate the activity of ERK5, but different from that seen for ERK1/2.     

Endothelin-1 induces the expression of C-reactive protein in rat vascular smooth muscle cells

Numerous studies have shown that both vasoconstrictive peptide endothelin-1 (ET-1) and inflammatory marker C-reactive protein (CRP) are implicated in the inflammatory process of atherosclerosis. The purpose of the present study was to observe effect of ET-1 on CRP production and the molecular mechanisms in rat vascular smooth muscle cells (VSMCs). The results showed that ET-1 was capable of stimulating VSMCs to produce CRP both in protein and in mRNA levels in vitro and in vivo. ET_A receptor antagonist BQ123, but not ET_B receptor antagonist BQ788, inhibited CRP production in VSMCs. In addition, ET-1 was able to elicit reactive oxygen species (ROS) generation and mitogen-activated protein kinase (MAPK) activation, and antioxidant pyrrolidine dithiocarbamate and p38MAPK inhibitor SB203580 inhibited ET-1-induced CRP expression. The results demonstrate that ET-1 induces CPR production in VSMCs via ET_A receptor followed by ROS and MAPK signal pathway, which may contribute to better understanding of the role of ET-1 in inflammatory activation of the vessel wall during atherogenesis.     

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.     

Pitavastatin attenuates the PDGF-induced LR11/uPA receptor-mediated migration of smooth muscle cells

Statins, inhibitors of HMG-CoA reductase, elicit various actions on vascular cells including the modulation of proliferation and migration of smooth muscle cells (SMCs). Here, we have elucidated the mechanism by which statins, in particular pitavastatin, attenuate the migration activity of SMCs. The expression of LR11, a member of the LDL receptor family and an enhancer of cell surface localization of urokinase-type plasminogen activator receptor (uPAR), is increased in cultured SMCs by treatment with PDGF-BB. Pitavastatin attenuates the PDGF-BB -induced surface expression of LR11 and uPAR. The increased migration of SMCs observed both upon overexpression of LR11 and via stimulation of secretion of soluble LR11 is not reversed by pitavastatin. In vivo studies showed that the SMCs expressing LR11 in plaques are almost congruent with intimal cells expressing nonmuscle myosin heavy chain (SMemb). Pitavastatin reduced the expression of LR11 and SMemb, and the levels of LR11, uPAR, and SMemb in cultured intimal SMCs were reduced to those seen in medial SMCs. We propose that this statin reduces PDGF-induced migration through the attenuation of the LR11/uPAR system in SMCs. Modulation of the LR11/uPAR system with statins suggests a novel treatment strategy for atherogenesis based on suppression of intimal SMC migration.     

A paradoxical pro-apoptotic effect of thrombin on smooth muscle cells

Whereas thrombin (below 10 nM) is a potent mitogen, recent studies report that exposure to higher doses of thrombin could lead to apoptosis of neurons and tumor cells. Our results show that prolonged exposure (> or = 24 h) to thrombin (50-100 nM) exerts a pro-apoptotic effect on cultured vascular smooth muscle cells (VSMCs). This phenomenon depends on thrombin serine-protease activity but is independent of PAR-1 and -4 activation and subsequent signaling. The parallel occurrence of cell retraction and cleavage of fibronectin suggests that thrombin-induced apoptosis is consecutive to pericellular proteolysis. These data point to a new potential action of thrombin in the cardiovascular system.     

Crystallizing nanoparticles derived from vascular smooth muscle cells contain the calcification inhibitor osteoprotegerin

Osteoprotegerin (OPG), a member of the TNF receptor superfamily, was initially found to modulate bone mass by blocking osteoclast maturation and function. Rodent models have also revealed a role for OPG as an inhibitor of vascular calcification. However, the precise mode of how OPG blocks mineralization is unclear. In this study, OPG was found in an in vitro assay to significantly inhibit calcification of vascular smooth muscle cells (VSMC) induced by high calcium/phosphate (Ca/P) treatment (p = 0.0063), although this effect was blunted at high OPG concentrations. By confocal microscopy, OPG was detected in VSMC in the Golgi, the same localization seen in osteoblasts, which express OPG in bone. Treatment of VSMC by minerals (Ca, P, or both) induced OPG mRNA expression as assessed by real-time quantitative PCR, and VSMC derived from atherosclerotic plaque material also exhibited higher OPG expression as compared to control cells (p < 0.05). Furthermore, OPG was detected by Western blotting in matrix vesicles (MV), nanoparticles that are released by VSMC with the capacity to nucleate mineral. In atherosclerotic arteries, OPG colocalized immunohistochemically with annexin VI, a calcium-dependent membrane and phospholipid binding protein found in MV. Thus, the calcification inhibitor OPG is contained in crystallizing MV and has a biphasic effect on VSMC: physiologic concentrations inhibit calcification, whereas high concentrations commonly seen in patients with vascular disease have no effect. Like other calcification inhibitors, OPG may be specifically loaded into these nanoparticles to be deposited at remote sites, where it acts to inhibit calcification.     

Toll-like receptor 3 mediates expression of clusterin/apolipoprotein J in vascular smooth muscle cells stimulated with RNA released from necrotic cells

Clusterin/Apolipoprotein J is a protein that is upregulated in a broad spectrum of diverse pathological processes. The predominant form is a secreted glycoprotein (sCLU) with cytoprotective and anti-inflammatory properties which shows enhanced expression in vascular smooth muscle cells (VSMC) following aortic injury and in atherosclerotic disease. Recent evidence indicates that during atherosclerosis, Toll-like receptors (TLRs) are activated in vascular cells by endogenous ligands. Here, we analyzed whether CLU expression in VSMC is controlled by TLRs, and stimulated by factors associated with or released by necrotic cells. Activation of TLR3 by the synthetic RNA analogue polyinosinic-polycytidylic acid (poly(I:C)) in CRL2018 VSMC and in mice led to induction of CLU mRNA and protein synthesis, respectively. In TLR3-deficient 10A yolk sac cells, induction of CLU by poly(I:C) challenge depended on the ectopic expression of human TLR3. In mice lacking the TLR3-signaling adaptor protein TRIF (TIR-domain-containing adaptor protein inducing IFN-β) CLU induction by poly(I:C) was abrogated. In addition to poly(I:C) CLU gene expression in CRL2018 cells was induced by purified cellular RNA and RNA present in necrotic cell lysate.Our data indicate that cellular RNA following its release from necrotic cells in atherosclerotic lesions can act as an endogenous TLR3 ligand to induce CLU expression in VSMC and in vivo. Thus, they expand the view on TLR2 and TLR4 as known pro-atherosclerotic effectors toward TLR3. Conclusively, TLR3 activation induces expression of cytoprotective and anti-inflammatory CLU by VSMC and mice, to potentially counteract atherosclerotic pathology.     

Epidermal growth factor up-regulates the expression of nestin through the Ras-Raf-ERK signaling axis in rat vascular smooth muscle cells

The contractile-synthetic phenotypic modulation of vascular smooth muscle cells (VSMCs) is a key event during atherosclerosis progression. Although many studies have reported possible cytokines and growth factors implicated to this process, the critical factors affecting the VSMC phenotype remain unclear due to the lack of early de-differentiation marker identifications. In this study, we showed that nestin, an intermediate filament protein, is expressed in primary cultures of rat VSMCs representing the synthetic phenotype and its expression is diminished as these cells re-differentiate after serum deprivation. However, the regulation of nestin expression was never reported despite its common usage as an early differentiation marker. Herein, we showed that nestin expression is regulated by epidermal growth factor (EGF) via de novo RNA and protein synthesis. Furthermore, signaling analyses revealed that the EGF-induced nestin re-expression is mediated through the activation of the Ras-Raf-ERK signaling axis. This is the first report to show that nestin expression is regulated by an extracellular signaling molecule.