Human Coronary Artery Smooth Muscle Research Articles

The Ran GTPase-activating protein (RanGAP1) is critically involved in smooth muscle cell differentiation, proliferation and migration following vascular injury: implications for neointima formation and restenosis.

Differentiation and dedifferentiation, accompanied by proliferation play a pivotal role for the phenotypic development of vascular proliferative diseases (VPD), such as restenosis. Increasing evidence points to an essential role of regulated nucleoporin expression in the choice between differentiation and proliferation. However, whether components of the Ran GTPase cycle, which is of pivotal importance for both nucleocytoplasmic transport and for mitotic progression, are subject to similar regulation in VPD is currently unknown. Here, we show that differentiation of human coronary artery smooth muscle cell (CASMC) to a contractile phenotype by stepwise serum depletion leads to significant reduction of RanGAP1 protein levels. The inverse event, dedifferentiation of cells, was assessed in the rat carotid artery balloon injury model, a well-accepted model for neointima formation and restenosis. As revealed by temporospatial analysis of RanGAP1 expression, neointima formation in rat carotid arteries was associated with a significant upregulation of RanGAP1 expression at 3 and 7 days after balloon injury. Of note, neointimal cells located at the luminal surface revealed persistent RanGAP1 expression, as opposed to cells in deeper layers of the neointima where RanGAP1 expression was less or not detectable at all. To gain first evidence for a direct influence of RanGAP1 levels on differentiation, we reduced RanGAP1 in human coronary artery smooth muscle cells by siRNA. Indeed, downregulation of the essential RanGAP1 protein by 50% induced a differentiated, spindle-like smooth muscle cell phenotype, accompanied by an upregulation of the differentiation marker desmin. Reduction of RanGAP1 levels also resulted in a reduction of mitogen induced cellular migration and proliferation as well as a significant upregulation of the cyclin-dependent kinase inhibitor p27KIP1, without evidence for cellular necrosis. These findings suggest that RanGAP1 plays a critical role in smooth muscle cell differentiation, migration and proliferation in vitro and in vivo. Appropriate modulation of RanGAP1 expression may thus be a strategy to modulate VPD development such as restenosis.

Modulation of Phosphorylation of Tocopherol and Phosphatidylinositol by hTAP1/SEC14L2-Mediated Lipid Exchange

The vitamin E derivative, alpha-tocopheryl phosphate (αTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with α-tocopherol (αT) kinase activity. Here, we characterize the production of αTP from αT and [γ-32P]-ATP in primary human coronary artery smooth muscle cells (HCA-SMC) using separation by thin layer chromatography (TLC) and subsequent analysis by Ultra Performance Liquid Chromatography (UPLC). In addition to αT, although to a lower amount, also γT is phosphorylated. In THP-1 monocytes, γTP inhibits cell proliferation and reduces CD36 scavenger receptor expression more potently than αTP. Both αTP and γTP activate the promoter of the human vascular endothelial growth factor (VEGF) gene with similar potency, whereas αT and γT had no significant effect. The recombinant human tocopherol associated protein 1 (hTAP1, hSEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to a putative αT kinase. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol-3-kinase gamma (PI3Kγ) indicating the formation of a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition of αT, βT, γT, δT or αTP differentially stimulates PI3Kγ, suggesting facilitated egress of sequestered PI from hTAP1 to the enzyme. It is suggested that the continuous competitive exchange of different lipophilic ligands in hTAPs with cell enzymes and membranes may be a way to make these lipophiles more accessible as substrates for enzymes and as components of specific membrane domains.

Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells by inducing a differentiated phenotype: role of peroxisome proliferator-activated receptor γ

The search for agents that are capable of preventing restenosis and reduce the risk of late thrombosis is of utmost importance. In this study we aim to evaluate the in vitro effects of ibuprofen on proliferation and migration of human coronary artery smooth muscle cells and on endothelial cells. Cell proliferation was evaluated by trypan blue exclusion. Cell migration was assessed by wound-healing 'scratch' assay and time-lapse video microscopy. Protein expression was assessed by immunoblotting, and morphology by immunocytochemistry. The involvement of the PPARγ pathway was studied with the agonist troglitazone, and the use of selective antagonists such as PGF2α and GW9662. We demonstrate that ibuprofen inhibits proliferation and migration of HCASMCs and induces a switch in HCASMCs towards a differentiated and contractile phenotype, and that these effects are mediated through the PPARγ pathway. Importantly we also show that the effects of ibuprofen are cell type-specific as it does not affect migration and proliferation of endothelial cells. Taken together, our results suggest that ibuprofen could be an effective drug for the development of novel drug-eluting stents that could lead to reduced rates of restenosis and potentially other complications of DES implantation.

Controlled delivery of fibroblast growth factor-9 from biodegradable poly(ester amide) fibers for building functional neovasculature.

For building functional vasculature, controlled delivery of fibroblast growth factor-9 (FGF9) from electrospun fibers is an appealing strategy to overcome challenges associated with its short half-life. FGF9 sustained delivery could potentially drive muscularization of angiogenic sprouts and help regenerate stable functional neovasculature in ischemic vascular disease patients. Electrospinning parameters of FGF9-loaded poly(ester amide) (PEA) fibers have been optimized, using blend and emulsion electrospinning techniques. In vitro PEA matrix degradation, biocompatibility, FGF9 release kinetics, and bioactivity of the released FGF9 were evaluated. qPCR was employed to evaluate platelet-derived growth factor receptor-β (PDGFRβ) gene expression in NIH-3T3 fibroblasts, 10T1/2 cells, and human coronary artery smooth muscle cells cultured on PEA fibers at different FGF9 concentrations. Loaded PEA fibers exhibited controlled release of FGF9 over 28days with limited burst effect while preserving FGF9 bioactivity. FGF9-loaded and unloaded electrospun fibers were found to support the proliferation of fibroblasts for five days even in serum-depleted conditions. Cells cultured on FGF9-supplemented PEA mats resulted in upregulation of PDGFRβ in concentration and cell type-dependent manner. This study supports the premise of controlled delivery of FGF9 from PEA electrospun fibers for potential therapeutic angiogenesis applications.

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.

Abstract 664: The Coronary Heart Disease--Associated Transcription Factor TCF21 Regulates Disease-Related Genes and May Contribute to the Migration of SMC Progenitors to the Fibrous Cap

Introduction: Recent genome-wide association studies have identified a number of genes that contribute to the risk for coronary heart disease, including TCF21, a bHLH transcription factor believed to serve a critical role in the differentiation of epicardial progenitor cells that give rise to coronary smooth muscle. Methods and Results: Results of immunolocalization and in situ hybridization studies showed that TCF21 is not expressed in medial smooth muscle cells (SMC) of adult human coronary arteries but primarily within the adventitia and within cells of unknown origin in atherosclerotic lesions. LacZ reporter animal studies with ApoE null background showed a shift in Tcf21 expression from adventitial and medial cells to the neointima, notably into the area below and within the forming fibrous cap, suggesting that Tcf21 expressing cells migrate into the forming atherosclerotic lesion. To investigate disease-related functions of TCF21 in SMCs, knockdown and overexpression studies were performed in human coronary artery smooth muscle cells, revealing an effect on migration, proliferation and SMC marker gene expression. Genome-wide RNA-seq studies in these cells with siRNA treatment identified putative TCF21 downstream genes and pathways involved in vascular development as well as atherosclerosis. Supporting ChIP-seq data suggests that these - as over half of all differentially expressed genes - are direct transcriptional targets. To elucidate the behavior of Tcf21 expressing cells in the development of disease lesions, we are employing an in vivo Tcf21 lineage tracing model. Preliminary data from these studies suggests that Tcf21 expressing cells give rise to fibrous cap SMCs, and ongoing studies are aimed at the further characterization of molecular pathways that are active in these Tcf21 expressing cells. Conclusions: The gene regulatory and expression profile of Tcf21 during disease progression strongly implies a key role in Coronary Heart Disease. Tcf21 is likely to play a role in the migration of SMC progenitors from the adventitia into the forming atherosclerotic plaque. As a source of differentiated SMCs of the fibrous cap Tcf21 may therefore promote plaque stability and protect from lesion rupture.

Abstract 141: Coronary Heart Disease-Associated Variation in TCF21 Disrupts a MicroRNA-224 Binding Site and miRNA-Mediated Regulation

Introduction: Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated variant in this region, rs12190287, resides in the 3’ untranslated region of the basic-helix-loop-helix transcription factor, TCF21 , and is predicted to alter the seed binding site for miR-224. Allelic imbalance studies demonstrated significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287. Hypothesis: We hypothesize that genetic variation at rs12190287 contributes to allele-specific TCF21 expression imbalance via miR-224 regulation, thus altering CHD risk. Methods and Results: Reporter assays in human coronary artery smooth muscle cells (HCASMC) showed that the disease-associated C allele is selectively targeted by miR-224, leading to reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb 2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor- and platelet derived growth factor signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis in vivo . Conclusions: Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for CHD at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21 , in conjunction with previous studies showing that this variant modulates transcriptional regulation through AP-1, suggests a unique bimodal level of complexity previously unreported for disease-associated variants.

Abstract 648: Visualizing the Genesis of Microcalcifications via Matrix Vesicle Aggregation: Novel Implications for Plaque Vulnerability

Background: Microcalcifications in the cap of vulnerable plaques contribute to plaque rupture. The mechanism by which microcalcifications form is poorly understood due to the inability to monitor initiating calcification events in vivo. We hypothesized that aggregation of cellular-derived matrix vesicles (MV) within atheroma lead to the formation of microcalcifications. Results: Transmission electron microscopy of ApoE-/- mouse plaques reveals MVs aggregating in regions of disrupted collagen (A). To image aggregation of calcifying MVs, we created a 3D collagen hydrogel system to simulate the atherosclerotic fibrous cap. Conditioned media from calcific human coronary artery smooth muscle cells was added to collagen hydrogels. Confocal microscopy reveals spherical microcalcifications (B, arrows) resembling those previously reported in human plaques. Super-resolution (< 200 nm) microscopy of the collagen hydrogels shows individual MVs aggregating (C, arrows) to form microcalcifications shown by a near-infrared calcium phosphate tracer (D). Scanning electron microscopy combined with x-ray spectroscopy confirms the presence of calcium phosphate mineral within MV aggregates (E, orange color). MV aggregation and calcification size is inversely related to the amount of collagen in our 3D model (p<0.05). Conclusion: To our knowledge, we are the first to directly visualize aggregation and calcification of MVs in real time. This approach presents a powerful tool to understand the genesis of microcalcifications from nucleating events. Aggregation of MVs within a thinning fibrous cap leads to the formation of microcalcifications, which further destabilize the cap.

Abstract 465: Deciphering the Genetic Regulation of ADAMTS7 , a Novel Locus for Coronary Artery Disease

Recent genome-wide association studies (GWAS) have identified an association between the ADAMTS7 locus and coronary artery disease (CAD) in humans. While ADAMTS7 is proposed to play a role in vascular smooth muscle cell (VSMC) migration and neointimal formation, the molecular regulation of human ADAMTS7 gene expression has not yet been explored. We assessed ADAMTS7 expression levels in primary mouse aortic smooth muscle cells (mAoSMC) as well as primary human coronary and human pulmonary artery smooth muscle cells (hCASMC, hPASMC) in response to treatment with various stimulatory agents. No differences in ADAMTS7 expression were observed upon treatment with PDGF, angiotensin II, and nicotine in any of the cell lines tested. However, TNFα upregulated ADAMTS7 by 4-fold in mAoSMC but not in human VSMCs while H 2 O 2 upregulated ADAMTS7 by 4-fold in hCASMC but not mAoSMCs. No agents modulated expression in hPASMC. Basal levels of ADAMTS7 varied among different VSMC lines; hCASMC had 2-fold greater levels of ADAMTS7 when compared to hPASMC and hAoSMC. These data demonstrate that ADAMTS7 is differentially regulated not only across different species, but also among different VSMC types, underscoring the complex genetic regulation of ADAMTS7 . In an attempt to elucidate important regulatory genomic regions controlling ADAMTS7 expression, we utilized data from the ENCODE project to identify regions surrounding ADAMTS7 that are enriched for regulatory domains, e.g. DNase hypsersensitivity (DHS) and H3K27 acetylation, in relevant cell types. We then overlayed data from CARDIoGRAM and C4D CAD GWAS to look for CAD-associated SNPs in these potential enhancers. Ultimately we selected 5 regions of interest (e.g., rs5029904 lies in region with increased H3K27 acetylation in ENCODE layered 7 cell lines, DHS peaks in serum fed and starved hAoSMCs, binding peaks for transcription factors via ENCODE ChIP-Seq experiments) for further studies in luciferase reporter assays multiple cell types to look for tissue specific expression mediated by these genomic regions. GWAS variants in these domains may affect the transcriptional regulation of ADAMTS7 , thus providing insight into the role for ADAMTS7 in human atherosclerosis.

Biomimetic l-aspartic acid-derived functional poly(ester amide)s for vascular tissue engineering

Functionalization of polymeric biomaterials permits the conjugation of cell signaling molecules capable of directing cell function. In this study, l-phenylalanine and l-aspartic acid were used to synthesize poly(ester amide)s (PEAs) with pendant carboxylic acid groups through an interfacial polycondensation approach. Human coronary artery smooth muscle cell (HCASMC) attachment, spreading and proliferation was observed on all PEA films. Vinculin expression at the cell periphery suggested that HCASMCs formed focal adhesions on the functional PEAs, while the absence of smooth muscle α-actin (SMαA) expression implied the cells adopted a proliferative phenotype. The PEAs were also electrospun to yield nanoscale three-dimensional (3-D) scaffolds with average fiber diameters ranging from 130 to 294nm. Immunoblotting studies suggested a potential increase in SMαA and calponin expression from HCASMCs cultured on 3-D fibrous scaffolds when compared to 2-D films. X-ray photoelectron spectroscopy and immunofluorescence demonstrated the conjugation of transforming growth factor-β1 to the surface of the functional PEA through the pendant carboxylic acid groups. Taken together, this study demonstrates that PEAs containing aspartic acid are viable biomaterials for further investigation in vascular tissue engineering.

Effect of inflammatory factors on cell proliferation and apoptosis in insulin-like grown factor 1-slienced human coronary artery smooth muscle cells

To study the effect of inflammatory factors on cell proliferation and apoptosis in insulin-like grown factor 1 (IGF1)-slienced human coronary artery smooth muscle cells (hCASMCs). We silenced the expression of IGF1 in hCASMCs using the lentivirus-mediated RNA interference technology. Blank control group and negative control group were set using the hCASMCs without the infection of a virus vector and the hCASMCs with the infection of a negative control virus vector, respectively. After the treatment of these cells with both tumor necrosis factor-α 50 ng/ml and interleukin-1β 40 ng/ml, the concentration of IGF1 in cell-culture medium was detected by enzyme-linked immunosorbent assay, and the proliferation and apoptosis were evaluated by MTT assay and flow cytometry. After the simulation with inflammatory factors, the concentration of IGF1 in the supernatant fluid of cultured IGF1-slienced hCASMCs was significantly lower than those in the blank control group and negative control group [(426.35±120.96) vs. (1 030.69±54.69) and (992.82±26.90)pg/ml, P=0.000). The proliferation of IGF1-slienced hCASMCs was substantially much less than the two control groups (0.302±0.011 vs. 0.401±0.028 and 0.302±0.011, F=37.628, P=0.000), and the apoptosis rate of IGF1-slienced hCASMCs was significant increased compared with the other two groups [(10.57±0.99)% vs. (0.19±0.13)% and (1.31±0.30)%, P=0.001]. Inflammatory factors can inhibit the cell proliferation and promote apoptosis after the knock-down of IGF1 in hCASMCs.

Suppression of cardio‐protective molecules by diabetic marker miR‐29: a mechanism for the high rate of cardiac damage in diabetes? (1086.6)

Diabetic marker miRNA‐29 (miR‐29) is up‐regulated by pro‐inflammatory cytokines and hyperglycemia. miR‐29 induced suppression of MCL‐1, a protein essential for mitochondrial function and cell survival, occurs in pancreatic β‐cell death. We hypothesized that conditions that increase miR‐29 levels in heart would promote suppression of MCL‐1 and increase cardiomyocyte cell death. Since Rapamycin (Rap) is known to promote diabetes, we further posited that Rap treatment (750 µg/kg/day; 6 weeks) would increase miR‐29 and suppress MCL‐1 in heart tissues of Zucker Diabetic Fatty (ZDF) rat, a rodent model for the progression of Type 2 Diabetes. Rap treatment attenuated plasma insulin (INS) levels implying development of insulinopenia; however, hyperglycemia persisted. q‐PCR showed a significant increase in miR‐29a, b, and c and suppression of MCL‐1 (p<0.01) in ZDF heart. Rap treatment also resulted in significant loss of cardiomyocytes as visualized by histopathological analysis by H & E and Masson's trichrome staining. Moreover, Rap treatment increased miR‐29 levels and suppressed MCL‐1 mRNA and protein levels in mouse atrial cardiomyocytes (10nM; 12hr) and in human coronary artery smooth muscle cells (20nM; 12hr). These data suggest that MCL‐1 regulation via miR‐29 family in INS‐responsive tissues is conserved across species and contributes to cardiomyocyte loss that can promote heart failure in diabetes.Grant Funding Source: NIH: 1R01HL118376‐01

Coronary Heart Disease-Associated Variation in TCF21 Disrupts a miR-224 Binding Site and miRNA-Mediated Regulation

Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated polymorphism in this region, rs12190287, resides in the 3′ untranslated region (3′-UTR) of TCF21, a basic-helix-loop-helix transcription factor, and is predicted to alter the seed binding sequence for miR-224. Allelic imbalance studies in circulating leukocytes and human coronary artery smooth muscle cells (HCASMC) showed significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287, consistent with this variant contributing to allele-specific expression differences. 3′ UTR reporter gene transfection studies in HCASMC showed that the disease-associated C allele has reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor-β (TGF-β) and platelet derived growth factor (PDGF) signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis. Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for coronary heart disease associated at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in conjunction with previous studies showing that this variant modulates transcriptional regulation through activator protein 1 (AP-1), suggests a unique bimodal level of complexity previously unreported for disease-associated variants.

Artichoke, Cynarin and Cyanidin Downregulate the Expression of Inducible Nitric Oxide Synthase in Human Coronary Smooth Muscle Cells

Artichoke (Cynara scolymus L.) is one of the world’s oldest medicinal plants with multiple health benefits. We have previously shown that artichoke leaf extracts and artichoke flavonoids upregulate the gene expression of endothelial-type nitric oxide synthase (eNOS) in human endothelial cells. Whereas NO produced by the eNOS is a vasoprotective molecule, NO derived from the inducible iNOS plays a pro-inflammatory role in the vasculature. The present study was aimed to investigate the effects of artichoke on iNOS expression in human coronary artery smooth muscle cells (HCASMC). Incubation of HCASMC with a cytokine mixture led to an induction of iNOS mRNA expression. This iNOS induction was concentration- and time-dependently inhibited by an artichoke leaf extract (1–100 µg/mL, 6 h or 24 h). Consistently, the artichoke leaf extract also reduced cytokine-induced iNOS promoter activation and iNOS protein expression. In addition, treatment of HCASMC with four well-known artichoke compounds (cynarin > cyanidin > luteolin ≈ cynaroside) led to a downregulation iNOS mRNA and protein expression, with cynarin being the most potent one. In conclusion, artichoke contains both eNOS-upregulating and iNOS-downregulating compounds. Such compounds may contribute to the beneficial effects of artichoke and may per se have therapeutic potentials.

In vitro evaluation of carbon-nanotube-reinforced bioprintable vascular conduits

Vascularization of thick engineered tissue and organ constructs like the heart, liver, pancreas or kidney remains a major challenge in tissue engineering. Vascularization is needed to supply oxygen and nutrients and remove waste in living tissues and organs through a network that should possess high perfusion ability and significant mechanical strength and elasticity. In this paper, we introduce a fabrication process to print vascular conduits directly, where conduits were reinforced with carbon nanotubes (CNTs) to enhance their mechanical properties and bioprintability. In vitro evaluation of printed conduits encapsulated in human coronary artery smooth muscle cells was performed to characterize the effects of CNT reinforcement on the mechanical, perfusion and biological performance of the conduits. Perfusion and permeability, cell viability, extracellular matrix formation and tissue histology were assessed and discussed, and it was concluded that CNT-reinforced vascular conduits provided a foundation for mechanically appealing constructs where CNTs could be replaced with natural protein nanofibers for further integration of these conduits in large-scale tissue fabrication.

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.

Identification and Initial Functional Characterization of a Human Vascular Cell–Enriched Long Noncoding RNA

Long noncoding RNAs (lncRNAs) represent a rapidly growing class of RNA genes with functions related primarily to transcriptional and post-transcriptional control of gene expression. There is a paucity of information about lncRNA expression and function in human vascular cells. Thus, we set out to identify novel lncRNA genes in human vascular smooth muscle cells and to gain insight into their role in the control of smooth muscle cell phenotypes. RNA sequencing (RNA-seq) of human coronary artery smooth muscle cells revealed 31 unannotated lncRNAs, including a vascular cell-enriched lncRNA (Smooth muscle and Endothelial cell-enriched migration/differentiation-associated long NonCoding RNA [SENCR]). Strand-specific reverse transcription polymerase chain reaction (PCR) and rapid amplification of cDNA ends indicate that SENCR is transcribed antisense from the 5' end of the FLI1 gene and exists as 2 splice variants. RNA fluorescence in situ hybridization and biochemical fractionation studies demonstrate SENCR is a cytoplasmic lncRNA. Consistent with this observation, knockdown studies reveal little to no cis-acting effect of SENCR on FLI1 or neighboring gene expression. RNA-seq experiments in smooth muscle cells after SENCR knockdown disclose decreased expression of Myocardin and numerous smooth muscle contractile genes, whereas several promigratory genes are increased. Reverse transcription PCR and Western blotting experiments validate several differentially expressed genes after SENCR knockdown. Loss-of-function studies in scratch wound and Boyden chamber assays support SENCR as an inhibitor of smooth muscle cell migration. SENCR is a new vascular cell-enriched, cytoplasmic lncRNA that seems to stabilize the smooth muscle cell contractile phenotype.

Postprandial triglyceride-rich lipoproteins promote invasion of human coronary artery smooth muscle cells in a fatty-acid manner through PI3k-Rac1-JNK signaling

The aim was to investigate the effect of postprandial triglyceride-rich lipoproteins (TRLs) with different fatty acid compositions on human coronary artery smooth muscle cell (hCASMC) invasion and to identify the molecular pathways involved. TRLs were isolated from the plasma of healthy volunteers after the ingestion of single meals enriched in MUFAs, saturated fatty acids (SFAs), or PUFAs. hCASMC invasion was analyzed using transwell chambers with Matrigel. TRLs-SFAs provoked the highest invasion, followed by TRLs-MUFAs and TRLs-PUFAs. Inhibition studies with Orlistat showed that invasion was dependent on the fatty acid composition of the TRLs. Fatty acids incorporated into the cell membranes strongly associated with cell invasion. Pull-down assays showed that TRLs-SFAs were able to increase Rac1 activity via inhibition of RhoA-dependent signaling. Chemical inhibition and siRNA studies showed that Rac1, PI3k, JNK, and MMP2 regulates TRL-SFA-induced hCASMC invasion. We demonstrate for the first time that TRLs induce hCASMCs invasion in a fatty acid dependent manner. This effect in TRLs-SFAs is mediated by the PI3k-Rac1-JNK, RhoA, and Rac1-MMP2 pathways. The ingestion of MUFA, compared to other dietary fatty acids such as SFA, could be considered as a nutritional strategy to reduce the atherosclerotic plaque formation.

Cell-Specific Expression of Uptake Transporters—A Potential Approach for Cardiovascular Drug Delivery Devices

Enhanced proliferation of human coronary artery smooth muscle cells (HCASMCs) and thereby formation of neointima is one of the factors contributing to failure of coronary stents. Even if the use of drug eluting stents (DES) and thereby the local delivery of cytotoxic compounds has significantly improved the clinical outcome, unselective cytotoxic effects are assumed to hamper clinical success. Novel pharmacological approaches are required to enhance cellular selectivity of locally delivered drugs. Cell specific overexpression of a drug transporter could be used to enhance cellular accumulation and therefore cell specificity. In the herein reported study we tested the possibility of cell specific transporter expression to enhance drug effects in HCASMCs. We generated adenoviral constructs to overexpress the organic cation transporter 1 (OCT1) under control of the promoter of SM22α, which had been previously reported as muscle cell specific gene. First the activity of the SM22α-promoter was assessed in various cell types supporting the notion of muscle cell specificity. Subsequently, the activity of the transporter was compared in infected HCAECs and HCASMCs revealing enhanced accumulation of substrate drugs in HCASMCs in presence of the SM22α-promoter. Testing the hypothesis that this kind of targeting might serve as a mechanism for cell-specific drug effects, we investigated the impact on paclitaxel treatment in HCASMC and HCAECs, showing significantly increased antiproliferative activity of this substrate drug on muscle cells. Taken together, our findings suggest that cell-specific expression of transport proteins serves as mechanism governing the uptake of cytotoxic compounds for a selective impact on targeted cells.

Regulation of Vascular Smooth Muscle Cell Phenotype in Three-Dimensional Coculture System by Jagged1-Selective Notch3 Signaling

The modulation of vascular smooth muscle cell (VSMC) phenotype is an essential element to fabricate engineered conduits of clinical relevance. In vivo, owing to their close proximity, endothelial cells (ECs) play a role in VSMC phenotype switching. Although considerable progress has been made in vascular tissue engineering, significant knowledge gaps exist on how the contractile VSMC phenotype is induced at the conclusion of the tissue fabrication process. The objectives of this study were as follows: (1) to establish ligand presentation modes on transcriptional activation of VSMC-specific genes, (2) to develop a three-dimensional (3D) coculture model using human coronary artery smooth muscle cells (HCASMCs) and human coronary artery endothelial cells (HCAECs) on porous synthetic scaffolds and, (3) to investigate EC-mediated Notch signaling in 3D cultures and the induction of the HCASMC contractile phenotype. Whereas transcriptional activation of VSMC-specific genes was not induced by presenting soluble Jagged1 and Jagged1 bound to protein G beads, a direct link between HCAEC-bound Jagged1 and HCASMC differentiation genes was observed. Our 3D culture results showed that HCASMCs seeded to scaffolds and cultured for up to 16 days readily attached, infiltrated the scaffold, proliferated, and formed dense confluent layers. HCAECs, seeded on top of an HCASMC layer, formed a distinct, separate monolayer with cell-type partitioning, suggesting that HCAEC growth was contact inhibited. While we observed EC monolayer formation with 200,000 HCAECs/scaffold, seeding 400,000 HCAECs/scaffold revealed the formation of cord-like structures akin to angiogenesis. Western blot analyses showed that 3D coculture induced an upregulation of Notch3 receptor in HCASMCs and its ligand Jagged1 in HCAECs. This was accompanied by a corresponding induction of the contractile HCASMC phenotype as demonstrated by increased expression of smooth muscle-α-actin (SM-α-actin) and calponin. Knockdown of Jagged1 with siRNA showed a reduction in SM-α-actin and calponin in cocultures, identifying a link between Jagged1 and the expression of contractile proteins in 3D cocultures. We therefore conclude that the Notch3 signaling pathway is an important regulator of VSMC phenotype and could be targeted when fabricating engineered vascular tissues.