Human Coronary Artery Smooth Muscle Research Articles

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>.

Abstract 139: Post-Translational Modifications of Connexins in Neointimal Formation

Intro Connexins are integrally involved in the process of cell division and atherosclerosis. Recently we identified that connexin 43 (Cx43) could significantly alter smooth muscle cell proliferation in vitro and in vivo. We now investigate the involvement of Cx43 in human neointimal formation and the mechanisms through which it controls cell cycle. Methods Protein interactions between Cx43 were identified in smooth muscle cells through co-immunoprecipitation, immuno-transmission electron microscopy and proximity ligation assays. Novel mice with alanine substitutions at MAPK phosphorylated serines (Cx43-MK4A) were investigated. Samples from diseased and non-diseased human coronary arteries were assessed for level of Cx43 phosphorylation and interactions. Ex vivo saphenous vein explant cultures were used to assess the contribution of Cx43 phosphorylation to human neointimal formation. In vitro studies were performed in human coronary artery smooth muscle cells and in mouse aortic smooth muscle cells from wild type and knockout mice. Results We demonstrate that levels of Cx43 are increased in human atherosclerotic plaques and that neointimal formation in human tissues corresponds to a significant increase in Cx43 phosphorylation and cyclin E interactions. In vitro, Cx43 facilitates trafficking of cyclin E to the nucleus in a manner that is dependent on its MAPK phosphorylation. Discussion Our studies suggest that in human vascular disease and neointimal formation Cx43 interactions with cyclin E are elevated. Further, Cx43 facilitates the transport of the cell cycle promoter cyclin E to the nucleus suggesting that this is a critical component of VSMC proliferation and therefore could be a novel target in the prevention of neointimal formation in human vascular disease.

Components of the interleukin-33/ST2 system are differentially expressed and regulated in human cardiac cells and in cells of the cardiac vasculature

Interleukin-33 (IL-33) is a recently described member of the IL-1 family of cytokines, which was identified as a ligand for the ST2 receptor. Components of the IL-33/ST2 system were shown to be expressed in normal and pressure overloaded human myocardium, and soluble ST2 (sST2) has emerged as a prognostic biomarker in myocardial infarction and heart failure. However, expression and regulation of IL-33 in human adult cardiac myocytes and fibroblasts was not tested before. In this study we found that primary human adult cardiac fibroblasts (HACF) and human adult cardiac myocytes (HACM) constitutively express nuclear IL-33 that is released during cell necrosis. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-1β significantly increased both IL-33 protein and IL-33 mRNA expression in HACF and HACM as well as in human coronary artery smooth muscle cells (HCASMC). The nuclear factor-κB (NF-κB) inhibitor dimethylfumarate inhibited TNF-α- and IL-1β-induced IL-33 production as well as nuclear translocation of p50 and p65 NF-κB subunits in these cells. Mitogen-activated protein/extracellular signal-regulated kinase inhibitor U0126 abrogated TNF-α-, IFN-γ-, and IL-1β-induced and Janus-activated kinase inhibitor I reduced IFN-γ-induced IL-33 production. We detected IL-33 mRNA in human myocardial tissue from patients undergoing heart transplantation (n=27) where IL-33 mRNA levels statistically significant correlated with IFN-γ (r=0.591, p=0.001) and TNF-α (r=0.408, p=0.035) mRNA expression. Endothelial cells in human heart expressed IL-33 as well as ST2 protein. We also reveal that human cardiac and vascular cells have different distribution patterns of ST2 isoforms (sST2 and transmembrane ST2L) mRNA expression and produce different amounts of sST2 protein. Both human macrovascular (aortic and coronary artery) and heart microvascular endothelial cells express specific mRNA for both ST2 isoforms (ST2L and sST2) and are a source for sST2 protein, whereas cardiac myocytes, cardiac fibroblasts and vascular SMC express only minor amounts of ST2 mRNA and do not secrete detectable amounts of sST2 antigen. In accordance with the cellular distribution of ST2 receptor, human cardiac fibroblasts and myocytes as well as HCASMC did not respond to treatment with IL-33, as recombinant human IL-33 did not induce NF-κB p50 and p65 subunits nuclear translocation or increase IL-6, IL-8, and monocyte chemoattractant protein (MCP-1) level in HACF, HACM and HCASMC. In summary, we found that endothelial cells seem to be the source of sST2 and the target for IL-33 in the cardiovascular system. IL-33 is expressed in the nucleus of human adult cardiac fibroblasts and myocytes and released during necrosis. Proinflammatory cytokines TNF-α, IFN-γ and IL-1β increase IL-33 in these cells in vitro, and IL-33 mRNA levels correlated with TNF-α and IFN-γ mRNA expression in human myocardial tissue.

Increased endothelin-1 responsiveness in human coronary artery smooth muscle cells exposed to 1,25-dihydroxyvitamin D3

Low blood concentrations of 25-hydroxyvitamin D(3) are associated with increased mortality, while some studies suggest improved cardiovascular outcomes with vitamin D(3) supplementation in chronic kidney disease. However, the physiological effects of vitamin D(3) on the cardiovascular system remain poorly understood making it difficult to determine whether vitamin D(3) supplementation might provide cardiovascular benefit or even cause harm. Thus here we investigated the effects of chronic 1,25-dihydroxyvitamin D(3) treatment on intracellular signaling in human coronary artery smooth muscle cells (HCASMCs) and found that 1,25-dihydroxyvitamin D(3) significantly potentiated endothelin (ET-1) signaling. Specifically, 1,25-dihydroxyvitamin D(3) (24-h pretreatment) caused a more than threefold enhancement in both ET-1-induced intracellular calcium mobilization and extracellular signal-regulated kinase (ERK) activation. This 1,25-dihydroxyvitamin D(3)-elicited signaling enhancement was not observed for either vasopressin or carbachol. With the use of endothelin receptor (ETR) isoform-selective antagonists, ETRA was found to be primarily responsible for the 1,25-dihydroxyvitamin D(3)-induced ET-1 responsiveness and yet ETRA mRNA expression and protein abundance were unaltered following 1,25-dihydroxyvitamin D(3) treatment. While there was an increase in ETRB mRNA expression in response to 1,25-dihydroxyvitamin D(3), the protein abundance of ETRB was again unchanged. Finally, ETRA/ETRB heterodimerization was not detected in HCASMCs in either the absence or presence of 1,25-dihydroxyvitamin D(3). Together, these data show for the first time that 1,25-dihydroxyvitamin D(3) enhances endothelin responsiveness in HCASMCs and that the effect is mediated through ETRA.

ASSA13-03-31 In Vitro Investigations on the Effects of Arsenic Trioxide on Human Coronary Artery Smooth Muscle Cells, Endothelial Cells and EMAP-II

ObjectiveTo investigate the effects of arsenic trioxide on human coronary artery smooth muscle cells, endothelial cells and expres-sion of EMAP-II in the extracellular matrix.MethodsHuman coronary artery smooth muscle cell line...

Characterization of tocopherol kinase activity in primary human coronary artery smooth muscle cells

The vitamin E derivative, alpha‐tocopheryl phosphate (αTP), is detectable in tissues in small quantity (30–300 nM) suggesting the existence of enzyme(s) with α‐tocopherol (αT) kinase activity. Here, we have characterized the production of αTP from αT and [γ‐32P]‐ATP in primary human coronary artery smooth muscle cells (HCA‐SMC) using thin layer chromatography (TLC) followed by ultra performance liquid chromatography (UPLC). In addition to αT, γT is also phosphorylated. γTP inhibits proliferation of THP‐1 cells and reduces CD36 expression more potently than αTP (10 μM). The recombinant human tocopherol associated protein (TAP1/SEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to the putative αT kinase in the correct spatial orientation. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol‐3‐kinase gamma (PI3Kγ) potentially by sequestering the substrate phosphatidylinositol (PI). Indeed, the addition αT or αTP to the reaction stimulates PI3Kγ, most likely by facilitating egress of PI from hTAP1 and its binding to the enzyme. It is suggested that the continuous mutual exchange of different ligands in the metabolic nanoreactor formed by hTAPs may be a way to make these lipophiles more accessible as substrates to enzymes and as constituents to specific membrane domains. Supported by USDA Contract #58–1950‐0–014.

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.

Anti‐angiogenic and anti‐tumor effects of TAK‐593, a potent and selective inhibitor of vascular endothelial growth factor and platelet‐derived growth factor receptor tyrosine kinase

We recently reported that TAK-593, a novel imidazo[1,2-b]pyridazine derivative, is a highly potent and selective inhibitor of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor tyrosine kinase families. Moreover, TAK-593 exhibits a uniquely long-acting inhibitory profile towards VEGF receptor 2 (VEGFR2) and PDGF receptor β (PDGFRβ). In this study, we demonstrated that TAK-593 potently inhibits VEGF- and PDGF-stimulated cellular phosphorylation and proliferation of human umbilical vein endothelial cells and human coronary artery smooth muscle cells. TAK-593 also potently inhibits VEGF-induced tube formation of endothelial cells co-cultured with fibroblasts. Oral administration of TAK-593 exhibited strong anti-tumor effects against various human cancer xenografts along with good tolerability despite a low level of plasma exposure. Even after the blood and tissue concentrations of TAK-593 decreased below the detectable limit, a pharmacodynamic marker (phospho VEGFR2) was almost completely suppressed, indicating that its long duration of enzyme inhibition might contribute to the potent activity of TAK-593. Immunohistochemical staining indicated that TAK-593 showed anti-proliferative and pro-apoptotic effects on tumors along with a decrease of vessel density and inhibition of pericyte recruitment to microvessels in vivo. Furthermore, dynamic contrast-enhanced magnetic resonance imaging revealed that TAK-593 reduced tumor vessel permeability prior to the onset of anti-tumor activity. In conclusion, TAK-593 is an extremely potent VEGFR/PDGFR kinase inhibitor whose potent anti-angiogenic activity suggests therapeutic potential for the treatment of solid tumors.

Aldosterone Increases Early Atherosclerosis and Promotes Plaque Inflammation Through a Placental Growth Factor‐Dependent Mechanism

BackgroundAldosterone levels correlate with the incidence of myocardial infarction and mortality in cardiovascular patients. Aldosterone promotes atherosclerosis in animal models, but the mechanisms are poorly understood.Methods and ResultsAldosterone was infused to achieve pathologically relevant levels that did not increase blood pressure in the atherosclerosis‐prone apolipoprotein E–knockout mouse (ApoE−/−). Aldosterone increased atherosclerosis in the aortic root 1.8±0.1‐fold after 4 weeks and in the aortic arch 3.7±0.2‐fold after 8 weeks, without significantly affecting plaque size in the abdominal aorta or traditional cardiac risk factors. Aldosterone treatment increased lipid content of plaques (2.1±0.2‐fold) and inflammatory cell content (2.2±0.3‐fold), induced early T‐cell (2.9±0.3‐fold) and monocyte (2.3±0.3‐fold) infiltration into atherosclerosis‐prone vascular regions, and enhanced systemic inflammation with increased spleen weight (1.52±0.06‐fold) and the circulating cytokine RANTES (regulated and normal T cell secreted; 1.6±0.1‐fold). To explore the mechanism, 7 genes were examined for aldosterone regulation in the ApoE−/− aorta. Further studies focused on the proinflammatory placental growth factor (PlGF), which was released from aldosterone‐treated ApoE−/− vessels. Activation of the mineralocorticoid receptor by aldosterone in human coronary artery smooth muscle cells (SMCs) caused the release of factors that promote monocyte chemotaxis, which was inhibited by blocking monocyte PlGF receptors. Furthermore, PlGF‐deficient ApoE−/− mice were resistant to early aldosterone‐induced increases in plaque burden and inflammation.ConclusionsAldosterone increases early atherosclerosis in regions of turbulent blood flow and promotes an inflammatory plaque phenotype that is associated with rupture in humans. The mechanism may involve SMC release of soluble factors that recruit activated leukocytes to the vessel wall via PlGF signaling. These findings identify a novel mechanism and potential treatment target for aldosterone‐induced ischemia in humans.

Subcellular localization of tissue factor and human coronary artery smooth muscle cell migration

Tissue factor (TF) is the most relevant physiological trigger of thrombosis. Additionally TF is a transmembrane receptor with cell signaling functions. The aim of this study was to investigate TF subcellular localization, function and signaling in human coronary artery smooth muscle cell migration. Coronary arteries and primary cultures of vascular smooth muscle cells (HVSMC) were obtained from human explanted hearts. Wound repair and Boyden chamber assays were used to measure migration in vitro. TF-pro-coagulant activity (TF-PCA) was measured in extracted cellular membranes. Analysis of TF distribution was performed by confocal microscopy. A nucleofector device was used for TF and protease activated receptor 2 (PAR2) silencing. mRNA levels were analyzed by RT-PCR. In migrating HVSMC TF translocates to the leading edge of the cells showing an intense patch-like staining in the lamellipodia. In the migrating front TF colocalizes with filamin (FLN) in the polarized lipid rafts. TF-PCA was increased in migrating cells. Silencing of the TF gene inhibits RSK-induced FLN-Ser-2152 phosphorylation, down-regulates CDC42, RhoA, and Rac1 protein expression and significantly inhibits cell migration. Silencing PAR2 also inhibits cell migration; however, silencing both TF and PAR2 induces a significantly higher effect on migration. Smooth muscle cells expressing TF have been identified in non-lipid-rich human coronary artery atherosclerotic plaques. TF translocates to the cell front in association with cytoskeleton proteins and regulates HVSMC migration by mechanisms dependent and independent of factor (F)VIIa/PAR2. These results extend the functional role of TF to smooth muscle cell trafficking in vessel wall remodeling.

Bindarit inhibits human coronary artery smooth muscle cell proliferation, migration and phenotypic switching.

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30 ng/mL) or fetal bovine serum (5%). Bindarit (100–300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal formation.

Opposing roles of STAT-1 and STAT-3 in regulating vascular endothelial growth factor expression in vascular smooth muscle cells

Increased microvessel density in atherosclerotic plaques plays a major role in promoting plaque destabilization resulting in increased risk of stroke and myocardial infarction. Previously we have shown that expression of the inflammatory cytokine, Oncostatin-M (OSM), in human atherosclerotic plaques correlated with increased microvessel density, indicating a role for OSM in promoting plaque angiogenesis. The purpose of this study was to determine the mechanism by which OSM regulates Vascular Endothelial Growth Factor (VEGF) expression in human coronary artery smooth muscle cells. Using shRNA and overexpression studies, we have shown that the transcription factor, STAT-1 inhibited VEGF expression, while STAT-3 promoted the expression of VEGF. We further show that the mechanism by which STAT-1 and STAT-3 regulates VEGF expression is through modulation of Hypoxia Inducible Factor-1α (HIF-1α). STAT-1 suppresses HIF-1α expression, whereas STAT-3 positively regulates HIF-1α expression. These results provide evidence that activated STAT-1 and STAT-3 regulate VEGF expression indirectly, by modulating HIF-1α activity.

Development and characterization of a coronary polylactic acid stent prototype generated by selective laser melting

In-stent restenosis is still an important issue and stent thrombosis is an unresolved risk after coronary intervention. Biodegradable stents would provide initial scaffolding of the stenosed segment and disappear subsequently. The additive manufacturing technology Selective Laser Melting (SLM) enables rapid, parallel, and raw material saving generation of complex 3- dimensional structures with extensive geometric freedom and is currently in use in orthopedic or dental applications. Here, SLM process parameters were adapted for poly-L-lactid acid (PLLA) and PLLA-co-poly-ε-caprolactone (PCL) powders to generate degradable coronary stent prototypes. Biocompatibility of both polymers was evidenced by assessment of cell morphology and of metabolic and adhesive activity at direct and indirect contact with human coronary artery smooth muscle cells, umbilical vein endothelial cells, and endothelial progenitor cells. γ-sterilization was demonstrated to guarantee safety of SLM-processed parts. From PLLA and PCL, stent prototypes were successfully generated and post-processing by spray- and dip-coating proved to thoroughly smoothen stent surfaces. In conclusion, for the first time, biodegradable polymers and the SLM technique were combined for the manufacturing of customized biodegradable coronary artery stent prototypes. SLM is advocated for the development of biodegradable coronary PLLA and PCL stents, potentially optimized for future bifurcation applications.

034 Endothelial Nitric Oxide Synthase-Overexpressing Human Early Outgrowth Endothelial Progenitor Cells Decrease Human Coronary Artery Smooth Muscle Cell Migration Through Paracrine Functions

Arterial restenosis, which occurs in up to 20% of angioplasty patients, is characterized by an excessive vascular smooth muscle cell proliferation resulting from the removal of the endothelial cell lining. Circulating endothelial progenitor cells (EPCs) have the ability to re-colonize and repair the damaged vascular endothelium, reducing restenosis. Nitric oxide (NO) contributes to mobilization and functional activity of EPCs, and estrogen has been shown to increase circulating EPC levels and accelerate the reendothelialization process by EPCs in mice. Moreover NO is important for transducing estrogen-dependent signalling and reendothelialization. We hypothesized that overexpressing endothelial nitric oxide synthase (eNOS), or treatment with estrogen, would potentiate the beneficial effects of EPCs in the context of restenosis. We found that native human early outgrowth EPCs (hEPCs) did not have any effect on human coronary artery smooth muscle cell (HCASMC) proliferation and migration In vitro, evaluated by BrdU incorporation and wound scratch assay respectively. In contrast, the NO donor SNAP significantly decreased the proliferation and migration of HCASMCs. Thereafter, hEPCs were either transfected with a human eNOS plasmid or stimulated with 17β-estradiol (E2) prior to being co-cultured with HCASMCs. Total eNOS protein and eNOS phosphorylation levels were increased by 3- to 3.5-fold in eNOS-transfected or E2-stimulated hEPCs, evaluated by western blot. This was associated with a 3-fold increase in NO production, performed by DAF-FM diacetate immunofluorescence (p<0.05). In eNOS-overexpressing hEPCs, enhanced bcl-2/bax ratio and reduced Annexin V/propidium iodide labeling indicated increased survival. Interestingly, we observed a significant (p<0.05) decrease in HCASMC migration when co-cultured with eNOS-overexpressing hEPCs, by 23%, or with E2-stimulated hEPCs, by 56%. However, HCASMC proliferation was not affected by either eNOS-overexpressing or E2-stimulated hEPCs.These results suggest that overexpressing eNOS in hEPCs increases their survival and enhances their capacity to modulate HCASMC migration through paracrine effects.Réseau de thérapie cellulaire et tissulaire Arterial restenosis, which occurs in up to 20% of angioplasty patients, is characterized by an excessive vascular smooth muscle cell proliferation resulting from the removal of the endothelial cell lining. Circulating endothelial progenitor cells (EPCs) have the ability to re-colonize and repair the damaged vascular endothelium, reducing restenosis. Nitric oxide (NO) contributes to mobilization and functional activity of EPCs, and estrogen has been shown to increase circulating EPC levels and accelerate the reendothelialization process by EPCs in mice. Moreover NO is important for transducing estrogen-dependent signalling and reendothelialization. We hypothesized that overexpressing endothelial nitric oxide synthase (eNOS), or treatment with estrogen, would potentiate the beneficial effects of EPCs in the context of restenosis. We found that native human early outgrowth EPCs (hEPCs) did not have any effect on human coronary artery smooth muscle cell (HCASMC) proliferation and migration In vitro, evaluated by BrdU incorporation and wound scratch assay respectively. In contrast, the NO donor SNAP significantly decreased the proliferation and migration of HCASMCs. Thereafter, hEPCs were either transfected with a human eNOS plasmid or stimulated with 17β-estradiol (E2) prior to being co-cultured with HCASMCs. Total eNOS protein and eNOS phosphorylation levels were increased by 3- to 3.5-fold in eNOS-transfected or E2-stimulated hEPCs, evaluated by western blot. This was associated with a 3-fold increase in NO production, performed by DAF-FM diacetate immunofluorescence (p<0.05). In eNOS-overexpressing hEPCs, enhanced bcl-2/bax ratio and reduced Annexin V/propidium iodide labeling indicated increased survival. Interestingly, we observed a significant (p<0.05) decrease in HCASMC migration when co-cultured with eNOS-overexpressing hEPCs, by 23%, or with E2-stimulated hEPCs, by 56%. However, HCASMC proliferation was not affected by either eNOS-overexpressing or E2-stimulated hEPCs. These results suggest that overexpressing eNOS in hEPCs increases their survival and enhances their capacity to modulate HCASMC migration through paracrine effects. Réseau de thérapie cellulaire et tissulaire

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.

Osteoblast-like Differentiation of Cultured Human Coronary Artery Smooth Muscle Cells by Bone Morphogenetic Protein Endothelial Cell Precursor-derived Regulator (BMPER)

Differentiation of vascular smooth muscle cells (SMCs) into osteoblast-like cells is considered to be a mechanism of vascular calcification. However, regulators of osteoblast-like differentiation of vascular SMCs are not fully elucidated. Here, we investigated the expression of bone morphogenetic protein (BMP)-binding endothelial cell precursor-derived regulator (BMPER), a vertebrate homologue of Drosophila crossveinless-2, in vascular SMCs and the role and mode of action of BMPER in osteoblast-like differentiation of human coronary artery SMCs (HCASMCs). BMPER was expressed in cultured human vascular SMCs, including HCASMCs. Silencing of endogenous BMPER expression by an RNA interference technique inhibited osteoblast-like differentiation of HCASMCs, as evaluated by up-regulation of osteoblast markers such as alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2), by down-regulation of a SMC marker α-smooth muscle actin (αSMA), and by mineralization. Treatment with recombinant BMPER enhanced, whereas BMP-2 reduced osteoblast-like differentiation. BMPER antagonized BMP-2-induced phosphorylation of Smad 1/5/8, suggesting that the effect of BMPER was mediated by antagonizing the action of BMP. BMPER increased IκBα phosphorylation and NF-κB activity and specific NF-κB decoy oligonucleotides deteriorated osteoblast-like differentiation of HCASMCs by BMPER. In human coronary artery with atherosclerotic plaque containing calcification, the BMPER-positive signals were observed in the neointimal and medial SMCs in the vicinity of the plaque. These findings indicate that BMPER is a novel regulator of the osteoblast-like differentiation of HCASMCs.

Fibrous biodegradable l-alanine-based scaffolds for vascular tissue engineering

In vascular tissue engineering, three-dimensional (3D) biodegradable scaffolds play an important role in guiding seeded cells to produce matrix components by providing both mechanical and biological cues. The objective of this work was to fabricate fibrous biodegradable scaffolds from novel poly(ester amide)s (PEAs) derived from l-alanine by electrospinning, and to study the degradation profiles and its suitability for vascular tissue-engineering applications. In view of this, l-alanine-derived PEAs (dissolved in chloroform) were electrospun together with 18-30% w/w polycaprolactone (PCL) to improve spinnability. A minimum of 18% was required to effectively electrospin the solution while the upper value was set in order to limit the influence of PCL on the electrospun PEA fibres. Electrospun fibre mats with average fibre diameters of ~0.4 µm were obtained. Both fibre diameter and porosity increased with increasing PEA content and solution concentration. The degradation of a PEA fibre mat over a period of 28 days indicated that mass loss kinetics was linear, and no change in molecular weight was found, suggesting a surface erosion mechanism. Human coronary artery smooth muscle cells (HCASMCs) cultured for 7 days on the fibre mats showed significantly higher viability (p < 0.0001), suggesting that PEA scaffolds provided a better microenvironment for seeded cells compared with control PCL fibre mats of similar fibre diameter and porosity. Furthermore, elastin expression on the PEA fibre mats was significantly higher than the pure PEA discs and pure PCL fibre mat controls (p < 0.0001). These novel biodegradable PEA fibrous scaffolds could be strong candidates for vascular tissue-engineering applications.

The role of Ras-ERK-IL-1β signaling pathway in upregulation of elastin expression by human coronary artery smooth muscle cells cultured in 3D scaffolds

Incorporation of endogenous elastin, a key structural component of the vascular extracellular matrix (ECM), is an important requirement for engineered vascular tissues. In addition to providing elastic recoil of the tissue, elastin influences cell function and promotes cell signaling by interacting with specific cell surface receptors. Although progress has been made in understanding the mechanisms of in vivo elastin expression and incorporation into fibers, it is notably absent from engineered vessels. Recently we showed that the three-dimensional (3D) scaffold topography was able to upregulate elastin synthesis by human coronary artery smooth muscle cells (HCASMC). The present study was undertaken to explore the molecular mechanisms responsible for 3D scaffold-induced elastin gene transcription. Here, we show several lines of evidence that signal transduction pathway leading to elastin gene expression by HCASMC cultured in synthetic 3D scaffolds to be strikingly different from two-dimensional (2D) surfaces. In 3D scaffolds, α5β1 integrin engagement by HCASMC was significantly reduced and the putative focal adhesion kinase (FAK) was poorly phosphorylated concomitant with FAK and protein tyrosine kinase Pyk2 downregulation. FAK-associated adhesion proteins vinculin and paxillin were also significantly downregulated by the 3D scaffold topography. Furthermore, contrary to 2D cultures, HCASMC cultured on 3D scaffolds had no Rho activation suggesting pliability of the elastomeric synthetic scaffold. Elastin expression in 3D cultures followed Ras-ERK1/2 signal transduction pathway and was further dependent on endogenously expressed interleukin-1β (IL-1β). Blocking of ERK1/2 activation using a pharmacologic inhibitor reduced both elastin and IL-1β gene expressions in 3D cultures. Transient transfection of IL-1β using siRNA, however, did not affect ERK1/2 activation but downregulated elastin gene expression suggesting that endogenous IL-1β acts downstream from ERK1/2. Taken together, results of the present study provide evidence that endogenous IL-1β play a role in elastin gene upregulation and, that this upregulation is mediated by the Ras-ERK1/2 pathway in 3D cultures.

Abstract 268: Resolvin D1 Inhibits Neointimal Formation After Vascular Injury in Mice

Background: Resolvin D1 (RvD1), a docosahexaenoic acid (DHA) - derived lipid mediator generated during the resolution of inflammation, possesses potent anti-inflammatory properties. The present study uses a well defined carotid ligation model to investigate the role of RvD1 in the attenuation of neointimal hyperplasia. Methods: Left common carotid artery ligation was performed in C57BL/6 mice, RvD1 (10 ng/day) or vehicle control was delivered intravenously for 8 consecutive days. Standard morphometric analysis was performed at 28 days post-ligation. Immunohistochemical characterization of leukocyte infiltration and biochemical characterization of tissue reactive oxygen species were performed at 1, 4, 7, 10 days post-ligation. Chemotaxis experiments were performed using isolated human neutrophils, human coronary artery smooth muscle cells (SMC), and murine thoracic aorta SMC in a standard boyden chamber assay. Results: RvD1 treatment afforded an early dampened inflammatory response with reduced infiltration of neutrophils and macrophages and lower detectable levels of tissue myeloperoxidase and superoxide within the first 7 days post-ligation. Morphometric analysis at 28 days post-ligation confirmed a markedly attenuated intimal area (8.37 vs. 33.06, P &lt; 0.01) and intimal to media ratio (0.23 vs. 0.83, P &lt; 0.01) in RvD1 animals when compared to control. In addition, intimal SMCs were significantly reduced in RvD1 treatment vs. control (30.91 vs. 41.02, P &lt; 0.01). In vitro chemotaxis experiments indicated RvD1 can significantly inhibit both neutrophil (1.39 vs. 3.6, P &lt; 0.05) and SMC (1.67 vs. 2.32, P &lt; 0.05) migration index to inflammatory stimulus leukotriene B4. Conclusions: Our results identify the therapeutic potential of RvD1 to attenuate the early inflammatory response, smooth muscle cell migration, and subsequent hyperplasia associated with pathological remodeling following vascular wall insult.

Essential Roles of Raf/Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway, YY1, and Ca2+ Influx in Growth Arrest of Human Vascular Smooth Muscle Cells by Bilirubin

The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations, bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypophosphorylation on amino acids S608 and S780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum-stimulated human vascular smooth muscle primary cell cultures, bilirubin favors growth arrest, and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered retinoblastoma protein profile of hypophosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5 S and 45 S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition.