Smooth Muscle Cell Phenotypic Switching Research Articles

Mechanisms of aortic dissection smooth muscle cell phenotype switch

ObjectiveTo investigate the expression of Nanog homeobox (NANOG) in thoracic aortic dissection (TAD) and the role of NANOG in regulating human aortic vascular smooth muscle cells (VSMCs) phenotype switch. MethodsAortic specimens were collected from 20 patients undergoing TAD and 10 controls. VSMCs were isolated by adherent cultivation approach. The expression of NANOG, osteopontin (OPN), and VSMCs phenotype markers were determined by quantitative real-time polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence. Cell counting, scratch wound-healing assay, Transwell migration, and apoptosis assays were used for cell function assessment. Deoxyribonucleic acid–protein binding detection was performed by chromatin immunoprecipitation. ResultsOur experiment results showed that NANOG and OPN were highly expressed in TAD aortic wall and VSMCs, both accompanying VSMCs phenotype switch. Overexpression of NANOG induced the up-regulation of VSMCs synthetic marker matrix metalloproteinase 2 and the down-regulation of VSMCs contractile markers α-smooth muscle actin and smooth muscle 22α. Overexpression of NANOG also enhanced the proliferation, migration, and antiapoptosis capabilities of VSMCs. The results also showed that these functions of NANOG was via OPN and NANOG directly up-regulated OPN by binding to its promoter region. ConclusionsOur study suggests that NANOG is highly expressed in TAD aortic wall and VSMCs. Increased NANOG promotes VSMCs phenotype switch by directly up-regulating OPN through binding to its promoter region.

C1q/TNF-related protein 9 inhibits the cholesterol-induced Vascular smooth muscle cell phenotype switch and cell dysfunction by activating AMP-dependent kinase.

Vascular smooth muscle cells (VSMCs) switch to macrophage‐like cells after cholesterol loading, and this change may play an important role in the progression of atherosclerosis. C1q/TNF‐related protein 9 (CTRP9) is a recently discovered adipokine that has been shown to have beneficial effects on glucose metabolism and vascular function, particularly in regard to cardiovascular disease. The question of whether CTRP9 can protect VSMCs from cholesterol damage has not been addressed. In this study, the impact of CTRP9 on cholesterol‐damaged VSMCs was observed. Our data show that in cholesterol‐treated VSMCs, CTRP9 significantly reversed the cholesterol‐induced increases in pro‐inflammatory factor secretion, monocyte adhesion, cholesterol uptake and expression of the macrophage marker CD68. Meanwhile, CTRP9 prevented the cholesterol‐induced activation of the TLR4–MyD88–p65 pathway and upregulated the expression of proteins important for cholesterol efflux. Mechanistically, as siRNA‐induced selective gene ablation of AMPKα1 abolished these effects of CTRP9, we concluded that CTRP9 achieves these protective effects in VSMCs through the AMP‐dependent kinase (AMPK) pathway.

MiR-125a-5p Modulates Phenotypic Switch of Vascular Smooth Muscle Cells by Targeting ETS-1

MicroRNAs are key regulators of vascular smooth muscle cells (VSMCs) phenotypic switch, one of the main events responsible for bare metal in-stent restenosis after percutaneous coronary intervention. miR-125a-5p is an important modulator of differentiation, proliferation, and migration in different cell types; however, its role in VSMCs is still unknown. The aim of this study was to evaluate the role of miR-125a-5p in VSMCs phenotypic switch. Our results suggest that miR-125a-5p is highly expressed in VSMCs, but it is down-regulated after vascular injury in vivo. Its overexpression is sufficient to reduce VSMCs proliferation and migration, and it is able to promote the expression of selective VSMCs markers such as alpha smooth muscle actin, myosin heavy chain 11, and smooth muscle 22 alpha. Interestingly, miR-125a-5p directly targets ETS-1, a transcription factor implicated in cell proliferation and migration and is crucial in PDGF-BB pathway in VSMCs. Thus, miR-125a-5p in this context inhibits PDGF-BB pathway and is therefore a potential regulator of VSMCs phenotypic switch.

Vessel wall morphology is equivalent for different artery types and localizations of advanced human aneurysms.

Aneurysm formation occurs most frequently as abdominal aortic aneurysm (AAA), but is also seen in other localizations like thoracic or peripheral aneurysm. While initial mechanisms for aneurysm induction remain elusive, observations from AAA samples show transmural inflammation with proteolytic imbalance and repair mechanisms triggered by the innate immune system. However, limited knowledge exists about aneurysm pathology, especially for others than AAA. We compared 42 AAA, 15 popliteal, 3 ascending aortic, five iliac, two femoral, two brachial, one visceral and two secondary aneurysms to non-aneurysmatic controls by histologic analysis, immunohistochemistry and cytokine expression. Muscular and elastic type arteries show a uniform way of aneurysm formation. All samples show similar morphology. The changes compared to controls are distinct and include matrix remodeling with smooth muscle cell phenotype switch and angiogenesis, adventitial lymphoid cell accumulation and M1 macrophage homing together with neutrophil inflammation. Inflammatory cytokines are up-regulated accordingly. Comparative analysis of different disease entities can identify characteristic pathomechanisms. The phenotype of human advanced aneurysm disease is observed for elastic and muscular type arteries, does not differ between disease localizations and might, thus, be a unique response of the vasculature to the still unknown trigger of aneurysm formation.

Abstract 264: Protein Disulfide Isomerase A1 is a Central Hub for Redox Regulation of VSMC Phenotype

Objective: Vascular smooth muscle cell (VSMC) phenotype switch depends on extrinsic/intrinsic cues including NOX NADPH oxidase-linked redox signaling. Growth factor-triggered NOX1 expression/activity requires the chaperone oxidoreductase protein disulfide isomerase-A1 (PDI). Acute PDI overexpression induces agonist-independent NOX1 expression. PDI is required for VSMC migration and cytoskeleton organization, and extracellular PDI supports expansive vascular remodeling via cytoskeleton reshaping. Such PDI effects led us to hypothesize that PDI may orchestrate VSMC phenotypic alterations. Approach and Results: In primary VSMC, PDI silencing spontaneously decreased differentiation marker expression. Transfection with a doxycycline-inducible lentiviral vector encoding PDI showed that sustained PDI overexpression (72h) enhanced actin branching pattern vs. baseline (anisotropy index, 0.103±0.019 vs. 0.220±0.027, 72h vs. 0h, mean±SEM, N=5, P<0.05), increased cell length and induced expression of differentiation marker calponin (2-fold, 72h vs 0h, N=5, P<0.05), alpha-actin and smoothelin, which were abrogated upon catalase incubation. Intracellular superoxide enhanced upon 48h of PDI overexpression (2-hydroxyethidium levels, 0.998±0.102 vs. 2.887±0.227 AU, N=4, P<0.05) and was NOX1-dependent, based on inhibition with GKT136901 (by 48%) or NOXA1ds peptide (by 54%) (N=6, P<0.05). Increased NOX1 mRNA occurred early after PDI overexpression (74%, 24h vs. 0h, N=4, P<0.05), while NOX4 mRNA was upregulated only after long-term PDI induction (100%, 72h vs. 0h, N=4, P<0.05). In rabbit restenosis model exhibiting strong PDI upregulation (12-fold at day 14 after injury), ex vivo PDI silencing 7 or 14 days after injury reversed PCNA expression, while promoting increased NOX1 and decreased NOX4 mRNA levels (+54% and -45%, respectively, siPDI vs. siSCR, N=3, P<0.05 for both). Conclusions: While short-term PDI overexpression supports NOX1 activation/expression, sustained PDI overexpression drives NOX4 expression and VSMC differentiation. Effects on cytoskeleton, NOX1/4 activation and temporal control of NOX1/4 expression suggest a central role for PDI as a hub for redox-mediated VSMC phenotype regulation.

Abstract 237: The Role of Lymphocyte Specific Protein-1 in the Phenotypic Switch of Smooth Muscle Cells After Arterial Injury

Background: After vascular injury, vascular smooth muscle cells (SMCs) switch from a differentiated contractile state to synthetic de-differentiated phenotype which contributes to the pathophysiology of restenosis. Experimental data generated by our lab indicate that TGF-β downregulates contractile proteins and stimulates migration. To understand how TGF-β promotes SMC phenotypic switch in injured arteries, we performed an Affymetrix Array analysis and identified Lymphocyte Specific Protein-1 (LSP1) among other upregulated genes. LSP1 is known to play a role in neutrophil extravasation, however the role of LSP1 within SMCs is unknown. We hypothesize that LSP1 contributes to SMC pathophysiological behavior through changes in cell architecture and migration in-vivo and in-vitro. Methods and Results: After carotid artery angioplasty, male Sprague-Dawley rats were sacrificed at 3, 7, and 14 days after injury for immunohistochemistry. Immunofluorescence staining revealed a unique upregulation of LSP1 within the neointima, media, and adventitia at 7 and 14 days, but not at 3 days after injury. Confocal images revealed that the LSP1 positive cells minimally express α-SMA (Pierson’s Coefficient, r=.017). Additional characterization experiments using immune cell markers CD3 and CD45 show no co-localization with LSP1 positive cells. To mimic the in-vivo neointimal cells and vascular injury induced de-differentiation in-vitro , rat A10 cells were treated with solvent or PDGF-bb (10 ng/mL). Quantitative RT-PCR demonstrated an upregulation of LSP1 mRNA after 24 hrs of PDGF-BB stimulation. Using Western Blotting, we confirm an upregulation of LSP1 protein after 48 hrs of PDGF-BB stimulation. Lastly, we performed nuclear and cytoplasmic fractionation followed by Western Blotting which demonstrated that LSP1 is remained within cytoplasmic fraction of the A10 cell after treatment with PDGF-BB. Conclusion: These results demonstrate that LSP1 is increased in-vivo after balloon injury, and in-vitro after PDGF-BB stimulation. Experiments to characterize the identity of these LSP1 cells in-vivo are in process, with future in-vitro experiments to focus on the role of LSP1 phosphorylation as a part of cytoskeletal remodeling and cellular migration.

Myocardin inhibited the gap protein connexin 43 via promoted miR-206 to regulate vascular smooth muscle cell phenotypic switch

Myocardin is regarded as a key mediator for the change of smooth muscle phenotype. The gap junction protein connexin 43 (Cx43) has been shown to be involved in vascular smooth muscle cells (VSMCs) proliferation and the development of atherosclerosis. However, the role of myocardin on gap junction of cell communication and the relation between myocardin and Cx43 in VSMC phenotypic switch has not been investigated. The goal of the present study is to investigate the molecular mechanism by which myocardin affects Cx43-regulated VSMC proliferation. Data presented in this study demonstrated that inhibition of the Cx43 activation process impaired VSMC proliferation. On the other hand, overexpression miR-206 inhibited VSMC proliferation. In additon, miR-206 silences the expression of Cx43 via targeting Cx43 3′ Untranslated Regions. Importantly, myocardin can significantly promote the expression of miR-206. Cx43 regulates VSMCs' proliferation and metastasis through miR-206, which could be promoted by myocardin and used as a marker for diagnosis and a target for therapeutic intervention. Thus myocardin affected the gap junction by inhibited Cx43 and myocardin-miR-206-Cx43 formed a loop to regulate VSMC phenotypic switch.

PPAR\u03b2/\u03b4, a Novel Regulator for Vascular Smooth Muscle Cells Phenotypic Modulation and Vascular Remodeling after Subarachnoid Hemorrhage in Rats

Cerebral vascular smooth muscle cell (VSMC) phenotypic switch is involved in the pathophysiology of vascular injury after aneurysmal subarachnoid hemorrhage (aSAH), whereas the molecular mechanism underlying it remains largely speculative. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) has been implicated to modulate the vascular cells proliferation and vascular homeostasis. In the present study, we investigated the potential role of PPARβ/δ in VSMC phenotypic switch following SAH. Activation of PPARβ/δ by GW0742 and adenoviruses PPARβ/δ (Ad-PPARβ/δ) significantly inhibited hemoglobin-induced VSMC phenotypic switch. However, the effects of PPARβ/δ on VSMC phenotypic switch were partly obstacled in the presence of LY294002, a potent inhibitor of Phosphatidyl-Inositol-3 Kinase-AKT (PI3K/AKT). Furthermore, following study demonstrated that PPARβ/δ-induced PI3K/AKT activation can also contribute to Serum Response Factor (SRF) nucleus localization and Myocardin expression, which was highly associated with VSMC phenotypic switch. Finally, we found that Ad-PPARβ/δ positively modulated vascular remodeling in SAH rats, i.e. the diameter of basilar artery and the thickness of vessel wall. In addition, overexpression of PPARβ/δ by adenoviruses significantly improved neurological outcome. Taken together, this study identified PPARβ/δ as a useful regulator for VSMC phenotypic switch and vascular remodeling following SAH, providing novel insights into the therapeutic strategies of delayed cerebral ischemia.

Docosahexaenoic acid inhibits hypoxia-induced pulmonary arterial smooth muscle cells phenotype switching by inhibiting NFATc1 signaling

Objective: To explore the molecular mechanism of docosahexaenoic acid (DHA) on regulating the phenotype switching of hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs). Methods: The PASMCs were isolated from Sprague Dawley rats. PASMCs were divided into five groups: normal control group, hypoxia group (1%O(2, )94%N(2, )5% CO(2) stimulation for 12 hours), hypoxia+ DHA group (10 μmol/L DHA pretreatment followed by 12 hours hypoxia), hypoxia+ DHA+ NFATc1 overexpression group (transfection of the NFATc1 lentivirus for 24 hours, followed by hypoxia stimulation for 12 hours after 10 μmol/L DHA treatment), and hypoxia+ DHA+ siNFATc1 group (transfection the siNFATc1 for 24 hours, followed by hypoxia stimulation for 12 hours after 10 μmol/L DHA treatment). The hypoxia stimulation was achieved by use of a special hypoxia incubator (1%O(2, )94%N(2, )5%CO(2)). The expressions of NFATc1 of various groups were determined by qRT-PCR and Western blot. The expression of α-SMA was determined by immunofluorescence staining, qRT-PCR and Western blot. The expression of SM22 was determined by qRT-PCR. The proliferation of PASMC was determined by EDU staining. Results: The mRNA and protein expression levels of NFATc1 were significantly upregulated in hypoxia group compared with the normal control group (P<0.05), while hypoxia-induced upregulation of NFTAc1 could be significantly downregulated by DHA treatment (P<0.05). The α-SMA positive cell number, protein and mRNA levels of α-SMA and the mRNA level of SM22 were significantly lower in the hypoxia group than in normal control group, which could be significantly reversed by DHA, the protective effects could then be abolished by NFATc1 overexpression. Above indices were significantly lower in the hypoxia+ DHA+ siNFATc1 group than in hypoxia+ DHA+ NFATc1 overexpression group (P<0.05). The proliferation of PASMCs was significantly higher in the hypoxia group than in the control group (P<0.05), and which could be significantly reduced by DHA (P<0.05), and the protective effect of DHA could be significantly abolished by overexpression of NFATc1 (P<0.05). The proliferation of PASMCs was significantly lower in the hypoxia+ DHA+ siNFATc1 group than in the hypoxia+ DHA+ overexpression NFATc1 group (P<0.05). Conclusion: DHA could prevent hypoxia-induced PASMCs phenotype switching and proliferation by inhibiting NFATc1 signaling.

MicroRNA-182 prevents vascular smooth muscle cell dedifferentiation via FGF9/PDGFRβ signaling.

The abnormal phenotypic transformation of vascular smooth muscle cells (SMCs) causes various proliferative vascular diseases. MicroRNAs (miRNAs or miRs) have been established to play important roles in SMC biology and phenotypic modulation. This study revealed that the expression of miR-182 was markedly altered during rat vascular SMC phenotypic transformation in vitro. We aimed to investigate the role of miR-182 in the vascular SMC phenotypic switch and to determine the potential molecular mechanisms involved. The expression of miR-182 gene was significantly downregulated in cultured SMCs during dedifferentiation from a contractile to a synthetic phenotype. Conversely, the upregulation of miR-182 increased the expression of SMC-specific contractile genes, such as α-smooth muscle actin, smooth muscle 22α and calponin. Additionally, miR-182 overexpression potently inhibited SMC proliferation and migration under both basal conditions and under platelet-derived growth factor-BB stimulation. Furthermore, we identified fibroblast growth factor 9 (FGF9) as the target gene of miR-182 for the phenotypic modulation of SMCs mediated through platelet-derived growth factor receptor β (PDGFRβ) signaling. These data suggest that miR-182 may be a novel SMC phenotypic marker and a modulator that may be used to prevent SMC dedifferentiation via FGF9/PDGFRβ signaling.

Serum Amyloid A Induces a Vascular Smooth Muscle Cell Phenotype Switch through the p38 MAPK Signaling Pathway.

Atherosclerosis is an important pathological condition which is accompanied by a vascular smooth muscle cell (VSMC) phenotype switch toward a synthetic phenotype. As an acute-phase protein, Serum Amyloid A (SAA) is thought to have a close relationship to atherosclerosis development. However, no study has investigated the direct effect of SAA on the VSMC phenotype switch, as well as the underlying mechanisms. The purpose of our study was to explore the effect of SAA on the VSMC phenotype switch and the potential mechanisms involved. In our study, we found that SAA induced the VSMC phenotype switch which reduced expression of the smooth muscle cell (SMC) marker and enhanced expression of the matrix synthesis related marker. The proliferative ability of VSMCs was also increased by SAA treatment. Furthermore, our research found that SAA activated the ERK1/2 and p38 MAPK signaling pathways. Finally, by applying the ERK1/2 and p38 inhibitors, U0126 and SB203580, we demonstrated that the SAA-induced VSMC phenotype switch was p38-dependent. Taken together, these results indicated that SAA may play an important role in promoting the VSMC phenotype switch through the p38 MAPK signaling pathway.

Aortic dissection is associated with reduced polycystin-1 expression, an abnormality that leads to increased ERK phosphorylation in vascular smooth muscle cells.

The vascular smooth muscle cell (VSMC) phenotypic switch is a key pathophysiological change in various cardiovascular diseases, such as aortic dissection (AD), with a high morbidity. Polycystin-1 (PC1) is significantly downregulated in the VSMCs of AD patients. PC1 is an integral membrane glycoprotein and kinase that regulates different biological processes, including cell proliferation, apoptosis, and cell polarity. However, the role of PC1 in intracellular signaling pathways remains poorly understood. In this study, PC1 downregulation in VSMCs promoted the expression of SM22, ACTA2 and calponin 1 (CNN1) proteins. Furthermore, PC1 downregulation in VSMCs upregulated phospho-MEK, phospho-ERK and myc, but did not change phospho-JNK and phospho-p38. These findings suggest that the MEK/ERK/myc signaling pathway is involved in PC1-mediated human VSMC phenotypic switch. Opposite results were observed when an ERK inhibitor was used in VSMCs downregulated by PC1. When the C-terminal domain of PC1 (PC1 C-tail) was overexpressed in VSMCs, the expression levels of phosphor-ERK, myc, SM22, ACTA2 and CNN1 proteins were downregulated. The group with the overexpressed mutant protein (S4166A) in the PC1 C-tail showed similar results to the group with the downregulated PC1 in VSMCs. These results suggest that the Ser at the 4166 site in PC1 is crucial in the PC1 mediated MEK/ERK/myc signaling pathway, which might be the key pathophysiological cause of AD.

The Yin-Yang Dynamics of DNA Methylation Is the Key Regulator for Smooth Muscle Cell Phenotype Switch and Vascular Remodeling.

DNA methylation plays an important role in chronic diseases such as atherosclerosis, yet the mechanisms are poorly understood. The objective of our study is to indicate the regulatory mechanisms of DNA methylation in vascular smooth muscle cells (VSMCs) and its roles in atherosclerosis. In ApoE-/- mice fed a Western diet, DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, significantly attenuated atherosclerotic lesions (20.1±2.2% versus 30.8±7.5%; P=0.016) and suppressed DNA methyltransferase activity and concomitantly decreased global 5-methylcytosine content in atherosclerotic lesions of ApoE-/- mice. Using a carotid ligation model, we found that 5-aza-2'-deoxycytidine also dramatically inhibited neointimal formation (intimal area: 2.25±0.14×104 versus 4.07±0.22×104 μm2; P<0.01). Abnormal methylation status at the promoter of ten-eleven translocation 2, one of the key demethylation enzymes in mammals, was ameliorated after 5-aza-2'-deoxycytidine treatment, which in turn caused an increase in global DNA hydroxymethylation and 5-hydroxymethylcytosine enrichment at the promoter of Myocardin. In vitro, 5-aza-2'-deoxycytidine treatment or DNA methyltransferase 1 knockdown decreased global 5-methylcytosine content and restored Myocardin expression in VSMCs induced by platelet-derived growth factor, thus preventing excessive VSMCs dedifferentiation, proliferation, and migration. Furthermore, DNA methyltransferase 1 binds to ten-eleven translocation 2 promoter and is required for ten-eleven translocation 2 methylation in VSMCs. The inhibitory effects of DNA demethylation on global 5-methylcytosine content and ten-eleven translocation 2 hypermethylation in atherosclerotic aorta can recover 5-hydroxymethylcytosine enrichment at the Myocardin promoter and prevent VSMC dedifferentiation and vascular remodeling.

Matrix metalloproteinase (MMP)-2 decreases calponin-1 levels and contributes to arterial remodeling in early hypertension

Increased matrix metalloproteinase (MMP)-2 is implicated in the vascular remodeling of hypertension. Calponin-1 is a contractile protein, and its absence is associated with vascular smooth muscle cell (VSMC) phenotype switch, which leads to migration and remodeling. We evaluated whether increased MMP-2 activity precedes chronic vascular remodeling by decreasing calponin-1 and inducing VSMC proliferation. Sham or two kidney-one clip (2K1C) rats were treated with doxycycline at 30mg/kg/day. Systolic blood pressure was increased in the 2K1C rats after 1 and 2weeks post-surgery, and doxycycline was effective to reduce it only at 2weeks of hypertension (p<0.05). Increased activity of MMP-2 was observed in aortas from 2K1C at 1 and 2weeks of hypertension, followed by increased VSMC proliferation, and those effects were abolished by treating 2K1C rats with doxycycline (p<0.05). Increased aortic media to lumen ratio started to emerge in 2K1C rats at 1week of hypertension, and it was established by 2weeks. MMP-2 and calponin-1 co-localized in the cytosol of VSMC. Aortas from 2K1C rats showed a significant reduction in calponin-1 levels at 1week of hypertension, and doxycycline prevented its loss (p<0.05). However, at 2weeks of hypertension, calponin-1 was upregulated in 2K1C (p<0.05 vs. Sham groups). The mRNA levels of calponin-1 were not altered in the aortas of 2K1C at 1week of hypertension. MMP-2 may contribute to the post-translational decrease in calponin-1, thus culminating in hypertension-induced maladaptive arterial remodeling.

Slug Is Increased in Vascular Remodeling and Induces a Smooth Muscle Cell Proliferative Phenotype.

ObjectivePrevious studies have confirmed Slug as a key player in regulating phenotypic changes in several cell models, however, its role in smooth muscle cells (SMC) has never been assessed. The purpose of this study was to evaluate the expression of Slug during the phenotypic switch of SMC in vitro and throughout the development of vascular remodeling.Methods and ResultsSlug expression was decreased during both cell-to-cell contact and TGFβ1 induced SMC differentiation. Tumor necrosis factor-α (TNFα), a known inductor of a proliferative/dedifferentiated SMC phenotype, induces the expression of Slug in SMC. Slug knockdown blocked TNFα-induced SMC phenotypic change and significantly reduced both SMC proliferation and migration, while its overexpression blocked the TGFβ1-induced SMC differentiation and induced proliferation and migration. Genome-wide transcriptomic analysis showed that in SMC, Slug knockdown induced changes mainly in genes related to proliferation and migration, indicating that Slug controls these processes in SMC. Notably, Slug expression was significantly up-regulated in lungs of mice using a model of pulmonary hypertension-related vascular remodeling. Highly remodeled human pulmonary arteries also showed an increase of Slug expression compared to less remodeled arteries.ConclusionsSlug emerges as a key transcription factor driving SMC towards a proliferative phenotype. The increased Slug expression observed in vivo in highly remodeled arteries of mice and human suggests a role of Slug in the pathogenesis of pulmonary vascular diseases.

MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension.

BackgroundVascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis.Methods and ResultsIn SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis.ConclusionsOur findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH.

Abstract 13: Targeting the PERK Pathway of ER Stress Response for Endothelium Protection and Restenosis Prevention: A Paradigm for Developing Anti-thrombogenic Stents

Introduction: Cardiovascular disease is the leading cause of mortality and morbidity in the US. Reconstructions often fail due to the recurrent lumen narrowing, or restenosis. Stents eluting rapamycin or paclitaxel are deployed to inhibit the excessive proliferation of smooth muscle cells (SMCs), which is the core component of restenosis. However, these drugs cause collateral damage to endothelial cells (ECs) leading to stent thrombosis. To address this, our group initiated the first campaign of drug screening for compounds that selectively inhibit SMC proliferation without causing EC dysfunction. We recently identified lead compounds as such, which are inhibitors for protein kinase RNA-like endoplasmic reticulum kinase (PERK), an endoplasmic reticulum (ER) stress sensor. Here we evaluated PERK as a target for intervention of both SMC pathophysiology and EC dysfunction. Methods: Rat carotid artery balloon angioplasty was performed as a restenosis model. PDGF-BB and TNF-a were applied to primary human aortic SMCs and ECs, respectively, to mimic the in vivo pathogenic stimuli. Results: In balloon-injured arteries, both PERK phosphorylation and the expression of its downstream transcription factor ATF4 were increased compared to sham control. PERK activation was also observed in vitro in both SMCs and ECs stimulated with PDGF-BB and TNF-a, respectively. In SMCs, either selective inhibition (1μM GSK2606414) or siRNA knockdown of PERK abolished PDGF-BB induced de-differentiation and proliferation. In ECs, PERK antagonism abrogated TNF-a induced growth impairment and EC secretion of Tissue Factor (TF), which is the key initiator of thrombogenesis. Finally, in a pilot experiment, peri-adventitial application of GSK2606414 (25mg/kg, n=2 rats) reduced intima/media ratio by 80% and increased lumen area by ~ 2 fold compared to vehicle control (n=2) at 4 weeks post injury. Conclusion: Our results indicate an important role of PERK activation in promoting SMC phenotype switching and EC dysfunction in vitro as well as restenosis in vivo . Thus, PERK targeting represents a potential strategy to simultaneously achieve restenosis prevention and endothelium protection, with a long-term goal of developing anti-thrombogenic drug-eluting stents.

IOX1, a JMJD2A inhibitor, suppresses the proliferation and migration of vascular smooth muscle cells induced by angiotensinII by regulating the expression of cell cycle-related proteins.

The epigenetic modification of vascular smooth muscle cell (VSMC) phenotypic switching, proliferation, migration, apoptosis and extracellular matrix synthesis is known to occur in atherosclerosis. The aim of the present study was to investigate the effects of IOX1, a Jumonji domain-containing 2A (JMJD2A) inhibitor, on regulation of the cell cycle in angiotensin II (Ang II)-stimulated VSMCs and to elucidate the possible mechanisms involved. The proliferation and migration of the Ang II-stimulated VSMCs in the presence or absence of IOX1 were evaluated in vitro. Flow cytometric analysis was used to determine the effects of IOX1 on cell cycle progression. RT-qPCR and western blot analysis were carried out to measure the expression levels of cell cycle-related genes. The trimethylation of histone H3 lysine 9 (H3K9me3) at the promoters of these genes was detected by chromatin immunoprecipitation (ChIP) assay. We confirmed that the JMJD2A levels were increased, whereas the H3K9me3 levels were decreased in the Ang II-stimulated VSMCs. The inhibition of JMJD2A by IOX1 suppressed the Ang II-induced cell proliferation, migration and cell cycle progression by inhibiting cyclin D1 expression and increasing p21 expression. The underlying mechanisms were related to the restoration of the H3K9me3 levels at the promoters of these genes. In conclusion, the findings of our study indicate that IOX1 exerts its anti-proliferative and anti-migratory effects by regulating the expression of the cell cycle-related proteins, cyclin D1 and p21.

Abstract P131: Mef2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension

Background: In pulmonary arterial hypertension (PAH), smooth muscle cell (SMC) phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C- Myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides a basis for exploring their modulation of MEF2C-MYOCD signaling, and, in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 signaling axis. Methods and Results: In SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was upregulated approx. ~1.5 fold compared to controls (p&lt;0.05). These increases in miR-214 were paralleled by downregulation of MEF2C, MYOCD and SMC-specific contractile proteins, leiomodin1 and smoothelin. Of these, leiomodin1 was directly targeted by the miR. MicroRNA-214 overexpression mimicked the PAH profile, downregulating MEF2C (1±0.054 vs 0.696±0.026, p&lt;0.05) and leiomodin1 (1± 0.051 vs 0.281±0.095, for scrambled control vs miR-214 mimic, p&lt;0.05). Hypoxia significantly reduced expression of SMC-specific contractile proteins, leiomodin1 and calponin1 (5 of 10 percent), and smoothelin (approx. 3 of 10 percent), and miR-214 antagomiR abrogated hypoxia-induced suppression of the contractile phenotype. We also found that hypoxia-induced hPASMC proliferation was significantly attenuated by the anti-miR (approx. 2-fold less compared to hypoxia control). Further, anti-miR-214 also restored PAH-PASMCs to a contractile (approx.5 of 10 percent reversal of MEF2C and leiomodin1 expression), and less proliferative phenotype seen during vascular homeostasis. Conclusions: Our findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-leiomodin1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH.

Abstract P132: MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension

Vascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a differentiated contractile to a synthetic state contributes to disease progression. While SMC contractile phenotype is reportedly maintained by a MEF2C-Myocardin (MYOCD) transcription factor interplay, its molecular control is poorly understood. MicroRNAs (miRs) have emerged as modulators of many cellular processes and some evidence associates them to MEF2C-MYOCD signaling. It is, therefore, plausible that miRs can regulate the synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in PAH is mediated by miR-214 via repression of signaling by MEF2C-MYOCD and downstream contractile proteins leiomodin1 and smoothelin. Using qRT-PCR, the levels of miR-214 expression were shown to be upregulated in pulmonary artery SMC (PASMCs) from PAH- vs. control human subjects as well as in commercially obtained human PASMC (hPASMCs) exposed to hypoxia (~1.5 fold, p&lt;0.05). These increases in miR-214 were paralleled by downregulation of MEF2C, MYOCD, and SMC-specific contractile proteins, leiomodin1 and smoothelin. MicroRNA-214 overexpression mimicked the PAH profile, downregulating MEF2C (1.00±0.054 vs. 0.696±0.026, p&lt;0.05) and leiomodin1 (1.00± 0.051 vs. 0.281±0.095, p&lt;0.05) protein levels for control vs miR-214 mimic, respectively. Hypoxia significantly reduced expression of SMC-specific contractile proteins leiomodin1, calponin1 (~50%) and smoothelin (~30%), and miR-214 antagomiR abrogated this response. We also investigated whether miR-214 participates in the induction of hPASMC proliferation, and found that hypoxia-induced hPASMC proliferation was significantly attenuated by the anti-miR (~2-fold). Further, anti-miR-214 restored PAH-PASMCs to a contractile (~50% reversal of MEF2C and leiomodin1 expression) and less proliferative phenotype. Our data illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-leiomodin1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes in vascular hyperproliferative disorders including PAH.