Human Coronary Artery Smooth Muscle Cells Proliferation Research Articles

Ginsenoside Rb1 protects human vascular smooth muscle cells against resistin-induced oxidative stress and dysfunction.

Resistin has been shown to play a key role in inducing vascular smooth muscle cells (VSMCs) malfunction in the atherosclerosis progression. Ginsenoside Rb1 is the main component of ginseng, which has been used for thousands of years and has been reported to have a powerful vascular protective effect. The aim of this study was to explore the protective effect of Rb1 on VSMCs dysfunction induced by resistin. In the presence or absence of Rb1, human coronary artery smooth muscle cells (HCASMC) were treated at different time points with or without 40 ng/ml resistin and acetylated low-density lipoprotein (acetylated LDL). Cell migration and proliferation were analyzed using wound healing test and CellTiter Aqueous Cell Proliferation Assay (MTS) test, respectively. Intracellular reactive oxygen species (ROS) (H2DCFDA as a dye probe) and superoxide dismutase (SOD) activities were measured by a microplate reader and the differences between groups were compared. Rb1 significantly reduced resistin-induced HCASMC proliferation. Resistin increased HCASMC migration time-dependently. At 20 µM, Rb1 could significantly reduce HCASMC migration. Resistin and Act-LDL increased ROS production to a similar level in HCASMCs, while Rb1 pretreated group reversed the effects of resistin and acetyl-LDL. Besides, the mitochondrial SOD activity was significantly reduced by resistin but was restored when pretreated with Rb1. We confirmed the protection of Rb1 on HCASMC and suggested that the mechanisms involved might be related to the reduction of ROS generation and increased activity of SOD. Our study clarified the potential clinical applications of Rb1 in the control of resistin-related vascular injury and in the treatment of cardiovascular disease.

Exploration of N6-Methyladenosine Profiles of mRNAs and the Function of METTL3 in Atherosclerosis.

Objectives: N6-methylladenosine (m6A) modification has not been fully studied in atherosclerosis. The objectives of this study were to investigate differentially expressed m6A methylated peaks and mRNAs, along with the regulatory role of methyltransferase 3 (METTL3) in pathological processes of atherosclerosis. Methods: The pathological models of human coronary artery smooth muscle cells (HCASMCs) were induced in vitro. The differentially expressed mRNAs and m6A peaks were identified by RNA-Seq and meRIP-Seq. The potential mechanisms were analyzed via bioinformatic assays. Methylases expression was tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) in HCASMCs, and by immunohistochemical assays in 40 human coronary arteries. The knockdown of METTL3 expression in cells was performed by siRNA transfection, and cell proliferation and migration were detected after transfection. Results: We identified 5121 m6A peaks and 883 mRNAs that were expressed differentially in the pathological processes of HCASMCs. Bioinformatic analyses showed that the different m6A peaks were associated with cell growth and cell adhesion, and the 883 genes showed that the extracellular matrix and PI3K/AKT pathway regulate the processes of HCASMCs. Additionally, 10 hub genes and 351 mRNAs with differential methylation and expression levels were found. METTL3 was upregulated in the arteries with atherosclerotic lesions and in the proliferation and migration model of HCASMCs, and pathological processes of HCASMCs could be inhibited by the knockdown of METTL3. The mechanisms behind regulation of migration and proliferation reduced by siMETTL3 are concerned with protein synthesis and energy metabolism. Conclusions: These results revealed a new m6A epigenetic method to regulate the progress of atherosclerosis, which suggest approaches for potential therapeutic interventions that target METTL3 for the prevention and treatment of coronary artery diseases.

Serum from Patients with Severe Alcoholic Liver Cirrhosis Inhibits Proliferation and Migration of Human Coronary Artery Smooth Muscle Cells.

Liver cirrhosis has been associated with an increased risk of coronary artery disease and clinical complications following percutaneous coronary revascularization. The present study is based on the hypothesis that cirrhosis may influence intimal hyperplasia following PCI. Sera from 10 patients with alcoholic liver cirrhosis and 10 age-matched healthy controls were used to stimulate cultured human coronary artery smooth muscle cells (HCASMC) for 48 h. HCASMC proliferation, migration, gene expression and apoptosis were investigated. Serum concentrations of growth factors and markers of liver function were also determined in patients and healthy controls. Treatment of HCASMC with patient sera reduced cell proliferation and migration (p < 0.05 vs. healthy controls), whereas apoptosis was unaffected (p = 0.160). Expression of genes associated with a synthetic vascular smooth muscle cell phenotype was decreased in cells stimulated with serum from cirrhotic patients (RBP1, p = 0.001; SPP1, p = 0.003; KLF4, p = 0.004). Platelet-derived growth factor-BB serum concentrations were lower in patients (p = 0.001 vs. controls). The results suggest the presence of circulating factors in patients with alcoholic liver cirrhosis affecting coronary smooth muscle cell growth. These findings may have implications for clinical outcomes following percutaneous coronary revascularization in these patients.

Shexiang Baoxin Pills Inhibited Proliferation and Migration of Human Coronary Artery Smooth Muscle Cells via PI3K/AKT/mTOR Pathway.

Background: Proliferation and migration of smooth muscle cells in the coronary artery contribute to the deterioration of coronary artery disease (CAD).Aim: This research was designed to study the function of Shexiang Baoxin pills (SBPs) on the proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) and their mechanism.Methods: Oxidized low-density lipoprotein (ox-LDL) was applied to stimulate the proliferation and migration of HCASMCs. The function of ox-LDL and SBP on HCASMCs was evidenced by the cell counting kit-8 assay, cell cycle, and Transwell assay. Network pharmacology was employed to predict the potential targets and pathways of SBP on CAD. Western blot assay and molecular docking were conducted to validate the potential targets and pathways.Results: The current research revealed that 2.5 mg/L SBP significantly inhibited the proliferation and migration of HCASMCs. Besides, network pharmacology revealed 11 candidate targets. Molecular docking and Western blot assay validated that the activation of the top 2 targets STAT3 and MAPK14 was associated with the inhibition of HCASMCs. Moreover, the Western blot assay also detected that HCASMCs treated with ox-LDL promoted the phosphorylation of the PI3K/AKT/mTOR pathway, and SBP inhibited the activation of the PI3K/AKT/mTOR pathway in HCASMCs stimulated by ox-LDL.Conclusion: This study demonstrated that the treatment of CAD using SBP may result from the suppression of the proliferation and migration of HCASMCs. The mechanism of this function partly resulted from relieving the phosphorylation of targets STAT3 and MAPK14 and the PI3K/AKT/mTOR pathway. This study enhanced our comprehension of SBP and provides new targets for the treatment of CAD.

LncRNA Landscape of Coronary Atherosclerosis Reveals Differentially Expressed LncRNAs in Proliferation and Migration of Coronary Artery Smooth Muscle Cells.

We aimed to investigate differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in atherosclerosis and validate the expression of lncRNAs and co-expressed target genes in proliferation and migration models of human coronary artery smooth muscle cells (HCASMCs). Ten coronary artery specimens from a subject who died from a heart attack were employed. The pathological analysis was analyzed by hematoxylin and eosin (H&E) staining, and the lncRNAs and mRNAs were identified by RNA sequencing. Bioinformatic analyses were performed to predict possible mechanisms. The proliferation and migration of HCASMCs were induced with oxidized low-density lipoprotein (ox-LDL). Differentially expressed lncRNAs and mRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). In this study, 68 lncRNAs and 222 mRNAs were identified differentially expressed in atherosclerosis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the Fanconi anemia pathway may be involved in atherosclerosis. GON4L was found to be the co-localized target gene of LNC_000439, and 14 genes had high correlations with the expression of seven lncRNAs. In addition, nine lncRNA–miRNA–mRNA networks were constructed, and 53 co-expressed gene modules were detected with weighted gene co-expression network analysis (WGCNA). LNC_000684, LNC_001046, LNC_001333, LNC_001538, and LNC_002115 were downregulated, while LNC_002936 was upregulated in proliferation and migration models of HCASMCs. In total, six co-expressed mRNAs were upregulated in HCASMCs. This study suggests that the differentially expressed lncRNAs identified by RNA sequencing and validated in smooth muscle cells may be a target for regulating HCASMC proliferation and migration in atherosclerosis, which will provide a new diagnostic basis and therapeutic target for the treatment of cardiovascular diseases.

G protein-coupled estrogen receptor GPR30 exerts vasoprotective effects in apolipoprotein E-deficient mice

IntroductionGPR30 is an intracellular transmembrane G protein-coupled receptor that mediates non-genomic estrogen signaling. The GPR30 agonist G-1 modulates glucose homeostasis and vascular function. However, its impact on vascular inflammation and atherogenesis has not yet been investigated in the atherosclerotic apolipoprotein E-deficient(ApoE-/-) mouse model.Material and methodsApoE-/- mice were fed a high-cholesterol diet for 7 weeks while being treated with the selective GPR30 agonist G-1 (n=6-7). After the treatment period, vascular relaxation capacity, vascular oxidative stress, and atherosclerotic plaque burden were assessed. In vitro, reactive oxygen species, expression levels of the angiotensin II type1(AT1) receptor, and proliferation rate were quantified in human coronary artery smooth muscle cells(HCASMC).ResultsG-1 significantly improved glucose tolerance in vivo (142.2±8.1mg/dl vs. 204.6±13.3mg/dl), G-1 reduced vascular oxidative stress (221±88RLU/s/mg vs.1,983±885RLU/s/mg) and improved endothelium-dependent vasodilation (relaxation to 35.1±4.5% vs.63.0±4.6%). Furthermore, treatment with G-1 significantly reduced the atherosclerotic plaque burden of female ApoE-/- mice (56.5±3.7% vs.75.5±2.9%). In vitro, G-1 provoked a significant downregulation of the AT1 receptor in HCASMC (0.67±0.09-fold). Furthermore, G-1 blunted angiotensin II-induced ROS production by HCASMC (817±7RLU/s/mg vs.1,625±105 RLU/s/mg) and diminished HCASMC proliferation (-26.8±2.7% vs.+50.4±1.7%).ConclusionsSelective GPR30 activation improves glucose tolerance in vivo and decreases vascular ROS production in vitro and in vivo. In vitro, the antioxidant effect might be mediated by downregulation of the AT1 receptor. In vivo, the antioxidant effect of G-1 is associated with an improved endothelial function and a reduced atherosclerotic plaque burden in ApoE-deficient mice, indicating beneficial vascular effects of GPR30 activation. GPR30 agonism might thus be a compelling treatment strategy against atherosclerosis.

The adipokine vaspin is associated with decreased coronary in-stent restenosis in vivo and inhibits migration of human coronary smooth muscle cells in vitro.

BackgroundPercutaneous coronary intervention represents the most important treatment modality of coronary artery stenosis. In-stent restenosis (ISR) is still a limitation for the long-term outcome despite the introduction of drug eluting stents. It has been shown that adipokines directly influence vessel wall homeostasis by influencing the function of endothelial cells and arterial smooth muscle cells. Visceral adipose tissue-derived serpin vaspin was recently identified as a member of serine protease inhibitor family and serveral studies could demonstrate a relation to metabolic diseases. The aim of this study was to investigate a role of vaspin in the development of in-stent restenosis in vivo and on migration of smooth muscle cells and endothelial cells in vitro.MethodsWe studied 85 patients with stable coronary artery disease who underwent elective and successful PCI with implatation of drug eluting stents. Blood samples were taken directly before PCI. Vaspin plasma levels were measured by specific ELISA. ISR was evaluated eight months later by coronary angiography. Human coronary artery smooth muscle cells (HCASMC) and human umbilical vein endothelial cells (HUVEC) migration was analyzed by an in-vitro migration assay with different concentrations (0.004ng/mL up to 40ng/mL) of vaspin as well as by an scratch assay. For proliferation an impedance measurement with specialiced E-Plates was performed.ResultsDuring the follow up period, 14 patients developed ISR. Patients with ISR had significantly lower vaspin plasma levels compared to patients without ISR (0.213 ng/ml vs 0.382 ng/ml; p = 0.001). In patients with plasma vaspin levels above 1.35 ng/ml we could not observe any restenosis. There was also a significant correlation of plasma vaspin levels and late lumen loss in the stented coronary segments. Further we could demonstrate that vaspin nearly abolishes serum induced migration of HCASMC (100% vs. 9%; p<0.001) in a biphasic manner but not migration of HUVEC. Proliferation of HCASMC and HUVEC was not modulated by vaspin treatment.ConclusionWe were able to show that the adipokine vaspin selectively inhibits human coronary SMC migration in vitro and has no effect on HUVEC migration. Vaspin had no effect on proliferation of HUVEC which is an important process of the healing of the stented vessel. In addition, the occurrence of ISR after PCI with implantation of drug eluting stents was significantly associated with low vaspin plasma levels before intervention. Determination of vaspin plasma levels before PCI might be helpful in the identification of patients with high risk for development of ISR after stent implantation. In addition, the selective effects of vaspin on smooth muscle cell migration could potentially be used to reduce ISR without inhibition of re-endothelialization of the stented segment.

P720Atheroprotective effects of 17beta-estradiol are mediated by PPARgamma in human coronary artery smooth muscle cells

Abstract Background 17β-estradiol (E2) mediates vasculoprotection in various preclinical and clinical models of atherosclerosis and neointimal hyperplasia. However, the molecular mechanisms underlying these effects are still not fully elucidated. Previous studies have demonstrated the essential role of the peroxisome-proliferator-activated-receptor-γ (PPARγ) in mediating vasculoprotective effects of E2 in vivo. The aim of the current study was to investigate whether PPARγ is implicated in mediating vasculoprotective mechanisms of E2 in human coronary artery smooth muscle cells (HCASMC). Methods Primary HCASMC were purchased and stimulated with E2 [10 nM], the selective estrogen receptor α (ERα) agonist propylpyrazole triol (PPT) [50 nM] and the selective ERα antagonist methyl-piperidino-pyrazole (MPP) [1 μM], respectively. Changes in PPARγ mRNA and protein expression upon stimulation of ERα were assessed by qPCR and Western blot analyses. Nuclear PPARγ protein expression and DNA binding affinity was assessed after the isolation of the nuclear protein fraction. Hereafter, HCASMC were incubated with E2, PPARγ-antagonist GW9662 [1 μM – 30 μM], or both. HCASMC proliferation was assessed by nuclear BrdU staining and reactive oxygen species (ROS) formation was assessed by L-012- and DCF-DA assays. Results E2 significantly increased PPARγ expression in HCASMC (1.95±0.41 –fold; n=5; p=0.0335). This effect was mimicked by ERα agonist PPT (1.63±0.27 –fold; n=7; p=0.0489) and was abrogated by co-incubation with ERα antagonist MPP (1.17±0.18 –fold; n=3; pvs. control &gt;0.05). Nuclear PPARγ expression was enhanced by E2 (1.53±0.16 –fold; n=4; pvs. control = 0.0074; Fig. 2A) whereas PPARγ's DNA binding activity to PPRE remained unchanged upon stimulation with E2 (0.94±0.11 –fold; n=4; pvs. control &gt;0.05). Pharmacological inhibition of PI3K/Akt by LY294002 abrogated E2-induced expression of PPARγ (0.24±0.09 –fold; n=3; pvs. E2 = 0.0017), arguing for a PI3K/Akt-dependent activation by E2. The role of PPARγ in mediating vasculoprotective effects of E2 was assessed in functional assays using PPARγ-antagonist GW9662. E2 diminished HCASMC proliferation which was restored by GW9662. While E2 only slightly decreased ROS production by HCASMC, GW9662 significantly increased ROS levels (1,036±169 RLU x s–1 x cell–1 versus 561±99 RLU x s–1 x cell–1; n=5–6; p=0.0287). Conclusion In summary, the present study identifies PPARγ as a downstream mediator of E2-related atheroprotective effects in HCASMC. 17β-estradiol regulates vascular PPARγ-expression in HCASMC via the ERα receptor and the PI3K/Akt pathway. PPARγ agonism might be a promising therapeutic strategy to prevent cardiovascular events in postmenopausal women with depleted E2 plasma levels. Acknowledgement/Funding This work was supported by the Bonfor program of the University of Bonn [grant number O-109.0057 to JJ].

Abstract 16886: Caspase-4/11 Mediates Intima Formation by Increasing Inflammation and Smooth Muscle Cell Migration and Proliferation

Introduction: Intima formation after vessel injury occurs via vascular smooth muscle cell(VSMC) proliferation, migration, dedifferentiation and inflammatory response. Recently, it was demonstrated that caspase-4 (Casp4, human)/caspase11(Casp11, mouse) increases the synthesis and release of inflammatory factors in macrophages, indicating a potential role of Casp4/Casp11 in intima formation. However, to date, the biological role of Casp4/Casp11 in vivo has not been investigated. Therefore, we hypothesize that Casp4/Casp11 deficiency will inhibit intima formation. Method and Results: We measured Casp4 expression and location by immunohistochemistry (IHC) staining in human coronary artery intima lesions from coronary artery disease patients. Surprisingly, we found Casp4 expression was remarkably increased in smooth muscle cells (SMC) and macrophages. We observed similar phenomena of Casp11 expression and location in intima of complete carotid ligation mouse model. These findings imply an important role of Casp4/11 in SMC function. Thus, we performed complete carotid ligation on Casp11 wild type (WT) and knockout (KO) mice.Morphometric analysis demonstrated that Casp11 depletion robustly attenuated intima area and intima/media ratio (I/M) compared with WT mice . Inflammation was quantified by CD45 IHC staining . SMC proliferation was detected by PCNA staining and there was a marked decrease of PCNA positive SMCs. In vitro, Casp4 expression was time- and dose-dependently increased by platelet-derived growth factor(PDGF), angiotensin II(AngII) and TNF-α in human coronary artery SMCs(HCASMCs). Depletion of Casp4 by siRNA decreased expression of cytokines, e.g. IL-1α, IL-1β, IL-6, as well as protein expression of adhesion factors (VCAM-1, ICAM-1, MCP-1) induced by PDGF, AngII or TNF-α. Moreover, HCASMC proliferation stimulated by PDGF was measured by EdU cell proliferation assay and was inhibited by 65% after knockdown of Casp4. Wound scratch assay demonstrated impaired HCASMC migration in Casp4 depleted cells. However, Casp4 has no effects on HCASMCs dedifferentiation. Mechanistically, Casp4 knockdown significantly inhibited NF-κB signaling. Conclusions: Casp4/11 promotes intima formation by increasing SMC proliferation, migration and inflammation through NF κB signaling. Specific inhibition of the Casp4/11 will provide novel therapeutic strategy for vascular remodeling related diseases.

MiR-365b-3p inhibits the cell proliferation and migration of human coronary artery smooth muscle cells by directly targeting ADAMTS1 in coronary atherosclerosis.

Abnormal proliferation and migration of vascular smooth muscle cells serves a crucial role in the development of atherosclerosis. Previous studies have suggested that some microRNAs (miRs) are involved in this process; however, the associated underlying molecular mechanism is unclear. In present study, human coronary artery smooth muscle cells (HCASMCs) were used to explore the function of miR-365b-3p in the coronary atherosclerosis. It was indicated that platelet-derived growth factor-BB (PDGF-BB) treatment inhibited miR-365b-3p expression and upregulated the expression of a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) in HCASMCs. Subsequently, miR-365b-3p mimic was transfected in HCASMCs to explore the function of this miR. The results of reverse transcription-quantitative polymerase chain reaction and western blot analysis indicated that overexpression of miR-365b-3p significantly downregulated ADAMTS1 expression. Functional assay results revealed that overexpression of miR-365b-3p significantly attenuated PDGF-BB-induced proliferation and migration of HCASMCs. Furthermore, the dual-luciferase reporter assay results confirmed that ADAMTS1 is a direct target gene of miR-365b-3p. This discovery proposed a novel channel of communication between ADAMTS1 and HCASMCs, and suggests a potential therapeutic approach for coronary atherosclerosis.

Convulxin, a C-type lectin-like protein, inhibits HCASMCs functions via WAD-motif/integrin-αv interaction and NF-κB-independent gene suppression of GRO and IL-8

Convulxin (CVX), a C-type lectin-like protein (CLPs), is a potent platelet aggregation inducer. To evaluate its potential applications in angiogenic diseases, the multimeric CVX were further explored on its mode of actions toward human coronary artery smooth muscle cells (HCASMCs). The N-terminus of β-chain of CVX (CVX-β) contains a putative disintegrin-like domain with a conserved motif upon the sequence comparison with other CLPs. Importantly, native CVX had no cytotoxic activity as examined by electrophoretic pattern. A Trp-Ala–Asp (WAD)-containing octapeptide, MTWADAEK, was thereafter synthesized and analyzed in functional assays. In the case of specific integrin antagonists as positive controls, the anti-angiogenic effects of CVX on HCASMCs were investigated by series of functional analyses. CVX showed to exhibit multiple inhibitory activities toward HCASMCs proliferation, adhesion and invasion with a dose- and integrin αvβ3-dependent fashion. However, the WAD-octapeptide exerting a minor potency could also work as an active peptidomimetic. In addition, flow cytometric analysis demonstrated both the intact CVX and synthetic peptide can specifically interact with integrin-αv on HCASMCs and CVX was shown to have a down-regulatory effect on the gene expression of CXC-chemokines, such as growth-related oncogene and interleukin-8. According to nuclear factor-κB (NF-κB) p65 translocation assay and Western blotting analysis, the NF-κB activation was not involved in the signaling events of CVX-induced gene expression. In conclusion, CVX may act as a disintegrin-like protein via the interactions of WAD-motif in CVX-β with integrin-αv on HCASMCs and it also is a gene suppressor with the ability to diminish the expression of two CXC-chemokines in a NF-κB-independent manner. Indeed, more extensive investigations are needed and might create a new avenue for the development of a novel angiostatic agent.

Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D.

The goal of this study was to determine whether and how adenosine affects the proliferation of human coronary artery smooth muscle cells (HCASMCs). In HCASMCs, 2-chloroadenosine (stable adenosine analogue), but not N(6)-cyclopentyladenosine, CGS21680, or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide, inhibited HCASMC proliferation (A2B receptor profile). 2-Chloroadenosine increased cAMP, reduced phosphorylation (activation) of ERK and Akt (protein kinases known to increase cyclin D expression and activity, respectively), and reduced levels of cyclin D1 (cyclin that promotes cell-cycle progression in G1). Moreover, 2-chloroadenosine inhibited expression of S-phase kinase-associated protein-2 (Skp2; promotes proteolysis of p27(Kip1)) and upregulated levels of p27(Kip1) (cell-cycle regulator that impairs cyclin D function). 2-Chloroadenosine also inhibited signaling downstream of cyclin D, including hyperphosphorylation of retinoblastoma protein and expression of cyclin A (S phase cyclin). Knockdown of A2B receptors prevented the effects of 2-chloroadenosine on ERK1/2, Akt, Skp2, p27(Kip1), cyclin D1, cyclin A, and proliferation. Likewise, inhibition of adenylyl cyclase and protein kinase A abrogated 2-chloroadenosine's inhibitory effects on Skp2 and stimulatory effects on p27(Kip1) and rescued HCASMCs from 2-chloroadenosine-mediated inhibition. Knockdown of p27(Kip1) also reversed the inhibitory effects of 2-chloroadenosine on HCASMC proliferation. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 μmol/L for 7 days) downregulated vascular expression of Skp2, upregulated vascular expression of p27(Kip1), and reduced neointima hyperplasia by 71% (P<0.05; neointimal thickness: control, 37 424±18 371 pixels; treated, 10 352±2824 pixels). In conclusion, the adenosine/A2B receptor/cAMP/protein kinase A axis inhibits HCASMC proliferation by blocking multiple signaling pathways (ERK1/2, Akt, and Skp2) that converge at cyclin D, a key G1 cyclin that controls cell-cycle progression.

Angiotensin II Downregulates MicroRNA-145 to Regulate Kruppel-like Factor 4 and Myocardin Expression in Human Coronary Arterial Smooth Muscle Cells under High Glucose Conditions.

MicroRNA (miR)-145 is the most abundant miR in vascular smooth muscle cells (VSMCs). However, the effect of hyperglycemia on the regulation of miR-145 is unknown. We hypothesized that the hyperglycemic condition activates a proinflammatory response that mediates the expression of miR-145 in VSMCs. We investigated whether miR-145 serves as a critical regulator to regulate the downstream proliferation factors (including Kruppel-like factor 4 [Klf4] and myocardin) in VSMCs under hyperglycemic conditions. Human coronary artery smooth muscle cells (HCASMCs) were cultured under high glucose conditions. Sustained high glucose at 25 mmol/L significantly decreased the expression of miR-145 in HCASMCs. High glucose significantly increased angiotensin II (Ang II) secretion from HCASMCs and Ang II suppressed miR-145 expression in HCASMCs. Ang II repression of miR145 expression resulted in increased Klf4 and decreased myocardin expression under conditions of high glucose. Overexpression of miR-145 significantly decreased Klf4 and increased myocardin expression and inhibited HCASMC proliferation and migration induced by a high glucose state. Balloon injury of the carotid artery in diabetic rats was performed to investigate miR-145, Klf and myocardin expression. The expression of miR-145 was maximally increased at 7 d after carotid injury and gradually declined thereafter. Overexpression of miR-145 and treatment with valsartan reversed Klf4 and myocardin protein expression induced by balloon injury and improved vascular injury. In conclusion, our study reveals that Ang II downregulates miR-145 to regulate Klf4 and myocardin expression in HCASMCs under high glucose conditions. Ang II plays a critical role in the regulation of miR-145 under hyperglycemic conditions.

Abstract 518: Differential Effects of Alcohol and Its Primary Metabolite Acetaldehyde on Vascular Smooth Muscle Cell Notch Signaling and Growth

Moderate consumption of alcohol (ethanol/EtOH) is a negative risk factor for atherosclerosis while binge drinking and chronic alcohol abuse is associated with increased cardiovascular disease and mortality. As the balance between the levels of EtOH itself and its primary metabolite, acetaldehyde, is likely different in these two drinking groups, we assessed the hypothesis that differential effects of EtOH and acetaldehyde on vascular smooth muscle cell (SMC) Notch signaling and growth may be key in mediating the effects of various drinking patterns on cardiovascular disease. Human coronary artery smooth muscle cells (HCASMC) were treated (24 h) with either EtOH (25 mM) or acetaldehyde (10 or 25 μM). We confirmed our previously reported inhibitory effects of EtOH on Notch signaling and subsequently on the growth of HCASMC. In contrast, acetaldehyde treatment stimulated HCASMC proliferation (determined by cell counts) and increased proliferating cell nuclear antigen expression, concomitant with stimulation of Notch signaling as determined by increased Notch target gene (HRT 1-3) mRNA levels. Interaction of a ligand (e.g., Dll4) with the Notch receptor initiates proteolytic cleavage by γ-secretase, resulting in the release of the active Notch-intracellular domain (NICD). A fluorogenic peptide cleavage assay demonstrated almost complete inhibition by EtOH of Dll4-stimulated γ-secretase activity in a solubilized membrane fraction from HCASMC (similar to the effect of the control inhibitor, DAPT), but no effect of acetaldehyde treatment. Similarly, EtOH, but not acetaldehyde, affected the association and distribution of the γ-secretase catalytic subunit, presenilin-1, with lipid rafts as determined by dual fluorescent labeling and confocal microscopic visualization (Alexa 488-conjugated cholera toxin B subunit for lipid rafts, and Alexa 594-conjugated anti-presenilin-1 antibody). In conclusion, EtOH and acetaldehyde have opposite effects on SMC Notch signaling and growth. These differential effects of alcohol and its metabolite may be relevant in mediating the opposing effects of different drinking patterns (e.g., daily moderate vs weekend binge) on cardiovascular disease.

Oxidized low-density lipoprotein increases the proliferation and migration of human coronary artery smooth muscle cells through the upregulation of osteopontin

Smooth muscle cell (SMC) proliferation and migration are known to play a critical role in the development of atherosclerosis. Oxidized low-density lipoprotein (oxLDL) is involved in the generation of atherosclerotic lesions. Recent studies have indicated that oxLDL is a well-established risk factor for atherosclerosis that induces vascular smooth muscle cell (VSMC) proliferation and migration; however, the exact mechanisms involved have not been fully elucidated. In this study, the proliferation of human coronary artery smooth muscle cells (HCASMCs) was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell migration was determined by Transwell assay. Osteopontin (OPN), matrix metaloproteinase-9 (MMP-9) and αvβ3 integrin expression were measured by mRNA and western blot analysis. OPN and MMP-9 knockdown cells were established through transfection with OPN siRNA or MMP-9 siRNA, respectively. Our results revealed that oxLDL makredly promoted HCASMC proliferation and migration in a dose-dependent manner. Further experiments demonstrated that oxLDL upregulated the expression of OPN and oxLDL. Cell proliferation and migration were markedly reduced following the knockdown of the OPN gene in the HCASMCs. We then found that treatment with oxLDL induced a concentration-dependent increase in MMP-9 mRNA and protein levels in the HCASMCs. These effects were partially abrogated by silencing OPN expression or blocking the αvβ3 integrin pathway. Moreover, cells treated with MMP-9 siRNA or αvβ3 antibody showed lower proliferation and migration rates. This study provides direct in vitro evidence that the exposure of HCASMCs to oxLDL induces the activation of OPN, leading to higher protein levels of MMP-9, and to an increased proliferation and migration of HCASMCs.

FABP4 Induces Vascular Smooth Muscle Cell Proliferation and Migration through a MAPK-Dependent Pathway

PurposeThe migration and proliferation of vascular smooth muscle cells play crucial roles in the development of atherosclerotic lesions. This study examined the effects of fatty acid binding protein 4 (FABP4), an adipokine that is associated with cardiovascular risk, endothelial dysfunction and proinflammatory effects, on the migration and proliferation of human coronary artery smooth muscle cells (HCASMCs). Methods and ResultsA DNA 5-bromo-2′-deoxy-uridine (BrdU) incorporation assay indicated that FABP4 significantly induced the dose-dependent proliferation of HCASMCs with a maximum stimulatory effect at 120 ng/ml (13% vs. unstimulated cells, p<0.05). An anti-FABP4 antibody (40 ng/ml) significantly inhibited the induced cell proliferation, demonstrating the specificity of the FABP4 proliferative effect. FABP4 significantly induced HCASMC migration in a dose-dependent manner with an initial effect at 60 ng/ml (12% vs. unstimulated cells, p<0.05). Time-course studies demonstrated that FABP4 significantly increased cell migration compared with unstimulated cells from 4 h (23%vs. 17%, p<0.05) to 12 h (74%vs. 59%, p<0.05). Pretreatment with LY-294002 (5 µM) and PD98059 (10 µM) blocked the FABP4-induced proliferation and migration of HCASMCs, suggesting the activation of a kinase pathway. On a molecular level, we observed an up-regulation of the MAPK pathway without activation of Akt. We found that FABP4 induced the active forms of the nuclear transcription factors c-jun and c-myc, which are regulated by MAPK cascades, and increased the expression of the downstream genes cyclin D1 and MMP2, CCL2, and fibulin 4 and 5, which are involved in cell cycle regulation and cell migration. ConclusionsThese findings indicate a direct effect of FABP4 on the migration and proliferation of HCASMCs, suggesting a role for this adipokine in vascular remodelling. Taken together, these results demonstrate that the FABP4-induced DNA synthesis and cell migration are mediated primarily through a MAPK-dependent pathway that activates the transcription factors c-jun and c-myc in HCASMCs.

188 DRUG-ELUTING STENTS CONTAINING IBUPROFEN: A NOVEL STRATEGY TO REDUCE RESTENOSIS AND PREVENT LATE IN-STENT THROMBOSIS

<h3>Introduction</h3> Drug-eluting stents (DES) have reduced the rates of restenosis to less than 10%; however, increased incidence of late in-stent thrombosis, is now a major clinical problem of using DES for its consequences in terms of morbidity and mortality. Interference with the process of re-endothelialization appears to play a major role in this latent complication. Thus, the search for molecular compounds capable of preventing restenosis that also can reduce the risk of late in-stent thrombosis is of utmost importance. Previously, we have shown that certain non-steroidal anti-inflammatory drugs (NSAIDs) are capable of inhibiting rat vascular smooth muscle cell proliferation, a key element in the process of restenosis post stent deployment. Here, we evaluated whether proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) is reduced by the NSAID, ibuprofen, whether this correlated with changes in the phenotype of HCASMCs and the potential molecular mechanisms involved. Also, we investigated whether human coronary artery endothelial cell (HCAEC) migration was affected by ibuprofen. <h3>Methods</h3> Cell proliferation was evaluated by trypan blue exclusion. Cell migration was assessed by wound healing assay and by time lapse videomicroscopy. Protein expression was assessed by immunoblotting and morphology by immunocytochemistry. The involvement of the PPARγ pathway was studied with the selective agonist, troglitazone, and with the PPARγ antagonists, PGF2α and GW9662. <h3>Results</h3> We show that ibuprofen inhibited proliferation as well as migration of HCASMCs in a dose-dependent manner (IC₅₀ of 680 and 410 µM, respectively). Interestingly, we found that ibuprofen induced a switch in HCASMCs towards a differentiated phenotype, with a characteristic spindle shape, and a significant increase in the expression of contractile protein markers, e.g. SMα-actin, SM22α and F-actin. PPARγ antagonists, PGF2α and GW9662, almost completely abrogated the proliferative and migratory responses of HCASMCs, as well the changes in morphology. However, PPARγ antagonists did not affect the expression pattern of contractile proteins, suggesting that these effects are mediated by a PPARγ-independent pathway. Importantly, ibuprofen did not affect migration of HCAECs even at high doses (up to 1000 µM), suggesting that the effects of ibuprofen on HCASMCs are selective. Interestingly, the protein levels of PPARγ are higher in HCAECs compared to those in HCASMCs. <h3>Conclusions</h3> Taken together, our results suggest that ibuprofen could be an effective treatment for the development of novel DES. Its effects on migration and proliferation of HCASMCs, and induction of a differentiated phenotype, could translate into reduced rates of restenosis, whilst the lack of an effect on HCAEC migration could result in a reduced risk of late in-stent thrombosis. Further studies in animal models will be required to evaluate the performance of an ibuprofen DES <i>in vivo</i>.

Human Leukocyte Antigen-G Inhibits Human Coronary Artery Smooth Muscle Cell Proliferation

Purpose Heart transplantation is currently the accepted therapy for patients with end-stage heart failure. However, long-term survival is limited by morbidity due to cardiac allograft vasculopathy (CAV). Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein with restricted tissue expression, plays an essential role in immune tolerance and has been inversely associated with acute cellular rejection and cardiac allograft vasculopathy after heart transplantation. The proliferation of human coronary artery smooth muscle cells (HCASMC) within the intimal layer of the blood vessel might represent the most critical cellular event associated with CAV. The concentric intimal thickening with narrowing of the vessel lume leads to obstructive lesions. Our objective was to evaluate for the first time the antiproliferative properties of the HLA-G molecule on HCASMC. Methods and Materials Commercially available primary HCASMC (n=8) were seeded for 24h, and then exposed to recombinant human HLA-G at various concentrations (100-1000 ng/ml). Proliferation was measured at various time points (24-120 hours) post-treatment by automated cell counting. Results At 24 and 48 hours of treatment there was no significant difference in proliferation between the groups. At 120 hours post-treatment, the proliferation of cells treated with HLA-G at 500 and 1000ng/ml was significantly lower ( * p=0.041 and * p=0.004, respectively) than that of the untreated, control group. At 120 hours there was also a significant decrease in proliferation of the cells treated with 1000ng/ml when compared to the group exposed to 100ng/ml ( * p=0.020). The viability of the cells was similar at all time-points. Conclusions We have shown for the first time that HLA-G inhibits HCASMC proliferation in an in vitro setting. The use of HLA-G in reducing smooth muscle cell proliferation within the coronary artery may represent a promising and novel therapeutic strategy to protect against allograft vasculopathy following heart transplantation.

379 Human Leukocyte Antigen-G Inhibits Human Coronary Artery Smooth Muscle Cell Proliferation

Heart transplantation is currently the accepted therapy for patients with end-stage heart failure. However, long-term survival is limited by morbidity due to cardiac allograft vasculopathy (CAV). Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein with restricted tissue expression, plays an essential role in immune tolerance and has been inversely associated with acute cellular rejection and cardiac allograft vasculopathy after heart transplantation. The proliferation of human coronary artery smooth muscle cells (HCASMC) within the intimal layer of the blood vessel might represent the most critical cellular event associated with CAV. The result of HCASMC proliferation is a concentric intimal thickening with narrowing of the vessel lumen which eventually leads to obstructive lesions. Our objective was to evaluate for the first time the antiproliferative properties of the HLA-G molecule on HCASMC. Commercially available primary HCASMC (n=8) were seeded for 24h in culture medium containing 5% fetal bovine serum. The cells were then exposed to recombinant human HLA-G at various concentrations (100-1000 ng/ml). Proliferation was measured at various time points (24-120 hours) post-treatment by automated cell counting. After 24 hours of treatment there was no significant difference in proliferation between the groups. At 48 hours post-treatment the proliferation of cells treated with HLA-G at 1000ng/ml was significantly lower (p=.017) than that of the untreated, control group. Similarly, at 120 hours post-treatment, the cells treated with 1000ng/ml of HLA-G displayed significantly lower (p=.043) proliferation than the controls. The viability of the cells was similar at all time-points. We have shown for the first time that HLA-G inhibits HCASMC proliferation in an in vitro setting. Our results are in agreement with previous clinical studies which have shown that the presence of HLA-G pre- and post-transplantation correlates with reduced incidence of acute rejection and confers protection against CAV. The use of HLA-G in reducing smooth muscle cell proliferation within the coronary artery may represent a promising and novel therapeutic strategy to protect against allograft vasculopathy following heart transplantation.

613 Everolimus induces HLA-G expression in human coronary artery smooth muscle cells: Impact on graft rejection and cardiac allograft vasculopathy

Human leukocyte antigen-G (HLA-G) is a non-classical MHC I protein that plays an important role in immune tolerance. Its expression in serum and endomyocardial biopsies of heart transplant patients is associated with a lower incidence of graft rejection and cardiac allograft vasculopathy (CAV). However, the mechanisms of HLA-G expression remain vague. Previous clinical studies found that patients treated with everolimus expressed significantly higher levels of HLA-G in the blood plasma. Our objective was to determine if everolimus upregulates HLA-G in human coronary artery smooth muscle cells (HCASMC), explaining a potential mechanism of HLA-G expression after heart transplant. We also tested if the soluble HLA-G inhibits HCASMC proliferation, a main cause of intimal hyperplasia associated with CAV. Commercially available HCASMC (n = 4) were cultured in media containing 5% fetal bovine serum. After reaching full confluence, the cells were exposed to various doses of everolimus (0.1-100 ng/ml) for 24 hours. HLA-G expression was assessed by Western blot. We also cultured HCASMC (n = 8) in conditioned media from Jeg-3 cells, choriocarcinoma cells known to release soluble HLA-G. Cell proliferation was measured at various time points (24-120 hours) post treatment. There was a significant dose-dependent increase in HLA-G expression in the cells treated with 100ng/ml of everolimus when compared to the control group (P = 0.012) (Figure) . Interestingly, there was no significant difference in proliferation between the cells treated with HLA-G conditioned media and the control group. We have shown for the first time that everolimus induces HLA-G expression in HCASMC in a dose-dependent manner. We have also shown that soluble HLA-G does not inhibit HCASMC proliferation in an in-vitro setting. Induction of HLA-G expression in HCASMC may represent a promising and novel therapeutic strategy to protect against graft rejection after heart transplantation. Although higher HLA-G levels protect against graft rejection, our results suggest that HLA-G does not inhibit SMC proliferation, implying that other cellular pathways may play a role in reducing CAV formation post transplant.