Human Coronary Artery Smooth Muscle Research Articles

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro

Neutrophil gelatinase-associated lipocalin (NGAL), a protein found in activated neutrophils, is expressed in kidney tubule cells in response to noxious stimuli, and is thus recognized as a marker of acute kidney injury. Recent studies have suggested that NGAL could also have pathophysiological importance in cardiovascular diseases. The aim of the present study was to examine NGAL expression in human carotid endarterectomy tissues exvivo as well as the effects of NGAL in the main cell types involved in atherogenesis, namely in human macrophages, endothelial cells, and smooth muscle cells invitro. NGAL protein was analyzed in human endarterectomy samples from patients with asymptomatic and symptomatic carotid stenosis by immunofluorescence, and NGAL mRNA expression was detected using RealTime-PCR. Human monocyte derived macrophages (MDM), human coronary artery smooth muscle cells (HCASMC), and human umbilical vein endothelial cells (HUVEC) were treated with recombinant human (rh) NGAL at different concentrations. Interleukin (IL)-6, IL-8, and monocyte chemo-attractant protein-1 (MCP-1) were determined by specific enzyme linked immunosorbent assays (ELISAs) in culture supernatants of such treated cells. Expression of NGAL protein was demonstrated by macrophages, smooth muscle cells, and endothelial cells in human carotid atherosclerotic tissue. NGAL mRNA expression was detected at a higher rate in atherosclerotic tissue of patients with symptomatic carotid stenosis (in 70%; n=19) compared with asymptomatic patients (in 37%; n=20, p<.001). Treatment of MDM, HCASMC, and HUVEC with rhNGAL led to a significant (p<0.05) and concentration dependent increase of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 in all cell types analyzed. By induction of pro-inflammatory mediators in human macrophages, smooth muscle cells and endothelial cells, NGAL, which is predominantly expressed in atherosclerotic plaques of symptomatic patients, could be involved in creating the local and systemic pro-inflammatory environment characteristic for atherosclerosis.

Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress

Background/Aims: Smooth muscle cells may dedifferentiate into the synthetic phenotype and promote atherosclerosis. Here, we explored the role of myoendothelial gap junctions in phenotypic switching of human coronary artery smooth muscle cells (HCASMCs) co-cultured with human coronary artery endothelial cells (HCAECs) exposed to shear stress. Methods: HCASMCs and HCAECs were seeded on opposite sides of Transwell inserts, and HCAECs were exposed to laminar shear stress of 12 dyn/cm² or 5 dyn/cm². The myoendothelial gap junctions were evaluated by using a multi-photon microscope. Results: In co-culture with HCAECs, HCASMCs exhibited a contractile phenotype, and maintained the expression of differentiation markers MHC and H1-calponin. HCASMCs and HCAECs formed functional intercellular junctions, as evidenced by colocalization of connexin(Cx)40 and Cx43 on cellular projections inside the Transwell membrane and biocytin transfer from HCAECs to HCASMCs. Cx40 siRNA and 18-α-GA attenuated protein expression of MHC and H1-calponin in HCASMCs. Shear stress of 5 dyn/cm² increased Cx43 and decreased Cx40 expression in HCAECs, and partly inhibited biocytin transfer from HCAECs to HCASMCs, which could be completely blocked by Cx43 siRNA or restored by Cx40 DNA transfected into HCAECs. The exposure of HCAECs to shear stress of 5 dyn/cm² promoted HCASMC phenotypic switching, manifested by morphological changes, decrease in MHC and H1-calponin expression, and increase in platelet-derived growth factor (PDGF)-BB release, which was partly rescued by Cx43 siRNA or Cx40 DNA or PDGF receptor signaling inhibitor. Conclusions: The exposure of HCAECs to shear stress of 5 dyn/cm² caused the dysfunction of Cx40/Cx43 heterotypic myoendothelial gap junctions, which may be replaced by homotypic Cx43/Cx43 channels, and induced HCASMC transition to the synthetic phenotype associated with the activation of PDGF receptor signaling, which may contribute to shear stress-associated arteriosclerosis.

Endothelial nitric oxide synthase induces heat shock protein HSPA6 (HSP70B′) in human arterial smooth muscle cells

Endothelial nitric oxide synthase (eNOS) is the major source of nitric oxide (NO) production in blood vessels. One of the pleitropic functions of eNOS derived NO is to inhibit vascular smooth muscle cell proliferation in the blood vessel wall, and whose dysfunction is a primary cause of atherosclerosis and restenosis. In this study there was an interest in examining the gene profile of eNOS adenoviral (Ad-eNOS) transduced human coronary artery smooth muscle cells (HCASMC) to further understand the eNOS inhibitory effect on smooth muscle cell proliferation. To this aim a whole genome wide analysis of eNOS transduced HCASMCs was performed. A total of 19 genes were up regulated, and 31 genes down regulated in Ad-eNOS transduced HCASMCs compared to cells treated with an empty adenovirus. Noticeably, a cluster of HSP70 gene family members was amongst the genes up regulated. Quantitative PCR confirmed that transcripts for HSPA1A (HSP70A), HSPA1B (HSP70B) and HSPA6 (HSP70B′) were elevated 2, 1.7 and 14-fold respectively in Ad-eNOS treated cells. The novel gene HSPA6 was further explored as a potential mediator of eNOS signaling in HCASMC. Immunoblotting showed that HSPA6 protein was induced by Ade-NOS. To functionally examine the effect of HSPA6 on SMCs, an adenovirus harboring the HSPA6 gene under the control of a constitutive promoter was generated. Transduction of HCASMCs with Ad-HSPA6 inhibited SMC proliferation at 3 and 6 days post serum growth stimulation, and paralleled the Ad-eNOS inhibition of SMC growth. The identification in this study that HSPA6 overexpression inhibits SMC proliferation coupled with the recent finding that inhibition of HSP90 has a similar effect, progresses the field of targeting HSPs for vascular repair.

Abstract 17671: TCF21 Regulates Coronary Artery Disease Causing Aryl-hydrocarbon Receptor Gene Expression and its Downstream Pathway Activation by Environmental Ligands

Background: Recently, the TCF21 gene was linked to the highly replicated coronary artery disease (CAD) associated polymorphism in the 6q23 locus. TCF21 is especially compelling as a causal gene since it is a transcription factor that is known to play a critical role in the development of coronary arteries. One downstream target gene identified through preliminary studies was AHR. AHR is a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (eg. cigarette smoke), and has recently been associated with CAD in mice and humans. We set out to identify the TCF21 downstream pathways that are related to CAD. Methods: We performed chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA-sequencing using primary human coronary artery smooth muscle cells (HCASMC) to survey the targets of TCF21 binding and the regulated downstream pathways. The peaks from ChIP-Seq were identified using MACSv2 according to ENCODE parameters. RNA-Seq was analyzed using edgeR to identify the differentially expressed genes. The effect of TCF21 on Aryl-hydrocarbon receptor (AHR) pathway activation was tested using knockdown and overexpression lentivirus transduction of HCASMC followed by 2,3,7,8-Tetrachlorodibenxo- p -dioxin (TCDD) treatment and qPCR. Results: RNA-Seq studies identified AHR as a significantly regulated mRNA downstream of TCF21 (p=1.4x10 -5 ). Also, the ChIP-Seq identified multiple TCF21 binding peaks in the AHR gene. We found a significant overlap of the AHR and TCF21 binding peaks (p&lt;1x10 -15 ) and genes differentially regulated by TCF21 and AHR (p=1x10 -5 ), among publicly available datasets. Furthermore, we found that the TCF21 level modulated the magnitude of the ligand-dependent activation of AHR pathway genes by TCDD. Conclusion: We identified a novel interaction between TCF21 and AHR, a gene involved in environmental xenobiotic response that was recently associated with CAD. The functional link between TCF21 and AHR provides an opportunity to explore gene by environment interactions in the pathogenesis of CAD.

Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

BackgroundTranscription of the myristoylated alanine‐rich C kinase substrate (MARCKS) is upregulated in animal models of intimal hyperplasia. MARCKS knockdown inhibits vascular smooth muscle cell (VSMC) migration in vitro; however, the mechanism is as yet unknown. We sought to elucidate the mechanism of MARCKS‐mediated motility and determine whether MARCKS knockdown reduces intimal hyperplasia formation in vivo.Methods and Results MARCKS knockdown blocked platelet‐derived growth factor (PDGF)‐induced translocation of cortactin to the cell cortex, impaired both lamellipodia and filopodia formation, and attenuated motility of human coronary artery smooth muscle cells (CASMCs). Activation of the small GTPases, Rac1 and Cdc42, was prevented by MARCKS knockdown. Phosphorylation of MARCKS resulted in a transient shift of MARCKS from the plasma membrane to the cytosol. MARCKS knockdown significantly decreased membrane‐associated phosphatidylinositol 4,5‐bisphosphate (PIP 2) levels. Cotransfection with an intact, unphosphorylated MARCKS, which has a high binding affinity for PIP 2, restored membrane‐associated PIP 2 levels and was indispensable for activation of Rac1 and Cdc42 and, ultimately, VSMC migration. Overexpression of MARCKS in differentiated VSMCs increased membrane PIP 2 abundance, Rac1 and Cdc42 activity, and cell motility. MARCKS protein was upregulated early in the development of intimal hyperplasia in the murine carotid ligation model. Decreased MARKCS expression, but not total knockdown, attenuated intimal hyperplasia formation.Conclusions MARCKS upregulation increases VSMC motility by activation of Rac1 and Cdc42. These effects are mediated by MARCKS sequestering PIP 2 at the plasma membrane. This study delineates a novel mechanism for MARCKS‐mediated VSMC migration and supports the rational for MARCKS knockdown to prevent intimal hyperplasia.

GW26-e1244 Effects of Lipopolysaccharide on the Growth and Proliferation of Human Coronary Artery Smooth Muscle Cells in Vitro

To explore the safe range of lipopolysaccharide(LPS) density and provide the experimental data for the related research of human coronary artery smooth muscle cells (HCASMC) associated with inflammation, we investigated the effects of different ratios of LPS at different points on the growth of

GW26-e1243 Inhibitory Effects of Hirudin on the Growth and Proliferation of Human Coronary Artery Smooth Muscle Cells and Endothelial Cells in Vitro

GW26-e1243 Inhibitory Effects of Hirudin on the Growth and Proliferation of Human Coronary Artery Smooth Muscle Cells and Endothelial Cells in Vitro

GW26-e0757 Inhibitory Effects of Luofengning-0 Formula on the Growth and Proliferation of Human Coronary Artery Smooth Muscle Cells and Endothelial Cells in Vitro: An Exploration of a New Type of Complex Monomer of Chinese Herbs Eluting Stents

GW26-e0757 Inhibitory Effects of Luofengning-0 Formula on the Growth and Proliferation of Human Coronary Artery Smooth Muscle Cells and Endothelial Cells in Vitro: An Exploration of a New Type of Complex Monomer of Chinese Herbs Eluting Stents

Tumor-targeted and pH-controlled delivery of doxorubicin using gold nanorods for lung cancer therapy.

BackgroundIn lung cancer, the efficacy of conventional chemotherapy is limited due to poor drug accumulation in tumors and nonspecific cytotoxicity. Resolving these issues will increase therapeutic efficacy.MethodsGNR-Dox-Tf-NPs (gold nanorod-doxorubicin-transferrin-nanoparticles) were prepared by different chemical approaches. The efficacy of these nanoparticles was carried out by cell viability in lung cancer and primary coronary artery smooth muscle cells. The receptor-mediated endocytosis studies were done with human transferrin and desferrioxamine preincubation. The GNR-Dox-Tf nanoparticles induced apoptosis, and DNA damage studies were done by Western blot, H2AX foci, and comet assay.ResultsWe developed and tested a gold nanorod-based multifunctional nanoparticle system (GNR-Dox-Tf-NP) that carries Dox conjugated to a pH-sensitive linker and is targeted to the transferrin receptor overexpressed in human lung cancer (A549, HCC827) cells. GNR-Dox-Tf-NP underwent physicochemical characterization, specificity assays, tumor uptake studies, and hyperspectral imaging. Biological studies demonstrated that transferrin receptor-mediated uptake of the GNR-Dox-Tf-NP by A549 and HCC827 cells produced increased DNA damage, apoptosis, and cell killing compared with nontargeted GNR-Dox-NP. GNR-Dox-Tf-NP-mediated cytotoxicity was greater (48% A549, 46% HCC827) than GNR-Dox-NP-mediated cytotoxicity (36% A549, 39% HCC827). Further, GNR-Dox-Tf-NP markedly reduced cytotoxicity in normal human coronary artery smooth muscle cells compared with free Dox.ConclusionThus, GNR-Dox-Tf nanoparticles can selectively target and deliver Dox to lung tumor cells and alleviate free Dox-mediated toxicity to normal cells.

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

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

Fluid shear stress upregulates placental growth factor in the vessel wall via NADPH oxidase 4.

Placental growth factor (PLGF), a potent stimulator of arteriogenesis, is upregulated during outward arterial remodeling. Increased fluid shear stress (FSS) is a key physiological stimulus for arteriogenesis. However, the role of FSS in regulating PLGF expression is unknown. To test the hypothesis that FSS regulates PLGF expression in vascular cells and to identify the signaling pathways involved, human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells were cultured on either side of porous Transwell inserts. HCAEC were then exposed to pulsatile FSS of 0.07 Pa ("normal," mimicking flow through quiescent collaterals), 1.24 Pa ("high," mimicking increased flow in remodeling collaterals), or 0.00 Pa ("static") for 2 h. High FSS increased secreted PLGF protein ∼1.4-fold compared with static control (n = 5, P < 0.01), while normal FSS had no significant effect on PLGF. Similarly, high flow stimulated PLGF mRNA expression nearly twofold in isolated mouse mesenteric arterioles. PLGF knockdown using siRNA revealed that HCAEC were the primary source of PLGF in cocultures (n = 5, P < 0.01). Both H2O2 and nitric oxide production were increased by FSS compared with static control (n = 5, P < 0.05). N(G)-nitro-l-arginine methyl ester (100 μM) had no significant effect on the FSS-induced increase in PLGF. In contrast, both catalase (500 U/ml) and diphenyleneiodonium (5 μM) attenuated the effects of FSS on PLGF protein in cocultures. Diphenyleneiodonium also blocked the effect of high flow to upregulate PLGF mRNA in isolated arterioles. Further studies identified NADPH oxidase 4 as a source of reactive oxygen species for this pathway. We conclude that FSS regulates PLGF expression via NADPH oxidase 4 and reactive oxygen species signaling.

Abstract P002: Aldosterone Promotes the Release of miRNA-Containing Exosomes from Endothelial Cells, Leading to Uptake by Smooth Muscle Cells

Background: Aldosterone increases exocytosis from endothelial cells, which may contribute to the detrimental actions of aldosterone in the vascular system. We propose a role for exocytosis of exosomes; these small extracellular particles contain host cell genetic material and once released they can circulate in the blood and transfer their contents (e.g. miRNAs) to recipient cells. We aimed to examine the effect of aldosterone on exosome release from endothelial cells, the miRNA content of exosomes and finally, exosome trafficking of miRNA from endothelial cells to smooth muscle cells. Methods and Results: Exosomes were isolated via ultracentrifugation from the media of human coronary artery endothelial cells (HCAECs) following incubation with aldosterone (100nM) or vehicle (DMSO). Exosomes were analyzed using western blot analysis testing for exosome and cell markers, and by nanoparticle-tracking analysis (NanoSight); aldosterone increased exosome concentration by 2.65 fold (19.1x1010 particles/mL) compared to the vehicle control (7.2x1010 particles/mL). RNA was extracted from isolated exosomes and host HCAECs and levels of miR-24 measured by qRT-PCR. miR-24 levels were unaffected by aldosterone stimulation in HCAECs (0.58±0.19; p=0.07) but were significantly increased in exosomes isolated from the media (2.35±0.07 fold; p=0.03). To test exosome paracrine trafficking, HCAECs were transfected with a c.elegans miRNA (cel-miR-39) and incubated adjacent to human coronary artery smooth muscle cells (HCASMCs) separated by a 0.4 μm filter, with or without aldosterone. Levels of cel-miR-39 in HCASMCs increased significantly (2.42±0.52 fold; p=0.02) in aldosterone-stimulated cells, compared to control. Blocking transcription with Dactinomycin (2.07±0.61 fold; p&gt;0.05) or blocking the mineralocorticoid receptor had no effect on aldosterone-mediated trafficking (3.43±1.49 fold; p&gt;0.05). Conclusion: This study demonstrates that aldosterone: 1) increases exosome secretion from endothelial cells, 2) increases miR-24 content of exosomes and 3) contributes to an increased uptake of exosome-packaged microRNAs by smooth muscle cells. Together, this represents a novel mechanism by which aldosterone contributes to vascular disease.

PP61 - Nrf2-mediated protection by the copper-bis(thiosemicarbazone) complex Cu(II)ATSM in human vascular smooth muscle cells

Copper(II)-diacetyl-bis(N4-methylthiosemi-carbazone) [Cu(II)ATSM] was developed as a selective positron emission tomography agent for imaging hypoxia, however the mechanism of its intracellular retention remains to be elucidated. Recent studies have highlighted the neuroprotective effects of Cu(II)ATSM against nitrosative damage in a mouse model of amyotrophic lateral sclerosis. As Cu(II)ATSM has also been shown to reduce peroxynitrite levels and oxidative stress in vitro, we sought to assess the effects of Cu(II)ATSM on activation of the Nrf2 defence pathway and protection against angiotensin II (Ang II) mediated apoptosis in human coronary artery smooth muscle cells (HCASMC). Treatment of HCASMC with Ang II (200nM, 12h) significantly increased apoptosis detected by annexin V fluorescence (P Supported by Heart Research UK (NET01/13).

Involvement of Rho-associated protein kinase (ROCK) and bone morphogenetic protein-binding endothelial cell precursor-derived regulator (BMPER) in high glucose-increased alkaline phosphatase expression and activity in human coronary artery smooth muscle cells.

BackgroundVascular calcification is an independent risk factor for cardiovascular disease. Diabetes mellitus increases the incidence of vascular calcification; however, detailed molecular mechanisms of vascular calcification in diabetes mellitus remain unknown. We recently reported that bone morphogenetic protein-binding endothelial cell precursor-derived regulator (BMPER) regulates osteoblast-like trans-differentiation of human coronary artery smooth muscle cells (HCASMCs).MethodsWe investigated the effect of a hydroxymethylglutaryl-coenzyme A reductase inhibitor (statin), commonly used in patients with atherosclerotic diseases and diabetes mellitus, on alkaline phosphatase (ALP) mRNA expression in aortas of streptozotocin-induced diabetic mice. We also investigated the effects of the statin, Rho-associated protein kinase (ROCK) inhibitors and BMPER knockdown on ALP mRNA expression and activity in HCASMCs cultured in high glucose-containing media.ResultsAlkaline phosphatase mRNA expression was increased in aortas of streptozotocin-induced diabetic mice, and the increase was inhibited by rosuvastatin. ALP mRNA expression and activity were increased in HCASMCs cultured in high glucose-containing media, and the increases were suppressed by rosuvastatin. This suppression was reversed by the addition of mevalonate or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate. High glucose-increased ALP mRNA expression and activity were suppressed by ROCK inhibitors. Moreover, BMPER mRNA expression was increased in diabetic mouse aortas and in HCASMCs cultured in high glucose-containing media, but was not inhibited by rosuvastatin or ROCK inhibitors. Knockdown of BMPER suppressed high glucose-increased ALP activity, but not ROCK activity in HCASMCs.ConclusionsThere are at least two independent pathways in high glucose-induced ALP activation in HCASMCs: the Rho–ROCK signaling pathway and the BMPER-dependent pathway.

Myocardin Family Members Drive Formation of Caveolae.

Caveolae are membrane organelles that play roles in glucose and lipid metabolism and in vascular function. Formation of caveolae requires caveolins and cavins. The make-up of caveolae and their density is considered to reflect cell-specific transcriptional control mechanisms for caveolins and cavins, but knowledge regarding regulation of caveolae genes is incomplete. Myocardin (MYOCD) and its relative MRTF-A (MKL1) are transcriptional coactivators that control genes which promote smooth muscle differentiation. MRTF-A communicates changes in actin polymerization to nuclear gene transcription. Here we tested if myocardin family proteins control biogenesis of caveolae via activation of caveolin and cavin transcription. Using human coronary artery smooth muscle cells we found that jasplakinolide and latrunculin B (LatB), substances that promote and inhibit actin polymerization, increased and decreased protein levels of caveolins and cavins, respectively. The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB. Overexpression of myocardin and MRTF-A caused 5-10-fold induction of caveolins whereas cavin-1 and cavin-2 were induced 2-3-fold. PACSIN2 also increased, establishing positive regulation of caveolae genes from three families. Full regulation of CAV1 was retained in its proximal promoter. Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs. Viral transduction of myocardin increased the density of caveolae 5-fold in vitro. A decrease of CAV1 was observed concomitant with a decrease of the smooth muscle marker calponin in aortic aneurysms from mice (C57Bl/6) infused with angiotensin II. Human expression data disclosed correlations of MYOCD with CAV1 in a majority of human tissues and in the heart, correlation with MKL2 (MRTF-B) was observed. The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.

Simvastatin Increases Fibulin-2 Expression in Human Coronary Artery Smooth Muscle Cells via RhoA/Rho-Kinase Signaling Pathway Inhibition.

The composition and structure of the extracellular matrix (ECM) in the vascular wall and in the atherosclerotic plaque are important factors that determine plaque stability. Statins can stabilize atherosclerotic plaques by modulating ECM protein expression. Fibulins are important components of the ECM. We evaluated the in vitro effect of simvastatin on the expression of fibulin-1, -2, -4 and -5 in human coronary artery smooth muscle cells (SMCs) and the mechanisms involved. Cells were incubated with simvastatin (0.05–1 μM), mevalonate (100 and 200 μM), geranylgeranyl pyrophosphate (GGPP) (15 μM), farnesyl pyrophosphate (FPP) (15 μM), the Rho kinase (ROCK) inhibitor Y-27632 (15 and 20 μM), the Rac-1 inhibitor (another member of Rho family) NSC23766 (100 μM), arachidonic acid (a RhoA/ROCK activator, 25–100 μM) and other fatty acids that are not activators of RhoA/ROCK (25–100 μM). Gene expression was analyzed by quantitative real-time PCR, and fibulin protein levels were analyzed by western blotting and ELISA. Simvastatin induced a significant increase in mRNA and protein levels of fibulin-2 at 24 hours of incubation (p<0.05), but it did not affect fibulin-1, -4, and -5 expression. Mevalonate and GGPP were able to reverse simvastatin’s effect, while FPP did not. In addition, Y-27632, but not NSC23766, significantly increased fibulin-2 expression. Furthermore, activation of the RhoA/ROCK pathway with arachidonic acid decreased fibulin-2 mRNA. Simvastatin increased mRNA levels and protein expression of the ECM protein fibulin-2 through a RhoA and Rho-Kinase-mediated pathway. This increase could affect the composition and structure of the ECM.

Activation of Transcription Factor GAX and Concomitant Downregulation of IL-1β and ERK1/2 Modulate Vascular Smooth Muscle Cell Phenotype in 3D Fibrous Scaffolds.

Since vascular smooth muscle cells (VSMCs) display phenotypic plasticity in response to changing environmental cues, understanding the molecular mechanisms underlying the phenotypic modulation mediated by a three-dimensional (3D) scaffold is important to engineer functional vasculature. Following cell seeding into 3D scaffolds, the synthetic phenotype is desired to enable cells to expand rapidly and produce and assemble extracellular matrix components, but must revert to a quiescent contractile phenotype after tissue fabrication to impart the contractile properties found in native blood vessels. This study shows that 3D electrospun fibrous scaffolds regulate human coronary artery smooth muscle cells (HCASMCs) toward a more synthetic phenotype characterized by reduced contractile markers, such as smooth muscle alpha-actin and calponin. The reduction in contractile markers expression was mediated by endogenously expressed proinflammatory cytokine interleukin-1β (IL-1β). 3D topography transiently induces concomitant upregulation of IL-1β and MAPK ERK1/2 through nuclear factor-κB-dependent signaling pathway. An early burst of expression of IL-1β is essential for suppression of the homeobox transcription factor Gax and related cyclin-dependent kinase inhibitor p21(Cip1), which are key regulators for cells exiting from cell cycle. Our findings provide new insights for understanding signaling mechanisms of HCASMCs in electrospun 3D fibrous scaffolds, which have considerable value for application in vascular tissue engineering.

Immunomodulatory polymeric scaffold enhances extracellular matrix production in cell co-cultures under dynamic mechanical stimulation

Despite the importance of immune cells in regulating the wound healing process following injury, there are few examples of synthetic biomaterials that have the capacity to push the body’s immune cells toward pro-regeneration phenotypes, and fewer still that are designed with the intention of achieving this immunomodulatory character. While monocytes and their derived macrophages have been recognized as important contributors to tissue remodeling in vivo, this is primarily believed to be due to their ability to regulate other cell types. The ability of monocytes and macrophages to generate tissue products themselves, however, is currently not well appreciated within the field of tissue regeneration. Furthermore, while monocytes/macrophages are found in remodeling tissue that is subjected to mechanical loading, the effect this biomechanical strain on monocytes/macrophages and their ability to regulate tissue-specific cellular activity has not been understood due to the complexity of the many factors involved in the in vivo setting, hence necessitating the use of controlled in vitro culture platforms to investigate this phenomenon. In this study, human monocytes were co-cultured with human coronary artery smooth muscle cells (VSMCs) on a tubular (3mm ID) degradable polyurethane scaffold, with a unique combination of non-ionic polar, hydrophobic and ionic chemistry (D-PHI). The goal was to determine if such a synthetic matrix could be used in a co-culture system along with dynamic biomechanical stimulus (10% circumferential strain, 1Hz) conditions in order to direct monocytes to enhance tissue generation, and to better comprehend the different ways in which monocytes/macrophages may contribute to new tissue production. Mechanical strain and monocyte co-culture had a complementary and non-mitigating effect on VSMC growth. Co-culture samples demonstrated increased deposition of sulphated glycosaminoglycans (GAGs) and elastin, as well as increases in the release of FGF-2, a growth factor that can stimulate VSMC growth, while dynamic culture supported increases in collagen I and III as well as increased mechanical properties (elastic modulus, tensile strength) vs. static controls. Macrophage polarization toward an M1 state was not promoted by the biomaterial or culture conditions tested. Monocytes/macrophages cultured on D-PHI were also shown to produce vascular extracellular matrix components, including collagen I, collagen III, elastin, and GAGs. This study highlights the use of synthetic biomaterials having immunomodulatory character in order to promote cell and tissue growth when used in tissue engineering strategies, and identifies ECM deposition by monocytes/macrophages as an unexpected source of this new tissue. Statement of SignificanceThe ability of biomaterials to regulate macrophage activation towards a wound healing phenotype has recently been shown to support positive tissue regeneration. However, the ability of immunomodulatory biomaterials to harness monocyte/macrophage activity to support tissue engineering strategies in vitro holds enormous potential that has yet to be investigated. This study used a monocyte co-culture on a degradable polyurethane (D-PHI) to regulate the response of VSMCs in combination with biomechanical strain in a vascular tissue engineering context. Results demonstrate that immunomodulatory biomaterials, such as D-PHI, that support a desirable macrophage activation state can be combined with biomechanical strain to augment vascular tissue production in vitro, in part due to the novel and unexpected contribution of monocytes/macrophages themselves producing vascular ECM proteins.

Down-regulation of hsa-miR-1264 contributes to DNMT1-mediated silencing of SOCS3.

Previously we found decreased expression of SOCS3 in neointimal hyperplastic region following balloon angioplasty in atherosclerotic micro swine. In our recent in vitro studies using human coronary artery smooth muscle cells (HCASMC), we observed the inhibition of SOCS3 expression in the presence of both TNF-α and IGF-1, correlating with the in vivo findings in microswine. We also reported that two independent mechanisms, JAK/STAT3/NFκB and promoter methylation of SOCS3 were responsible for TNF-α and IGF-1 induced SOCS3 inhibition. In this study, using miRNA array and gene expression approaches, we explored the molecular mechanisms involved in the above SOCS3 repression and identified several miRNAs that are associated with the regulation of SOCS3 expression. Our miRNA expression profiling revealed profound down-regulation of two specific miRNAs, hsa-miR-758 and hsa-miR-1264, whose expression levelswere decreased by 8-10 folds inHCASMCs thatwere treated with both TNF-α and IGF-1. This was accompanied with a significant up-regulation of three specific miRNAs, hsa-miR-155, hsa-miR-146b-5p and hsa-miR-146a, which showed about 3-7 fold increases in their expression levels. Importantly, we also found that the miRNA hsa-miR-1264 targets DNA methyltransferase-1 (DNMT1) transcripts by binding to its 3'UTR region to affect its expression. Expression of hsa-miR-1264 in HCASMCs not only resulted in decreased DNMT1 mRNA transcripts but it also increased SOCS3 expression. The treatment with TNF-α and IGF-1 resulted in drastic decrease in hsa-miR-1264 levels with no change in the expression of DNMT1. Consequently, the DNMT1 activity caused hypermethylation in the CpG island of the SOCS3 promoter region and inhibited its expression. This could be a causative epigenetic mechanism associated with TNF-α and IGF-1 induced smooth muscle cell proliferation involved in the pathogenesis of coronary artery hyperplasia and restenosis.

Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci.

To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including “growth factor binding,” “matrix interaction,” and “smooth muscle contraction.” We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology.