Vascular Smooth Muscle Cells Phenotypic Modulation Research Articles

Simvastatin Decreases Stent‐Induced In‐Stent Restenosis Rate Via Downregulating the Expression of PCNA and Upregulating that of p27kip1

The inhibition of the proliferation and migration of vascular smooth muscle cells (VSMC) is one cholesterol-independent effect of statins that could lower the rate of in-stent restenosis (ISR), even if the exact mechanism remains unclear. The aim of this study was to explore the exact inhibitory mechanisms of simvastatin on ISR in vivo. Forty-five rabbits were randomized into 3 feeding groups of equal size (n=15): normal rabbit chow (normal group), a high-cholesterol diet (control group), and a simvastatin-enriched high-cholesterol diet (simvastatin group). Balloon de-endothelialization was first performed in the control and simvastatin groups after 3 days, followed by stent deployment at week 14. All rabbits were killed at week 18, and the histological changes of the ISR segments were observed. The expressions of cyclin-dependent kinase inhibitor p27 (p27kip1), proliferating cell nuclear antigen (PCNA), and α-smooth muscle (α-SM) actin were measured. In the simvastatin group compared to the control group, the neointimal thickness, neointimal area, and degree of stenosis decreased, while the residual lumen area increased significantly (P<0.05). Moreover, the expression of α-SM actin in the control group decreased by 55.4% compared to the normal group, while it increased by 29.7% with respect to the simvastatin group (P<0.05). Finally, the expression of p27kip1 increased, while that of PCNA decreased significantly in the simvastatin group compared to the control group (P<0.05). Simvastatin may inhibit VSMC phenotype modulation and proliferation by downregulating the expression of PCNA and upregulating that of p27kip1.

192 MODULATED EXOSOME SECRETION BY VASCULAR SMOOTH MUSCLE CELLS IS A NOVEL REGULATORY MECHANISM OF VASCULAR CALCIFICATION

Vascular calcification is a regulated pathological process similar to bone formation which is mediated by vascular smooth muscle cells (VSMCs) undergoing osteogenic transdifferentiation. Initiation of vascular calcification occurs in small membrane-bound matrix vesicles (MVs), secreted by VSMCs into the extracellular matrix however the mechanisms regulating MV biogenesis and secretion are unclear. Fetuin-A, a circulating protein abundant in MVs was used to trace the origin of VSMC-derived MVs. Alexa488-labelled fetuin-A was rapidly uptaken by human VSMCs and appeared in the late endosomal system and multivesicular bodies (MVBs) indicating that MVs are secreted from the endosomal compartment similar to exosomes; extracellular vesicles originating from MVBs and secreted by the range of cells. Biochemical analysis of MVs showed that they were enriched with exosomal markers, CD9 and CD63. Furthermore, inhibition of sphingomyelin phosphodiesterase 3 (SMPD3), which in involved in exosome biogenesis abrogated MV secretion by VSMCs confirming that MVs represent exosomes. Calcifying conditions induced exosome secretion by VSMCs and this was accompanied by elevated SMPD3 expression. Importantly, an inhibition of SMPD3 prevented VSMC calcification. Phenotypic modulation of VSMCs and loss of the contractile phenotype resulted in elevated exosome secretion while contractile VSMCs secreted significantly less exosomes. Notably, elevated extracellular calcium rapidly induced calcification of synthetic VSMCs whilst contractile VSMCs did not calcify. In agreement with this data, abundant immunohistochemical staining for CD63 was observed only in atherosclerotic human aorta in close association with calcified areas with little staining detected in the healthy vessel wall. Taken together this study demonstrates that MVs originate from MVBs and are secreted via the exosomal pathway. Loss of the contractile phenotype and mineral imbalance promote VSMC calcification by enhanced exosome secretion. Targeting the mechanisms of VSMC exosome secretion and/or their loading with calcification inhibitors may provide novel therapeutic interventions aimed for the prevention of vascular calcification.

MicroRNA-31 controls phenotypic modulation of human vascular smooth muscle cells by regulating its target gene cellular repressor of E1A-stimulated genes

Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. The cellular repressor of E1A-stimulated genes (CREG) has been shown to play an important role in phenotypic modulation of VSMCs. However, the mechanism regulating CREG upstream signaling remains unclear. MicroRNAs (miRNAs) have recently been found to play a critical role in cell differentiation via target-gene regulation. This study aimed to identify a miRNA that binds directly to CREG, and may thus be involved in CREG-mediated VSMC phenotypic modulation. Computational analysis indicated that miR-31 bound to the CREG mRNA 3′ untranslated region (3′-UTR). miR-31 was upregulated in quiescent differentiated VSMCs and downregulated in proliferative cells stimulated by platelet-derived growth factor and serum starvation, demonstrating a negative relationship with the VSMC differentiation marker genes, smooth muscle α-actin, calponin and CREG. Using gain-of-function and loss-of-function approaches, CREG and VSMC differentiation marker gene expression levels were shown to be suppressed by a miR-31 mimic, but increased by a miR-31 inhibitor at both protein and mRNA levels. Notably, miR-31 overexpression or inhibition affected luciferase expression driven by the CREG 3′-UTR containing the miR-31 binding site. Furthermore, miR-31-mediated VSMC phenotypic modulation was inhibited in CREG-knockdown human VSMCs. We also determined miR-31 levels in the serum of patients with coronary artery disease (CAD), with or without in stent restenosis and in healthy controls. miR-31 levels were higher in the serum of CAD patients with restenosis compared to CAD patients without restenosis and in healthy controls. In summary, these data demonstrate that miR-31 not only directly binds to its target gene CREG and modulates the VSMC phenotype through this interaction, but also can be an important biomarker in diseases involving VSMC phenotypic modulation. These novel findings may have extensive implications for the diagnosis and therapy of a variety of proliferative vascular diseases.

Spry1 and Spry4 Differentially Regulate Human Aortic Smooth Muscle Cell Phenotype via Akt/FoxO/Myocardin Signaling

BackgroundChanges in the vascular smooth muscle cell (VSMC) contractile phenotype occur in pathological states such as restenosis and atherosclerosis. Multiple cytokines, signaling through receptor tyrosine kinases (RTK) and PI3K/Akt and MAPK/ERK pathways, regulate these phenotypic transitions. The Spry proteins are feedback modulators of RTK signaling, but their specific roles in VSMC have not been established.Methodology/Principal FindingsHere, we report for the first time that Spry1, but not Spry4, is required for maintaining the differentiated state of human VSMC in vitro. While Spry1 is a known MAPK/ERK inhibitor in many cell types, we found that Spry1 has little effect on MAPK/ERK signaling but increases and maintains Akt activation in VSMC. Sustained Akt signaling is required for VSMC marker expression in vitro, while ERK signaling negatively modulates Akt activation and VSMC marker gene expression. Spry4, which antagonizes both MAPK/ERK and Akt signaling, suppresses VSMC differentiation marker gene expression. We show using siRNA knockdown and ChIP assays that FoxO3a, a downstream target of PI3K/Akt signaling, represses myocardin promoter activity, and that Spry1 increases, while Spry4 decreases myocardin mRNA levels.ConclusionsTogether, these data indicate that Spry1 and Spry4 have opposing roles in VSMC phenotypic modulation, and Spry1 maintains the VSMC differentiation phenotype in vitro in part through an Akt/FoxO/myocardin pathway.

Curcumin Regulates VSMC Phenotype Transition via Modulation of Notch and Wnt Signaling Pathways

Abstract Preclinical Research In the development of cardiovascular diseases, vascular smooth muscle cells (VSMCs) undergo transition from a contractile to a synthetic phenotype. Notch‐ and Wnt‐dependent signaling pathways play a critical role during this process. Curcumin has potential for clinical use including anti‐inflammatory and antitumor actions. However, the effect of curcumin on VSMC phenotype transition remains unclear. In VSMCs isolated from the thoracic aorta of Sprague Dawley rats, curcumin (5–20 μM) produced a concentration‐dependent inhibition of serum‐elicited VSMC migration. Furthermore, curcumin, at the dose of 20 μM, partially reversed the repression of VSMC markers induced by serum stimulation, an effect associated with attenuation of Jagged‐1 and Notch‐1 levels and downregulation of the downstream gene Hey‐1. Downregulation of Wnt‐4 was also observed after curcumin treatment in the presence of serum, which was followed by inhibition of nuclear β‐catenin translocation and cyclin D1 expression. These data suggest that curcumin is a potent regulator of VSMCs phenotype transition and may play a critical role in regulating these events after vascular injury.

Inactivation of Serum Response Factor Contributes To Decrease Vascular Muscular Tone and Arterial Stiffness in Mice

Vascular smooth muscle (SM) cell phenotypic modulation plays an important role in arterial stiffening associated with aging. Serum response factor (SRF) is a major transcription factor regulating SM genes involved in maintenance of the contractile state of vascular SM cells. We investigated whether SRF and its target genes regulate intrinsic SM tone and thereby arterial stiffness. The SRF gene was inactivated SM-specific knockout of SRF (SRF(SMKO)) specifically in vascular SM cells by injection of tamoxifen into adult transgenic mice. Fifteen days later, arterial pressure and carotid thickness were lower in SRF(SMKO) than in control mice. The carotid distensibility/pressure and elastic modulus/wall stress curves showed a greater arterial elasticity in SRF(SMKO) without modification in collagen/elastin ratio. In SRF(SMKO), vasodilation was decreased in aorta and carotid arteries, whereas a decrease in contractile response was found in mesenteric arteries. By contrast, in mice with inducible SRF overexpression, the in vitro contractile response was significantly increased in all arteries. Without endothelium, the contraction was reduced in SRF(SMKO) compared with control aortic rings owing to impairment of the NO pathway. Contractile components (SM-actin and myosin light chain), regulators of the contractile response (myosin light chain kinase, myosin phosphatase target subunit 1, and protein kinase C-potentiated myosin phosphatase inhibitor) and integrins were reduced in SRF(SMKO). SRF controls vasoconstriction in mesenteric arteries via vascular SM cell phenotypic modulation linked to changes in contractile protein gene expression. SRF-related decreases in vasomotor tone and cell-matrix attachment increase arterial elasticity in large arteries.

MicroRNA-145 Restores Contractile Vascular Smooth Muscle Phenotype and Coronary Collateral Growth in the Metabolic Syndrome

Transient, repetitive occlusion stimulates coronary collateral growth (CCG) in normal animals. Vascular smooth muscle cells (VSMCs) switch to synthetic phenotype early in CCG, then return to contractile phenotype. CCG is impaired in the metabolic syndrome. We determined whether impaired CCG was attributable to aberrant VSMC phenotypic modulation by miR-145-mediated mechanisms, and whether restoration of physiological miR-145 levels in metabolic syndrome (JCR rat) improved CCG. CCG was stimulated by transient, repetitive left anterior descending artery occlusion and evaluated after 9 days by coronary blood flow measurements (microspheres). miR-145 was delivered to JCR VSMCs via adenoviral vector (miR-145-Adv). In JCR rats, miR-145 was decreased late in CCG (≈ 2-fold day 6; ≈ 4-fold day 9 versus SD), which correlated with decreased expression of smooth muscle-specific contractile proteins (≈ 5-fold day 6; ≈ 10-fold day 9 versus SD), indicative of VSMCs' failure to return to the contractile phenotype late in CCG. miR-145 expression in JCR rats (miR-145-Adv) on days 6 to 9 of CCG completely restored VSMCs contractile phenotype and CCG (collateral/normal zone flow ratio was 0.93 ± 0.09 JCR+miR-145-Adv versus 0.12 ± 0.02 JCR versus 0.87 ± 0.02 SD). Restoration of VSMC contractile phenotype through miR-145 delivery is a highly promising intervention for restoration of CCG in the metabolic syndrome.

Abstract TMP26: Binding of KLF4 and HDAC2 to The Promoter Region of SM22alpha Mediates Phenotypic Modulation In vitro and In vivo: Implications in Cerebral Aneurysm Formation

Cerebral Vascular Smooth Muscle Cell (VSMC) phenotypic modulation appears to play an important role in cerebral aneurysm formation and progression, yet the molecular mechanisms involved remain unknown. We investigated the role of inflammatory cytokine, Tumor Necrosis Factor-alpha (TNF-α) and cigarette smoke in directly mediating phenotypic modulation in VSMC. We hypothesize that this may, in part, occur through the binding of the transcription factor, KLF4, to the promoter region of SM22alpha, thereby downregulating VSMC differentiation and contractile genes. This may be critical in the pathogenesis of cerebral aneurysm formation. Methods: Cultured cerebral VSMC from rat Circle of Willis were treated with increasing doses of TNF-α (10ng/ml &amp; 50ng/ml) and cigarette smoke extract (CSE; 10ug/ml &amp; 40ug/ml) from 2 to 24hrs. VSMC were transfected with siRNA specific to KLF4. In vivo experiments included application of pluronic gel (containing TNF-α &amp; CSE) to rat carotid artery adventitia. Expression of SM22alpha and KLF4 was measured with qPCR. Chromatin Immunoprecipitation (ChIP) was performed both in vitro and in vivo after treatment (TNF-α and CSE) and incubation with anti-KLF4, anti-HDAC2, anti-H4Ac and anti-H3K9Ac. Results: Both TNF-α and CSE independently suppress SM22alpha and increase expression of KLF4 in vitro and in vivo. siKLF4 inhibits the effects of TNF-α and CSE on KLF4 and SM22alpha. ChIP assays demonstrate that KLF4 binds directly to the promoter region of SM22alpha and further recruits HDAC2 to the promoter region. This complex leads to histone modifications leading to decreased acetylation of H4 and H3K9, resulting in decreased expression of SM22alpha. Conclusion: KLF4 is a key mediator of TNF-α and CSE induced phenotypic modulation of cerebral VSMC. Phenotypic modulation has been implicated in cerebral aneurysm formation in humans. KLF4 dependent regulation of cerebral VSMC phenotypic modulation may be an underlying molecular mechanism involved in the pathogenesis of cerebral aneurysm formation and a target for future therapies.

Characterization of transcriptional and posttranscriptional properties of native and cultured phenotypically modulated vascular smooth muscle cells

Various in vitro models are used for studying phenotypic modulation of vascular smooth muscle cells (VSMCs) and the established culture of vascular smooth muscle cells (cVSMCs) is most often used for this purpose. On the other hand, vascular interstitial cells (VICs) are native phenotypically modulated VSMCs present in blood vessels under normal physiological conditions. The aim of this work has been to compare the difference in expression of a number of VSMC-specific markers, which are commonly used for the characterisation of phenotypic modulation of VSMCs, between freshly dispersed VSMCs, VICs and cVSMCs from rat abdominal aorta. Our experiments show that VICs are present in the rat aorta and express markers of VSMCs. Both VICs and cVSMCs display the presence of sparse individual stress fibres enriched in alpha smooth muscle actin (αSM-actin), whereas in VSMCs, this protein is more densely packed. Compared with contractile VSMCs, both VICs and cVSMCs display decreased expression of VSMC-specific markers such as smoothelin, myosin light chain kinase and SM22α; however, the expression of two major cytoskeletal and contractile proteins (smooth muscle myosin heavy chain and αSM-actin) was downregulated in cVSMCs but not in VICs compared with contractile VSMCs. These results suggest different mechanisms for the phenotypic modulation of cVSMCs and VICs. VICs might therefore represent a novel convenient model for studying molecular mechanisms that govern the phenotypic modulation of VSMCs.

Myeloid Cell 5-Lipoxygenase Activating Protein Modulates the Response to Vascular Injury

Human genetics have implicated the 5-lipoxygenase enzyme in the pathogenesis of cardiovascular disease, and an inhibitor of the 5-lipoxygenase activating protein (FLAP) is in clinical development for asthma. Here we determined whether FLAP deletion modifies the response to vascular injury. Vascular remodeling was characterized 4 weeks after femoral arterial injury in FLAP knockout mice and wild-type controls. Both neointimal hyperplasia and the intima/media ratio of the injured artery were significantly reduced in the FLAP knockouts, whereas endothelial integrity was preserved. Lesional myeloid cells were depleted and vascular smooth muscle cell (VSMC) proliferation, as reflected by bromodeoxyuridine incorporation, was markedly attenuated by FLAP deletion. Inflammatory cytokine release from FLAP knockout macrophages was depressed, and their restricted ability to induce VSMC migration ex vivo was rescued with leukotriene B(4). FLAP deletion restrained injury and attenuated upregulation of the extracellular matrix protein, tenascin C, which affords a scaffold for VSMC migration. Correspondingly, the phenotypic modulation of VSMC to a more synthetic phenotype, reflected by morphological change, loss of α-smooth muscle cell actin, and upregulation of vascular cell adhesion molecule-1 was also suppressed in FLAP knockout mice. Transplantation of FLAP-replete myeloid cells rescued the proliferative response to vascular injury. Expression of lesional FLAP in myeloid cells promotes leukotriene B(4)-dependent VSMC phenotypic modulation, intimal migration, and proliferation.

Abstract 372: Regulation of Nox1 and Nox2 by Cigarette Smoke in Cerebral Vascular Smooth Muscle Cells: A Role of Oxidative Stress in Cerebral Aneurysm Formation

Objectives: Cigarette smoke is one of the most significant environmental risk factor for cerebral aneurysm formation and progression. Factors including inflammation and matrix degradation are considered to be important in aneurysm formation. Reactive Oxygen Species (ROS) play a pathophysiological role in vascular inflammation, proliferation, migration and angiogenesis. Among the major sources of ROS in vascular smooth muscle cells (VSMCs) are NAD(P)H oxidase (Nox). We investigated the role of Cigarette Smoke Extract (CSE) in regulation of Nox1 and Nox2 isoforms in cerebral VSMCs both in vitro and in vivo. Methods: Rat cerebral VSMCs were treated with CSE at 10 and 40 ug/ml for 4 and 24 hours. The level of water soluble components of cigarette smoke and nicotine levels present in the CSE overlap with plasma levels of these constituents in human smokers. RNA was extracted and qPCR was performed. Cerebral VSMCs were transfected with siNox1 and siNox2 for 24 and 72 hours and further incubated with CSE for 24 hours. As part of in vivo studies, Pluronic Gel (40% w/v) containing 0.2-0.8 mg/ml of CSE was applied to the adventitial surface of rat carotid arteries for 24 hours. Vessels were harvested for mRNA quantification. Results: CSE decreased the expression of VSMC marker genes and myocardin, increased expression of KLF4 (a transcription factor) and proinflammatory/matrix remodeling marker genes including MMPs, VCAM1 and MCP1. In addition CSE increased the expression of both Nox1 and Nox2 at 4 and 24 hours but was more significant at 4h. Preliminary studies with siNox1 and siNox2 decreased the expression of KLF4, and reversed inflammatory markers gene expression caused by CSE stimulation. siNox1 increased the expression of marker genes including alpha-Actin and 22-alpha. Expression of Nox1 and Nox2 was markedly increased following in vivo application of pluronic gel. Conclusions: These findings indicate that cerebral VSMCs contain Nox1 and Nox2, are potently regulated by CSE and play an important role in NAD(P)H oxidase driven ROS production. CSE induces marked phenotypic modulation of vertebral VSMC while concomitantly increasing expression of Nox1 and Nox2. Furthermore, alteration of Nox expression abrogates aspects of CSE induced VSMC phenotypic modulation. These molecular changes implicate oxidative stress in vascular pathology underlying cerebral aneurysms and may provide a potential target for future therapeutic strategies.

Abstract 374: Epigenetic Mechanisms Regulate TNF-α-Induced Phenotypic Modulation in Cerebral Vascular Smooth Muscle Cells: Implications for Cerebral Aneurysm Biology

Objectives: Inflammation and cerebral Vascular Smooth Muscle Cell (VSMC) phenotypic modulation appear to be involved in cerebral aneurysm formation. Tumor Necrosis Factor-alpha (TNF-α) is found in increased quantity in ruptured and un-ruptured cerebral aneurysms. Our previous work demonstrates that TNF-α potently induces cerebral VSMC phenotypic modulation . We explored the underlying mechanisms involved in these changes. Methods: Rat cerebral VSMCs were treated with TNF-alpha at 50 ng/ml (based on preliminary data) for 2, 6 and 24 hours. Cells at 80-90% confluence were fixed with formaldehyde. DNA was sheared using sonication. Chromatin immune-precipitation was performed using the following antibodies: 1) anti-KLF4 (Krupple Like Factor 4), 2) anti-HDAC2 (Histone Deacetylase 2), 3) anti-H3K9Ac (Histone 3 Lysine 9 Acetylation), 4) anti-H3K27triMe (Histone 3 Lysine 27 Tri-Methylation) and 5) anti-H4Ac (Histone 4 Acetylation). qPCR was performed with primers specific to CArG containing promoter regions of differentiation marker genes {(alpha-Actin and Myosin Heavy Chain (MHC)} and Myocardin. Results were normalized against input DNA. As part of in vivo experiments Pluronic Gel (40% w/v) containing TNF-alpha at 30-60 ug/ml was applied to rat carotid vessels for 6-8 hours. Vessels were harvested and frozen in liquid nitrogen and chromatin immune-precipitation was performed. Results: TNF-alpha stimulation promoted profound phenotypic modulation of cerebral VSMCs. We demonstrated an increased percent enrichment of alpha-actin, MHC and Myocardin promoters with KLF4, HDAC2, and H3K27triMe antibodies and decreased enrichment with H3K9Ac antibody. H4Ac antibody showed decreased enrichment of alpha-Actin and MHC promoter in vivo. Hence, there is evidence of direct binding of the transcription factor, KLF4, to marker gene promoter regions. Along with direct effects, KLF4 exerts indirect effects via binding with HDAC2 which deactylates H3 and H4 histones. Although, KLF4 demonstrated increased binding at 24 hours but HDAC2 did not. HDAC2 binding was more significant at early time points. These effects promote heterochromatin formation and decrease SRF binding to VSMC gene CArG box chromatin which in turn results in decreased transcription of marker genes and phenotypic modulation. Conclusions: We have demonstrated underlying molecular mechanisms involved in repression of cerebral VSMCs marker genes following exposure to TNF-alpha. TNF-alpha has a role in inflammation, matrix degradation and phenotypic modulation. This suggests a novel mechanism whereby epigenetic control of chromatin structure may play an important role in regulating VSMC gene expression in response to inflammatory cytokines like TNF-alpha which has implications for the pathogenesis of cerebral aneurysms.

Abstract 373: Cigarette Smoke Produces Phenotypic Modulation of Cerebral Vascular Smooth Muscle Cells: Role of KLF4 and Epigenetic Control

Objectives: Cigarette smoke is one of the most important environmental factors associated with cerebral aneurysm formation and progression. Cigarette smoke causes phenotypic modulation of Cerebral Vascular Smooth Muscle Cells (VSMCs) which is considered an important underlying mechanism in cerebral aneurysm formation/progression. We studied epigenetic changes caused by Cigarette Smoke Extract (CSE) in VSMC differentiation marker genes. Methods: Rat cerebral VSMCs were treated with CSE at 40 ug/ml (optimal dosage based on preliminary data) dissolved in HEPES buffer (biological activity comparable to soluble components of cigarette smoke in humans) for 2 hours. Cells at 80-90% confluence were fixed with formaldehyde. DNA was sheared using sonication. Chromatin immune-precipitation was performed using following antibodies: 1) anti-KLF4 (Krupple Like Factor 4), 2) anti-HDAC2 (Histone Deacetylase 2), 3) anti-H3K9Ac (Histone 3 Lysine 9 Acetylation), 4) anti-H3K27triMe (Histone 3 Lysine 27 Tri-Methylation) and 5) anti-H4Ac (Histone 4-Acetylation). qPCR was performed with primers specific to CArG containing promoter regions of differentiation marker genes {(alpha-Actin and Myosin Heavy Chain (MHC)} and Myocardin. Results were normalized against input DNA. As part of in vivo experiments Pluronic Gel (40% w/v) containing CSE at 0.8 mg/ml was applied to rat carotid vessels for 6-8 hours. Vessels were harvested and frozen in liquid nitrogen and chromatin immune-precipitation was performed. Results: CSE stimulation promoted a non-differentiated phenotype of cerebral VSMCs. We demonstrated a marked increase in percent enrichment of alpha-actin, MHC and Myocardin promoters with KLF4, HDAC2, and H3K27triMe antibodies and decreased enrichment with H3K9Ac antibody. Although H4Ac antibody showed decreased enrichment at the alpha-Actin promoter, this was not observed for MHC and Myocardin promoters. This was consistent for both in vivo and in vitro studies. Conclusions: We previously demonstrated that CSE decreases expression of cerebral VSMCs marker genes and SRF co-activator, Myocardin and increases expression of transcription factor, KLF4. Further it promotes a pro-inflammatory phenotype. Decreased expression of the above mentioned genes is a result of direct binding of KLF4 to the CArG containing promoter region. Further, KLF4 recruits HDAC2 which deacetylases H3K9 and H4 causing DNA compaction thereby repressing transcription. Our results provide insight into the underlying molecular mechanisms involved in CSE-induced VSMC phenotypic modulation and provide future potential therapeutic targets applicable to cerebral aneurysms.

Ginsenoside Rb1 inhibits the carotid neointimal hyperplasia induced by balloon injury in rats via suppressing the phenotype modulation of vascular smooth muscle cells

This study aims to investigate the effects of ginsenoside Rb1 on vascular intimal hyperplasia in rats and explore the mechanisms. The rat vascular neointimal hyperplasia model was made by rubbing the endothelia of carotid artery with a balloon and Rb1 (10 and 30mg/kg/day) was given the day after surgery for 14 consecutive days. The neointimal hyperplasia level and the degree of vascular smooth muscle cells (VSMCs) proliferation were evaluated by histopathology and by calculating the proliferating cell nuclear antigen (PCNA) positive expression percentage; protein expressions of PCNA, phosphorylation extracellular signal-regulated kinase 1/2 (pERK1/2), smooth muscle α-actin (SM α-actin), and the mRNA expressions of proto-oncogene c-myc, SM α-actin, SM-emb (embryonic smooth muscle myosin heavy chain) and p38 MAPK were detected by immunohistochemistry and Real Time RT-PCR, respectively. Compared with the endothelia rubbing model group, Rb1 10 and 30mg/kg/day medication significantly ameliorated the neointimal hyperplasia (P<0.05), and decreased the positive expression percentage of PCNA(P<0.05). Rb1 medication also significantly decreased the elevated protein expression of pERK1/2 and the mRNA expression of c-myc(P<0.05), and tended to reduce the expression of p38 MAPK mRNA. Endothelial rubbing increased the SM-emb mRNA expression, but decreased the expression of SM α-actin mRNA which was reversed by Rb1 (P<0.05). The results indicate that Rb1 inhibits the vascular neointimal hyperplasia induced by balloon-injury in rats via suppressing the VSMC proliferation, which may be involved in part the inhibition of pERK1/2 protein and related to its inhibition on VSMC phenotype modulation.

The vascular smooth muscle cell in arterial pathology: a cell that can take on multiple roles

Vascular smooth muscle cells (VSMCs) are the stromal cells of the vascular wall, continually exposed to mechanical signals and biochemical components generated in the blood compartment. They are involved in all the physiological functions and the pathological changes taking place in the vascular wall. Owing to their contractile tonus, VSMCs of resistance vessels participate in the regulation of blood pressure and also in hypertension. VSMCs of conduit arteries respond to hypertension-induced increases in wall stress by an increase in cell protein synthesis (hypertrophy) and extracellular matrix secretion. These responses are mediated by complex signalling pathways, mainly involving RhoA and extracellular signal-regulated kinase1/2. Serum response factor and miRNA expression represent main mechanisms controlling the pattern of gene expression. Ageing also induces VSMC phenotypic modulation that could have influence on cell senescence and loss of plasticity and reprogramming. In the early stages of human atheroma, VSMCs support the lipid overload. Endocytosis/phagocytosis of modified low-density lipoproteins, free cholesterol, microvesicles, and apoptotic cells by VSMCs plays a major role in the progression of atheroma. Migration and proliferation of VSMCs in the intima also participate in plaque progression. The medial VSMC is the organizer of the inwardly directed angiogenic response arising from the adventitia by overexpressing vascular endothelial growth factor in response to lipid-stimulated peroxisome proliferator-activated receptor-γ, and probably also the organizer of the adventitial immune response by secreting chemokines. VSMCs are also involved in the response to proteolytic injury via their ability to activate blood-borne proteases, to secrete antiproteases, and to clear protease/antiprotease complexes.

Abstract 112: Cigarette Smoke Directly Produces a Pro-inflammatory/matrix remodeling Phenotype in Cerebral Vascular Smooth Muscle Cells via KLF4: A Potential Mechanism for Cerebral Aneurysm Pathogenesis

Cigarette smoke is one of the most significant environmental risk factor for cerebral aneurysm formation. Inflammation, matrix degradation and vascular smooth muscle cell phenotypic modulation are thought to be important in aneurysm pathology yet molecular pathogenic mechanisms are unknown. We investigated the role of cigarette smoke in producing phenotypic modulation and inflammation in cerebral vascular smooth muscle cells (VSMCs) focusing on a potential pivotal role of the transcription factor, KLF4. Methods: Rat cerebral VSMCs were treated with Cigarette Smoke Extract (CSE) at 4 and 40 ug/ml for 4, 24 and 48 hours. The level of water soluble components of cigarette smoke and nicotine levels present in the CSE overlap with plasma levels of these constituents in human smokers. SMC marker genes (SM-alpha-Actin, 22-alpha and MHC), transcription co-activator, Myocardin, KLF4 and inflammatory/matrix remodeling genes (MMP-2, 3, 9, VCAM-1, MCP-1, and iNOS) were measured using qPCR. siRNA against KLF4 and non-specific siGFP were transfected in cultured VSMCs to examine a potential critical role for KFL4 in this process. As part of in vivo experiments, Pluronic Gel (40% w/v) containing 0.2 - 0.8 mg/ml CSE was applied to the adventitial surface of rat carotid arteries for 24-48 hours. Results: CSE significantly induced KLF4 expression at 4 hours (p&lt;0.05 vs. baseline) preceding phenotypic modulation of VSMCs. CSE directly produced marked phenotypic modulation with decreased expression of SMC marker genes and myocardin while markedly increasing expression of inflammatory/matrix remodeling genes (p&lt;0.05 vs. baseline). Strikingly, this profound phenotypic modulation was reversed with siKLF4. Similarly, decreased expression of marker genes and Myocardin with concomitant upregulation of KLF4 and inflammatory/matrix remodeling genes was noted following in vivo pluronic gel application containing CSE. The most notable increase was noted in MMPs, MCP-1, IL-1 and TNF-alpha expression (all p&lt;0.05 vs. control). Conclusions: Cigarette smoke directly produces marked phenotypic modulation of cerebral vascular smooth muscle cells in vitro and in vivo promoting a pro-inflammatory/matrix remodeling phenotype. These effects appear to be mediated via KLF4. Inflammation, accelerated matrix remodeling and VSMC phenotypic modulation are implicated in human cerebral aneurysms. The molecular mechanisms activated by cigarette smoke outlined here may have critical implications for human cerebral aneurysm pathogenesis and may represent important targets for future therapy. Fig: Gene Expression.

Abstract 110: Tumor Necrosis Factor-alpha Induces Phenotypic Modulation in Cerebral Vascular Smooth Muscle Cells via KLF4: Implications for Cerebral Aneurysm Pathology

Inflammation and vascular smooth muscle cell (VSMC) phenotypic modulation appears to be important in cerebral aneurysm formation/progression yet molecular pathological mechanisms remain unknown. We investigated a potential direct role of Tumor Necrosis Factor-alpha (TNF-alpha) mediated via the transcription factor, KLF4, in inducing VSMC phenotypic modulation and inflammation in vitro and in vivo, which may be critical in the pathogenesis of cerebral aneurysms. Methods: Rat cerebral VSMCs were treated with TNF-alpha (5 and 50 ng/ml) for 24 hours. The expression of KLF4, VSMCs marker genes (SM-alpha-Actin, SM-22alpha and SM-MHC), the SRF co-activator, Myocardin, and inflammatory/ matrix remodeling genes (MMP-3, VCAM-1, iNOS, and MCP-1) was measured using qPCR. Cells were transfected with siRNA against KLF4 to examine the role of KLF4 as a direct mediator of phenotypic modulation. To investigate in vivo effects, pluronic gel (40% w/v) containing TNF-alpha (30-60 ug/ml) was applied to carotid vessels in 7-week-old rats for 6 and 24 hours. Carotid vessels were harvested and qPCR performed. In another experiment, aneurysm induction surgery was performed in rats using a standard model (unilateral carotid ligation/induction of hypertension). The circle of Willis cerebral vessels were harvested and qPCR performed comparing treated and control animals. Results: TNF-alpha treatment produced a dose-dependent decrease in SMC marker gene and myocardin expression while potently inducing expression of KLF4 and the aforementioned inflammatory/matrix remodeling genes (p&lt;0.05 vs. baseline) demonstrating a profound direct role of TNF-alpha in inducing cerebral VSMC phenotypic modulation in vitro. This phenotypic modulation was markedly abrograted using siKLF4. In vivo, TNF-alpha applied to carotid vessels produced a marked increase in KLF4 expression at 6 hours and a subsequent decrease in SMC marker gene expression and increase in expression of inflammatory/matrix remodeling genes at 24 hours (p&lt;0.05 treated vs. control). Similarly, increased expression of KLF4 and TNF-alpha was noted in the Circle of Willis in vivo two weeks after aneurysm induction surgery. Decreased marker gene expression along with increased inflammatory/matrix remodeling marker gene expression was also seen (all p&lt;0.05 vs. control). Conclusion: TNF-alpha appears to potently induce phenotypic modulation of cerebral VSMCs producing a pro-inflammatory phenotype with elevation of matrix remodeling genes both in vitro and in vivo. KLF4 appears to be a critical mediator of this process. As inflammation (and specifically TNF-alpha) and matrix remodeling have been implicated in the pathogenesis of human cerebral aneurysms, the molecular mechanisms outlined herein may represent a novel target for future therapy.

Naringenin inhibits vascular smooth muscle cell function involving reactive oxygen species production modulation and NF-κB activity suppression

ObjectiveGrowth factor and oxidative stress-mediated phenotypic modulation, migration and proliferation of vascular smooth muscle cells (VSMCs) play a pivotal role in the pathogenesis of atherosclerosis and restenosis. Naringenin, a flavonoid...

Beyond Phosphorylation

The vasculature is composed of endothelial cells, vascular smooth muscle cells (VSMCs), fibroblasts, and immune cells with closely integrated functions. It is exposed to a variable environment of pulsatile flow and changing blood pressure, plus fluctuations in nutrient, oxidative, and cytokine stress. Adaptation to this changing environment is critical for sustained vascular health. VSMCs undergo a phenotype change in response to vascular injury, including migration, proliferation, and matrix production, also known as VSMC activation or phenotypic modulation. VSMC activation contributes to the progression of atherosclerosis and postangioplasty restenosis. Understanding the endogenous regulators of vascular plasticity and adaptation has revealed potential targets for pharmacological intervention to prevent vascular stenosis. For example, Ross established cAMP as a critical signaling pathway for the maintenance of VSMC quiescence.1 Since that time, agents that increase cAMP, such as phosphodiesterase inhibitors, have demonstrated usefulness as vasodilators, with clinical implications for pulmonary hypertension.2,3 Intracellular accumulation of cAMP signals to protein kinase A and the cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac; simultaneous activation of both of these pathways by cAMP is essential for the antiproliferative impact on VSMCs.3,4 …

Effects of rosuvastatin on the coordinated proteomic response of human coronary smooth muscle cells to low density lipoproteins

Introduction The clinical benefits of statins in reducing coronary events have been related to repopulation of the acellular core of atheromatous plaques with vascular smooth muscle cells (VSMC) and plaque stabilization. Intimal low-density lipoproteins (LDL) have been associated with VSMC phenotypic modulation and plaque destabilization. We have recently reported that LDL impair vascular remodelling because of changes affecting cytoskeleton proteins. Here, we used a proteomic approach to study whether rosuvastatin reverses the effects induced by atherogenic concentrations of LDL in human VSMC. Methods and Results VSMC treated with/without 10 μM rosuvastatin were incubated in the presence/absence of 100 μg/mL LDL for 24 h. Using 2DE and MALDI-ToF MS, we identified 39 non-redundant proteins (52 spots) with differential expression in LDL-treated cells. Thirteen of these proteins were reverted to normal levels by rosuvastatin. Thus, four proteins showed decreased intensity, six showed increased intensity, and three multispot proteins changed their distribution pattern in the rosuvastatin group. These proteins were identified as components of endoplasmic reticulum, mitochondrion, nucleus, cell membrane, cytoskeleton, and cytoplasm. In LDL-VSMC, rosuvastatin affected proteins involved in a variety of functions including cytoskeleton dynamics, chaperone activity, carbohydrate metabolism, protein biosynthesis and folding, kinase activity, redox processes, cytoprotective effects, and cell proliferation. Conclusions Our results demonstrate that the reversing effects of rosuvastatin in LDL-enriched VSMC affect functional proteins with a role in cellular metabolism and dynamics, which might contribute to the beneficial effects of statins by stabilizing lipid-rich atherosclerotic plaques in human atherosclerosis.