Smooth Muscle Cell Phenotypic Modulation Research Articles

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.

TNF-α Induces Phenotypic Modulation in Cerebral Vascular Smooth Muscle Cells: Implications for Cerebral Aneurysm Pathology

Little is known about vascular smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. Tumor necrosis factor-alpha (TNF-α) has been associated with aneurysms, but potential mechanisms are unclear. Cultured rat cerebral SMCs overexpressing myocardin induced expression of key SMC contractile genes (SM-α-actin, SM-22α, smooth muscle myosin heavy chain), while dominant-negative cells suppressed expression. Tumor necrosis factor-alpha treatment inhibited this contractile phenotype and induced pro-inflammatory/matrix-remodeling genes (monocyte chemoattractant protein-1, matrix metalloproteinase-3, matrix metalloproteinase-9, vascular cell adhesion molecule-1, interleukin-1 beta). Tumor necrosis factor-alpha increased expression of KLF4, a known regulator of SMC differentiation. Kruppel-like transcription factor 4 (KLF4) small interfering RNA abrogated TNF-α activation of inflammatory genes and suppression of contractile genes. These mechanisms were confirmed in vivo after exposure of rat carotid arteries to TNF-α and early on in a model of cerebral aneurysm formation. Treatment with the synthesized TNF-α inhibitor 3,6-dithiothalidomide reversed pathologic vessel wall alterations after induced hypertension and hemodynamic stress. Chromatin immunoprecipitation assays in vivo and in vitro demonstrated that TNF-α promotes epigenetic changes through KLF4-dependent alterations in promoter regions of myocardin, SMCs, and inflammatory genes. In conclusion, TNF-α induces phenotypic modulation of cerebral SMCs through myocardin and KLF4-regulated pathways. These results demonstrate a novel role for TNF-α in promoting a pro-inflammatory/matrix-remodeling phenotype, which has important implications for the mechanisms behind intracranial aneurysm formation.

Thin-Capped Atheromata With Reduced Collagen Content in Pigs Develop in Coronary Arterial Regions Exposed to Persistently Low Endothelial Shear Stress

The mechanisms promoting the focal formation of rupture-prone coronary plaques in vivo remain incompletely understood. This study tested the hypothesis that coronary regions exposed to low endothelial shear stress (ESS) favor subsequent development of collagen-poor, thin-capped plaques. Coronary angiography and 3-vessel intravascular ultrasound were serially performed at 5 consecutive time points in vivo in 5 diabetic, hypercholesterolemic pigs. ESS was calculated along the course of each artery with computational fluid dynamics at all 5 time points. At follow-up, 184 arterial segments with previously identified in vivo ESS underwent histopathologic analysis. Compared with other plaque types, eccentric thin-capped atheromata developed more in segments that experienced lower ESS during their evolution. Compared with lesions with higher preceding ESS, segments persistently exposed to low ESS (<1.2 Pa) exhibited reduced intimal smooth muscle cell content; marked intimal smooth muscle cell phenotypic modulation; attenuated procollagen-I gene expression; increased gene and protein expression of the interstitial collagenases matrix-metalloproteinase-1, -8, -13, and -14; increased collagenolytic activity; reduced collagen content; and marked thinning of the fibrous cap. Eccentric thin-capped atheromata, lesions particularly prone to rupture, form more frequently in coronary regions exposed to low ESS throughout their evolution. By promoting an imbalance of attenuated synthesis and augmented collagen breakdown, low ESS favors the focal evolution of early lesions toward plaques with reduced collagen content and thin fibrous caps-2 critical determinants of coronary plaque vulnerability.

CTP synthase, a smooth muscle‐sensitive therapeutic target for effective vascular repair after injury

Neointima formation due to smooth muscle cell (SMC) proliferation following injury is a major medical challenge in cardiovascular intervention. However, anti–neointimal drugs often block re‐endothelialization as a side effect. Identification of SMC sensitive therapeutic targets will potentially conquer this medical barrier. In the present study, we identified CTP synthase (CTPS) as one of these potential targets. Blockade of CTPS function by shRNA or its specific inhibitor suppressed SMC proliferation and neointima formation. In contrast, targeting CTPS treatments had no side effects on endothelial cell (EC) in vitro, and promoted reendothelialization in vivo. Mechanistically, blockade of CTPS pathway activated CTP synthesis salvage pathway in ECs but not in SMCs, which sustained EC proliferation. Meanwhile, blockade of CTPS function induced SMC phenotypic modulation into the contractile phenotype, which characterized by the expression of pro‐angiogenic factors while inhibiting anti‐angiogenic factors in SMCs, leading to enhanced EC proliferation. In summary, for the first time, our study discovered a fundamental difference between SMC and EC in their CTP biosynthesis during pathological proliferation which provided a novel strategy to selectively block SMC proliferation while not disturbing or even promoting reendothelialization for effective vascular repair after injury.

CTP synthase, a smooth muscle‐sensitive therapeutic target for effective vascular repair after injury

Neointima formation due to smooth muscle cell (SMC) proliferation following injury is a major medical challenge in cardiovascular intervention. However, anti–neointimal drugs often block re‐endothelialization as a side effect. Identification of SMC‐sensitive therapeutic targets will potentially conquer this medical barrier. In the present study, we identified CTP synthase (CTPS) as one of these potential targets. Blockade of CTPS function by shRNA or its specific inhibitor suppressed SMC proliferation and neointima formation. In contrast, targeting CTPS treatments had no side effects on endothelial cell (EC) in vitro, and promoted re‐endothelialization in vivo. Mechanistically, blockade of CTPS pathway activated CTP synthesis salvage pathway in ECs but not in SMCs, which sustained EC proliferation. Meanwhile, blockade of CTPS function induced SMC phenotypic modulation into the contractile phenotype, which characterized by the expression of pro‐angiogenic factors while inhibiting anti‐angiogenic factors in SMCs, leading to enhanced EC proliferation. In summary, for the first time, our study discovered a fundamental difference between SMC and EC in their CTP biosynthesis during pathological proliferation which provided a novel strategy to selectively block SMC proliferation while not disturbing or even promoting re‐endothelialization for effective vascular repair after 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.

Activation of PPAR-α induces cell cycle arrest and inhibits transforming growth factor-β1 induction of smooth muscle cell phenotype in 10T1/2 mesenchymal cells

Transforming growth factor-β1 (TGF-β1) regulates the cell cycle and the differentiation of mesenchymal cells into smooth muscle cells (SMCs). However, the precise intracellular signaling pathways involved in these processes have not been fully clarified. It has also been shown that there is an increase in TGF-β1 expression in human atherosclerotic plaques. Furthermore, peroxisome proliferator-activated receptors (PPARs) and their agonists have recently gained more attention in the study of the pathogenesis of atherosclerosis. In this study, we examined the role of PPARs in the TGF-β1-mediated cell cycle control and SMC phenotypic modulation of C3H10T1/2 (10T1/2) mesenchymal cells. The results showed the following: (1) the PI3K/Akt/p70S6K signaling cascade is involved in TGF-β1-induced differentiation of 10T1/2 cells into cells with a SMC phenotype. (2) PPAR-α agonists (i.e., WY14,643 and clofibrate), but not a PPAR-δ/β agonist (GW501516) or PPAR-γ agonist (troglitazone), inhibit TGF-β1-induced SMC markers and the DNA binding activity of serum response factor (SRF) in 10T1/2 cells. (3) WY14,643 and clofibrate inhibit the TGF-β1 activation of the Smad3/Akt/P70S6K signaling cascade. (4) TGF-β1-induced cell cycle arrest at the G0/G1 phases is mediated by Smad3 in 10T1/2 cells. (5) The PPAR-α-mediated 10T1/2 cell cycle arrest at the G0/G1 phases is TGF-β receptor independent. These results suggest that PPAR-α mediates cell cycle control and TGF-β1-induced SMC phenotypic changes in 10T1/2 cells.

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.

Lipid Phosphate Phosphatase 3 Negatively Regulates Smooth Muscle Cell Phenotypic Modulation to Limit Intimal Hyperplasia

The lipid phosphate phosphatase 3 (LPP3) degrades bioactive lysophospholipids, including lysophosphatidic acid and sphingosine-1-phosphate, and thereby terminates their signaling effects. Although emerging evidence links lysophosphatidic acid to atherosclerosis and vascular injury responses, little is known about the role of vascular LPP3. The goal of this study was to determine the role of LPP3 in the development of vascular neointima formation and smooth muscle cells (SMC) responses. We report that LPP3 is expressed in vascular SMC after experimental arterial injury. Using gain- and loss-of-function approaches, we establish that a major function of LPP3 in isolated SMC cells is to attenuate proliferation (extracellular signal-regulated kinases) activity, Rho activation, and migration in response to serum and lysophosphatidic acid. These effects are at least partially a consequence of LPP3-catalyzed lysophosphatidic acid hydrolysis. Mice with selective inactivation of LPP3 in SMC display an exaggerated neointimal response to injury. Our observations suggest that LPP3 serves as an intrinsic negative regulator of SMC phenotypic modulation and inflammation after vascular injury, in part, by regulating lysophospholipid signaling. These findings may provide a mechanistic link to explain the association between a PPAP2B polymorphism and coronary artery disease risk.

Cyclic stretch-induced thrombin generation by rat vascular smooth muscle cells is mediated by the integrin αvβ3 pathway

Vascular smooth muscle cell (VSMC) phenotypic modulation plays a pivotal role in atherothrombotic diseases. Thrombin generation at the surface of VSMCs and activation of integrin mechanotransduction pathways represent potential mechanisms. Here, we examine whether mechanical stretch increases thrombin generation on cultured rat aortic VSMCs. The integrin α(v)β(3) antagonist peptide (cRGDPV) dose-dependently decreased thrombin generation without stretch. Static stretch (5%, 1 Hz) failed to modify the thrombin-forming capacity of VSMCs, whereas 10% cyclic stretch during 60 and 360 min enhanced integrin α(v)β(3) expression and thrombin generation at the surface of VSMCs by 30% without inducing apoptosis. Cyclic stretch also stimulated Src phosphorylation, cleavage of talin, and binding of prothrombin to VSMCs. Upregulation of α(v)β(3) expression, Src phosphorylation, and enhanced thrombin generation by cyclic stretch were abolished by cRGDPV and silencing RNA (siRNA) against α(v) as well as by selective inhibition of integrin α(v)β(3) inside-out signalling by a talin-siRNA. Complete abolition of stretch-induced VSMC-supported thrombin generation by the RGT peptide, which disrupts the interaction of Src with the β(3) cytoplasmic tail, demonstrates the link between outside-in pathways involving β(3)-Src interaction and thrombin activity dependent on inside-out signalling. These data show that the contribution of cyclic stretch to VSMC-supported thrombin generation is driven by the integrin α(v)β(3) signalling pathway and suggest a role for pulsatility-induced intramural thrombin in VSMC-dependent vascular remodelling.

Biology of Intracranial Aneurysms: Role of Inflammation

Intracranial aneurysms (IAs) linger as a potentially devastating clinical problem. Despite intense investigation, our understanding of the mechanisms leading to aneurysm development, progression and rupture remain incompletely defined. An accumulating body of evidence implicates inflammation as a critical contributor to aneurysm pathogenesis. Intracranial aneurysm formation and progression appear to result from endothelial dysfunction, a mounting inflammatory response, and vascular smooth muscle cell phenotypic modulation producing a pro-inflammatory phenotype. A later final common pathway appears to involve apoptosis of cellular constituents of the vessel wall. These changes result in degradation of the integrity of the vascular wall leading to aneurysmal dilation, progression and eventual rupture in certain aneurysms. Various aspects of the inflammatory response have been investigated as contributors to IA pathogenesis including leukocytes, complement, immunoglobulins, cytokines, and other humoral mediators. Furthermore, gene expression profiling of IA compared with control arteries has prominently featured differential expression of genes involved with immune response/inflammation. Preliminary data suggest that therapies targeting the inflammatory response may have efficacy in the future treatment of IA. Further investigation, however, is necessary to elucidate the precise role of inflammation in IA pathogenesis, which can be exploited to improve the prognosis of patients harboring IA.

Simulated Bladder Pressure Stimulates Human Bladder Smooth Muscle Cell Proliferation via the PI3K/SGK1 Signaling Pathway

A mechanical stimulus on detrusor tissue is critical to bladder outlet obstruction progression and functional bladder tissue engineering. A hypothesis is that mechanical stimulus triggers human bladder smooth muscle cell proliferation. To help better understand this relationship of bladder function to growth at the cellular level we used a novel method of applying cyclic hydrodynamic pressure that simulates the bladder cycle to cell cultures. We detected the proliferation response of human bladder smooth muscle cells (4310, ScienCell™) to different pressures as well as the signal transduction mechanism of this process. Human bladder smooth muscle cells cultured in scaffolds underwent 4 pressures (0, 100, 200 and 300 cm H(2)O) for 24 hours, as controlled by a BioDynamic® bioreactor. We then used flow cytometry to examine cell cycle distribution, and polymerase chain reaction and immunoblot to quantify SGK1 and AKT expression and activation in each group. SGK1 was silenced in human bladder smooth muscle cells using small interfering RNA to validate the role of SGK1 in mediating pressure induced cell proliferation. Compared with the 0 cm H(2)O control group, human bladder smooth muscle cells in the 200 and 300 cm H(2)O groups showed increased cell proliferation. SGK1 expression and activity were also increased while AKT, another downstream signal of PI3K, did not change significantly. SGK1 silencing abolished the increases in cell proliferation induced by pressure. To our knowledge we provide the first report of cyclic hydrodynamic pressure stimulating the proliferation of human bladder smooth muscle cells cultured in scaffolds. The signal transduction mechanism for this process is involved with the PI3K/SGK1 and not the PI3K/AKT signaling pathway.

Abstract 519: Calmodulin Is a Marker of a Distinct Smooth Muscle Cell Population Recruited by Plaque-Derived Macrophages from the Human Carotid Artery Media

Background: Different experimental observations suggest the expansion of a distinct medial smooth muscle cell (SMC) subset during atheromatous plaque establishment and restenosis. Objectives: We investigated the presence and features of such SMC subset in cell cultures derived from human carotid endarterectomy (CEA) specimens; moreover, we aimed at finding markers that could play a role in SMC behavior. Methods and Results: CEA specimens comprised a grossly undiseased (UP) and a diseased portion (DP). The UP contained a thin intimal thickening with the underlying media and the DP an atherosclerotic plaque with the underlying media. Cell cultures were initiated by tissue explantation. From plaque tissues only macrophage-derived foam cells were retrieved. From medial tissues 2 distinct SMC phenotypes were identified: 1) large SMCs, flat with a monolayered growth pattern, isolated from the UP media; 2) small SMCs, fusiform and growing in multilayers, isolated from both the UP and DP media only when cocultured with plaque-derived macrophages. Small SMCs displayed higher proliferative and migratory activities and a poor level of differentiation compared with large SMCs. Two-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry showed that calmodulin (CaM), a calcium-binding protein involved in cell-cycle regulation, was mainly expressed in small SMCs. This result was confirmed using a specific CaM antibody. Coculture of large SMCs with plaque-derived macrophages induced a switch to the small phenotype and was associated with increased CaM expression. Consistently, the specific CaM inhibitor W-7 hampered the large-to-small switch and predominantly inhibited the proliferation of small SMCs. In vivo, CaM was markedly expressed in atherosclerotic plaques whereas it was barely detectable in the media. Conclusions: Plaque-derived macrophages promote the selective migration of a distinct SMC subpopulation exhibiting features of an “atheroma-prone” phenotype. CaM is a marker of small SMCs in vitro and of plaque SMCs in vivo, and plays a role in SMC phenotypic modulation. Further studies on these SMC populations can be instrumental in understanding and influencing the evolution of atherosclerosis.

Abstract 176: Extracellular S100A4 Is a Key Modulator of Arterial Smooth Muscle Cell Phenotypic Transition

Background: It has been proposed that smooth muscle cells (SMCs) from the arterial wall are heterogeneous and that only a subset of medial SMCs are prone to accumulate into the intima leading to atheromatous plaque formation. 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 a marker of R-SMCs in vitro and of intimal SMCs both in pig and human. S100A4 is an intracellular Ca2+ binding protein that can also be secreted; it has extracellular functions probably via the receptor for advanced glycation end products (RAGE). Objective: Investigate the role of S100A4 in SMC phenotypic change, a process characteristic of atherosclerotic plaque formation. Methods and results: Transfection of a human S100A4-containing plasmid in S-SMCs (practically devoid of S100A4) led to overexpression of S100A4 in approximately 10% of SMCs, release of S100A4, and a transition towards a R-phenotype of the whole SMC population. Moreover, 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 increased proliferative and migratory activities as well as downregulation of SMC differentiation markers (i.e. alpha-smooth muscle actin and smooth muscle myosin heavy chains). It yielded activation of NF-kappa B without changing RAGE mRNA baseline level. Blockade of extracellular S100A4 in R-SMCs with S100A4 neutralizing antibody induced a transition from R- to S-phenotype, decreased proliferative activity and upregulated SMC differentiation markers. In contrast, silencing of S100A4 mRNA in R-SMCs and during platelet-derived growth factor-BB-induced S-to-R-phenotypic change did not affect SMC morphology in spite of decreased proliferative activity. Conclusions: Our results show that extracellular S100A4 modulates SMC phenotypic changes and is essential for the establishment of the R-phenotype. It could be a new target to prevent SMC accumulation during atherosclerosis and restenosis.

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.

3,3′Diindolylmethane Suppresses Vascular Smooth Muscle Cell Phenotypic Modulation and Inhibits Neointima Formation after Carotid Injury

Background3, 3′diindolylmethane (DIM), a natural phytochemical, has shown inhibitory effects on the growth and migration of a variety of cancer cells; however, whether DIM has similar effects on vascular smooth muscle cells (VSMCs) remains unknown. The purpose of this study was to assess the effects of DIM on the proliferation and migration of cultured VSMCs and neointima formation in a carotid injury model, as well as the related cell signaling mechanisms.Methodology/Principal FindingsDIM dose-dependently inhibited the platelet-derived growth factor (PDGF)-BB-induced proliferation of VSMCs without cell cytotoxicity. This inhibition was caused by a G0/G1 phase cell cycle arrest demonstrated by fluorescence-activated cell-sorting analysis. We also showed that DIM-induced growth inhibition was associated with the inhibition of the expression of cyclin D1 and cyclin-dependent kinase (CDK) 4/6 as well as an increase in p27Kip1 levels in PDGF-stimulated VSMCs. Moreover, DIM was also found to modulate migration of VSMCs and smooth muscle-specific contractile marker expression. Mechanistically, DIM negatively modulated PDGF-BB-induced phosphorylation of PDGF-recptorβ (PDGF-Rβ) and the activities of downstream signaling molecules including Akt/glycogen synthase kinase(GSK)3β, extracellular signal-regulated kinase1/2 (ERK1/2), and signal transducers and activators of transcription 3 (STAT3). Our in vivo studies using a mouse carotid arterial injury model revealed that treatment with 150 mg/kg DIM resulted in significant reduction of the neointima/media ratio and proliferating cell nuclear antigen (PCNA)-positive cells, without affecting apoptosis of vascular cells and reendothelialization. Infiltration of inflammatory cells was also inhibited by DIM administration.ConclusionThese results demonstrate that DIM can suppress the phenotypic modulation of VSMCs and neointima hyperplasia after vascular injury. These beneficial effects on VSMCs were at least partly mediated by the inhibition of PDGF-Rβ and the activities of downstream signaling pathways. The results suggest that DIM has the potential to be a candidate for the prevention of restenosis.

Atherosclerosis development is differentially regulated in whole genome vs. bone marrow‐derived haploinsufficient NFAT5/ApoE null mice

We have previously shown that the transcription factor, nuclear factor of activated T‐cells 5 (NFAT5), plays a role in vascular smooth muscle cell phenotypic modulation in acute injury, but the role of NFAT5 in atherosclerosis is unknown. Our main objectives were to determine if genome‐wide NFAT5 haploinsufficency (NFAT5+/−) altered atherosclerotic development and to determine whether NFAT5 expression in bone marrow (BM)‐derived cells affected lesion progression. NFAT5+/−ApoE−/− mice were placed on a high fat diet for 20 wks and analysis of the thoracic aorta and aortic arch established that genome‐wide NFAT5 haploinsufficiency reduced atherosclerotic lesion formation by 73% and 43%, respectively, but did not alter lesion size in the aortic root. BM transplant studies revealed that transplantation of NFAT5+/−ApoE−/− BM into NFAT5+/+ApoE−/− mice resulted in a similar 86% reduction in lesion formation of the thoracic aorta, yet produced an unexpected 37% increase in total lesion area of the aortic root. Immunohistochemical analysis of root lesions revealed that mice transplanted with NFAT5‐deficient BM had increased macrophage investment of the lesion. We have identified that NFAT5 expression in BM‐derived cells is critical for the regulation of lesion morphometry and composition, and we show that NFAT5 deficiency in macrophages leads to their increased investment of aortic root lesions.

Impact of Endothelial Cells on 3D Cultured Smooth Muscle Cells in a Biomimetic Hydrogel

For the development of vascular tissue engineering, the impact of endothelial cells (ECs) on smooth muscle cell (SMC) spreading, proliferation, and differentiation is explored in the current study using a coculture model. In this coculture model, SMCs were encapsulated in a biomimetic hydrogel based on methacrylated dextran-graft-lysine (Dex-MA-LA) and methacrylamide-modified gelatin (Gel-MA), and exposed to a monolayer of ECs. With EC coculture, SMC proliferation in 3D hydrogel was promoted at initial period, and the formation of denser cellular networks was enhanced. ECs dynamically modulated SMC phenotype by promoting a more contractile SMC phenotype initially (on day 2), indicated by the upregulated expression of contractile genes α-actin, calponin, smooth muscle-myosin heavy chain (SM-MHC), and smoothelin; however, the onset of maximum expressions was delayed by ECs. Full differentiation of SMCs was not obtained even with EC coculture. Higher level of platelet-derived growth factor (PDGF)-BB and latent transforming growth factor (TGF)-β1 were detected in medium of coculture. These biochemical cues together with the physical cue of tensional force within cellular networks may be responsible for the dynamic modulation of SMC phenotype in coculture. Synthesis of elastin was promoted by ECs at transcriptional level. The formation of denser cellular networks and synthesis of elastin suggest that coculture with ECs is a potential method to construct functional vessel media layer in vitro.

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms.

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.