Primary Human Smooth Muscle Cells Research Articles

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.

Alpha1‐adrenergic receptor subtype specific activation of ERK in human primary smooth muscle cells (1065.4)

Three α1‐adrenergic receptor (AR) subtypes have been cloned and characterized, the α1A‐,α1B‐, and α1D‐AR. Evidence suggests functional effects of receptor activation in various tissues is subtype selective. For example, ERK activation in cardiomyoctyes appears to be α1A‐AR dependent, and may be cardioprotective in heart failure. We examined α1‐AR expression and subtype specific ERK activation in human primary arterial smooth muscle cells. Simultaneous co‐expression of all three subtypes was demonstrated by western blot, and further confirmed by RT‐PCR utilizing subtype specific primers. ERK activation was assessed by western blot. Preliminary data suggests ERK activation is blocked by α1D‐AR selective antagonists and is not induced by α1A‐AR selective agonists. If α1A‐AR selective agents were used in heart failure, our data would suggest that ERK activation in peripheral vasculature would be minimal, therefore mitigating concern of α1A‐AR mediated vascular injury as a result of therapy. However, additional subtype selective effects will need to be better defined by future studies.

Autotaxin mRNA and protein expression and regulation by salmeterol xinafoate and fluticasone propionate in lung cells (660.13)

Autotaxin (ATX) is the lyso‐phospholipase D enzyme that catalyzes the final step in the synthesis of the lipid mediator lysophosphatidic acid (LPA). ATX, LPA, and LPA receptors in the lung are all strongly associated with the pathology of pulmonary fibrosis, and increasing evidence implicates these same mediators as contributors to asthma. Accordingly, we hypothesized that the asthma drugs salmeterol xinafoate (SX), a long‐acting beta‐2 agonist bronchodilator, and fluticasone propionate (FP), an anti‐inflammatory steroid, would reduce the expression of ATX in lung cells. Cells were treated for 72 hr in the absence or presence of the drugs, alone or in combination. ATX protein assessed by Western blotting of culture medium and ATX mRNA assessed by qPCR were not detected in primary human bronchial epithelial cells or the Beas‐2B bronchial epithelial cell line. ATX protein but not mRNA was detected for human A549 alveolar adenocarcinoma cells and mouse LA4 cells, both of which represent alveolar rather than airway epithelium. Both SX and FP decreased ATX protein expression in these alveolar cells. Stronger ATX protein and mRNA signals were observed in mesenchymal cells, both primary human airway smooth muscle (HASM) cells and the HFL‐1 human fetal lung fibroblast cell line. In these cells, the level of mRNA was decreased moderately by SX, to a greater extent by FP, and decreased yet further by SX + FP. However, ATX protein was minimally decreased or not at all, despite the clear decrease in mRNA. These data reveal interesting cell type‐specific differences in ATX mRNA and protein expression, and they show that asthma therapeutic agents can reduce the expression of ATX in at least some of these cells, which may contribute to their overall therapeutic benefit in patients with asthma.Grant Funding Source: Supported by investigator‐initiated grant 115165 from GlaxoSmithKline

Airway mesenchymal cell death by mevalonate cascade inhibition: Integration of autophagy, unfolded protein response and apoptosis focusing on Bcl2 family proteins

HMG-CoA reductase, the proximal rate-limiting enzyme in the mevalonate pathway, is inhibited by statins. Beyond their cholesterol lowering impact, statins have pleiotropic effects and their use is linked to improved lung health. We have shown that mevalonate cascade inhibition induces apoptosis and autophagy in cultured human airway mesenchymal cells. Here, we show that simvastatin also induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in these cells. We tested whether coordination of ER stress, autophagy and apoptosis determines survival or demise of human lung mesenchymal cells exposed to statin. We observed that simvastatin exposure activates UPR (activated transcription factor 4, activated transcription factor 6 and IRE1α) and caspase-4 in primary human airway fibroblasts and smooth muscle cells. Exogenous mevalonate inhibited apoptosis, autophagy and UPR, but exogenous cholesterol was without impact, indicating that sterol intermediates are involved with mechanisms mediating statin effects. Caspase-4 inhibition decreased simvastatin-induced apoptosis, whereas inhibition of autophagy by ATG7 or ATG3 knockdown significantly increased cell death. In BAX−/−/BAK−/− murine embryonic fibroblasts, simvastatin-triggered apoptotic and UPR events were abrogated, but autophagy flux was increased leading to cell death via necrosis. Our data indicate that mevalonate cascade inhibition, likely associated with depletion of sterol intermediates, can lead to cell death via coordinated apoptosis, autophagy, and ER stress. The interplay between these pathways appears to be principally regulated by autophagy and Bcl-2-family pro-apoptotic proteins. These findings uncover multiple mechanisms of action of statins that could contribute to refining the use of such agent in treatment of lung disease.

Hypoxia Exerts Dualistic Effects on Inflammatory and Proliferative Responses of Healthy and Asthmatic Primary Human Bronchial Smooth Muscle Cells

BackgroundFor oxygen supply, airway wall cells depend on diffusion though the basement membrane, as well as on delivery by micro-vessels. In the asthmatic lung, local hypoxic conditions may occur due to increased thickness and altered composition of the basement membrane, as well as due to edema of the inflamed airway wall.ObjectiveIn our study we investigated the effect of hypoxia on proliferation and pro-inflammatory and pro-angiogenic parameter production by human bronchial smooth muscle cells (BSMC). Furthermore, conditioned media of hypoxia-exposed BSMC was tested for its ability to induce sprout outgrowth from endothelial cells spheroids.MethodsBSMC were cultured in RPMI1640 (5% FCS) under normoxic (21% O2) and hypoxic (1% and 5% O2) conditions. Proliferation was determined by cell count and Western blot analysis for cyclin E and Proliferating Cell Nuclear Antigen (PCNA). Secretion of IL-6, IL-8, ENA-78 and VEGF-A was analyzed by ELISA. BSMC conditioned medium was tested for its angiogenic capacity by endothelial cell (EC)-spheroid in vitro angiogenesis assay.ResultsProliferation of BSMC obtained from asthmatic and non-asthmatic patients was significantly reduced in the presence of 1% O2, whereas 5% O2 reduced proliferation of asthmatic BSMC only. Hypoxia induced HIF-1α expression in asthmatic and non-asthmatic BSMC, which coincided with significantly increased release of IL-6, IL-8 and VEGF-A, but not ENA-78. Finally, endothelial sprout outgrowth from EC spheroids was increased when exposed to hypoxia conditioned BSMC medium.ConclusionHypoxia had dualistic effects on proliferative and inflammatory responses of asthmatic and non-asthmatic BSMC. First, hypoxia reduced BSMC proliferation. Second, hypoxia induced a pro-inflammatory, pro-angiogenic response. BSMC and EC may thus be promising new targets to counteract and/or alleviate airway wall remodeling.

MicroRNA‐10a controls airway smooth muscle cell proliferationviadirect targeting of the PI3 kinase pathway

Airway smooth muscle (ASM) cells play important physiological roles in the lung, and abnormal proliferation of ASM directly contributes to the airway remodeling during development of lung diseases such as asthma. MicroRNAs are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation; however, little is known about the precise role of microRNAs in the proliferation of the ASM. Here we report that a specific microRNA (miR-10a) controls ASM proliferation through directly inhibiting the phosphoinositide 3-kinase (PI3K) pathway. Next-generation sequencing identified miR-10a as the most abundant microRNA expressed in primary human airway smooth muscle (HASM) cells, accounting for > 20% of all small RNA reads. Overexpression of miR-10a reduced mitogen-induced HASM proliferation by ∼50%, whereas inhibition of miR-10a increased HASM proliferation by ∼40%. Microarray profiling of HASM cells expressing miR-10a mimics identified 52 significantly down-regulated genes as potential targets of miR-10a, including the catalytic subunit α of PI3K (PIK3CA), the central component of the PI3K pathway. MiR-10a directly suppresses PIK3CA expression by targeting the 3'-untranslated region (3'-UTR) of the gene. Inhibition of PIK3CA by miR-10a reduced V-akt murine thymoma viral oncogene homolog 1 (AKT) phosphorylation and blunted the expression of cyclins and cyclin-dependent kinases that are required for HASM proliferation. Together, our study identifies a novel microRNA-mediated regulatory mechanism for PI3K signaling and ASM proliferation and further suggests miR-10a as a potential therapeutic target for lung diseases whose etiology resides in abnormal ASM proliferation.

The role of vascular-derived perlecan in modulating cell adhesion, proliferation and growth factor signaling

Smooth muscle cell proliferation can be inhibited by heparan sulfate proteoglycans whereas the removal or digestion of heparan sulfate from perlecan promotes their proliferation. In this study we characterized the glycosaminoglycan side chains of perlecan isolated from either primary human coronary artery smooth muscle or endothelial cells and determined their roles in mediating cell adhesion and proliferation, and in fibroblast growth factor (FGF) binding and signaling. Smooth muscle cell perlecan was decorated with both heparan sulfate and chondroitin sulfate, whereas endothelial perlecan contained exclusively heparan sulfate chains. Smooth muscle cells bound to the protein core of perlecan only when the glycosaminoglycans were removed, and this binding involved a novel site in domain III as well as domain V/endorepellin and the α2β1 integrin. In contrast, endothelial cells adhered to the protein core of perlecan in the presence of glycosaminoglycans. Smooth muscle cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains and promoted the signaling of FGF2 but not FGF1. Also endothelial cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains, but in contrast, promoted the signaling of both growth factors. Based on this differential bioactivity, we propose that perlecan synthesized by smooth muscle cells differs from that synthesized by endothelial cells by possessing different signaling capabilities, primarily, but not exclusively, due to a differential glycanation. The end result is a differential modulation of cell adhesion, proliferation and growth factor signaling in these two key cellular constituents of blood vessels.

Aged Garlic Extract and One of the Constituent, (+)-(2S,3R)-Dehydrodiconiferyl Alcohol, Inhibits Alkaline Phosphatase Activity Induced by Inflammation Factors in Human Vascular Smooth Muscle Cells

Previous clinical study showed that aged garlic extract (AGE) has a preventive effect of vascular calcification in patients with coronary artery diseases. It has been reported that vascular smooth muscle cells (VSMC) and inflammatory cells including macrophages and lymphocytes migrate to sub-intimal region where atherosclerotic plaques are formed, and VSMC can differentiate into osteoblast-like cells, which are represented by the induction of alkaline phosphatase (ALP). We found that primary human coronary artery smooth muscle cells (HCASMC) showed the increased ALP activity, when cultured in the medium containing ascorbic acid, β-glycerophosphate, dexamethasone (IM), and supplemented with conditioned medium from macrophages (MCM). Then we tested the effect of AGE subdivided fractions and several compounds found in AGE, and then found that ((+)-(2S,3R)-Dehydrodiconiferyl alcohol, a dilignol compound existed in hydrophobic fraction of AGE, inhibited ALP activity in HCASMC.

Mapping Mechanical Properties of the Extra Cellular Matrix Surrounding Cells Cultured in 3D

There is a preponderance of evidence for roles of bulk stromal stiffness in cell regulation, but little is about the mechanical microenvironment. To investigate these roles we culture cells within 3D extracellular matrix (ECM) hydrogels. We implement automated optical tweezers active microrheology (aAMR) to probe extracellular stiffness and map it in the volume surrounding cells. Our aAMR uses optical forces acting on microbeads to measure the complex response function of the ECM surrounding each bead. Hundreds of beads are probed in one hour to map the mechanical landscape and to seek correlations between local stiffness and cell properties such as contractility, signaling and differentiation. For example, we will present our study of primary human aortic smooth muscle cells (AoSMCs) cultured within a collagen hydrogel. We observed a range of stiffness from over 200 Pa to less than 20 Pa, with the stiffer regions near the leading edge of the cell. Such stiffening is consistent with the non-linear stress-strain relationship of natural tissue in which stiffness increases with stretch. After treatment with inhibitors of contractility such as blebbistatin or the ROCK Inhibitor Y-27632, we observed cell relaxation coincident with a distribution of softer values ranging from 100 to ∼10 Pa. Such leading edge contractile force-dependent stiffness is consistent with our earlier findings for primary mesenchymal stem cells and an invasive capillary morphogenesis model, in which only several beads could be measured in one hour due to the notable complexity of performing AMR. In contrast, our aAMR densely measures ECM stiffness, chronically, and is a new research tool for studying the interplay between viscoelastic properties, forces and signaling at the ECM-cell interface.

Integration of proteomic and transcriptomic profiles identifies a novel PDGF-MYC network in human smooth muscle cells

Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the proliferation, migration and synthetic activities of smooth muscle cells that characterize physiologic and pathologic tissue remodeling in hollow organs. However, neither the molecular basis of PDGFR-regulated signaling webs, nor the extent to which specific components within these networks could be exploited for therapeutic benefit has been fully elucidated. Expression profiling and quantitative proteomics analysis of PDGF-treated primary human bladder smooth muscle cells identified 1,695 genes and 241 proteins as differentially expressed versus non-treated cells. Analysis of gene expression data revealed MYC, JUN, EGR1, MYB, RUNX1, as the transcription factors most significantly networked with up-regulated genes. Forty targets were significantly altered at both the mRNA and protein levels. Proliferation, migration and angiogenesis were the biological processes most significantly associated with this signature, and MYC was the most highly networked master regulator. Alterations in master regulators and gene targets were validated in PDGF-stimulated smooth muscle cells in vitro and in a model of bladder injury in vivo. Pharmacologic inhibition of MYC and JUN confirmed their role in SMC proliferation and migration. Network analysis identified the diaphanous-related formin 3 as a novel PDGF target regulated by MYC and JUN, which was necessary for PDGF-stimulated lamellipodium formation. These findings provide the first systems-level analysis of the PDGF-regulated transcriptome and proteome in normal smooth muscle cells. The analyses revealed an extensive cohort of PDGF-dependent biological processes and connected key transcriptional effectors to their regulation, significantly expanding current knowledge of PDGF-stimulated signaling cascades. These observations also implicate MYC as a novel target for pharmacological intervention in fibroproliferative expansion of smooth muscle, and potentially in cancers in which PDGFR-dependent signaling or MYC activation promote tumor progression.

New vitamin D less-calcemic analog affect human bone cell line and cultured vascular smooth muscle cells similar to other less-calcemic analogs

Primary cultures of human bone and vascular cells respond to vitamin D treatment by modulation of cell proliferation measured by DNA synthesis (DNA) and energy metabolism measured by creatine kinase specific activity (CK) via binding to vitamin D receptors (VDR) which are expressed in these cells. Vitamin D compounds also modulate the response to estradiol-17β (E2) and the expression mRNAs of estrogen receptors (ERα and ERβ), VDR, 25-hydroxy vitamin D3 1-α hydroxylase (1OHase) and lipoxygenases (12LO and 15LO). We now compared our newly synthesized analog: 1α,25-dihydroxy-9-methylene-19-norvitamin D3 JK152 (JK), on bone and vascular cells compared to other analogs. Human bone cell line SaOS2 respond to JK by increased DNA and stimulated CK dose-dependently, similar to the less-calcemic analogs CB 1093 (CB) and EB 1089 (EB). JK also up-regulated the response to E2 in terms of DNA and CK. JK inhibited DNA synthesis and increased CK in primary human vascular smooth muscle cells (VSMC) dose-dependently similar to EB and CB. JK up regulated the response to E2 in terms of CK with no effect on DNA. JK similar to CB and EB stimulated mRNA expression of VDR and ERα, 12LO and 15LO, with no effect on ERβ and 1OHase mRNA expression in SaOS2 measured by real time PCR. Similar treatments of VSMC with JK, CB and EB stimulated 12LO and 15LO, VDR and ERα mRNA expression with no effect on ERβ and 1OHase mRNA expression. The results presented here demonstrate that the new vitamin D less-calcemic analog JK is similar to other analogs in its effects on human cultured cells and therefore may be used in combined hormone replacement treatment (HRT) both in vitro and in vivo.

MicroRNA-138 Regulates Hypoxia-Induced Endothelial Cell Dysfunction By Targeting S100A1

The Ca2+ sensor S100A1 is essential for proper endothelial cell (EC) nitric oxide (NO) synthase (eNOS) activation. S100A1 levels are greatly reduced in primary human microvascular ECs subjected to hypoxia, rendering them dysfunctional. However mechanisms that regulate S100A1 levels in ECs are unknown. Here we show that ECs transfected with a S100A1–3′ untranslated region (UTR) luciferase reporter construct display significantly reduced gene expression when subjected to low oxygen levels or chemical hypoxia. Bioinformatic analysis suggested that microRNA -138 (MiR-138) could target the 3′UTR of S100A1. Patients with critical limb ischemia (CLI) or mice subjected to femoral artery resection (FAR) displayed increased MiR-138 levels and decreased S100A1 protein expression. Consistent with this finding, hypoxia greatly increased MiR-138 levels in ECs, but not in skeletal muscle C2C12 myoblasts or differentiated myotubes or primary human vascular smooth muscle cells. Transfection of a MiR-138 mimic into ECs reduced S100A1–3 ‘UTR reporter gene expression, while transfection of an anti MiR-138 prevented the hypoxia-induced downregulation of the reporter gene. Deletion of the 22 nucleotide putative MiR-138 target site abolished the hypoxia-induced loss of reporter gene expression. Knockdown of Hif1-α mediated by siRNA prevented loss of hypoxia-induced reporter gene expression. Conversely, specific activation of Hif1-α by a selective prolyl-hydroxylase inhibitor (IOX2) reduced reporter gene expression even in the absence of hypoxia. Finally, primary ECs transfected with a MiR-138 mimic displayed reduced tube formation when plated onto Matrigel matrix and expressed less NO when stimulated with VEGF. These effects were reversed by gene transfer of S100A1 using recombinant adenovirus. We conclude that hypoxia-induced MiR-138 is an essential mediator of EC dysfunction via its ability to target the 3′UTR of S100A1.

Von Willebrand Factor Inhibits Mature Smooth Muscle Gene Expression through Impairment of Notch Signaling

Von Willebrand factor (vWF), a hemostatic protein normally synthesized and stored by endothelial cells and platelets, has been localized beyond the endothelium in vascular disease states. Previous studies have implicated potential non-hemostatic functions of vWF, but signaling mechanisms underlying its effects are currently undefined. We present evidence that vWF breaches the endothelium and is expressed in a transmural distribution pattern in cerebral small vessel disease (SVD). To determine the potential molecular consequences of vWF permeation into the vessel wall, we also tested whether vWF impairs Notch regulation of key smooth muscle marker genes. In a co-culture system using Notch ligand expressing cells to stimulate Notch in A7R5 cells, vWF strongly inhibited both the Notch pathway and the activation of mature smooth muscle gene promoters. Similar repressive effects were observed in primary human cerebral vascular smooth muscle cells. Expression of the intracellular domain of NOTCH3 allowed cells to bypass the inhibitory effects of vWF. Moreover, vWF forms molecular complexes with all four mammalian Notch ectodomains, suggesting a novel function of vWF as an extracellular inhibitor of Notch signaling. In sum, these studies demonstrate vWF in the vessel wall as a common feature of cerebral SVD; furthermore, we provide a plausible mechanism by which non-hemostatic vWF may play a novel role in the promotion of vascular disease.

Up-regulation of KCa3.1 promotes human airway smooth muscle cell phenotypic modulation

Airway smooth muscle (ASM) cell phenotype modulation, characterized by reversible switching between contractile and proliferative phenotypes, is considered to contribute to proliferative diseases such as allergic asthma and chronic obstructive pulmonary disease (COPD). KCa3.1 has been suggested to be involved in regulating ASM cell activation, proliferation, and migration. However, little is known regarding the exact role of KCa3.1 in ASM cell phenotypic modulation. To elucidate the role of KCa3.1 in regulating ASM cell phenotypic modulation, we investigated the effects of KCa3.1 channels on ASM contractile marker protein expression, proliferation and migration of primary human bronchial smooth muscle (BSM) cells. We found that PDGF increased KCa3.1 channel expression in BSM cells with a concomitant marked decrease in the expression of contractile phenotypic marker proteins including smooth muscle myosin heavy chain (SMMHC), smooth muscle α-actin (α-SMA), myocardin and KCa1.1. These changes were significantly attenuated by the KCa3.1 blocker, TRAM-34, or gene silencing of KCa3.1. Pharmacological blockade or gene silencing of KCa3.1 also suppressed PDGF-induced human BSM cell migration and proliferation accompanied by a decrease in intracellular free Ca2+ levels as a consequence of membrane depolarization, resulting in a reduction in cyclin D1 level and cell cycle arrest at G0-G1 phase. Additionally, PDGF-induced up-regulation of KCa3.1 and down-regulation of BSM contractile marker proteins were regulated by the ERK inhibitor U0126 and the AKT inhibitor LY294002. These findings highlight a novel role for the KCa3.1 channel in human BSM cell phenotypic modulation and provide a potential target for therapeutic intervention for proliferative airway diseases.

Docosahexaenoic acid (DHA)-induced heme oxygenase-1 attenuates cytotoxic effects of DHA in vascular smooth muscle cells

ObjectiveDocosahexaenoic acid (DHA), a member of n-3 polyunsaturated fatty acids (n-3 PUFA) is a potent regulator of molecular events implicated in cardiovascular health. In a previous study we found that Ca2+-dependent oxidative stress is the central and initial event responsible for induction of unfolded protein response (UPR), cell cycle arrest and apoptosis in DHA treated primary human smooth muscle cells isolated from small pulmonary artery (hPASMC). In the present study we examined the impact of heme oxygenase (HO)-1, induced by DHA, on DHA-induced oxidative stress, UPR, cell proliferation and apoptosis in hPASMC. Methods & resultsDHA led to a time- and concentration-dependent increase in HO-1 mRNA and protein levels in hPASMC. The DHA-induced HO-1 upregulation could be attenuated by preincubation of cells with a strong antioxidant Tempol or by siRNA-mediated depletion of nuclear factor erythroid 2-related factor-2 (Nrf2). In DHA-treated hPASMC, depletion of HO-1 by siRNA-mediated silencing resulted in increased levels of reactive oxygen species (ROS) and increased duration of UPR, the latter revealed by monitoring of spliced X-box binding protein 1 (XBP-1) variant. Moreover, HO-1 silencing augmented apoptosis in DHA-treated hPASMC as found by increased numbers of cleaved caspase-3-positive cells. HO-1 silencing did not affect proliferation of hPASMC exposed to DHA. ConclusionOur results indicate that DHA-induced, ROS-dependent upregulation of HO-1 attenuates oxidative stress, UPR and apoptosis in DHA-treated hPASMC.

Assessment of a novel, capsid-modified adenovirus with an improved vascular gene transfer profile

BackgroundCardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression.MethodsVascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3).ResultsCompared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies.ConclusionsWe have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer.

G-protein coupled receptor 35 activation mediates human vascular smooth muscle migration

Introduction: GPR35 is a poorly characterised G protein-coupled receptor endogenously activated by kynurenic acid and selectively coupled to Gα13, a G protein involved in regulation of cellular motility and proliferation via the Rho kinase pathway. The role of GPR35 in cardiovascular disease (CVD) is poorly explored, though it has been proposed to regulate blood pressure in rodents. Ligands which activate GPR35 are orthologue selective however, we have identified potent novel human GPR35 specific ligands; agonist pamoic acid and antagonists, CID-2745687 and ML145 for further dissection of GPR35 pharmacology. Based on GPR35's coupling to Gα13, here we investigated the role of GPR35 in primary human saphenous vein vascular smooth muscle cell (HSVSMC) migration and proliferation; a pathological process integral to the development of vein graft failure and restenosis. Methods: Human selective GPR35 ligands were pharmacologically characterised in β-arrestin translocation assays via bioluminenscence resonence energy transfer (BRET) in cells stably expressing FLAG-hGPR35-eYFP (Flp-In TREx 293). Endogenous expression of GPR35 was quantified in HSVSMC via QRT-PCR and migration was measured in HSVSMCs stimulated with 10-500nM pamoic acid, +/- CID-2745687 and ML145 (100nM) or ROCK1/2 inhibitor Y-27632 (10μM) in scratch wound assays using ImageJ. HSVSMC proliferation was assessed via MTS assay. Changes in HSVSMC morphology and actin-cytoskeletal structure were assessed via tetramethyl rhodamine isothiocyanate (TRITC) actin phalloidin staining. Results: BRET assays in FLAG-hGPR35-eYFP cells demonstrated that pamoic acid was a potent, human selective GPR35 agonist (pEC50 7.28±0.007) while CID-2745687 (pIC50 7.16±0.12) and ML145 (pIC50 8.08±0.12) were potent antagonists. QRT-PCR demonstrated HSVSMC endogenously express GPR35 (ΔCT=19.97±0.23, normalised to 18s). Analysis of the actin cytoskeleton revealed GPR35 activation induced a rearrangement of actin filaments consistent with HSVSMC migration. Quantification of HSVSMC migration revealed the GPR35 agonist pamoic acid induced a 30% increase in migration (p 0.05 vs -serum control). GPR35 activation did not affect HSVSMC proliferation. Conclusion: Here, we have described a novel role for GPR35 activation in the migratory capacity of HSVSMC via its downstream effector ROCK1/2. This suggests GPR35 might be a therapeutic target in the setting of vein graft failure and restenosis.

Differential regulation of extracellular matrix and soluble fibulin-1 levels by TGF-β₁ in airway smooth muscle cells.

Fibulin-1 (FBLN-1) is a secreted glycoprotein that is associated with extracellular matrix (ECM) formation and rebuilding. Abnormal and exaggerated deposition of ECM proteins is a hallmark of many fibrotic diseases, such as chronic obstructive pulmonary disease (COPD) where small airway fibrosis occurs. The aim of this study was to investigate the regulation of FBLN-1 by transforming growth factor beta 1 (TGF-β1) (a pro-fibrotic stimulus) in primary human airway smooth muscle (ASM) cells from volunteers with and without COPD. Human ASM cells were seeded at a density of 1×104 cells/cm2, and stimulated with or without TGF-β1 (10 ng/ml) for 72 hours before FBLN-1 deposition and soluble FBLN-1 were measured. Fold change in FBLN-1 mRNA was measured at 4, 8, 24, 48, 72 hours. In some experiments, cycloheximide (0.5 µg/ml) was used to assess the regulation of FBLN-1 production. TGF-β1 decreased the amount of soluble FBLN-1 both from COPD and non-COPD ASM cells. In contrast, the deposition of FBLN-1 into the ECM was increased in ASM cells obtained from both groups. TGF-β1 did not increase FBLN-1 gene expression at any of the time points. There were no differences in the TGF-β1 induced FBLN-1 levels between cells from people with or without COPD. Cycloheximide treatment, which inhibits protein synthesis, decreased both the constitutive release of soluble FBLN-1, and TGF-β1 induced ECM FBLN-1 deposition. Furthermore, in cycloheximide treated cells addition of soluble FBLN-1 resulted in incorporation of FBLN-1 into the ECM. Therefore the increased deposition of FBLN-1 by ASM cells into the ECM following treatment with TGF-β1 is likely due to incorporation of soluble FBLN-1 rather than de-novo synthesis.

Polyethyieneimine siRNA complexes released from coated electrospun polyethylene terephthalate bypass graft materials facilitate gene silencing in infiltrating primary human aortic smooth muscle cells

Polyethyieneimine siRNA complexes released from coated electrospun polyethylene terephthalate bypass graft materials facilitate gene silencing in infiltrating primary human aortic smooth muscle cells

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.