Treatment Of Smooth Muscle Cells Research Articles

Abstract 13977: Insulin-Like Growth Factor Binding Protein 7 is Associated With Development of Cell Senescence and Progression of Coronary Atherosclerosis

Introduction: Cellular senescence, an irreversible cell cycle arrest, has been associated with atherosclerosis. Senescent cells (SC) secrete proinflammatory molecules contributing to disease progression. Insulin-like growth factor binding protein 7 (IGFBP7) binds IGF-1 and also interacts with IGF-1 receptor, suppressing intracellular IGF-1 signaling. IGFBP7 has been linked to heart failure-associated cell senescence. Hypothesis: We hypothesize that SC secrete IGFBP7, and IGFBP7 contributes to atherogenesis. Methods: Premature cell senescence was induced by treatment of smooth muscle cells (SMC) with H2O2. IGFBP7 levels were measured by immunoblotting of cell lysates and conditioned media (CM) and by immunohistochemistry in Apoe-null mice and in Rapacz hypercholesterolemic pigs. Binding of IGFBP7 with IGF-1 receptor was quantified by proximity ligation assay. Results: H2O2-treated SMC have marked increase in expression of the senescence markers beta-galactosidase (SA-βGal) and γH2A.X, and 3-fold (P<0.05) increase in IGFBP7 in cell CM. Abundant senescent SMC (αSMA + /SA-βGal + ) were detected in fibrous cap’s plaque in murine innominate artery and these cells had increased IGFBP7 compared to SMC in the vascular media. Increased IGFBP7 levels (1.8-fold increase, P<0.05) were also detected in porcine coronary plaque and IGFBP7 upregulation correlated with elevated IGFBP7/IGF-1 receptor binding. These data suggest a mechanistic link between senescence, IGFBP7 upregulation and atherogenesis. SMC were incubated with CM from senescent SMC (or non-senescent controls) to determine if SC-secreted IGFBP7 inhibits IGF-1 signaling. Preincubation with CM from SC inhibited IGF-1-induced Akt phosphorylation, however co-incubation with anti-IGFBP7 antibody reversed this effect, indicating that IGFBP7 in the CM mediates the inhibitory effect on IGF-1 signaling. Conclusions: We have shown that SMC senescence induced IGFBP7 and that high IGFBP7 levels were associated with senescent SMC in the atherosclerotic plaque. IGFBP7 inhibits IGF-1 signaling in vitro and binds IGF-1 receptor in plaque SMC. Our results suggest a novel role of IGFBP7 in senescence and in atherogenesis and identify IGFBP7 as a potential target for anti-atherogenic therapy.

Monocyte adhesive hyaluronan matrix induced by hyperglycemia in diabetic lung injuries

Infiltrated pre-inflammatory monocytes and macrophages have important roles in the induction of diabetic lung injuries, but the mechanism mediating their infiltration is still unclear. Here, we showed that airway smooth muscle cells (SMCs) activated monocyte adhesion in response to hyperglycemic glucose (25.6mM) by significantly increasing hyaluronan (HA) in the cell matrix, with concurrent 2- to 4-fold increases in adhesion of U937 monocytic-leukemic cells. The HA-based structures were attributed directly to the high-glucose and not to increased extracellular osmolality, and they required growth stimulation of SMCs by serum. Treatment of SMCs with heparin in high-glucose induces synthesis of a much larger HA matrix, consistent with our observations in the glomerular SMCs. Further, we observed increases in tumor necrosis factor-stimulated gene-6 (TSG-6) expression in high-glucose and high-glucose plus heparin cultures, and the heavy chain (HC)-modified HA structures existed on the monocyte-adhesive cable structures in high-glucose and in high-glucose plus heparin-treated SMC cultures. Interestingly, these HC-modified HA structures were unevenly distributed along the HA cables. Further, the invitro assay with recombinant human TSG-6 and the HA14 oligo showed that heparin has no inhibitory activity on the TSG-6-induced HC-transfer to HA, consistent with the results from SMC cultures. These results support the hypothesis that hyperglycemia in airway smooth muscle induces the synthesis of a HA matrix that recruits inflammatory cells and establishes a chronic inflammatory process and fibrosis that lead to diabetic lung injuries.

Abstract 158: The Histone Demethylase JMJD3 Regulates Smooth Muscle Differentiation And Is Associated With Blood Pressure

Hypertension is associated with significant morbidity and mortality in the United States, and despite its prevalence an unmet need exists for more personalized targeted therapies. Blood pressure is regulated by environmental and genetic factors, and genome wide association studies (GWAS) have identified numerous genes in the human population associated with hypertension. Recently, one GWAS identified the single nucleotide polymorphism rs62059712 that is associated with systolic blood pressure in humans. rs62059712 is located upstream of the gene JMJD3, a histone demethylase that our lab has studied in a variety of disease contexts, and the rs62059712 major C allele (MAF 0.92) is associated with increased blood pressure in humans. Based on our analysis of data from the UCSC genome browser, we found that rs62059712 is located in a DNase Hypersensitive Site marked by H3K27Ac, suggesting it may regulate JMJD3 transcription. In luciferase assays, the fragment containing rs62059712 displayed transcription activity in smooth muscle cells (SMC), and the major C allele exhibited increased luciferase activity compared to the minor T allele in mouse aortic SMC (p<0.05). Notably, rs62059712 is in linkage disequilibrium with rs74480102, which did not display allele-specific transcription activity in SMC. siRNA knockdown of JMJD3 in mouse aortic SMC significantly decreased SMC-specific gene expression (p<0.05), and SMC marker gene expression was also reduced by treatment of SMC with the JMJD3 selective inhibitor GSK4J (p<0.05). Likewise, SMC contractile gene expression was reduced in aortas from SM22Cre;JMJD3 flox/flox mice harboring SMC-specific deletion of JMJD3. Further, angiotensin II increased JMJD3 expression in human aortic SMC. Finally, in chromatin immunoprecipitation assays in mouse aortic SMC, JMJD3 was significantly enriched at smooth muscle gene promoters (p<0.05). Our results identify the blood pressure-associated gene JMJD3 as a key epigenetic regulator of smooth muscle differentiation. Additionally, we show that the rs62059712 major C allele, which is associated with increased blood pressure, increases JMJD3 transcription activity, and likely expression, to drive increased downstream contractile smooth muscle gene expression.

Abstract 466: Fhl5 Enhances Vascular Calcification By Regulating Smooth Muscle Cell Transcriptional Networks

Coronary artery calcium (CAC) is a hallmark of coronary artery disease (CAD) with increased CAC levels associated with increased risk for CAD related morbidity and mortality. The development of CAC is a pathological process driven by the modulation of smooth muscle cells (SMCs) and maladaptive extracellular matrix remodeling. In the most recent multi-ancestry meta-analysis of more than 35,000 individuals, FHL5 (Four-and-a-half LIM domains 5) was prioritized as a novel CAC gene through Bayesian colocalization and epigenomic analyses. scRNA-seq studies of human coronary and carotid arteries revealed high enrichment of FHL5 gene expression in SMC and pericyte populations. These preliminary studies motivate our hypothesis that FHL5 functions as a transcriptional regulator of vascular remodeling pathways associated with increased CAC risk. Following treatment of SMCs with a calcifying cocktail, stable FHL5 overexpression promoted the osteogenic SMC phenotypic transition, characterized by increased levels of osteogenic activators and reduced expression of SMC contractile markers. This osteogenic gene signature correlated with increased SMC calcification, consistent with the direction of effect of the CAC risk allele. To provide insights into the transcriptional program regulated by FHL5 that contributes to the vascular calcification phenotype, we mapped FHL5 binding sites in SMCs using the CUT&RUN method. We identified 17,201 FHL5 binding sites that overlapped the active enhancer mark H3K27ac. FHL5 target genes were enriched in pathways linked to vascular remodeling, such as cell adhesion and extracellular matrix organization. Moreover, risk variants for pulse pressure, a metric of arterial stiffness that is highly correlated with CAC, was enriched in FHL5 binding sites. Lastly, integration with FHL5 trans-eQTL target genes in STARNET arterial tissues revealed a network of CAD-associated candidate genes regulated by FHL5 in vitro and in vivo that impact SMC proliferation, migration, and calcification. Taken together, we provide evidence that FHL5 regulates a network of genes linked to arterial stiffness, which may explain the underlying mechanism of its genetic association with CAC in individuals from the general population.

Myometrial-derived CXCL12 promotes lipopolysaccharide induced preterm labour by regulating macrophage migration, polarization and function in mice.

Preterm birth is a major contributor to neonatal mortality and morbidity. Infection results in elevation of inflammation‐related cytokines followed by infiltration of immune cells into gestational tissue. CXCL12 levels are elevated in preterm birth indicating it may have a role in preterm labour (PTL); however, the pathophysiological correlations between CXCL12/CXCR4 signalling and premature labour are poorly understood. In this study, PTL was induced using lipopolysaccharide (LPS) in a murine model. LPS induced CXCL12 RNA and protein levels significantly and specifically in myometrium compared with controls (3‐fold and 3.5‐fold respectively). Highest levels were found just before the start of labour. LPS also enhanced the infiltration of neutrophils, macrophages and T cells, and induced macrophage M1 polarization. In vitro studies showed that condition medium from LPS‐treated primary smooth muscle cells (SMC) induced macrophage migration, M1 polarization and upregulated inflammation‐related cytokines such as interleukin (IL)‐1, IL‐6 and tumor necrosis factor alpha (TNF‐α). AMD3100 treatment in pregnant mice led to a significant decrease in the rate of PTL (70%), prolonged pregnancy duration and suppressed macrophage infiltration into gestation tissue by 2.5‐fold. Further, in‐vitro treatment of SMC by AMD3100 suppressed the macrophage migration, decreased polarization and downregulated IL‐1, IL‐6 and TNF‐α expression. LPS treatment in pregnant mice induced PTL by increasing myometrial CXCL12, which recruits immune cells that in turn produce inflammation‐related cytokines. These effects stimulated by LPS were completely reversed by AMD3100 through blocking of CXCL12/CXCR4 signalling. Thus, the CXCL12/CXCR4 axis presents an excellent target for preventing infection and inflammation‐related PTL.

Endothelial cell-derived pro-fibrotic factors increase TGF-β1 expression by smooth muscle cells in response to cycles of hypoxia-hyperoxia

BackgroundThe vascular pathology of peripheral artery disease (PAD) encompasses abnormal microvascular architecture and fibrosis in response to ischemia-reperfusion (I/R) cycles. We aimed to investigate the mechanisms by which pathological changes in the microvasculature direct fibrosis in the context of I/R. MethodsPrimary human aortic endothelial cells (ECs) were cultured under cycles of normoxia-hypoxia (NH) or normoxia-hypoxia-hyperoxia (NHH) to mimic I/R. Primary human aortic smooth muscle cells (SMCs) were cultured and treated with media from the ECs. FindingsThe mRNA and protein expression of the pro-fibrotic factors platelet derived growth factor (PDGF)-BB and connective tissue growth factor (CTGF) were significantly upregulated in ECs undergoing NH or NHH cycles. Treatment of SMCs with media from ECs undergoing NH or NHH cycles led to significant increases in TGF-β1, TGF-β pathway signaling intermediates, and collagen expression. Addition of neutralizing antibodies against PDGF-BB and CTGF to the media blunted the increases in TGF-β1 and collagen expression. Treatment of SMCs with PAD patient-derived serum also led to increased TGF-β1 levels. InterpretationIn an in-vitro model of I/R, which recapitulates the pathophysiology of PAD, increased secretion of PDGF-BB and CTGF by ECs was shown to be predominantly driving TGF-β1-mediated expression by SMCs. These cell culture experiments help elucidate the mechanism and interaction between ECs and SMCs in microvascular fibrosis associated with I/R. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of I/R. FundingNational Institute on Aging at the National Institutes of Health grant number R01AG064420. Research in contextEvidence before this study: Previous studies in gastrocnemius biopsies from peripheral artery disease (PAD) patients showed that transforming growth factor beta 1 (TGF-β1), the most potent inducer of pathological fibrosis, is increased in the vasculature of PAD patients and correlated with collagen deposition. However, the exact cellular source of TGF-β1 remained unclear. Added value of this study: Exposing cells to cycles of normoxia-hypoxia-hyperoxia (NHH) resulted in pathological changes that are consistent with human PAD. This supports the idea that the use of NHH may be a reliable, novel in vitro model of PAD useful for studying associated pathophysiological mechanisms. Furthermore, pro-fibrotic factors (PDGF-BB and CTGF) released from endothelial cells were shown to induce a fibrotic phenotype in smooth muscle cells. This suggests a potential interaction between these cell types in the microvasculature that drives increased TGF-β1 expression and collagen deposition. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of ischemia-reperfusion.

Endothelial Progenitor Cells Modulate the Phenotype of Smooth Muscle Cells and Increase Their Neointimal Accumulation Following Vascular Injury

Smooth muscle cells (SMCs) are the main driver of neointima formation and restenosis following vascular injury. In animal models, endothelial progenitor cells (EPCs) accelerate endothelial regeneration and reduce neointima formation after arterial injury; however, EPC-capture stents do not reduce target vessel failure compared with conventional stents. Here we examined the influence of EPCs on features of SMCs pivotal for their impact on injury-induced neointima formation including proliferation, migration, and phenotype switch. EPCs, their conditioned medium, and EPC-derived microparticles induced proliferation of SMCs while limiting their apoptosis. In transwell membrane experiments and scratch assays, EPCs stimulated migration of SMCs and accelerated their recovery from scratch-induced injury. Treatment of SMCs with an EPC-derived conditioned medium or microparticles triggered transformation of SMCs toward a synthetic phenotype. However, co-cultivation of EPCs and SMCs enabling direct cell-cell contacts preserved their original phenotype and protected from the transformative effect of SMC cholesterol loading. Adhesion of EPCs to SMCs was stimulated by SMC injury and reduced by blocking CXCR2 and CCR5. Interaction of EPCs with SMCs modulated their secretory products and synergistically increased the release of selected chemokines. Following carotid wire injury in athymic mice, injection of EPCs resulted not only in reduced neointima formation but also in altered cellular composition of the neointima with augmented accumulation of SMCs. EPCs stimulate proliferation and migration of SMCs and increase their neointimal accumulation following vascular injury. Furthermore, EPCs context-dependently modify the SMC phenotype with protection from the transformative effect of cholesterol when a direct cell-cell contact is established.

Effect of cytokine treatment on the expression and secretion of brain derived neurotrophic factor in the smooth muscle of the rat colon.

The production of pro-inflammatory cytokines and chemokines is increased during inflammatory bowel disease (IBD). Previously, it was demonstrated that brain derived neurotrophic factor (BDNF) expression is increased in experimental models of colitis. BDNF is partially responsible for the structural and functional changes that take place during IBD. However, the exact mechanisms underlying the upregulation of BDNF during gut inflammation are unknown. The aim of the present study was to determine the effects of direct treatment of smooth muscle cells with inflammatory cytokines on the synthesis and secretion of BDNF. BDNF expression and secretion levels were measured using ELISA kits on tissue lysates and on incubation media used to culture the rat colon smooth muscle tissues treated for 24 h with either tumor necrosis factor (TNF)-α or interleukin (IL)-1β. Compared with the control tissue samples, treatment with TNF-α and IL-1β resulted in a significant increase in the protein expression levels of BDNF in the incubated smooth muscle tissue. TNF-α and IL-1β also stimulated the secretion of BDNF. Chelation of intracellular Ca2+ with BABTA-AM prevented the TNF-α and IL-1β-induced increase in BDNF protein expression and secretion levels. Furthermore, inhibition of protein kinase A (PKA) significantly reduced BDNF expression levels when treated with cytokines but not secretion. In conclusion, proinflammatory cytokines that are upregulated during IBD, directly stimulated BDNF expression and secretion in a Ca2+ dependent manner. Considering the ability of BDNF to enhance smooth muscle contraction and pain sensation, this autocrine loop may partially explain the characteristic hypercontractility and hypersensitivity associated with IBD.

Canagliflozin inhibits vascular smooth muscle cell proliferation and migration: Role of heme oxygenase-1

Recent cardiovascular outcome trials found that sodium-glucose cotransporter-2 (SGLT2) inhibitors reduce cardiovascular disease and mortality in type 2 diabetic patients; however, the underlying mechanisms are not fully known. Since the proliferation and migration of vascular smooth muscle cells (SMCs) contributes to the development of arterial lesions, we hypothesized that SGLT2 inhibitors may exert their beneficial cardiovascular effects by inhibiting the growth and movement of vascular SMCs. Treatment of rat or human aortic SMCs with clinically relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin, inhibited cell proliferation and migration. The inhibition of SMC growth by canagliflozin occurred in the absence of cell death, and was associated with the arrest of SMCs in the G0/G1 phase of the cell cycle and diminished DNA synthesis. Canagliflozin also resulted in the induction of heme oxygenase-1 (HO-1) expression, and a rise in HO activity in vascular SMCs, whereas, empagliflozin or dapagliflozin had no effect on HO activity. Canagliflozin also activated the HO-1 promoter and this was abrogated by mutating the antioxidant responsive element or by overexpressing dominant-negative NF-E2-related factor-2 (Nrf2). The induction of HO-1 by canagliflozin relied on reactive oxygen species (ROS) formation and was negated by antioxidants. Finally, silencing HO-1 expression partially rescued the proliferative and migratory response of canagliflozin-treated SMCs, and this was reversed by carbon monoxide and bilirubin. In conclusion, the present study identifies canagliflozin as a novel inhibitor of vascular SMC proliferation and migration. Moreover, it demonstrates that canagliflozin stimulates the expression of HO-1 in vascular SMCs via the ROS-Nrf2 pathway, and that the induction of HO-1 contributes to the cellular actions of canagliflozin. The ability of canagliflozin to exert these pleiotropic effects may contribute to the favorable clinical actions of the drug and suggest an extra potential benefit of canagliflozin relative to other SGLT2 inhibitors.

Canagliflozin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration

Cardiovascular disease is the major cause of morbidity and mortality in diabetes. Although numerous factors contribute to the development of diabetes‐associated cardiovascular disease, excessive proliferation and migration of vascular smooth muscle cells (SMCs) plays a critical role. Sodium‐glucose cotransporter 2 (SGLT2) inhibitors are the latest approved class of glucose lowering drugs. By blocking glucose uptake in the proximal tubule of the kidney, SGLT2 inhibitors induce glycosuria leading to decreases in fasting and postprandial glycemia. Notably, large multicenter clinical trials have demonstrated that SGLT2 inhibitors reduce cardiovascular disease and mortality in patients with type 2 diabetes. The mechanism underlying the cardiovascular benefits of SGLT2 inhibitors is not fully known; however, direct effects on vascular SMCs have not been considered. In the present study, we investigated the effect of three different SGLT2 inhibitors on vascular SMC function. Treatment of rat and human aortic SMCs with canagliflozin resulted in a potent, concentration‐dependent inhibition of cell proliferation and DNA synthesis. The anti‐proliferative effect of canagliflozin was observed at clinically relevant concentrations, and this was not seen with other SGLT2 inhibitors, including empagliflozin and dapagliflozin. The inhibition of SMC growth by canagliflozin occurred in the absence of cell death and was associated with the arrest of SMCs in the G0/G1 phase of the cell cycle. In addition, scratch‐wound assays found that vascular SMC migration was blocked by canagliflozin in a concentration‐dependent manner, whereas empagliflozin or dapagliflozin had no effect on SMC motility. In conclusion, the present study identified canagliflozin as a robust inhibitor of vascular SMC proliferation and migration. These inhibitory effects of canagliflozin are detected at pharmacologically relevant concentrations and are unique for this drug and not seen with other SGLT2 inhibitors. The ability of canagliflozin to exert these pleiotropic effects on vascular SMC function may contribute to the salutary cardiovascular actions of this drug in patients with type 2 diabetes.Support or Funding InformationSupported by American Diabetes Association grant #1‐17‐IBS‐290.

Abstract 883: Nadph Oxidase Nox1-dependent Endocytosis and Activation is Regulated by a Conserved Trafficking Motif in Vascular Smooth Muscle Cells

We previously reported that TNF-α-mediated Nox1-dependent redox signaling of vascular smooth muscle cells (SMC) requires endocytosis. However, the molecular mechanisms regulating Nox1 endocytosis are not known. The goal of this study was to identify motifs in the Nox1 protein sequence necessary for Nox1 endocytosis and reactive oxygen species (ROS) generation. Immunoprecipitation of Nox1, after treatment of SMC with TNF-α, showed it to be associated with Early Endosome Antigen 1 (EEA1) and Ras-related protein Rab-5A (Rab5), markers of early endosomes. Analysis of the Nox1 protein sequence identified two conserved putative trafficking motifs, Leucine-Leucine (LL), and Valine-Leucine-Valine (VLV). Biotinylation of SMC plasma membrane proteins under non-stimulated conditions suggested increased levels of Nox1 VLV mutant in the plasma membrane, whereas levels of Nox1 LL mutant where comparable to wildtype (WT) Nox1. Furthermore, cells expressing the VLV mutant showed impaired endocytosis of Nox1 following TNF-α as compared to cells expressing WT Nox1 or the LL mutant. SMC were treated with TNF-α in the presence of Oxyburst, a non-cell permeable redox-sensitive fluorophore whose internalization requires endocytosis. Cells expressing WT or Nox1 LL, but not Nox1 VLV, exhibited punctate intracellular fluorescence, indicating endosomal ROS production. Next, we examined whether the VLV motif is required for Nox1-dependent SMC migration. Transwell migration of SMC was similar in cells expressing WT or the LL Nox1 mutant; however, migration was decreased in cells expressing the Nox1 VLV mutant. Finally, we identified proteins whose association with Nox1 during endocytosis were dependent on the VLV motif. Nox1 WT and VLV mutant were cloned in frame with the biotin ligase BioID2. Following stimulation with TNF-α, Western blotting for anti-biotin, and subsequent mass spectrometry, identified several proteins directly associated with WT Nox1 but not Nox1 VLV. These findings suggest that endosomal compartmentalization of Nox1 and its redox signaling is regulated by a specific motif on Nox1 that is necessary for protein interactions. Targeted inhibition of Nox1 activation is a novel therapeutic strategy in the prevention of vascular disease.

Klf4, Klf2, and Zfp148 activate autophagy-related genes in smooth muscle cells during aortic aneurysm formation.

Abdominal aortic aneurysms (AAAs) are a progressive dilation of the aorta that is characterized by an initial influx of inflammatory cells followed by a pro‐inflammatory, migratory, proliferative, and eventually apoptotic smooth muscle cell phenotype. In recent years, the mechanisms related to the initial influx of inflammatory cells have become well‐studied; the mechanisms related to chronic aneurysm formation, smooth muscle cell apoptosis and death are less well‐characterized. Autophagy is a generally believed to be a protective cellular mechanism that functions to recycle defective proteins and cellular organelles to maintain cellular homeostasis. Our goal with the present study was to investigate the role of autophagy in smooth muscle cells during AAA formation. Levels of the autophagy factors, Beclin, and LC3 were elevated in human and mouse AAA tissue via both qPCR and immunohistochemical analysis. Confocal staining in human and mouse AAA tissue demonstrated Beclin and LC3 were present in smooth muscle cells during AAA formation. Treatment of smooth muscle cells with porcine pancreatic elastase or interleukin (IL)‐1β activated autophagy‐related genes in vitro while treatment with a siRNA to Kruppel‐like transcription factor 4 (Klf4), Kruppel‐like transcription factor 2 (Klf2) or Zinc‐finger protein 148 (Zfp148) separately inhibited activation of autophagy genes. Chromatin immunoprecipitation assays demonstrated that Klf4, Klf2, and Zfp148 separately bind autophagy genes in smooth muscle cells following elastase treatment. These results demonstrate that autophagy is an important mechanism related to Klfs in smooth muscle cells during AAA formation.

The phenotype of vascular smooth muscle cells co-cultured with endothelial cells is modulated by PDGFR-β/IQGAP1 signaling in LPS-induced intravascular injury.

Background Sepsis, a leading cause of death in intensive care units, is generally associated with vascular dysfunction. However, its pathophysiological process has not been fully clarified, lacking in-depth knowledge of its pathophysiological process may hinder the improvement of diagnosis and therapy for sepsis. Hence, as the key parts of the vascular wall, the interaction between endothelial cells (ECs) and smooth muscle cells (SMCs) under septic situation need to be further studied.Methods ECs and SMCs were co-cultured using Transwell plates. Lipopolysaccharide (LPS) was used to induce sepsis. A scratch-wound assay was used to assess cell migration, and western blotting was used to assess the level of redifferentiation of SMCs as well as the expression of PDGFR-β and IQGAP1.Results Co-culture with ECs reduced the redifferentiation of SMCs induced by LPS (10 μg/ml), which was characterized by increased migration ability and decreased expression of contractile proteins (e.g., SM22 and α-SMA). The production of TNF-α could decrease the level of PDGFR-β in SMCs. Treatment of SMCs with the PDGFR-β inhibitor imatinib (5 μM) was able to counteract LPS-induced SMC redifferentiation and reduce IQGAP1 protein expression, especially when SMCs were co-cultured with ECs.Conclusion The phenotype of vascular SMCs co-cultured with ECs was modulated by IQGAP1 through the PDGFR-β pathway, which may lead to vascular remodeling and homeostasis in LPS-induced intravascular injury. This pathway could be a novel target for the treatment of vascular damage.

Analysis of Combined Transcriptomes Identifies Gene Modules that Differentially Respond to Pathogenic Stimulation of Vascular Smooth Muscle and Endothelial Cells

Smooth muscle cells (SMCs) and endothelial cells (ECs) are vital cell types composing the vascular medial wall and the atheroprotective inner lining, respectively. Current treatments for cardiovascular disease inhibit SMC hyperplasia but compromise EC integrity, predisposing patients to thrombosis. Therapeutics targeting SMCs without collateral damage to ECs are highly desirable. However, differential (SMC versus EC) disease-associated regulations remain poorly defined. We conducted RNA-seq experiments to investigate SMC-versus-EC differential transcriptomic dynamics, following treatment of human primary SMCs and ECs with TNFα or IL-1β, both established inducers of SMC hyperplasia and EC dysfunction. As revealed by combined SMC/EC transcriptomes, after TNFα or IL-1β induction, 174 and 213 genes respectively showed greater up-regulation in SMCs than in ECs (SMC-enriched), while 117 and 138 genes showed greater up-regulation in ECs over SMCs (EC-enriched). Analysis of gene interaction networks identified central genes shared in the two SMC-enriched gene sets, and a distinct group of central genes common in the two EC-enriched gene sets. Significantly, four gene modules (subnetworks) were identified from these central genes, including SMC-enriched JUN and FYN modules and EC-enriched SMAD3 and XPO1 modules. These modules may inform potential intervention targets for selective blockage of SMC hyperplasia without endothelial damage.

Platelet extracellular vesicles induce a pro-inflammatory smooth muscle cell phenotype

ABSTRACTExtracellular vesicles (EVs) are mediators of cell communication during health and disease, and abundantly released by platelets upon activation or during ageing. Platelet EVs exert modulatory effects on immune and vascular cells. Platelet EVs may modulate the function of vascular smooth muscle cells (SMC). Platelet EVs were isolated from platelet-rich plasma and incubated with SMC in order to assess binding, proliferation, migration and pro-inflammatory phenotype of the cells. Platelet EVs firmly bound to resting SMC through the platelet integrin αIIbβ3, while binding also occurred in a CX3CL1–CX3CR1-dependent manner after cytokine stimulation. Platelet EVs increased SMC migration comparable to platelet derived growth factor or platelet factor 4 and induced SMC proliferation, which relied on CD40- and P-selectin interactions. Flow-resistant monocyte adhesion to platelet EV-treated SMC was increased compared with resting SMC. Again, this adhesion depended on integrin αIIbβ3 and P-selectin, and to a lesser extent on CD40 and CX3CR1. Treatment of SMC with platelet EVs induced interleukin 6 secretion. Finally, platelet EVs induced a synthetic SMC morphology and decreased calponin expression. Collectively, these data indicate that platelet EVs exert a strong immunomodulatory activity on SMC. In particular, platelet EVs induce a switch towards a pro-inflammatory phenotype, stimulating vascular remodelling.

Antrodia camphorata attenuates cigarette smoke-induced ROS production, DNA damage, apoptosis, and inflammation in vascular smooth muscle cells, and atherosclerosis in ApoE-deficient mice.

Cigarette smoke exposure activates several cellular mechanisms predisposing to atherosclerosis, including oxidative stress, dyslipidemia, and vascular inflammation. Antrodia camphorata, a renowned medicinal mushroom in Taiwan, has been investigated for its antioxidant, anti-inflammatory, and antiatherosclerotic properties in cigarette smoke extracts (CSE)-treated vascular smooth muscle cells (SMCs), and ApoE-deficient mice. Fermented culture broth of Antrodia camphorata (AC, 200-800 µg/mL) possesses effective antioxidant activity against CSE-induced ROS production. Treatment of SMCs (A7r5) with AC (30-120 µg/mL) remarkably ameliorated CSE-induced morphological aberrations and cell death. Suppressed ROS levels by AC corroborate with substantial inhibition of CSE-induced DNA damage in AC-treated A7r5 cells. We found CSE-induced apoptosis through increased Bax/Bcl-2 ratio, was substantially inhibited by AC in A7r5 cells. Notably, upregulated SOD and catalase expressions in AC-treated A7r5 cells perhaps contributed to eradicate the CSE-induced ROS generation, and prevents DNA damage and apoptosis. Besides, AC suppressed AP-1 activity by inhibiting the c-Fos/c-Jun expressions, and NF-κB activation through inhibition of I-κBα degradation against CSE-stimulation. This anti-inflammatory property of AC was accompanied by suppressed CSE-induced VEGF, PDGF, and EGR-1 overexpressions in A7r5 cells. Furthermore, AC protects lung fibroblast (MRC-5) cells from CSE-induced cell death. In vivo data showed that AC oral administration (0.6 mg/d/8-wk) prevents CSE-accelerated atherosclerosis in ApoE-deficient mice. This antiatherosclerotic property was associated with increased serum total antioxidant status, and decreased total cholesterol and triacylglycerol levels. Thus, Antrodia camphorata may be useful for prevention of CSE-induced oxidative stress and diseases. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 2070-2084, 2017.

Abstract 12983: mtDNA Damage in Concert With PCSK9 Expression Induces Apoptosis in Vascular Smooth Muscle Cells

Introduction: Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in inducing hypercholesterolemia and associated cardiovascular diseases. Autophagy is considered a protective and adaptive mechanism to promote cell survival, while apoptosis represents cell death program designed to remove injured cells. Exposure of vascular smooth muscle cells (SMCs) to noxious stimuli such as LPS or ox-LDL induces PCSK9 expression and apoptosis. Methods and Results: Treatment of human primary vascular SMCs to 20 ng/ml LPS significantly induced both mtDNA damage and PCSK9 expression. mtDNA damage induction with the autophagy inhibitor 3-methyladenine enhanced PCSK9 expression, whereas mtDNA damage inhibition with the autophagy inducer rapamycin reduced PCSK9 expression, indicating an interplay between autophagy, mtDNA damage and PCSK9. Inhibition of DNase II (siRNA) for reduced the extent of mtDNA damage and PCSK9 expression confirmed the role of mtDNA damage in PCSK9 expression. Next, we investigated the role of PCSK9 in the development of apoptosis. We observed that PCSK9 upregulation by hPCSK9 treatment induced and PCSK9 inhibition by siRNA transfection reduced apoptosis of SMCs (measured as TUNEL staining and expression of Bax, Bcl2 and cleaved caspase-3). Conclusions: These observations provide a compelling evidence for the role of mtDNA damage in PCSK9 release. Both collectively induce SMC apoptosis during inflammatory states characteristic of cardiovascular diseases, such as atherosclerosis.

Potential Role of Glycogen Synthase Kinase-3β in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.

Glycogen synthase kinase (GSK)-3β, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3β results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3β regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3β inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM α-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3β inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3β inhibitors were mimicked by transfection with GSK-3β siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3β mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM α-actin. As expected, overexpression of constitutively active or wild-type GSK-3β in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3β reduces myocardin transcriptional activity, suggesting a role for GSK-3β in myocardin transcriptional activity and smooth muscle differentiation.

Abstract 227: TGF-β/Smad3 Promotes Smooth Muscle Cell De-differentiation and Proliferation Through Crosstalk with the Wnt/β-Catenin Pathway

Introduction: Endovascular interventions are the first line treatment for patients with atherosclerotic disease. However, a significant number of these fail due to neointimal hyperplasia. The Wnt4/β-catenin pathway has been reported to play an important role in this process, but the mechanism underlying the up-regulation of this pathway after injury remains unclear. Our lab has previously demonstrated that following vascular injury, elevated TGF-β and its signaling protein, Smad3, exacerbate intimal hyperplasia in Smooth Muscle Cells (SMCs). Moreover, our Affymetrix array data revealed an upregulation (>3 fold) of several members of the Wnt family in response to TGF-β/Smad3 treatment of SMCs. Therefore, we hypothesize that the TGF-β/Smad3 axis is responsible for the activation of the Wnt/β-catenin signaling pathway that promotes neointimal hyperplasia. Methods and Results: To mimic the injury induced up-regulation of TGF-β/Smad3 in vitro, SMCs were infected with an adenovirus to increase Smad3 and then treated with recombinant TGF-β for 48 hrs. Real time PCR confirmed that Wnt expression increased substantially in response to TGF-β/Smad3 stimulation. Using western blotting, we found that SMCs treated with TGF-β/Smad3 had a significantly increased stabilization of β-catenin compared to SMCs treated with TGF-β alone. This effect was abolished by Niclosamide, a broad-spectrum Wnt inhibitor. Conversely, β-catenin stabilization increased after stimulation by a Wnt agonist and recombinant Wnts 2b, 4, 5a, and 9a, but not Wnt11. Importantly, we found that these Wnt ligands stimulated the expression of SMC de-differentiation markers including CXCR4, CD34, Sox18, and FGF1 to varying levels as measured by RT-PCR. Wnt4 and Wnt5a were also found to significantly promote SMC proliferation (21% and 27%, respectively, p<0.05) when measured by the Cell Titer Glo Assay. Conclusions: These results suggest that the neointima-producing Wnt/β-catenin pathway may be activated due to elevated TGF-β/Smad3 signaling and promote SMC de-differentiation and proliferation. Thus, the cross talk of these two pathways may provide a potential therapeutic target for preventing failure of endovascular interventions.

Autocrine and Paracrine Effects of VEGF‐A on PLGF in an In Vitro Model of the Vessel Wall

Placenta growth factor (PLGF) and vascular endothelial growth factor-A (VEGF-A) are important regulators of both physiological and pathological vascular remodeling. PLGF mediates these effects by activation and recruitment of monocytes to the vessel wall, while VEGF-A primarily acts upon endothelial cells (EC) inducing their proliferation and migration. We previously reported that human coronary EC primarily express PLGF, while human coronary smooth muscle cells (SMC) primarily express VEGF-A. In the vasculature, EC and SMC are in close proximity to each other. Thus, in study we tested the hypothesis that EC VEGF-A and/or PLGF production influences SMC VEGF-A and/or PLGF expression, and vice versa. EC and SMC were cultured on either side of a porous Transwell insert. SMC were cultured either on the bottom of the well (no cell-cell interaction) or on the opposite side of the insert (some cell-cell interaction). In both instances, coculture of EC and SMC increased PLGF in media relative to EC monoculture (~58%, n=5 p<0.01), but decreased VEGF-A in media compared to SMC monoculture (~72% n=5, p<0.01). Next, EC monolayers were treated with 100 ng/ml of exogenous VEGF-A or VEGF-E (a VEGFR-2 selective isoform). VEGF-A had no significant effect on PLGF. However, VEGF-E increased secreted PLGF (~30%, n=5, p<0.01). PLGF siRNA treatment of SMC increased secreted VEGF (~85% n=5, p<0.001) in SMC, suggesting an inhibitory effect of PLGF on VEGF expression. Furthermore, coculture of PLGF siRNA treated SMC with EC resulted in an increase in secreted PLGF compared to untreated cells (~25% n=5, p<0.05). The available evidence suggests that signaling between EC and SMC contributes to differential expression of PLGF and VEGF-A. Support: R01 HL084494 (PL).