Increase In Smooth Muscle Cell Proliferation Research Articles

Abstract 281: A20 (tnfaip3) Knockdown Protects From Abdominal Aortic Aneurysms By Limiting Extra-cellular Matrix Degradation And Maintaining Aortic Smooth Muscle Cell Mass

Background: Our lab has recently demonstrated that haploinsufficiency of A20/TNFAIP3, a potent anti-inflammatory/NFκB inhibitory gene, protects mice from abdominal aortic aneurysms (AAA) by enhancing TGFβ signaling and modulating smooth muscle cell (SMC) differentiation and apoptosis. Here, we explored the impact of A20 knockdown on TGFβ-mediated SMC proliferation and extra cellular matrix (ECM) degradation, both involved in AAA pathogenesis. Methods: AAA was induced by periadventitial elastase application to the infrarenal aorta of both A20 wild-type (WT) and A20 heterozygous (Het) 10-week-old mice who received 0.2% BAPN in their drinking water. Aortae were harvested 28 days after for immunohistochemistry (IHC) analysis of SMC proliferation and ECM markers. In vitro , human aortic SMC cells (HAoSMC) were transduced with rAd.ShRNA A20 (siA20), resulting in 50-70% A20 knockdown, or control rAd.ShRNA Scramble (siScr). HAoSMC were starved for 24 hours, then treated with TGFβ. Proliferation was analyzed by MTS assay, and cell extracts assayed by Western blot (WB) for cell-cycle markers. Results: A20 haplo-insufficiency protected from AAA formation by: 1) decreasing ECM degradation, as gauged by significantly lower MMP-9 expression (n=3, p<0.01), and 2) promoting vessel wall thickening by increasing SMC proliferation (ki67+ nuclei/vessel wall, n=4, p<0.01). Increased SMC proliferation in Het vs. WT aortae correlated with significantly lower expression of the cell-cycle brake, p21 (p21+ nuclei/vessel wall, n=3-7, p<0.05). Akin mouse aortae, A20 knockdown significantly increased TGFβ-mediated HAoSMC proliferation, assessed by the MTS assay (siA20 vs. siScr, n=5, p<0.05). This associated with significantly higher protein levels of the proliferation regulator phospho-ERK1/2 in siA20 vs. siScr-transduced HAoSMC (WB, n=4, p<0.001). Conclusion: Our findings qualify the protective effect of A20 knockdown against AAA development by preserving ECM integrity and promoting TGFβ-mediated SMC proliferation. These targets of A20 knockdown were validated in HAoSMC, further supporting our pursuit of A20-siRNA based therapies to prevent or limit progression of AAA.

Macrophage-derived PDGF-B induces muscularization in murine and human pulmonary hypertension.

Excess macrophages and smooth muscle cells (SMCs) characterize many cardiovascular diseases, but crosstalk between these cell types is poorly defined. Pulmonary hypertension (PH) is a lethal disease in which lung arteriole SMCs proliferate and migrate, coating the normally unmuscularized distal arteriole. We hypothesized that increased macrophage platelet-derived growth factor–B (PDGF-B) induces pathological SMC burden in PH. Our results indicate that clodronate attenuates hypoxia-induced macrophage accumulation, distal muscularization, PH, and right ventricle hypertrophy (RVH). With hypoxia exposure, macrophage Pdgfb mRNA was upregulated in mice, and LysM‑Cre mice carrying floxed alleles for hypoxia-inducible factor 1a, hypoxia-inducible factor 2a, or Pdgfb had reduced macrophage Pdgfb and were protected against distal muscularization and PH. Conversely, LysM‑Cre von-Hippel Lindaufl/fl mice had increased macrophage Hifa and Pdgfb and developed distal muscularization, PH, and RVH in normoxia. Similarly, Pdgfb was upregulated in macrophages from human idiopathic or systemic sclerosis–induced pulmonary arterial hypertension patients, and macrophage-conditioned medium from these patients increased SMC proliferation and migration via PDGF-B. Finally, in mice, orotracheal administration of nanoparticles loaded with Pdgfb siRNA specifically reduced lung macrophage Pdgfb and prevented hypoxia-induced distal muscularization, PH, and RVH. Thus, macrophage-derived PDGF-B is critical for pathological SMC expansion in PH, and nanoparticle-mediated inhibition of lung macrophage PDGF-B has profound implications as an interventional strategy for PH.

Flow with variable pulse frequencies accelerates vascular recellularization and remodeling of a human bioscaffold.

Despite significant advances in vascular tissue engineering, the ideal graft has not yet been developed and autologous vessels remain the gold standard substitutes for small diameter bypass procedures. Here, we explore the use of a flow field with variable pulse frequencies over the regeneration of an ex vivo-derived human scaffold as vascular graft. Briefly, human umbilical veins were decellularized and used as scaffold for cellular repopulation with human smooth muscle cells (SMC) and endothelial cells (EC). Over graft development, the variable flow, which mimics the real-time cardiac output of an individual performing daily activities (e.g., resting vs. exercising), was implemented and compared to the commonly used constant pulse frequency. Results show marked differences on SMC and EC function, with changes at the molecular level reflecting on tissue scales. First, variable frequencies significantly increased SMC proliferation rate and glycosaminoglycan production. These results can be tied with the SMC gene expression that indicates a synthetic phenotype, with a significant downregulation of myosin heavy chain. Additionally and quite remarkably, the variable flow frequencies motivated the re-endothelialization of the grafts, with a quiescent-like structure observed after 10 days of conditioning, contrasting with the low surface coverage and unaligned EC observed under constant frequency (CF). Besides, the overall biomechanics of the generated grafts (conditioned with both pulsed and CFs) evidence a significant remodeling after 55 days of culture, depicted by high burst pressure and Young's modulus. These last results demonstrate the positive recellularization and remodeling of a human-derived scaffold toward an arterial vessel.

4573 Characterization of vascular disease in an Acta2 mutant mouse model

OBJECTIVES/GOALS: ACTA2 R179 carriers present with early-onset stroke; occlusive lesions of the distal internal carotid artery and branches are filled with cells staining positive for smooth muscle cell (SMC) markers. We will identify pathways leading to increased SMC proliferation and migration and thus occlusion. METHODS/STUDY POPULATION: We generated an Acta2SMC-R179C/+ mouse model, which expresses the Acta2 R179C mutation in SMCs via the SM22a-Cre-Lox system. rt-PCR performed in aortic tissue confirms the presence of the mutation in the mutant mice and absence in mice with only the floxed allele (WT). We will determine phenotypic differences between mutant and WT brains using micro CT, vascular casting, histology, and immunostaining. We will characterize mutant SMC phenotype in culture by assessing expression of contractile genes and stem cell markers, proliferation, and migration. Single cell RNA (scRNA) sequencing of the brain will assess differential gene expression and cell populations between mutant and WT mice. RESULTS/ANTICIPATED RESULTS: Mutant mice have decreased blood pressure compared to WT mice from 8-24 weeks old, consistent with the phenotype seen in ACTA2 R179 patients. We expect to see occluded and straighter cerebrovascular arteries and white matter changes in the Acta2SMC-R179C/+ mice. iPSC-derived SMCs from patients show de-differentiation, continued expression of stem cell markers, and increased proliferation and migration. We expect to see a similar phenotype in Acta2SMC-R179C/+ mouse SMCs in culture. Via scRNA sequencing, we expect to see altered transcriptional profiles in mutant mice brains including upregulated proliferative pathways in SMCs, glial cell activation, and gene expression changes in neurons. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will contribute important information on the pathogenesis of the cerebrovascular disease in ACTA2 R179 patients. These results may aid in identifying treatments to prevent or decrease risk of developing strokes in those with known predisposition to cerebrovascular occlusive disease.

Divergent effects of canonical and non-canonical TGF-β signalling on mixed contractile-synthetic smooth muscle cell phenotype in human Marfan syndrome aortic root aneurysms.

Aortic root aneurysm formation is a cardinal feature of Marfan syndrome (MFS) and likely TGF‐β driven via Smad (canonical) and ERK (non‐canonical) signalling. The current study assesses human MFS vascular smooth muscle cell (SMC) phenotype, focusing on individual contributions by Smad and ERK, with Notch3 signalling identified as a novel compensatory mechanism against TGF‐β‐driven pathology. Although significant ERK activation and mixed contractile gene expression patterns were observed by traditional analysis, this did not directly correlate with the anatomic site of the aneurysm. Smooth muscle cell phenotypic changes were TGF‐β‐dependent and opposed by ERK in vitro, implicating the canonical Smad pathway. Bulk SMC RNA sequencing after ERK inhibition showed that ERK modulates cell proliferation, apoptosis, inflammation, and Notch signalling via Notch3 in MFS. Reversing Notch3 overexpression with siRNA demonstrated that Notch3 promotes several protective remodelling pathways, including increased SMC proliferation, decreased apoptosis and reduced matrix metalloproteinase activity, in vitro. In conclusion, in human MFS aortic SMCs: (a) ERK activation is enhanced but not specific to the site of aneurysm formation; (b) ERK opposes TGF‐β‐dependent negative effects on SMC phenotype; (c) multiple distinct SMC subtypes contribute to a ‘mixed’ contractile‐synthetic phenotype in MFS aortic aneurysm; and (d) ERK drives Notch3 overexpression, a potential pathway for tissue remodelling in response to aneurysm formation.

Abstract P166: Paracrine Endothelial Microrna214 Exosomes Modulate Smooth Muscle Cell Function in Pulmonary Arterial Hypertension

Background: Communication among various cell types is known to play a pivotal role in PAH. Endothelial cell (EC)-derived exosomes have emerged as important mediators of intercellular communication and are known to influence the function of recipient cells. A role for miR-214 in EC-derived exosomes in PAH, however, is entirely unknown. In this study, we hypothesize that hypoxia-induced EC injury releases exosomes enriched in miR-214 that mediates phenotype switching of smooth muscle cells (SMCs) resulting in increased SMC proliferation in PAH. Methods and Results: We subjected human pulmonary artery endothelial cells (hPAECs) to hypoxia vs. normoxia (norm) to recapitulate the PAH phenotype in vitro . Quantitative RT-PCR of hPAECs showed that hypoxia upregulated miR-214 by 1.51±0.24-fold (p<0.05 vs. norm). Moreover, exosomes derived from hypoxic hPAECs were found to be enriched in miR-214 (1.73±0.16-fold vs. norm, p<0.05). TEM and Nanotracking Analysis System determined the size of EC-derived exosomes to be within a characteristic 30-100nm. Furthermore, Z-stack confocal imaging demonstrated that FITC-PHK67-labeled EC-derived exosomes were taken up by hPASMCs. Western blot of hPASMCs treated with hypoxia-induced EC-derived exosomes showed upregulation of phospho-Akt (1.65±0.29-fold vs. norm, p <0.05), phospho-ERK1/2 (1.68±0.27-fold vs. norm, p <0.05), and Bcl2 (1.63±0.19-fold vs. norm, NS). Similar treatment also decreased SMC-specific genes, such as MYH11, calponin and smoothelin, indicating that EC-derived hypoxic exosomes can induce switching of PASMCs to a synthetic, pro-proliferative phenotype. Moreover, scratch assay demonstrated that application of EC-derived exosomes laden with exogenous miR-214 increased SMC migration (1.28±0.08-fold vs. control, p =0.05). Conclusions & Discussion: We found that miR-214 expression was induced in hypoxia both in ECs and in exosomes released from them. Furthermore, EC-derived hypoxic or miR-214-laden exosomes were able to transduce changes in SMC, which result in a proliferative and synthetic phenotype. These findings demonstrate that EC-derived miR-214 can direct a proliferative fate for smooth muscle cells, and may contribute to SMC-associated pathophysiological changes in PAH.

Protective Role of RNA Helicase DEAD-Box Protein 5 in Smooth Muscle Cell Proliferation and Vascular Remodeling.

RNA helicases, highly conserved enzymes, are currently believed to be not only involved in RNA modulation, but also in other biological processes. We recently reported that RNA helicase DDX (DEAD-box protein)-5 is required for maintaining the homeostasis of vascular smooth muscle cells (SMCs). However, the expression and function of RNA helicase in vascular physiology and disease is unknown. To investigate the role of RNA helicase in vascular diseases. We showed here that DDX-5 was the most abundant DEAD-box protein expressed in human and rodent artery, which mainly located in SMCs. It was demonstrated that DDX-5 levels were reduced in cytokine-stimulated SMCs and vascular lesions. DDX-5 knocking down or deficiency increased SMC proliferation and migration, whereas overexpression of DDX-5 prevented aberrant proliferation and migration of SMCs. Mechanistic studies revealed transcription factor GATA (GATA-binding protein)-6 as a novel downstream target of DDX-5, which directly interacted with GATA-6 and protected it from MDM (mouse double minute)-2-mediated degradation. Our ChIP assay identified a previously unreported binding of p27Kip1 promoter to GATA-6. DDX-5 increased the recruitment of GATA-6 to p27Kip1 promoter, which enhanced p27Kip1 expression and maintained SMC quiescence. Finally, we showed exacerbated neointima formation in DDX-5 SMC-deficient mice after femoral artery injury, whereas overexpression of DDX-5 potently inhibited vascular remodeling in balloon-injured rat carotid artery. These findings provide the first evidence for a role of RNA helicase DDX-5 in the protection against SMC proliferation, migration, and neointimal hyperplasia. Our data extend the fundamental role of RNA helicase beyond RNA modulation, which provides the basic information for new therapeutic strategies for vascular diseases.

Interferon Regulatory Factor 4 Inhibits Neointima Formation by Engaging Krüppel-Like Factor 4 Signaling.

The mechanisms underlying neointima formation remain unclear. Interferon regulatory factors (IRFs), which are key innate immune regulators, play important roles in cardiometabolic diseases. However, the function of IRF4 in arterial restenosis is unknown. IRF4 expression was first detected in human and mouse restenotic arteries. Then, the effects of IRF4 on neointima formation were evaluated with universal IRF4-deficient mouse and rat carotid artery injury models. We performed immunostaining to identify IRF4-expressing cells in the lesions. Smooth muscle cell (SMC)-specific IRF4-knockout (KO) and -transgenic (TG) mice were generated to evaluate the effects of SMC-IRF4 on neointima formation. We used microarray, bioinformatics analysis, and chromatin immunoprecipitation assay to identify the downstream signals of IRF4 and to verify the targets in vitro. We compared SMC-IRF4-KO/Krüppel-like factor 4 (KLF4)-TG mice with SMC-IRF4-KO mice and SMC-specific IRF4-TG/KLF4-KO mice with SMC-specific IRF4-TG mice to investigate whether the effect of IRF4 on neointima formation is KLF4-dependent. The effect of IRF4 on SMC phenotype switching was also evaluated. IRF4 expression in both the human and mouse restenotic arteries is eventually downregulated. Universal IRF4 ablation potentiates neointima formation in both mice and rats. Immunostaining indicated that IRF4 was expressed primarily in SMCs in restenotic arteries. After injury, SMC-IRF4-KO mice developed a thicker neointima than control mice. This change was accompanied by increased SMC proliferation and migration. However, SMC-specific IRF4-TG mice exhibited the opposite phenotype, demonstrating that IRF4 exerts protective effects against neointima formation. The mechanistic study indicated that IRF4 promotes KLF4 expression by directly binding to its promoter. Genetic overexpression of KLF4 in SMCs largely reversed the neointima-promoting effect of IRF4 ablation, whereas ablation of KLF4 abolished the protective function of IRF4, indicating that the protective effects of IRF4 against neointima formation are KLF4-dependent. In addition, IRF4 promoted SMC dedifferentiation. IRF4 protects arteries against neointima formation by promoting the expression of KLF4 by directly binding to its promoter. Our findings suggest that this previously undiscovered IRF4-KLF4 axis plays a key role in vasculoproliferative pathology and may be a promising therapeutic target for the treatment of arterial restenosis.

MicroRNA-33 protects against neointimal hyperplasia induced by arterial mechanical stretch in the grafted vein.

Mechanical factors play significant roles in neointimal hyperplasia after vein grafting, but the mechanisms are not fully understood. Here, we investigated the roles of microRNA-33 (miR-33) in neointimal hyperplasia induced by arterial mechanical stretch after vein grafting. Grafted veins were generated by the 'cuff' technique. Neointimal hyperplasia and cell proliferation was significantly increased, and miR-33 expression was decreased after 1-, 2-, and 4-week grafts. In contrast, the expression of bone morphogenetic protein 3 (BMP3), which is a putative target of miR-33, and the phosphorylation of smad2 and smad5, which are potential downstream targets of BMP3, were increased in the grafted veins. miR-33 mimics/inhibitor and dual luciferase reporter assay confirmed the interaction of miR-33 and BMP3. miR-33 mimics attenuated, while miR-33 inhibitor accelerated, proliferation of venous smooth muscle cells (SMCs). Moreover, recombinant BMP3 increased SMC proliferation and P-smad2 and P-smad5 levels, whereas BMP3-directed siRNAs had the opposite effect. Then, venous SMCs were exposed to a 10%-1.25 Hz cyclic stretch (arterial stretch) by using the FX4000 cyclic stretch loading system in vitro to mimic arterial mechanical conditions. The arterial stretch increased venous SMC proliferation and repressed miR-33 expression, but enhanced BMP3 expression and smad2 and smad5 phosphorylation. Furthermore, perivascular multi-point injection in vivo demonstrated that agomiR-33 not only attenuates BMP3 expression and smad2 and smad5 phosphorylation, but also slows neointimal formation and cell proliferation in grafted veins. These effects of agomiR-33 on grafted veins could be reversed by local injection of BMP3 lentivirus. The miR-33-BMP3-smad signalling pathway protects against venous SMC proliferation in response to the arterial stretch. miR-33 is a target that attenuates neointimal hyperplasia in grafted vessels and may have potential clinical applications.

MicroRNA MiR-199a-5p Regulates Smooth Muscle Cell Proliferation and Morphology by Targeting WNT2 Signaling Pathway

MicroRNA miR-199a-5p impairs tight junction formation, leading to increased urothelial permeability in bladder pain syndrome. Now, using transcriptome analysis in urothelial TEU-2 cells, we implicate it in the regulation of cell cycle, cytoskeleton remodeling, TGF, and WNT signaling pathways. MiR-199a-5p is highly expressed in the smooth muscle layer of the bladder, and we altered its levels in bladder smooth muscle cells (SMCs) to validate the pathway analysis. Inhibition of miR-199a-5p with antimiR increased SMC proliferation, reduced cell size, and up-regulated miR-199a-5p targets, including WNT2. Overexpression of WNT2 protein or treating SMCs with recombinant WNT2 closely mimicked the miR-199a-5p inhibition, whereas down-regulation of WNT2 in antimiR-expressing SMCs with shRNA restored cell phenotype and proliferation rates. Overexpression of miR-199a-5p in the bladder SMCs significantly increased cell size and up-regulated SM22, SM α-actin, and SM myosin heavy chain mRNA and protein levels. These changes as well as increased expression of ACTG2, TGFB1I1, and CDKN1A were mediated by up-regulation of the smooth muscle-specific transcriptional activator myocardin at mRNA and protein levels. Myocardin-related transcription factor A downstream targets Id3 and MYL9 were also induced. Up-regulation of myocardin was accompanied by down-regulation of WNT-dependent inhibitory Krüppel-like transcription factor 4 in miR-199a-5p-overexpressing cells. In contrast, Krüppel-like transcription factor 4 was induced in antimiR-expressing cells following the activation of WNT2 signaling, leading to repression of myocardin-dependent genes. MiR-199a-5p plays a critical role in the WNT2-mediated regulation of proliferative and differentiation processes in the smooth muscle and may behave as a key modulator of smooth muscle hypertrophy, which is relevant for organ remodeling.

Abstract 357: The Role of X-Box Binding Protein 1 Splicing in Smooth Muscle Cell Proliferation and Neointima Formation

Rationale: Smooth muscle cell (SMC) proliferation plays an important role in neointima formation in response to vascular injury. However, the underlying mechanism is still not well understood. As one of the key transducer in endoplasmic reticulum stress response, the X-box binding protein 1 (XBP1) has been demonstrated to contribute to endothelial cell proliferation, autophagic response and apoptosis, but less is known about its role in SMC growth. Methods and Results: In this study, we intend to study the role of XBP1 in SMC proliferation and its contribution to neointima formation in response to vascular injury. Platelet derived growth factor (PDGF) induced XBP1 splicing in SMCs via the interaction between PDGF receptor and inositol requiring enzyme 1 alpha (IRE1α). Transient over-expression of the spliced XBP1 (XBP1s) increased SMC proliferation, while knockdown of XBP1 or IRE1α abolished PDGF-induced SMC proliferation. XBP1s down-regulated calponin H1 gene expression at mRNA level through microRNAs targeting the 3’-terminal untranslated region. Reconstitution of calponin H1 via adenoviral transfer could ablate XBP1s-induced SMC proliferation. Proteomics analysis of secreted proteins revealed that over-expression of XBP1s in SMCs decreased the secretion of transforming growth factor β family. Addition of exogenous TGFβ3 abolished XBP1s-induced calponin H1 suppression and SMC proliferation. High levels of TGFβ3 and calponin H1 were detected in SMCs isolated from SM22-Cre/XBP1loxP mice. These SMCs showed retardation in proliferation. Importantly, in the femoral artery injury model, XBP1 deficiency in SMCs prevented the neointima formation. Conclusion: XBP1 splicing plays an important role in SMC proliferation via regulating calponin H1 expression. Targeting XBP1 splicing in SMC may provide a novel therapeutic strategy to intervene neointima formation.

Aldosterone Promotes Vascular Remodeling by Direct Effects on Smooth Muscle Cell Mineralocorticoid Receptors

Vascular remodeling occurs after endothelial injury, resulting in smooth muscle cell (SMC) proliferation and vascular fibrosis. We previously demonstrated that the blood pressure-regulating hormone aldosterone enhances vascular remodeling in mice at sites of endothelial injury in a placental growth factor-dependent manner. We now test the hypothesis that SMC mineralocorticoid receptors (MRs) directly mediate the remodeling effects of aldosterone and further explore the mechanism. A wire-induced carotid injury model was performed in wild-type mice and mice with inducible SMC-specific deletion of the MR. Aldosterone did not affect re-endothelialization after injury in wild-type mice. Deletion of SMC-MR prevented the 79% increase in SMC proliferation induced by aldosterone after injury in MR-Intact littermates. Moreover, both injury-induced and aldosterone-enhanced vascular fibrosis were attenuated in SMC-specific MR knockout mice. Further exploration of the mechanism revealed that aldosterone-induced vascular remodeling is prevented by in vivo blockade of the placental growth factor-specific receptor, type 1 vascular endothelial growth factor receptor (VEGFR1), the receptor for placental growth factor. Immunohistochemistry of carotid vessels shows that the induction of VEGFR1 expression in SMC after vascular injury is attenuated by 72% in SMC-specific MR knockout mice. Moreover, aldosterone induction of vascular placental growth factor mRNA expression and protein release are also prevented in vessels lacking SMC-MR. These studies reveal that SMC-MR is necessary for aldosterone-induced vascular remodeling independent of renal effects on blood pressure. SMC-MR contributes to induction of SMC VEGFR1 in the area of vascular injury and to aldosterone-enhanced vascular placental growth factor expression and hence the detrimental effects of aldosterone are prevented by VEGFR1 blockade. This study supports exploring MR antagonists and VEGFR1 blockade to prevent pathological vascular remodeling induced by aldosterone.

Abstract 18000: The Role of X-box Binding Protein 1 Splicing in Smooth Muscle Cell Proliferation and Neointima Formation

Background: Smooth muscle cell (SMC) proliferation plays an important role in neointima formation in response to vascular injury. However, the underlying mechanism is still not well understood. It has been well documented that endoplasmic reticulum (ER) stress plays a provoking role in cardiovascular disease including neointima formation. As one of the key transducer in ER stress response, the X-box binding protein 1 (XBP1) has been demonstrated to contribute to endothelial cell proliferation, autophagic response and apoptosis, but less is known about its role in SMC growth. Methods and Results: In this study, we intend to study the role of XBP1 in SMC proliferation and its contribution to neointima formation in response to vascular injury. Quantitative RT-PCR and immunohistological staining analysis revealed that XBP1 splicing was up-regulated in the arterial vasculature in aged ApoE deficient mice and in response to vascular injury in wild type mice with elevated XBP1 protein in the neointima. Platelet derived growth factor (PDGF) induced XBP1 splicing via the interaction between PDGF receptor and inositol requiring enzyme 1 alpha (IRE1α). Transient over-expression of the spliced XBP1 (XBP1s) increased SMC proliferation, while knockdown of XBP1 or IRE1α abolished PDGF-induced SMC proliferation. XBP1s down-regulated calponin H1 gene expression at mRNA and protein levels, which might be through the down-regulation of TGFβ3 signalling. Reconstitution of calponin H1 via adenoviral transfer could ablate XBP1s-induced SMC proliferation. Addition of exogenous TGFβ3 abolished XBP1s-induced calponin H1 suppression and SMC proliferation. High levels of TGFβ3 and calponin H1 were detected in SMCs isolated from SM22-Cre/XBP1 loxP mice. These SMCs showed retardation in proliferation as compared to SMCs from wild type litters. Importantly, in the femoral artery injury model, XBP1 deficiency in SMCs prevented the neointima formation. CONCLUSION: XBP1 splicing plays an important role in SMC proliferation via regulating calponin H1 expression. Targeting XBP1 splicing in SMC may provide a novel therapeutic strategy to intervene neointima formation.

Abstract 302: Smooth Muscle Cell Mineralocorticoid Receptors Directly Mediate Aldosterone-induced Remodeling After Vascular Injury Via Type 1 Vegf Receptors

Background and Significance Endothelial damage initiates vascular remodeling with smooth muscle cell (SMC) proliferation and vascular fibrosis. Excessive remodeling, as in restenosis after vascular procedures or atherosclerosis, limits blood flow to distal tissues. In animals, aldosterone (aldo) infusion enhances remodeling which is reversed by aldo antagonists, implicating the mineralocorticoid receptor (MR). We previously showed that MR is expressed in SMC and that aldo induces the type 1 VEGF receptor (VEGFR1) in injured vessels. Hypothesis and Results We hypothesize that aldo promotes pathological vascular remodeling by directly activating MR in SMC and promoting SMC proliferation via VEGFR1 signaling. We show in vivo that aldo does not affect re-endothelialization after injury supporting our focus on SMC. To test this hypothesis we measured aldo-induced remodeling after wire carotid injury in mice with inducible SMC-specific deletion of MR (SMC-MR-KO). The 80% increase in SMC proliferation with aldo in control littermates is completely prevented in SMC-MR-KO mice (p<0.005, n=11). To examine the mechanism, mouse carotid SMC were treated with aldo in vitro producing a dose dependent proliferative response (1,5,10 and100nM aldo, 9%±2, 17%±3, 21%±6, and 25%±7 vs control, respectively, p<0.05, n=8). The increase in proliferation with 10 nM aldo+IgG was completely inhibited by treatment with specific VEGFR1-blocking antibody (Ab, p=0.04, n=8). Immunohistochemistry of carotid vessels reveals that VEGFR1 is induced in SMC after injury and further enhanced by aldo (55%±0.5 increase with injury, 74%± 0.5 with aldo+injury vs uninjured control, p<0.05, n=8).We examined the role of VEGFR1 in vivo and VEGFR1 blockade significantly reduces aldo-induced SMC proliferation after injury (46%±4 decrease vs aldo+IgG, p<0.05, n=9). Tail cuff measurements reveal no difference in blood pressure with any of the treatments. Conclusions These data support that SMC MR directly mediates aldo-induced vascular remodeling independent blood pressure. The mechanism involves VEGFR1 that is induced on SMC in the area of injury. This study supports exploring VEGFR1-blocking drugs to prevent pathological vascular remodeling induced by aldo in humans.

Rare, Nonsynonymous Variant in the Smooth Muscle-Specific Isoform of Myosin Heavy Chain, MYH11 , R247C, Alters Force Generation in the Aorta and Phenotype of Smooth Muscle Cells

Mutations in myosin heavy chain (MYH11) cause autosomal dominant inheritance of thoracic aortic aneurysms and dissections. At the same time, rare, nonsynonymous variants in MYH11 that are predicted to disrupt protein function but do not cause inherited aortic disease are common in the general population and the vascular disease risk associated with these variants is unknown. To determine the consequences of the recurrent MYH11 rare variant, R247C, through functional studies in vitro and analysis of a knock-in mouse model with this specific variant, including assessment of aortic contraction, response to vascular injury, and phenotype of primary aortic smooth muscle cells (SMCs). The steady state ATPase activity (actin-activated) and the rates of phosphate and ADP release were lower for the R247C mutant myosin than for the wild-type, as was the rate of actin filament sliding in an in vitro motility assay. Myh11(R247C/R247C) mice exhibited normal growth, reproduction, and aortic histology but decreased aortic contraction. In response to vascular injury, Myh11(R247C/R247C) mice showed significantly increased neointimal formation due to increased SMC proliferation when compared with the wild-type mice. Primary aortic SMCs explanted from the Myh11(R247C/R247C) mice were dedifferentiated compared with wild-type SMCs based on increased proliferation and reduced expression of SMC contractile proteins. The mutant SMCs also displayed altered focal adhesions and decreased Rho activation, associated with decreased nuclear localization of myocardin-related transcription factor-A. Exposure of the Myh11(R247C/R247C) SMCs to a Rho activator rescued the dedifferentiated phenotype of the SMCs. These results indicate that a rare variant in MYH11, R247C, alters myosin contractile function and SMC phenotype, leading to increased proliferation in vitro and in response to vascular injury.

Wnt signalling in smooth muscle cells and its role in cardiovascular disorders

Vascular smooth muscle cells (SMCs) are the major cell type within blood vessels. SMCs exhibit low rates of proliferation, migration, and apoptosis in normal blood vessels. However, increased SMC proliferation, migration, and apoptosis rates radically alter the composition and structure of the blood vessel wall and contribute to cardiovascular diseases, such as atherosclerosis, and restenosis that occur after coronary artery vein grafting and stent implantation. Consequently, therapies that modulate SMC proliferation, migration, and apoptosis may be useful for treating cardiovascular diseases. The family of Wnt proteins, which were first identified in the wingless drosophila, has a well-established role in embryogenesis and development. It is now emerging that Wnt proteins also regulate SMC proliferation, migration, and survival. In this review article, we discuss recently emerging research that has revealed that Wnt proteins are important regulators of SMC behaviour via activation of β-catenin-dependent and β-catenin-independent Wnt signalling pathways.

Effect of hydrogen sulfide on restenosis of peripheral arteries after angioplasty

Peripheral artery disease (PAD) may lead to a poor quality of life. Although percutaneous transluminal angioplasty (PTA) is widely used for the treatment of PAD, restenosis remains a major drawback. Hydrogen sulfide (H2S) plays potential roles in many physiological processes, such as vasodilatation and inhibition of smooth muscle cell proliferation. However, little is known regarding its role in arterial restenosis. In this study, we induced atherosclerotic-like lesions in rabbits, and we treated the rabbits with balloon angioplasty (BA) in a similar manner as PTA performed in the clinic. The rabbits were treated with sodium hydrosulfide (NaHS, a donor of H2S) or DL-propargylglycine (PPG, an inhibitor of H2S synthase). Treatment with NaHS significantly inhibited arterial restenosis following BA by reducing the intimal area and the intima/media ratio, while PPG treatment had a tendency to result in more severe restenosis. NaHS treatment significantly reduced smooth muscle cell (SMC) proliferation and elevated SMC apoptosis in the neointima. In contrast, PPG induced a significant increase in SMC proliferation. In conclusion, H2S attenuates the progression of neointimal hyperplasia and inhibits restenosis after BA. This discovery may lead to potential novel therapies, which can improve the prognosis of PAD patients.

Divergent effects of 17-β-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-α-induced neointima formation

Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α). TNF-α can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protective effects of estradiol (E2). We therefore investigated the effects of TNF-α on human neointima formation and SMC/EC functions and any modulatory effects of E2.Saphenous vein (SV) segments were cultured in the presence of TNF-α (10ng/ml), E2 (2.5nM) or both in combination.Neointimal thickening was augmented by incubation with TNF-α, an effect that was abolished by co-culture with E2. TNF-α increased SV–SMC proliferation in a concentration-dependent manner that was optimal at 10ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1–50nM). Surprisingly, E2 itself at low concentrations (1 and 5nM) stimulated SV–SMC proliferation to a level comparable to that of TNF-α alone. SV–EC migration was significantly impaired by TNF-α (42% of control), and co-culture with E2 partially restored the ability of SV–EC to migrate and repair the wound. In contrast, TNF-α increased SV–SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-α potently induced ICAM-1 and VCAM-1 expression in both SV–EC and SV–SMC. However there was no modulation by E2 in either cell-type.In conclusion, TNF-α induced SV neointima formation, increased SMC proliferation and migration, impaired SV–EC migration and increased expression of adhesion molecules. E2 exerted distinct cell-type and function-specific modulation, the mechanisms underlying which are worthy of further detailed study.

YIA 5 Splicing of HDAC7 modulates smooth muscle cell proliferation and neointima formation through nuclear β-catenin translocation

Vascular smooth muscle cell (SMC) proliferation has an indispensable role in the pathogenesis of vascular disease, but the mechanism is not fully elucidated. The epigenetic enzyme histone deacetylase 7 (HDAC7)...

Splicing of Histone Deacetylase 7 Modulates Smooth Muscle Cell Proliferation and Neointima Formation Through Nuclear β-Catenin Translocation

Vascular smooth muscle cell (SMC) proliferation has an indispensable role in the pathogenesis of vascular disease, but the mechanism is not fully elucidated. The epigenetic enzyme histone deacetylase 7 (HDAC7) is involved in endothelial homeostasis and SMC differentiation and could have a role in SMC proliferation. In this study, we sought to examine the effect of 2 HDAC7 isoforms on SMC proliferation and neointima formation. We demonstrated that overexpression of unspliced HDAC7 (HDAC7u) could suppress SMC proliferation through downregulation of cyclin D1 and cell cycle arrest, whereas spliced HDAC7 (HDAC7s) could not. Small interfering RNA (siRNA)-mediated knockdown of HDAC7 increased SMC proliferation and induced nuclear translocation of β-catenin. Additional experiments showed that only HDAC7u could bind to β-catenin and retain it in the cytoplasm. Reporter gene assay and reverse transcription polymerase chain reaction revealed a reduction of β-catenin activity in cells overexpressing HDAC7u but not HDAC7s. Deletion studies indicated that the C-terminal region of HDAC7u is responsible for the interaction with β-catenin. However, the addition of amino acids to the N terminus of HDAC7u disrupted the binding, further strengthening our hypothesis that HDAC7s does not interact with β-catenin. The growth factor platelet-derived growth factor-BB increased the splicing of HDAC7 while simultaneously decreasing the expression of HDAC7u. Importantly, in an animal model of femoral artery wire injury, we demonstrated that knockdown of HDAC7 by siRNA aggravates neointima formation in comparison with control siRNA. Our findings demonstrate that splicing of HDAC7 modulates SMC proliferation and neointima formation through β-catenin nuclear translocation, which provides a potential therapeutic target in vascular disease.