Smooth Muscle Cell Phenotypic Modulation Research Articles

Abstract 140: Nuclear Targeting Apelin Induces Phenotypic Transition of Vascular Smooth Muscle Cells

Apelin, and its receptor APJ, are a peptidic system playing a crucial role in vascular diseases. However, the role of apelin in atherogenesis and smooth muscle cell (SMC) proliferation remains unclear. We isolated 2 distinct SMC phenotypes from porcine coronary artery: spindle-shaped (S) and rhomboid (R). Biological features of R-SMCs (i.e. enhanced proliferative and migratory activities as well as poor level of differentiation) explain their capacity to accumulate into the intima. We identified S100A4 as being a marker of the R-SMCs in vitro and of intimal SMCs, both in pig and human. The aim of our study was to investigate the effects of apelin on SMC phenotypic transition and S100A4 expression. Apelin was highly expressed in R-SMCs including in their nucleus. P-SORT software analysis of preproapelin sequence suggested that N-terminal truncated apelin may target the nucleus, and we confirmed this in SMCs by overexpression of mutated preproapelin-His-tag. Transfection of mutated preproapelin-His-tag encoding plasmid in differentiated S-SMCs induced a transition towards a R-phenotype associated with increased proliferative activity, downregulation of SMC differentiation markers, and increased nuclear expression and release of S100A4. In contrast, transfection of S-SMCs with wild type preproapelin-His-tag encoding plasmid did not induce nuclear targeting of apelin or S100A4, and did not change the S-phenotype. Stimulation of S-SMCs with PDGF-BB, known to induce a transition to the R-phenotype, yielded nuclear targeting of both apelin and S100A4. In vivo, Apelin was expressed in SMC nuclei of stent-induced intimal thickening while its expression in the media was mainly cytoplasmic. Our results suggest that nuclear targeting of apelin in SMCs acts on S100A4 expression and release, cell proliferation and differentiation. The pathophysiological consequences of this retargeting could be instrumental in the understanding of artherosclerosis.

ADAR1‐mediated RNA editing, a novel mechanism controlling phenotypic modulation of vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) phenotypic modulation plays a critical role in the development and progression of several proliferative cardiovascular diseases such as atherosclerosis, hypertension, restenosis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. A hallmark feature of the phenotypic modulation is the down‐regulation of SMC contractile genes. Platelet‐derived growth factor‐BB (PDGF‐BB), a well‐known stimulator of SMC phenotypic modulation, down‐regulates SMC gene expression via posttranscriptional regulation of the related genes. The post‐transcriptional mechanisms involved in SMC phenotypic gene expression, however, remain largely unknown. We found that the down‐regulation of SMC contractile genes is caused by abnormal RNA splicing of their precursor mRNAs (pre‐mRNAs). This abnormal pre‐mRNA editing is facilitated by adenosine deaminase acting on RNA (ADAR), which converts adenosines to inosines (A→I editing). PDGF‐BB induces ADAR1 while down‐regulating the expression of SMC myosin heavy chain (SMMHC) and calponin (CNN). Knockdown of ADAR1 by shRNA restores PDGF‐BB‐blocked SMMHC and CNN expression, demonstrating that ADAR1 plays an essential role in SMC phenotype modulation. In vivo animal studies show that SMMHC and CNN pre‐mRNA is accumulated while their mature mRNA is decreased in balloon‐injured rat carotid arteries. Moreover, ADAR1 is highly induced in media layer SMCs initially, and neointima SMCs subsequently following the injury. Our study suggests that ADAR1‐mediated RNA splicing may also play an important role in VSMC phenotypic modulation and vascular remodeling in vivo.

Prostaglandin E2 Inhibition Promotes Human Aortic Smooth Muscle Cell Differentiation

INTRODUCTIONIn a mouse model of abdominal aortic aneurysms (AAAs) induced by chronic angiotensin II (AngII) infusion, the effectiveness of cyclooxygenase‐2 (COX‐2) inhibition in attenuating AAA progression is associated with maintenance of a differentiated smooth muscle cell (SMC) phenotype. However, the mechanisms responsible for COX‐2‐dependent aortic SMC phenotypic modulation are unknown. Therefore, we assessed the hypothesis that COX‐2 reduces human aortic SMC (hASMC) differentiation by the action of prostaglandin E2 (PGE2).METHODSCultured hASMCs were treated with varying concentrations of AngII, PGE2, or inhibitors of COX‐2 and PGE2 synthetic pathway. The effects of these treatments on the expression of SMC differentiation markers and components of these pathways were examined.RESULTSCOX‐2 inhibition with celecoxib significantly reduced PGE2 levels and increased expression of alpha‐actin (marker of differentiation), whereas exogenous PGE2 treatment reduced hASMC differentiation. AngII treatment resulted in increased protein expression of the inducible PGE2 synthase ‐ microsomal PGE2 synthase (mPGES‐1) and decrease in alpha‐actin. Inhibition of mPGES‐1 and PGE2 by 15‐deoxyPGJ2 resulted in increased alpha‐actin whereas mPGES‐1 knockdown reduced PGE2 synthesis, and promoted hASMC differentiation.CONCLUSIONCOX‐2 inhibition by celecoxib may increase hASMC differentiation by inhibiting production of mPGES‐1‐derived PGE2. Specific inhibition of mPGES‐1 may serve as a potential target for attenuating AAA progression by maintaining a differentiated SMC phenotype.FUNDINGNIH HL083122

Heterogeneity in the Proliferative Capacity of Smooth Muscle Cells (SMCs)

In cardiovascular disease, artery walls remodel through an increase in SMC numbers. The predominant hypothesis for this is that SMCs in the tunica media undergo phenotypic modulation into a proliferative cell type. However, direct evidence for SMC phenotypic modulation is scant. We therefore exploited time‐lapse microscopy methods to track the fate of freshly isolated, contractile SMCs. As SMCs isolated from different smooth muscle (SM) tissues are heterogeneous in nature, we have used time lapse microscopy in combination with immunocytochemistry to investigate the proliferative capacity of SMCs from portal vein (PV), carotid artery (CA) and distal colon.The adventia/endothelium and mucosa/serosa were mechanically removed before isolating cells by enzymatic digestion and trituration. Highly elongated, contractile SMCs that stained for both SM α‐actin (SMA) and SM myosin heavy chain (SM‐MHC) were obtained from all tissues. Significantly, both colon and PV tissues also contained large numbers of spherical cells that did not stain for SMA or SM‐MHC (nonSMCs). In standard culture conditions, the SMCs showed limited proliferative capacity: with the exception of one SMC that divided once (out of 15 tracked cells), colon SMCs did not proliferate. Of 11 PV SMCs tracked, 7 SMCs did divide, though none progressed beyond the 3rd generation (daughter of daughter) by confluency. Conversely, the nonSMCs proliferated rapidly (reaching the 5th generation in 5 days) and dominated the resulting cultures. In contrast, all CA cells stained for both SMA and SM‐MHC i.e. no nonSMCs were present. Whilst most CA SMCs underwent apoptosis early in culture (27 of 31 cells), those that survived went on to proliferate at varying rates (up to the 6th generation in 5 days). These results illustrate the complexities involved in creating models of SMC proliferation.

Dedicator of cytokinesis 2, a novel regulator for smooth muscle phenotypic modulation and vascular remodeling.

Vascular smooth muscle cell (SMC) phenotypic modulation and vascular remodeling contribute to the development of several vascular disorders such as restenosis after angioplasty, transplant vasculopathy, and atherosclerosis. The mechanisms underlying these processes, however, remain largely unknown. The objective of this study is to determine the role of dedicator of cytokinesis 2 (DOCK2) in SMC phenotypic modulation and vascular remodeling. Platelet-derived growth factor-BB induced DOCK2 expression while modulating SMC phenotype. DOCK2 deficiency diminishes platelet-derived growth factor-BB or serum-induced downregulation of SMC markers. Conversely, DOCK2 overexpression inhibits SMC marker expression in primary cultured SMC. Mechanistically, DOCK2 inhibits myocardin expression, blocks serum response factor nuclear location, attenuates myocardin binding to serum response factor, and thus attenuates myocardin-induced smooth muscle marker promoter activity. Moreover, DOCK2 and Kruppel-like factor 4 cooperatively inhibit myocardin-serum response factor interaction. In a rat carotid artery balloon-injury model, DOCK2 is induced in media layer SMC initially and neointima SMC subsequently after vascular injury. Knockdown of DOCK2 dramatically inhibits the neointima formation by 60%. Most importantly, knockout of DOCK2 in mice markedly blocks ligation-induced intimal hyperplasia while restoring SMC contractile protein expression. Our studies identified DOCK2 as a novel regulator for SMC phenotypic modulation and vascular lesion formation after vascular injury. Therefore, targeting DOCK2 may be a potential therapeutic strategy for the prevention of vascular remodeling in proliferative vascular diseases.

Abstract 16650: Role of Copper Transport Proteins in VSMC Phenotypic Modulation and Vascular Remodeling

Vascular smooth muscle cell (VSMC) phenotypic modulation plays a critical role in vascular repair and vascular remodeling after injury. This involves transition from quiescent contractile (C) to de-differentiated phenotype (S)(C to S) induced by PDGF, followed by re-differentiation to contractile phenotype (S to C). Copper (Cu), an essential micronutrient, is involved in vascular remodeling, and its bioavailability is controlled by Cu transport proteins (importer CTR1 and exporter ATP7A) and Cu chaperone (antioxidant-1, Atox1), which activates Cu enzymes such as amine oxidase, SSAO. However, role of Cu transport proteins in VSMC phenotypic modulation is entirely unknown. Here we show that PDGF stimulation in primary VSMC induced de-differentiation (C to S) by downregulating contractile SMC protein, which was accompanied by a robust increase in Cu uptake and CTR1, ATP7A and SSAO levels, and inhibited by Cu chelators. Knockdown of CTR1, ATP7A, SSAO, but not siAtox1, suppressed PDGF-induced VSMC de-differentiation (69%, 72% and 92%, respectively). Of note, ATP7A depletion inhibited PDGF-induced SSAO while SSAO siRNA suppressed ATP7A induction, suggesting that there is a positive feedback loop whereby ATP7A-SSAO pathway promotes de-differentiation. Mechanistically, PDGF-induced expression of KLF4, a repressor of SMC protein expression, was blocked by depletion of CTR1, ATP7A and SSAO. We reported that Atox1 also functions as a Cu-dependent transcription factor independent of ATP7A. Consistently, Atox1 siRNA suppressed serum starvation-induced VSMC re-differentiation, and overexpression of Atox1 increased SMC contractile gene expression. In vivo vascular injury model showed that SSAO and Atox1 were increased in neointimal SMCs at early de-differentiation phase and at later re-differentiation phase, respectively, in response to injury. Summary and Conclusion: CTR1-ATP7A-SSAO pathway is involved in PDGF-induced VSMC de-differentiation via regulating KLF4 while Cu dependent transcription factor function of Atox1 promotes VSMC re-differentiation via regulating SMC contractile gene transcription. Cu transport proteins and SSAO are potential therapeutic targets for modulating VSMC phenotype switching and vascular remodeling.

Upregulation of intermediate-conductance Ca2+-activated K+ channels (KCNN4) in porcine coronary smooth muscle requires NADPH oxidase 5 (NOX5).

AimsNADPH oxidase (NOX) is the primary source of reactive oxygen species (ROS) in vascular smooth muscle cells (SMC) and is proposed to play a key role in redox signaling involved in the pathogenesis of cardiovascular disease. Growth factors and cytokines stimulate coronary SMC (CSMC) phenotypic modulation, proliferation, and migration during atherosclerotic plaque development and restenosis. We previously demonstrated that increased expression and activity of intermediate-conductance Ca2+-activated K+ channels (KCNN4) is necessary for CSMC phenotypic modulation and progression of stenotic lesions. Therefore, the purpose of this study was to determine whether NOX is required for KCNN4 upregulation induced by mitogenic growth factors.Methods and ResultsDihydroethidium micro-fluorography in porcine CSMCs demonstrated that basic fibroblast growth factor (bFGF) increased superoxide production, which was blocked by the NOX inhibitor apocynin (Apo). Apo also blocked bFGF-induced increases in KCNN4 mRNA levels in both right coronary artery sections and CSMCs. Similarly, immunohistochemistry and whole cell voltage clamp showed bFGF-induced increases in CSMC KCNN4 protein expression and channel activity were abolished by Apo. Treatment with Apo also inhibited bFGF-induced increases in activator protein-1 promoter activity, as measured by luciferase activity assay. qRT-PCR demonstrated porcine coronary smooth muscle expression of NOX1, NOX2, NOX4, and NOX5 isoforms. Knockdown of NOX5 alone prevented both bFGF-induced upregulation of KCNN4 mRNA and CSMC migration.ConclusionsOur findings provide novel evidence that NOX5-derived ROS increase functional expression of KCNN4 through activator protein-1, providing another potential link between NOX, CSMC phenotypic modulation, and atherosclerosis.

Extracellular S100A4 induces smooth muscle cell phenotypic transition mediated by RAGE

We identified S100A4 as a marker of rhomboid (R) smooth muscle cells (SMCs) in vitro (the synthetic phenotype, typical of intimal SMCs) in the porcine coronary artery and of intimal SMCs in vivo in both pigs and humans. S100A4 is an intracellular Ca2+ signaling protein and can be secreted; it has extracellular functions via the receptor for advanced glycation end products (RAGE). Our objective was to explore the role of S100A4 in SMC phenotypic change, a phenomenon characteristic of atherosclerotic plaque formation. Transfection of a human S100A4-containing plasmid in spindle-shaped (S) SMCs (devoid of S100A4) led to approximately 10% of S100A4-overexpressing SMCs, S100A4 release, and a transition towards a R-phenotype of the whole SMC population. Furthermore treatment of S-SMCs with S100A4-rich conditioned medium collected from S100A4-transfected S-SMCs induced a transition towards a R-phenotype, which was associated with decreased SMC differentiation markers and increased proliferation and migration by activating the urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). It yielded NF-κB activation in a RAGE-dependent manner. Blockade of extracellular S100A4 in R-SMCs with S100A4 neutralizing antibody induced a transition from R- to S-phenotype, decreased proliferative activity and upregulation of SMC differentiation markers. By contrast, silencing of S100A4 mRNA in R-SMCs did not change the level of extracellular S100A4 or SMC morphology in spite of decreased proliferative activity. Our results show that extracellular S100A4 plays a pivotal role in SMC phenotypic changes. It could be a new target to prevent SMC accumulation during atherosclerosis and restenosis. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Dedicator of cytokinesis 2, a novel regulator for vascular smooth muscle cell phenotype modulation and vascular remodeling (670.3)

The objective of this study is to determine the role of dedicator of cytokinesis 2 (DOCK2) in vascular smooth muscle cell (VSMC) phenotypic modulation and vascular remodeling. Platelet‐derived growth factor‐BB (PDGF‐BB) induced DOCK2 expression while modulating VSMC phenotype as indicated by reduced VSMC marker expression. DOCK2 overexpression inhibited while DOCK2 knockout enhanced SMC marker gene expression in primary VSMC. Mechanistically, DOCK2 regulated VSMC phenotype through suppression myocardin/serum response factor (SRF)‐mediated transcription of VSMC marker genes. DOCK2 blocked myocardin, but not SRF expression and inhibited myocardin‐activated α‐SMA promoter activity. DOCK2 was also essential for PDGF‐BB‐induced VSMC proliferation and migration. DOCK2 stimulated VSMC migration via induction of focal adhesion contact and stress fiber formation. By using both rat carotid artery balloon‐injury and mouse ligation injury models, we observed that DOCK2 was highly induced in injury‐induced neointima. Knockdown of DOCK2 dramatically inhibited the neointima formation by 60%. Most importantly, knockout of DOCK2 in mice markedly blocked ligation‐induced intimal hyperplasia. These data demonstrate that DOCK2 plays an important role in VSMC phenotypic modulation and vascular lesion formation following vascular injury.Grant Funding Source: HL093429 and HL107526 to Shi‐You Chen, Pre‐doctoral Fellowship Award 12PRE12030180 to Xia Guo

Down‐regulation of miR‐96 by bone morphogenetic protein signaling is critical for vascular smooth muscle cell phenotype modulation (LB202)

The bone morphogenetic protein (BMP) signaling pathway is critical for the induction and maintenance of contractile phenotype in vascular smooth muscle cells (VSMCs). Inactivation of BMP signaling is common in abnormalities in vascular development and in vascular proliferative conditions, such as pulmonary artery hypertension. Herein, we identify microRNA‐96 (miR‐96) as a modulator of the VSMC phenotype in response to BMP4 signaling. We show that miR‐96 is down‐regulated by BMP4 treatment, which results in the derepression of a novel target, Tribbles‐like protein 3 (Trb3). miR‐96 targets a partially complementary sequence localized in the 3’ UTR of Trb3. Trb3 is an essential positive regulator of the BMP signaling pathway and promotes contractile phenotype in VSMCs. In conclusion, our study demonstrates a novel mechanism of regulation of SMC‐specific gene expression and induction of a VSMC contractile phenotype by the BMP4 signaling pathway via suppression of the miR‐96‐Trb3 axis.

Down‐Regulation of miR‐96 by Bone Morphogenetic Protein Signaling is Critical for Vascular Smooth Muscle Cell Phenotype Modulation

The bone morphogenetic protein (BMP) signaling pathway is critical for the induction and maintenance of contractile phenotype in vascular smooth muscle cells (VSMCs). Inactivation of BMP signaling is common in abnormalities in vascular development and in vascular proliferative conditions, such as pulmonary artery hypertension. Herein, we identify microRNA-96 (miR-96) as a modulator of the VSMC phenotype in response to BMP4 signaling. We show that miR-96 is down-regulated by BMP4 treatment, which results in the derepression of a novel target, Tribbles-like protein 3 (Trb3). miR-96 targets a partially complementary sequence localized in the 3' UTR of Trb3. Trb3 is an essential positive regulator of the BMP signaling pathway and promotes contractile phenotype in VSMCs. In conclusion, our study demonstrates a novel mechanism of regulation of SMC-specific gene expression and induction of a VSMC contractile phenotype by the BMP4 signaling pathway via suppression of the miR-96-Trb3 axis.

Abstract W P365: Critical Role of Smooth Muscle Cell Phenotypic Modulation in Cerebral Aneurysm Formation and Rupture

Objective: Little is known about smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. TNF-α has been associated with aneurysms, but a direct role has not been established. Methods: Cultured cerebral SMC were treated with TNF-α for PCR, western blot, chromatin immune-precipitation (CHIP), and adenovirus promoter transfection. In vivo experiments were carried out in the following models: application of TNF-α to the surface of carotid arteries, cerebral model of hypertension and hemodynamic stress, and cerebral model of aneurysm formation and rupture. The TNF-α inhibitor 3,6’dithiothalidomide (DTH) was synthesized. Results: Cultured cerebral SMC over-expressing myocardin induced expression of key SMC contractile genes (SM-α-actin, SM-22α, SM-MHC), while dominant negative suppressed expression. TNF-α treatment inhibited this contractile phenotype and induced pro-inflammatory genes (MCP-1, MMPs, VCAM-1, IL-1β). TNF-α increased expression of KLF4 and KLF4 siRNA abrogated TNF-α induced phentotypic modulation. These mechanisms were confirmed in vivo following exposure of rat carotid arteries to TNF-α and early in a model of cerebral hypertension and hemodynamic stress prior to cerebral aneurysm formation. Treatment with DTH reversed these pathological vessel wall alterations. TNF-α knock-out mice and DTH pre-treatment decreased the incidence of aneurysm formation and rupture. As compared with sham mice, TNF-α expression was not significantly different in TNF-α knock-out mice or those pre-treated with DTH, but was elevated in unruptured and ruptured aneurysms. Initiation of DTH 7 days after aneurysm induction did not alter aneurysm incidence, but resulted in stabilization and decreased rupture. CHIP assays in vivo and in vitro demonstrated that TNF-α promotes epigenetic changes through KLF4 dependent alterations in promoter regions of myocardin, SMC’s, and inflammatory genes. Conclusion: TNF-α induces phenotypic modulation of cerebral SMC through myocardin and KLF4 regulated pathways. These results demonstrate a novel role for TNF-α in promoting a pro-inflammatory phenotype. These data suggests a critical role of TNF-α in the formation and rupture of aneurysms.

The Effects of Elastic Fiber Protein Insufficiency and Treatment on the Modulus of Arterial Smooth Muscle Cells

Elastic fibers are critical for the mechanical function of the large arteries. Mechanical effects of elastic fiber protein deficiency have been investigated in whole arteries, but not in isolated smooth muscle cells (SMCs). The elastic moduli of SMCs from elastin (Eln-/-) and fibulin-4 (Fbln4-/-) knockout mice were measured using atomic force microscopy. Compared to control SMCs, the modulus of Eln-/- SMCs is reduced by 40%, but is unchanged in Fbln4-/- SMCs. The Eln-/- SMC modulus is rescued by soluble or α elastin treatment. Altered gene expression, specifically of calponin, suggests that SMC phenotypic modulation may be responsible for the modulus changes.

The Transcription Factor TEAD1 Represses Smooth Muscle-specific Gene Expression by Abolishing Myocardin Function*♦

The TEAD (transcriptional enhancer activator domain) proteins share an evolutionarily conserved DNA-binding TEA domain, which binds to the MCAT cis-acting regulatory element. Previous studies have shown that TEAD proteins are involved in regulating the expression of smooth muscle α-actin. However, it remains undetermined whether TEAD proteins play a broader role in regulating expression of other genes in vascular smooth muscle cells. In this study, we show that the expression of TEAD1 is significantly induced during smooth muscle cell phenotypic modulation and negatively correlates with smooth muscle-specific gene expression. We further demonstrate that TEAD1 plays a novel role in suppressing expression of smooth muscle-specific genes, including smooth muscle α-actin, by abolishing the promyogenic function of myocardin, a key mediator of smooth muscle differentiation. Mechanistically, we found that TEAD1 competes with myocardin for binding to serum response factor (SRF), resulting in disruption of myocardin and SRF interactions and thereby attenuating expression of smooth muscle-specific genes. This study provides the first evidence demonstrating that TEAD1 is a novel general repressor of smooth muscle-specific gene expression through interfering with myocardin binding to SRF.

Phenotypic Modulation of Mesenteric Vascular Smooth Muscle Cells from Type 2 Diabetic Rats is Associated with Decreased Caveolin-1 Expression

Aims: Diabetes-induced vascular complications are associated with vascular smooth muscle cell (VSMC) phenotypic modulation, switching from a contractile to a synthetic-proliferative phenotype. Loss of caveolin-1 is involved with proliferation of VSMCs. We tested the hypothesis that mesenteric VSMCs from type 2 diabetic Goto-Kakizaki (GK) rat undergo phenotypic modulation and it is linked to decreased caveolin-1 expression. Methods: VSMCs were isolated from mesenteric arteries from GK rats and age-matched control Wistar rats. Western blotting was used to determine expression of target proteins such as caveolin-1, calponin (marker of differentiation), and proliferating cell nuclear antigen (PCNA, marker of proliferation). In addition, we measured intracellular reactive oxygen species (ROS) production using H2DCF-DA and activation of extracellular signal-regulated kinase (ERK1/2) by western blotting in VSMCs from GK stimulated with lipopolysaccharide (LPS), an endotoxin upregulated in diabetes. Results: Mesenteric VSMCs from diabetic GK rats exhibited decreased caveolin-1 and calponin expression and increased PCNA expression compared to control. Increased levels of ROS and phospho-ERK1/2 expression were also found in GK VSMCs. LPS augmented ROS and phosphorylated ERK1/2 levels to a greater extent in GK VSMCs than in control. Likewise, high glucose decreased caveolin-1 and calponin expression, increased PCNA expression and augmented ROS production in control mesenteric VSMCs. Conclusion: These results suggest that mesenteric VSMCs from diabetic GK rats undergo phenotypic modulation and it is associated with decreased caveolin-1 expression. These alterations may be due to enhanced inflammatory stimuli and glucose levels present in diabetic milieu.

Vascular Smooth Muscle Cells in Cerebral Aneurysm Pathogenesis

Vascular smooth muscle cells (SMC) maintain significant plasticity. Following environmental stimulation, SMC can alter their phenotype from one primarily concerned with contraction to a pro-inflammatory and matrix remodeling phenotype. This is a critical process behind peripheral vascular disease and atherosclerosis, a key element of cerebral aneurysm pathology. Evolving evidence demonstrates that SMCs and phenotypic modulation play a significant role in cerebral aneurysm formation and rupture. Pharmacological alteration of smooth muscle cell function and phenotypic modulation could provide a promising medical therapy to inhibit cerebral aneurysm progression. This study reviews vascular SMC function and its contribution to cerebral aneurysm pathophysiology.

MicroRNA/mRNA profiling and regulatory network of intracranial aneurysm

BackgroundIntracranial aneurysm (IA) is one of the most lethal forms of cerebrovascular diseases characterized by endothelial dysfunction, vascular smooth muscle cell phenotypic modulation, inflammation and consequently loss of vessel cells and extracellular matrix degradation. Besides environmental factors, genetics seem to be a very important factor in the genesis of this disease. Previous mRNA expression studies revealed a large number of differentially expressed genes between IA and control tissue. However, microRNAs (miRNA), small non-coding RNAs which are post-transcriptional regulators of gene expression, have been barely studied. Studying miRNAs could provide a hypothetical mechanism underlying rupture of IA.MethodsA microarray study was carried out to determine difference in microRNAs and mRNA between patients’ IA tissues and controls. Quantitative RT-PCR assay compared the expression level between two groups (14 IA domes vs. 14 controls) were used for validation. Validated miRNAs were analyzed using Ingenuity Pathway Analysis (IPA) to identify the networks and pathways.Results18 miRNAs were confirmed by qPCR to be robustly down-regulated in 14 ruptured IA patients including hsa-mir-133b, hsa-mir-133a, hsa-mir-1, hsa-mir-143-3p, hsa-mir-145-3p, hsa-mir-145-5p, hsa-mir-455-5p, hsa-mir-143-5p, hsa-mir-23b-3p etc., of which 11 miRNAs are clusters: hsa-mir-1/has-mir-133a, hsa-mir-143/hsa-mir-145, hsa-mir-23b/hsa-mir-24-1, and hsa-mir-29b-2/hsa-mir-29c. 12 predicted functions were generated using IPA which showed significant associations with migration of phagocytes, proliferation of mononuclear leukocytes, cell movement of mononuclear leukocytes, cell movement of smooth muscle cells etc.ConclusionThese data support common disease mechanisms that may be under miRNA control and provide exciting directions for further investigations aimed at elucidating the miRNA mechanisms and targets that may yield new therapies for IA.

Tumor Necrosis Factor-α Modulates Cerebral Aneurysm Formation and Rupture

Inflammation is a critical process behind cerebral aneurysm formation and rupture. Tumor necrosis factor alpha (TNF-α) is a key immune modulator that has been implicated in cerebral aneurysm pathophysiology. This may occur through TNF-α-mediated endothelial injury, smooth muscle cell phenotypic modulation, recruitment of macrophages, activation of chemotactic cytokines, upregulation of matrix remodeling genes, production of free radicals leading to oxidative stress, and ultimately cellular apoptosis. Recent studies have indicated that TNF-α may be a potential target for the development of novel medical therapies, but additional experimental data is needed to clarify the intricacies of TNF-α activation and its critical downstream targets in cerebral aneurysms. This review provides an update on the mechanisms underlying TNF-α-induced molecular modulation in cerebral aneurysms.

Up-regulation of KCa3.1 promotes human airway smooth muscle cell phenotypic modulation

Airway smooth muscle (ASM) cell phenotype modulation, characterized by reversible switching between contractile and proliferative phenotypes, is considered to contribute to proliferative diseases such as allergic asthma and chronic obstructive pulmonary disease (COPD). KCa3.1 has been suggested to be involved in regulating ASM cell activation, proliferation, and migration. However, little is known regarding the exact role of KCa3.1 in ASM cell phenotypic modulation. To elucidate the role of KCa3.1 in regulating ASM cell phenotypic modulation, we investigated the effects of KCa3.1 channels on ASM contractile marker protein expression, proliferation and migration of primary human bronchial smooth muscle (BSM) cells. We found that PDGF increased KCa3.1 channel expression in BSM cells with a concomitant marked decrease in the expression of contractile phenotypic marker proteins including smooth muscle myosin heavy chain (SMMHC), smooth muscle α-actin (α-SMA), myocardin and KCa1.1. These changes were significantly attenuated by the KCa3.1 blocker, TRAM-34, or gene silencing of KCa3.1. Pharmacological blockade or gene silencing of KCa3.1 also suppressed PDGF-induced human BSM cell migration and proliferation accompanied by a decrease in intracellular free Ca2+ levels as a consequence of membrane depolarization, resulting in a reduction in cyclin D1 level and cell cycle arrest at G0-G1 phase. Additionally, PDGF-induced up-regulation of KCa3.1 and down-regulation of BSM contractile marker proteins were regulated by the ERK inhibitor U0126 and the AKT inhibitor LY294002. These findings highlight a novel role for the KCa3.1 channel in human BSM cell phenotypic modulation and provide a potential target for therapeutic intervention for proliferative airway diseases.

Cigarette smoke modulates vascular smooth muscle phenotype: implications for carotid and cerebrovascular disease.

BackgroundThe role of smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation and pathogenesis of stroke has not been determined. Cigarette smoke is a major risk factor for atherosclerosis, but potential mechanisms are unclear, and its role in SMC phenotypic modulation has not been established.Methods and ResultsIn cultured cerebral vascular SMCs, exposure to cigarette smoke extract (CSE) resulted in decreased promoter activity and mRNA expression of key SMC contractile genes (SM-α-actin, SM-22α, SM-MHC) and the transcription factor myocardin in a dose-dependent manner. CSE also induced pro-inflammatory/matrix remodeling genes (MCP-1, MMPs, TNF-α, IL-1β, NF-κB). CSE increased expression of KLF4, a known regulator of SMC differentiation, and siKLF4 inhibited CSE induced suppression of SMC contractile genes and myocardin and activation of inflammatory genes. These mechanisms were confirmed in vivo following exposure of rat carotid arteries to CSE. Chromatin immune-precipitation assays in vivo and in vitro demonstrated that CSE promotes epigenetic changes with binding of KLF4 to the promoter regions of myocardin and SMC marker genes and alterations in promoter acetylation and methylation.ConclusionCSE exposure results in phenotypic modulation of cerebral SMC through myocardin and KLF4 dependent mechanisms. These results provides a mechanism by which cigarette smoke induces a pro-inflammatory/matrix remodeling phenotype in SMC and an important pathway for cigarette smoke to contribute to atherosclerosis and stroke.