Stimulation Of Smooth Muscle Cell Proliferation Research Articles

Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, Cardiotrophin-1 Expression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration.

Persistent oxidative stress and inflammation contribute causally to smooth muscle cell (SMC) proliferation and migration, the characteristic features of vascular proliferative diseases. Oxidatively modified low-density lipoproteins (OxLDL) elevate oxidative stress levels, inflammatory responses, and matrix metallopeptidase (MMP) activation, resulting ultimately in SMC migration, proliferation, and phenotype change. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Empagliflozin is an SGLT2 inhibitor and exerts pleiotropic cardiovascular protective effects, including antioxidant and anti-inflammatory effects. Here, we investigated (i) whether OxLDL regulates RECK expression, (ii) whether ectopic expression of RECK reverses OxLDL-induced SMC migration and proliferation, and (iii) whether pretreatment with empagliflozin reverses OxLDL-induced RECK suppression, MMP activation, and SMC migration, proliferation, and differentiation. Indeed, results show that OxLDL at pathophysiological concentration promotes SMC migration and proliferation via NF-κB/miR-30b-dependent RECK suppression. Moreover, OxLDL changed the SMC phenotype to a more pro-inflammatory type, and this effect is blunted by RECK overexpression. Further, treatment with empagliflozin reversed OxLDL-induced miR-30b induction, RECK suppression, MMP activation, SMC migration, proliferation, and proinflammatory phenotype changes. OxLDL-induced cardiotrophin (CT)-1 expression and CT-1 stimulated SMC proliferation and migration in part via leukemia inhibitory factor receptor (LIFR) and glycoprotein 130 (gp130). Ectopic expression of RECK inhibited these effects by physically associating with LIFR and gp130, as evidenced by immunoprecipitation/immunoblotting and double immunofluorescence. Importantly, empagliflozin inhibited CT-1-induced mitogenic and migratory effects. Together, these results suggest the therapeutic potential of sustaining RECK expression or empagliflozin in vascular diseases characterized by SMC proliferation and migration.

Improved in vitro endothelialization on nanostructured titania with tannin/glycosaminoglycan-based polyelectrolyte multilayers

Blood compatibility of cardiovascular implants is still a major concern. Rapid endothelialization of these implant surfaces has emerged as a promising strategy to enhance hemocompatibility and prevent complications such as thrombus formation and restenosis. The successful endothelialization of implant surfaces mostly depends on the migration of endothelial cells (ECs), the differentiation of stem cells, and the inhibition of smooth muscle cell (SMC) proliferation. Our previous study demonstrated that nanostructured titania surfaces modified with polyelectrolyte multilayers based on tanfloc (a cationic tannin derivative) and glycosaminoglycans (heparin and hyaluronic acid) have improved antithrombogenic properties. In this work, we used in vitro cell culture of ECs and SMCs to investigate the outcomes of these surface modifications on endothelialization. The cells were seeded on the surfaces, and their viability, adhesion, and proliferation were evaluated after 1, 3, and 5 days. Indirect immunofluorescent staining was used to determine the cellular expression of ECs through the presence of specific marker proteins after 7 and 10 days, and EC migration on the NT surfaces was also investigated. The surfaces modified with tanfloc and heparin showed enhanced EC adhesion, proliferation, and migration. However, SMC proliferation is not promoted by the surfaces. Therefore, these surfaces may promote endothelialization without stimulating SMC proliferation, which could improve the hemocompatibility without enhancing the risk of SMC proliferation leading to restenosis. The surface modification here proposed is a promising candidate to be used in cardiovascular applications due to enhanced antithrombogenic and endothelialization properties.

PDGF-BB-mediated activation of CREB in vascular smooth muscle cells alters cell cycling via Rb, FoxO1 and p27kip1

Introduction & AimThe vascular response to injury leads to the secretion of several factors, including platelet-derived growth factor (PDGF-BB). PDGF-BB stimulates smooth muscle cell (SMC) conversion to the synthetic phenotype, thereby enhancing proliferation and migration, and contributing to neointimal hyperplasia. Likewise, the cAMP response element binding protein (CREB) transcription factor has been shown to mediate SMC proliferation in response to various mitogens. We therefore investigated the contribution of CREB to PDGF-BB-dependent proliferation of SMCs with the intention of identifying signaling pathways involved both up and downstream of CREB activation. Methods & ResultsTreatments were performed on vascular SMCs from a porcine coronary artery explant model. The role of CREB was examined via adenoviral expression of a dominant-negative CREB mutant (kCREB) as well as inhibition of CREB binding protein (CBP). Involvement of the p27kip1 pathway was determined using a constitutively expressing p27kip1 adenoviral vector. PDGF-BB stimulated transient CREB phosphorylation on Ser-133 via ERK1/2-, PI3-kinase- and Src-dependent pathways. Expression of kCREB decreased PDGF-BB-dependent cell proliferation. PCNA expression and Rb phosphorylation were also inhibited by kCREB. These cell cycle proteins are controlled via p27kip1 expression in response to CREB-dependent post-translational modification of FoxO1. kCREB had no effect on Cyclin D1 expression, but did prevent PDGF-BB-induced Cyclin D1 nuclear translocation. An interaction inhibitor of CBP confirmed that Cyclin D1 is downstream of PDGF-BB and CREB. ConclusionCREB phosphorylation is required for SMC proliferation in response to PDGF-BB. This phenotypic change requires CBP and is mediated by Cyclin D1 and p27kip as a result of changes in FoxO1 activity.

Interaction among estrogen, IGF-1, and H2S on smooth muscle cell proliferation.

Both estrogen and hydrogen sulfide (H2S) inhibit the proliferation of vascular smooth muscle cells (SMCs) and development of atherosclerosis. In the absence of endogenous H2S as occurred in CSE-knockout (KO) mouse, however, estrogen stimulates the proliferation of vascular SMCs. The underlying mechanisms for this seemingly controversial vascular effect of estrogen are unclear. In the present study, we demonstrated that the stimulatory effect of estrogen on the proliferation of CSE-KO SMCs was suppressed by the inhibitor of insulin-like growth factor-1 receptor (IGF-1R) or knockdown of IGF-1R protein expression. Estrogen downregulated the expression of insulin-like growth factor-1 (IGF-1) and IGF-1R in aortic tissues or aortic SMCs isolated from WT and CSE-KO mice. Furthermore, endogenous H2S downregulated IGF-1R, but upregulated estrogen receptor (ER)-α, in aortic tissues or SMCs. ER-α and IGF-1R were co-located in SMCs and co-immunoprecipitated, which was decreased by H2S. Finally, both endogenous and exogenous H2S induced the S-sulfhydration of IGF-1R, but not ER-α, in WT-SMCs and CSE-KO SMCs, which underlies the decreased formation of IGF-1R/ER-α hybrid in the presence of H2S. Thus, the absence of H2S favors the interaction of estrogen with IGF-1R/ER-α hybrid to stimulate SMCs proliferation. The appreciation of a critical role of H2S in preventing estrogen-induced SMCs proliferation will help better understand the regulation of complex vascular effects of estrogen and sex-related cardiovascular diseases.

Identification and analyses of inhibitors targeting apolipoprotein(a) kringle domains KIV-7, KIV-10, and KV provide insight into kringle domain function

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 μm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6-2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).

Janus Kinase 3, a Novel Regulator for Smooth Muscle Proliferation and Vascular Remodeling.

Vascular remodeling because of smooth muscle cell (SMC) proliferation is a common process occurring in several vascular diseases, such as atherosclerosis, aortic aneurysm, post-transplant vasculopathy, restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completely understood. The objective of this study is to determine the role and mechanism of Janus kinase 3 (JAK3) in vascular remodeling and SMC proliferation. Platelet-derived growth factor-BB, an SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by short hairpin RNA, inhibits the SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Mechanistically, JAK3 promotes the phosphorylation of signal transducer and activator of transcription 3 and c-Jun N-terminal kinase in SMC, 2 signaling pathways known to be critical for SMC proliferation and vascular remodeling. Blockade of these 2 signaling pathways by their inhibitors impeded the JAK3-mediated SMC proliferation. In vivo, knockdown of JAK3 attenuates injury-induced neointima formation with attenuated neointimal SMC proliferation. Knockdown of JAK3 also induces neointimal SMC apoptosis in rat carotid artery balloon injury model. Our results demonstrate that JAK3 mediates SMC proliferation and survival during injury-induced vascular remodeling, which provides a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases.

Abstract 332: Inhibition of the Enhancer of Zeste Homolog Family Mitigates Intimal Hyperplasia in Rat Carotid Arteries

Rationale: The enhancer of zeste homologs 1 and 2 (EZH1 and EZH2) are histone-lysine N-methyltransferases that modify histones by methylation which leads to transcriptional repression. It has recently been shown that EZH1 and EZH2 play an important role in various cancers. Whether they play a role in recurrent vascular diseases is not known. Objective: We assessed whether EZH1 and EZH2 are important contributors to the development of intimal hyperplasia (IH) and restenosis. Methods and Results: Following rat carotid balloon angioplasty, EZH2 showed highest expression at day 3 post procedure and then decreased at day 7 in injured arteries, as determined by Western blotting. Change of EZH1 is less prominent. Dual inhibition of EZH1 and EZH2 through peri-adventitial administration of a selective inhibitor, UNC-1999, effectively inhibited intimal hyperplasia, with ~40% reduction in the ratio of intima to media (I/M). Moreover, in cultured primary rat smooth muscle cells (SMCs) as well as MOVAS cells, pretreatment with 5μM UNC-1999 resulted in a 60% decrease in cell proliferation and ~80% reduction of migration that were stimulated by PDGF-BB. Simultaneous knockdown of EZH1 and EZH2, as well as knockdown of their shared scaffold protein(EED), led to effective inhibition of proliferation and migration of SMCs. However, knockdown of EZH2 alone did not recapitulate the effects of dual inhibition of EZH1 and EZH2. Conclusions: This study demonstrates that dual inhibition of EZH1 and EZH2, both histone modifiers, mitigates intimal hyperplasia in vivo and attenuates PDGF-BB stimulated SMC proliferation and migration in vitro.

The MEK1 inhibitors UO126 and PD98059 block PDGF-AB induced phosphorylation of threonine 292 in porcine smooth muscle cells

PDGF-AB and FGF-2 (GFs) induce smooth muscle cell (SMC) proliferation which is indispensible for arteriogenesis. While there is common agreement that GFs stimulate SMC proliferation through phosphorylation (P-) of MEK1/2 at Ser218/222, we previously demonstrated that the MEK inhibitors PD98059 and UO126 did not inhibit P-Ser218/222 as originally proposed but caused strong hyperphosphorylation. Here, we demonstrate that GFs increased phosphorylation of MEK1 at Thr292 while UO126 and PD98059 blocked this phosphorylation. This was again surprising since phosphorylation of Thr292 is regarded as a negative feedback loop. Our findings suggest that inhibition of Thr292 phosphorylation in combination with hyperphosphorylation of Ser218/222 serves as an “off” switch of SMC proliferation and potentially of arteriogenesis.

Abstract 521: Janus Kinase 3 is a Novel Regulator for Smooth Muscle Proliferation and Vascularremodeling

Vascular remodeling due to smooth muscle cell (SMC) proliferation is a common process occurring in a number of vascular diseases such as atherosclerosis, aortic aneurysm, posttransplant vasculopathy, and restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completed understood. Our present study has identified Janus kinase 3 (JAK3), a member of the Janus kinase family, as a novel regulator for SMC proliferation. Platelet-derived growth factor (PDGF)-BB, a SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by shRNA inhibits SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Interestingly, JAK3 does not affect SMC contractile protein expression, suggesting that JAK3 mediate the proliferation, but not the phenotypic modulation of SMC. Mechanistically, JAK3 promotes SMC proliferation by regulating phosphorylation of Signal transducer and activator of transcription 3 and c-Jun N-terminal kinase. In vivo, by using a rat carotid balloon-injury model, we found that knockdown of JAK3 significantly attenuates injury-induced neointima formation. Importantly, JAK3 knockdown blocked the expression of proliferating cell nuclear antigen, suggesting that JAK3 is essential for SMC proliferation in vivo. Moreover, JAK3 knockdown prompts apoptosis in neointima SMC, indicating that JAK3 also stimulates SMC survival during neointima formation. Collectively, our data demonstrate that JAK3 mediates vascular remodeling by promoting both the SMC proliferation and survival.

A crosstalk between TGF-β/Smad3 and Wnt/β-catenin pathways promotes vascular smooth muscle cell proliferation

RationaleEndovascular interventions performed for atherosclerotic lesions trigger excessive vascular smooth muscle cell (SMC) proliferation leading to intimal hyperplasia. Our previous studies show that following endovascular injury, elevated TGF-β/Smad3 promotes SMC proliferation and intimal hyperplasia. Furthermore in cultured SMCs, elevated TGF-β/Smad3 increases the expression of several Wnt genes. Here we investigate a crosstalk between TGF-β/Smad3 and Wnt/β-catenin signaling and its role in SMC proliferation. Methods and resultsTo mimic TGF-β/Smad3 up-regulation in vivo, rat aortic SMCs were treated with Smad3-expressing adenovirus (AdSmad3) or AdGFP control followed by stimulation with TGF-β1 (or solvent). AdSmad3/TGF-β treatment up-regulated Wnt2b, Wnt4, Wnt5a, Wnt9a, and Wnt11 (confirmed by qRT-PCR and ELISA), and also increased β-catenin protein as detected by Western blotting. Blocking Wnt signaling using a Frizzled receptor inhibitor (Niclosamide) abolished TGF-β/Smad3-induced β-catenin stabilization. Increasing β-catenin through degradation inhibition (using SKL2001) or by adenoviral expression enhanced SMC proliferation. Furthermore, application of recombinant Wnt2b, Wnt4, Wnt5a, or Wnt9a, but not Wnt11, stabilized β-catenin and stimulated SMC proliferation as well. In addition, increased β-catenin was found in the neointima of injured rat carotid artery where TGF-β and Smad3 are known to be up-regulated. ConclusionsThese results suggest a novel mechanism whereby elevated TGF-β/Smad3 stimulates the secretion of canonical Wnts which in turn enhances SMC proliferation through β-catenin stabilization. This crosstalk between TGF-β/Smad3 and Wnt/β-catenin canonical pathways provides new insights into the pathophysiology of vascular SMCs linked to intimal hyperplasia.

Preventing in-stent restenosis using lipoprotein (a), lipid and cholesterol adsorbent materials

Atherosclerosis is one of the major cause of mortality in developed countries. The characteristic lesion of atherosclerosis is the atheroma or plaque that forms through thickening of the inner layer of the vessel wall (called the intima). The development of stent in 1980s revolutionised treatment of cardiovascular diseases, including atherosclerosis. However the advent of stenting was hindered by the new problem of in-stent restenosis. It was demonstrated that in-stent restenosis was the result of a new pathology in the form of neointimal hyperplasia, which was a maladaptive healing response to bare-metal stent implantation. Recent evidence suggests that although drug-eluting stent (DES) have reduced restenosis rates, important concerns have been raised regarding increased late stent thrombosis, myocardial infarction and death. With advances in nanotechnology and smart materials, covered stents has been proposed to overcome this problem. This is due to in-stent late restenosis and thromboses are mainly caused by smooth muscle cells (SMC) proliferation. Studies showed that there is a relation between high low-density lipoprotein (LDL) and lipoprotein (a) [Lp(a)] level in blood stream and chance of in-stent restenosis, moreover studies show that Lp(a) could stimulate SMC proliferation. We hypothesis development of covered stent with novel design and use of smart materials which could adsorb cholesterol and prevent contact between Lp(a) and vessel wall to overcome problem indicated in DES. In addition cost of stents will significantly reduce by elimination of drugs as well as complex manufacturing of the drug incorporation.

Chemerin promotes the proliferation and migration of vascular smooth muscle and increases mouse blood pressure.

Blood chemerin concentration shows positive correlation not only with body mass index and serum triglyceride level but also with systolic blood pressure. While it seems likely that chemerin influences vascular smooth muscle cell (SMC) proliferation and migration, which are crucial to the development of hypertension, this remains to be clarified. In the present study, we investigated whether chemerin controls SMC proliferation and migration in vitro and also affects blood pressure in vivo. In vitro, chemerin significantly stimulated rat mesenteric arterial SMC proliferation and migration, as determined by a cell counting assay and Boyden chamber assay, respectively. The migratory effect of chemerin was confirmed in human aortic SMCs. Chemerin significantly increased ROS production in SMCs and phosphorylation of Akt (Ser(473)) and ERK, as measured by fluorescent staining and Western blot analysis, respectively. Various inhibitors (ROS inhibitor: N-acetyl-l-cysteine, phosphatidylinositol 3-kinase inhibitor: LY-294002, MAPKK inhibitor: PD-98059, NADPH oxidase inhibitor: gp91 ds-tat, and xanthine oxidase inhibitor: allopurinol) as well as chemokine-like receptor 1 small interfering RNA significantly inhibited chemerin-induced SMC proliferation and migration. Furthermore, chemerin-neutralizing antibody prevented carotid neointimal hyperplasia in the mouse ligation model. In vivo, chronic chemerin treatment (6 μg/kg, 6 wk) increased systolic blood pressure as well as phosphorylation of Akt and ERK in the mouse isolated aorta. In summary, we, for the first time, demonstrate that chemerin/chemokine-like receptor 1 stimulates SMC proliferation and migration via ROS-dependent phosphorylation of Akt/ERK, which may lead to vascular structural remodeling and an increase in systolic blood pressure.

Nitric Oxide Stimulates Matrix Synthesis and Deposition by Adult Human Aortic Smooth Muscle Cells Within Three-Dimensional Cocultures

Vascular diseases are characterized by the over-proliferation and migration of aortic smooth muscle cells (SMCs), and degradation of extracellular matrix (ECM) within the vessel wall, leading to compromise in cell-cell and cell-matrix signaling pathways. Tissue engineering approaches to regulate SMC over-proliferation and enhance healthy ECM synthesis showed promise, but resulted in low crosslinking efficiency. Here, we report the benefits of exogenous nitric oxide (NO) cues, delivered from S-Nitrosoglutathione (GSNO), to cell proliferation and matrix deposition by adult human aortic SMCs (HA-SMCs) within three-dimensional (3D) biomimetic cocultures. A coculture platform with two adjacent, permeable 3D culture chambers was developed to enable paracrine signaling between vascular cells. HA-SMCs were cultured in these chambers within collagen hydrogels, either alone or in the presence of human aortic endothelial cells (HA-ECs) cocultures, and exogenously supplemented with varying GSNO dosages (0-100 nM) for 21 days. Results showed that EC cocultures stimulated SMC proliferation within GSNO-free cultures. With increasing GSNO concentration, HA-SMC proliferation decreased in the presence or absence of EC cocultures, while HA-EC proliferation increased. GSNO (100 nM) significantly enhanced the protein amounts synthesized by HA-SMCs, in the presence or absence of EC cocultures, while lower dosages (1-10 nM) offered marginal benefits. Multi-fold increases in the synthesis and deposition of elastin, glycosaminoglycans, hyaluronic acid, and lysyl oxidase crosslinking enzyme (LOX) were noted at higher GSNO dosages, and coculturing with ECs significantly furthered these trends. Similar increases in TIMP-1 and MMP-9 levels were noted within cocultures with increasing GSNO dosages. Such increases in matrix synthesis correlated with NO-stimulated increases in endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expression within EC and SMC cultures, respectively. Results attest to the benefits of delivering NO cues to suppress SMC proliferation and promote robust ECM synthesis and deposition by adult human SMCs, with significant applications in tissue engineering, biomaterial scaffold development, and drug delivery.

Urotensin-II Induces Vascular Smooth Muscle Cell Proliferation and Creb Phosporylation Through Store Operated Calcium Entry and EGFR Transactivation

Urotensin-II (U-II) is a vasoactive peptide with many effects in the cardiovascular system. It has been described to induce vascular smooth muscle cell (SMC) proliferation, and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling; however, its signalling pathway still remains unclear. The aim of this study was to elucidate the role of calcium (Ca2+)-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation, focussing our attention on store-operated Ca2+ entry (SOCE) pathway and epithelium growth factor receptor (EGFR) transactivation.We used 5-bromo-2-deoxyuridine (BrdU) labeling, molecular knockdown with small interfering RNA (siRNA) and immunofluorescence to study UII-promoted aortic SMC proliferation. Ca2+ mobilization assays were performed to study Ca2+ entry in cultured SMC.We found that UII enhanced intracellular Ca2+ concentration ([Ca2+]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as STIM1, Orai1, or TRPC1. Moreover, UII stimulated SMC proliferation and Ca2+/cAMP response element-binding protein (CREB) activation through SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Additionally, we determined that epithelium growth factor receptor (EGFR) transactivation, extracellular signal-regulated kinase (ERK) and Ca2+/calmodulin-dependent kinase (CaMK) signaling pathways were involved in UII-mediated Ca2+ influx, CREB activation and VSMCs proliferation.Our data show for the first time that UII-induced SMC proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1 and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.Acknowledgements: This study was supported by Spanish Ministry of Science and Innovation [BFU-2010-21043-C02-01; BFU-2010-21043-C02-02]; Instituto Carlos III [RD12/0042/0041, PI12/00941]; and The Andalusian Government [P10-CVI-6095; PI-0108-2012].

Simvastatin attenuates the additive effects of TNF-α and IL-18 on the connexin 43 up-regulation and over-proliferation of cultured aortic smooth muscle cells

Statin therapy is known to down-regulate inflammatory activities in atheromatous tissues of animals. The aims of this study were to examine the regulatory role of interleukin-18 (IL-18) in the connexin 43 (Cx43) and the proliferation of cultured aortic smooth muscle cells (SMCs) as well as to elucidate the underlying therapeutic mechanism of simvastatin. Vytorin therapy significantly alleviated high-cholesterol diet-induced hypercholesterolemia, suppressed neointimal hyperplasia, macrophage infiltration, and Cx43 and IL-18 expression in rabbit aortic walls. In vitro study using an aortic SMC line showed that IL-18 up-regulated constitutive Cx43 expression and potentiated tumor necrosis factor-α (TNF-α)-triggered Akt and MAPK signaling pathways. Simvastatin treatment alone reduced constitutive Cx43 levels and prevented the TNF-α-induced IL-18 up-regulation. Mechanistic investigation using kinase-specific inhibitors showed that simvastatin pretreatment attenuated TNF-α-elicited Akt and ERK1/2 phosphorylation, whereas PI3K and all MAPK activities were also implied in the additive effect of TNF-α and IL-18 on Cx43 up-regulation. Proliferation assay indicated that IL-18 stimulated SMC proliferation and synergized the TNF-α-stimulated cell proliferation. Likewise, simvastatin treatment suppressed the SMC over-proliferation induced not only by TNF-α alone, but also by simultaneous treatment with TNF-α and IL-18. The suppression of simvastatin in SMC proliferation was not mediated through mitochondrial related pro-apoptogenesis under both scenarios. In conclusion, simvastatin attenuates the additive effects of TNF-α and IL-18 on Cx43 up-regulation and over-proliferation of aortic SMCs, mainly through the blockade of Akt signaling pathway. These findings may fortify the rationale underlying the atheroprotective mechanism of statin therapy.

The RNA-Binding Protein RBPMS2 Regulates Development of Gastrointestinal Smooth Muscle

Gastrointestinal development requires regulated differentiation of visceral smooth muscle cells (SMCs) and their contractile activities; alterations in these processes might lead to gastrointestinal neuromuscular disorders. Gastrointestinal SMC development and remodeling involves post-transcriptional modification of messenger RNA. We investigated the function of the RNA-binding protein for multiple splicing 2 (RBPMS2) during normal development of visceral smooth muscle in chicken and expression of its transcript in human pathophysiological conditions. We used avian replication-competent retroviral misexpression approaches to analyze the function of RBPMS2 in vivo and in primary cultures of chicken SMCs. We analyzed levels of RBPMS2 transcripts in colon samples from pediatric patients with Hirschsprung's disease and patients with chronic pseudo obstruction syndrome (CIPO) with megacystis. RBPMS2 was expressed strongly during the early stage of visceral SMC development and quickly down-regulated in differentiated and mature SMCs. Misexpression of RBPMS2 in differentiated visceral SMCs induced their dedifferentiation and reduced their contractility by up-regulating expression of Noggin, which reduced activity of bone morphogenetic protein. Visceral smooth muscles from pediatric patients with CIPO expressed high levels of RBPMS2 transcripts, compared with smooth muscle from patients without this disorder. Expression of RBPMS2 is present in visceral SMC precursors. Sustained expression of RBPMS2 inhibits the expression of markers of SMC differentiation by inhibiting bone morphogenetic protein activity, and stimulates SMC proliferation. RBPMS2 transcripts are up-regulated in patients with CIPO; alterations in RBPMS2 function might be involved in digestive motility disorders, particularly those characterized by the presence of muscular lesions (visceral myopathies).

MicroRNA‐21 represses human cystathionine gamma‐lyase expression by targeting at specificity protein‐1 in smooth muscle cells

Cystathionine gamma-lyase (CSE) is the major H(2)S-generating enzyme in vascular smooth muscle cells (SMCs). CSE/H(2)S system contributes to the maintenance of SMC phenotype, and transcript factor specificity protein-1 (SP1) is a critical regulator of CSE expression during SMC differentiation. The involvements of microRNA-21 (miR-21) in cardiovascular pathophysiology have been known, however miR-21 regulation of CSE and SP1 as well as SMC phenotype are uncertain. Using quantitative real-time PCR, we demonstrated that the expression of miR-21 was upregulated in dedifferentiated human aorta SMCs (HASMCs) and injured mouse carotid arteries. To determine the potential roles of miR-21 in SP1-mediated CSE gene expression and SMC phenotypic change, we showed that miR-21 expression was upregulated by miR-21 precursor. Interestingly, miR-21 overexpression significantly repressed the protein expressions of both CSE and SP1, inhibited H(2)S production, stimulated SMC proliferation, and reduced SMC differentiation marker gene expression, respectively. The mRNA expression of CSE but not SP1 was inhibited by miR-21 precursor. Blockage of SP1 binding by mithramycin or inhibition of CSE activity by DL-propargylglycine did not change miR-21 expression. We further demonstrated that miR-21 repressed SP1 protein expression by directly targeting at SP1 3' untranslational regions, which in turn downregulated CSE mRNA expression and stimulated SMC proliferation. Take together, these results suggest that miR-21 participates in CSE/H(2)S-mediated-SMC differentiation by targeting SP1.

Abstract 427: Transforming Growth Factor β/Smad3 Stimulates Smooth Muscle Cell Migration and Proliferation Through a Signaling Pathway that Involves C-X-C Chemokine Receptor Type 4 Expression and Activation of Mitogen-Activated Protein Kinase

Introduction: Intimal hyperplasia is a complex process that requires vascular smooth muscle cell (SMC) migration and proliferation. We have previously shown that levels of transforming growth factor beta (TGF-β) and its signaling protein Smad3 are elevated following arterial injury and contribute to intimal hyperplasia. In this study we exam whether these effects might be mediated through a mechanism that involves the chemokine stromal cell derived factor (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4). Methods and Results: A microarray was used to better understand the signaling pathways through which TGF-β and Smad3 stimulate SMC proliferation and migration. Overexpression of Smad3 in SMCs followed by stimulation with TGF-β (5 ng/ml) for 6 hours resulted in significant alteration of ∼300 genes, the most dramatic effect being a >500 fold upregulation of CXCR4. Quantitative RT-PCR and Western blotting confirmed that Smad3/TGF- β increased CXCR4 transcription and protein expression by 155±9 and 24±5 -fold, respectively. Immunocytochemistry and flowcytometry analysis revealed a 6-fold increase in CXCR4 positive cells in TGF-β stimulated Smad3 overexpressing SMCs compared to control (P<0.05). TGF-β/Smad3 treated SMCs exhibited enhanced migration (4-fold VS control, P<0.01) in response to SDF-1α (100 ng/ml) which was eliminated after preincubation of SMCs with CXCR4 inhibitor AMD3100 (10 μM). Moreover, there was a 45±9% increase in proliferation (BrdU) in response to SDF-1α in TGF-β/Smad3 treated SMCs compared to GFP control (P<0.05). To explore the mechanism by which SDF-1α/CXCR4 produces proliferation and migration in SMCs, we studied the intracelluar signaling protein mitogen-activated protein kinase (MAPK), which is a known intermediate for both processes. Incubation of TGF-β/Smad3 SMCs with SDF-1α led to activation of MAPK (11-fold) as well as nuclear translocation of CXCR4 in a dose and time dependent manner. Conclusions: Our data demonstrate that TGF-β through Smad3 stimulates expression of CXCR4 in medial vascular SMCs. These SMCs in response to SDF-1α then proliferate and migrate to the subintimal layer. These findings suggest a novel pathway through which TGF-β enhances intimal hyperplasia at sites of arterial injury.

TGF-β and Smad3 modulate PI3K/Akt signaling pathway in vascular smooth muscle cells.

Transforming growth factor-β (TGF-β) is upregulated at the time of arterial injury; however, the mechanism through which TGF-β enhances the development of intimal hyperplasia is not clear. Recent studies from our laboratory suggest that in the presence of elevated levels of Smad3, TGF-β stimulates smooth muscle cell (SMC) proliferation. This is a novel phenomenon in that TGF-β has traditionally been known as a potent inhibitor of cellular proliferation. In these studies we explore the signaling pathways through which TGF-β mediates its proliferative effect in vascular SMCs. We found that TGF-β phosphorylates and activates Akt in a time-dependent manner, and this effect is significantly enhanced by overexpression of Smad3. Furthermore, both chemical and molecular inhibition of Smad3 can reverse the effect of TGF-β on Akt. Although we found numerous signaling pathways that might function as intermediates between Smad3 and Akt, p38 appeared the most promising. Overexpression of Smad3 enhanced p38 phosphorylation and inhibition of p38 with a chemical inhibitor or a small interfering RNA blocked TGF-β-induced Akt phosphorylation. Moreover, TGF-β/Smad3 enhancement of SMC proliferation was blocked by inhibition of p38. Phosphorylation of Akt by TGF-β/Smad3 was not dependent on gene expression or protein synthesis, and immunoprecipitation studies revealed a physical association among p38, Akt, and Smad3 suggesting that activation requires a direct protein-protein interaction. Our findings were confirmed in vivo where overexpression of Smad3 in a rat carotid injury model led to enhancement of p-p38, p-Akt, as well as SMC proliferation. Furthermore, inhibition of p38 in vivo led to decreased Akt phosphorylation and SMC proliferation. In summary, our studies reveal a novel pathway whereby TGF-β/Smad3 stimulates SMC proliferation through p38 and Akt. These findings provide a potential mechanism for the substantial effect of TGF-β on intimal hyperplasia and suggest new targets for chemical or molecular prevention of vascular restenosis.

Signaling Mechanisms Mediating Uridine Adenosine Tetraphosphate–induced Proliferation of Human Vascular Smooth Muscle Cells

Proliferation of vascular smooth muscle cells (SMCs) plays an important role in the development of atherosclerosis and restenosis. Extracellular mononucleotides, such as adenosine triphosphate and uridine-5'-triphosphate stimulate SMC proliferation. However, the effects of dinucleotides on SMC proliferation and their underlying signaling mechanisms are less well defined. Recently, increasing evidence suggests that the dinucleotide, uridine adenosine tetraphosphate (Up4A) plays a role in the regulation of cardiovascular function. We have previously demonstrated that Up4A stimulates DNA synthesis and proliferation of human SMCs. This study investigated the signaling mechanisms underlying the proliferative effect of Up4A. Up4A-induced increase in bromodeoxyuridine incorporation was blocked by the mammalian target of rapamycin inhibitor, rapamycin, and the MEK inhibitor, PD98059. Up4A-stimulated phosphorylation and kinase activity of S6 kinase (S6K) and Erk1/2 were inhibited by PD98059, whereas phosphorylation and kinase activity of S6K, but not Erk1/2, were inhibited by rapamycin. Up4A also increased the phosphorylation of Akt, which was blocked by the PI3-kinase inhibitor, LY294002. Up4A-stimulated activation of S6K, but not Erk1/2, was also prevented by LY294002. Furthermore, Up4A-stimulated phosphorylation and kinase activity of S6K and Erk1/2 were inhibited by the P2 receptor antagonist, suramin, but not by the P2X receptor antagonist, Ip5I. Up4A also stimulated an increase in the protein expression of cycle-dependent kinase 2, which was prevented by rapamycin, PD98059, and suramin. These results suggest that the signaling mechanisms underlying the Up4A-stimulated proliferation of SMCs are mediated by P2Y receptors and involve the PI3-K/Akt and mitogen-activated protein kinase pathways, leading to the independent activation of S6K and an increase in cycle-dependent kinase 2 expression. This work stresses the concept that dinucleotides, like mononucleotides, play potentially important roles in the regulation of vascular function.