Response Of Vascular Smooth Muscle Cells Research Articles

Trichostatin A prevents neointimal hyperplasia via activation of Krüppel like factor 4

The proliferation of vascular smooth muscle cells (VSMCs) is an integral part of the mechanism of vascular diseases such as restenosis. Post-translational modifications by histone deacetylase (HDAC) inhibitors play an important role in the regulation of gene expression by inducing cell cycle arrest. However, the role and mechanism of the HDAC inhibitor trichostatin A (TSA) on neointimal proliferation remain unknown. In this study, we investigated the effect and mechanism whereby TSA prevents the proliferation of VSMCs and neointimal hyperplasia induced by balloon injury in rat carotid artery. Local administration of TSA significantly prevented neointimal hyperplasia. TSA dramatically inhibited the proliferation and DNA synthesis of VSMCs in response to FBS or PDGF-BB. Overexpression of Krüppel like factor 4 (KLF4) blocked the cell proliferation and DNA synthesis, as determined by the MTT and [3H]thymidine incorporation assays, whereas knockdown of KLF4 resulted in an increase in VSMC proliferation. In VSMCs, TSA increased the mRNA level and protein expression of KLF4. Treatment with TSA or transfection of KLF4 increased the expression of both p21 and p27 and promoter activity. In addition, the anti-proliferative activity of TSA was recovered in KLF4-knockdown cells.These data demonstrate that TSA inhibits neointimal thickening and VSMC proliferation via activation of the KLF4/p21/p27 signaling pathway.

TNF-α response of vascular endothelial and vascular smooth muscle cells involve differential utilization of ASK1 kinase and p73

Atherosclerosis involves a specialized inflammatory process regulated by an intricate network of cytokine and chemokine signaling. Atherosclerotic lesions lead to the release of cytokines that can have multiple affects on various vascular cell functions either promoting lesion expansion or alternatively retard progression. Tumor necrosis factor-α (TNF-α) is one such cytokine that can activate both cell survival and cell death mechanisms simultaneously. Here we show that TNF-α induces apoptosis in human aortic endothelial cells (HAECs), while it promotes the proliferation of vascular smooth muscle cells (VSMCs). Both events involved the activation of the Rb-E2F1 transcriptional regulatory pathway. Stimulation of HAECs with TNF-α led to an increased expression of p73 protein and a reduction in the levels of p53. This involved apoptosis signal-regulating kinase 1 (ASK1)- mediated inactivation of Rb and its dissociation from the p73 promoter. In contrast, TNF-α stimulation of VSMCs enhanced the association of E2F1 with proliferative promoters like thymidylate synthase and cdc25A, while Rb was dissociated. ASK1 kinase has a critical role in the apoptotic process, as its depletion or dissociation from Rb reduced TNF-α-induced apoptosis. These results show that the cytokine TNF-α can elicit diametrically opposite responses in vascular endothelial cells and VSMCs, utilizing the Rb-E2F pathway.

PS196. Mitochondrial Heat Shock Proteins Regulate Vascular Smooth Muscle Cell Responses to Oxidant Stress and Determine Apoptotic Threshold

PS196. Mitochondrial Heat Shock Proteins Regulate Vascular Smooth Muscle Cell Responses to Oxidant Stress and Determine Apoptotic Threshold

Calcium Regulates Key Components of Vascular Smooth Muscle Cell–Derived Matrix Vesicles to Enhance Mineralization

Matrix vesicles (MVs) are specialized structures that initiate mineral nucleation during physiological skeletogenesis. Similar vesicular structures are deposited at sites of pathological vascular calcification, and studies in vitro have shown that elevated levels of extracellular calcium (Ca) can induce mineralization of vascular smooth muscle cell (VSMC)-derived MVs. To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress. Transmission electron microscopy showed that both nonmineralized and mineralized MVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites. In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes.

Ginsenoside Rb1 Inhibits Proliferation and Inflammatory Responses in Rat Aortic Smooth Muscle Cells

Ginsenoside Rb1, a known phytoestrogen, is a major pharmacologically active component in ginseng. The present study was designed to investigate the effect of ginsenoside Rb1 on fetal bovine serum (FBS)-induced proliferation and tumor necrosis factor-α (TNF-α)-evoked inflammatory responses in cultured rat aortic vascular smooth muscle cells (VSMCs). The data showed that Rb1 potently inhibited VSMC proliferation and cell growth induced by 5% FBS. These inhibitory effects were associated with G(1) cell cycle arrest and down-regulation of cell cycle proteins. Treatment with Rb1 reduced FBS-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, TNF-α-evoked inflammatory responses were inhibited by Rb1. Reporter gene assay indicated that Rb1 could transactivate ERβ especially. Moreover, Rb1-mediated inhibition of VSMCs proliferation was greatly blocked by transfection of ERβ siRNA. These results suggest that Rb1 inhibits FBS-induced proliferation and TNF-α-evoked inflammatory responses in VSMCs. The findings presented here highlight the possible therapeutic use of Rb1 in cardiovascular disease.

A Novel Multiwell Device to Study Vascular Smooth Muscle Cell Responses Under Cyclic Strain

Vascular smooth muscle cells (VSMCs) are constantly exposed to cyclic stretch in the body, which makes it beneficial to study the effects of cyclic stretch on VSMCs. In this study, we developed a poly(dimethyl siloxane) (PDMS) compact six-well device that can be used to study the combined effect of cyclic strain and various growth factors on cultured VSMCs. Cell adhesion, alignment, and proliferation under 10% or 20% cyclic strain at 1 Hz were studied using this surface-enhanced PDMS device. The combined effects of cyclic strain with either transforming growth factor-β, vascular endothelial growth factor, fibroblast growth factor, or epidermal growth factor on VSMC proliferation was also examined. Results showed that VSMCs adhered well on the surface-enhanced multiwell device and they aligned perpendicularly to the direction of the cyclic strain. Cell proliferation was inhibited by 10% cyclic strain at 1 Hz compared with static control. The mitogenic effects of the growth factor were less potent under either 10% or 20% cyclic strain. With simple modification to accommodate more wells, this device could potentially be a useful tool for more economical, high throughput screening application.

Nicotine Induces Pro-inflammatory Response in Aortic Vascular Smooth Muscle Cells Through a NFκB/Osteopontin Amplification Loop-Dependent Pathway

Nicotine has anti- and pro-inflammatory properties in various cells. Its role in aortic vascular smooth muscle cells (VSMC) was explored. Human aortic VSMC were cultured. After nicotine (1.0 μM) and/or pyrrolidinedithiocarbamic acid (PDTC, 50 μM) treatment, the activation of nuclear factor κB (NFκB) was investigated. The levels of pro-inflammatory cytokines, osteopontin (OPN), interleukin-6 (IL-6), and monocyte chemoattractant protein 1 (MCP-1) were also assessed. After OPN was downregulated by small interfering RNA (siRNA) transfection, the pro-inflammatory effect was reassessed. We found that NFκB was activated after nicotine administration. Nicotine upregulated OPN, IL-6, and MCP-1 expressions, and this effect attenuated after PDTC pretreatment. RNAi knocked down the OPN expression in nicotine-treated cells and abolished its pro-inflammatory effects. We concluded that nicotine induces a pro-inflammatory response in VSMC through a NFκB/osteopontin amplification loop-dependent pathway.

Are contraction and adhesion activated simultaneously by Angiotensin II in vascular smooth muscle?

Administration of angiotensin (AII) at 10-7 M induces a rapid vasoconstrictor response. We propose that vascular smooth muscle cell (VSMC) contraction in response to AII is accompanied by changes in adhesion to the extracellular matrix (ECM). We hypothesized that integrin mediated ECM adhesion would be altered during AII mediated VSMC contraction. Initial whole cell adhesion studies, using a cell adhesion assay, demonstrated no appreciable difference in cell adhesion following 5–60 min AII treatment. However, studies were repeated using atomic force microscopy (AFM), which provides high temporal resolution and sensitivity for adhesion force detection. Low (p3~p4) and late passage (p7~p11) VSMCs isolated from 100–150 um diameter skeletal muscle arterioles were studied. AFM cantilever tips coated with fibronectin (FN) were allowed to interact with the cell surface for an initial 30 min control recording, and an additional 30–60 min following application of 10-7 M AII. AII induced a marked increase in VSMC elasticity, after 30 min treatment. The increase in cellular elasticity was preceded by an initial rapid (sec) change in cell shape (i.e. early contractile event). Adhesion data indicated an increase in average force and number of adhesion events, post AII. Our results suggest that integrin mediated adhesion changes occur during the mechanical response of VSMC to AII. (NIH R01HL102472 and P01HL095486)

Rosiglitazone Regulates C-reactive Protein–induced Inflammatory Responses Via Glucocorticoid Receptor–mediated Inhibition of p38 Mitogen-activated Protein Kinase–Toll-like Receptor 4 Signal Pathway in Vascular Smooth Muscle Cells

C-reactive protein (CRP) activates toll-like receptor 4 (TLR4) to initiate inflammatory response involved in the pathogenesis of atherosclerosis through mitogen-activated protein kinase (MAPK) signal pathways. Rosiglitazone, a synthetic peroxisome proliferator-activated receptor γ (PPARγ) agonist, is considered to be an important inhibitor of the inflammatory response. The present study was to explore the effect of rosiglitazone on the CRP-induced inflammatory responses and the related signal pathway in vascular smooth muscle cells (VSMCs). The results showed that rosiglitazone reduced the expressions of proinflammatory cytokines, such as vascular endothelial growth factor-A and inducible nitric oxide synthase, and enhanced the expression or activation of anti-inflammatory transcription factors including PPARγ and glucocorticoid receptor (GR) in VSMCs in response to CRP. The further investigations indicated that rosiglitazone inhibited CRP-induced TLR4 expression and p38 MAPK phosphorylation in VSMCs, and TLR4 knockdown potentiated the inhibitory effects of rosiglitazone on vascular endothelial growth factor-A and inducible nitric oxide synthase expressions. In addition, GR antagonist RU486 but not PPARγ inhibitor GW9662 remarkably weakened the inhibitory effects of rosiglitazone on CRP-induced TLR4 expression and p38 phosphorylation in VSMCs. But GW9662 did not affect rosiglitazone-induced GR phosphorylation. These suggest that rosiglitazone exerts its anti-inflammatory effect through activating GR and subsequently inhibiting p38 MAPK-TLR4 signaling pathway in CRP-stimulated VSMCs.

STAT1-mediated signal integration between IFNγ and LPS leads to increased EC and SMC activation and monocyte adhesion

Inflammation plays an important role in host defenses against infectious agents and injury, but it also contributes to the pathophysiology of atherosclerosis. Signal transducer and activated transcription 1 (STAT1) has been identified as a point of convergence for the cross talk between the pro-inflammatory cytokine interferon γ (IFNγ) and the Toll-like receptor-4 (TLR4) ligand LPS in immune cells. However, there is no information available on the role of STAT1 in TLR4-mediated progression of atherosclerosis and on potential synergism between lipopolysaccharides (LPS) and IFNγ signaling in cells from the vasculature. Cultured human microvascular endothelial cells (HMECs) exposed to LPS activated STAT1 in a delayed manner that was inhibited by cycloheximide treatment. Pretreatment of HMECs as well as primary vascular smooth muscle cells (VSMCs) with IFNγ followed by LPS resulted in a significant increase in STAT1 phosphorylation compared with both factors alone. Increased STAT1 protein levels, strictly mediated by IFNγ, correlated with the augmented STAT1 phosphorylation that was absent in TLR4(-/-) cells. As assessed by PCR, Western analysis, and ELISA, this coincided with increased expression of the chemokine interferon gamma-induced protein 10 kDa (IP-10) and the adhesion molecule ICAM-1 in a TLR4-dependent manner.The STAT1-inhibitor fludarabine markedly reduced these effects as well as IFNγ and LPS-dependent adhesion of U937 cells to endothelial cells, emphasizing the potential importance of STAT1 in the integration of both signals. With the established roles of IFNγ and TLRs in atherosclerotic pathology, the STAT1-dependent signal integration between IFNγ and TLR in ECs and VSMCs in response to exogenous and endogenous atherogenic ligands could result in amplification of pro-inflammatory responses in the damaged vessel and be a novel mechanism involved in the initiation and progression of atherosclerosis.

Tissue Factor Pathway Inhibitor

To investigate in women older than 60 whether aortic stiffness or pulse pressure (PP) is associated with selected procoagulant or anticoagulant factors and to examine whether pulsatile stretch influences these factors in human vascular smooth muscle cells (VSMCs) in vitro. Aortic pulse wave velocity (PWV) and carotid PP were studied in 123 apparently healthy postmenopausal women. PWV, PP, von Willebrand factor, and free tissue factor pathway inhibitor (TFPI), but not mean arterial pressure, increased with age. Free TFPI and PWV were positively correlated, even after adjustment for age and PP and other confounding parameters. In vitro, 5% or 10% pulsatile stretch (at 1 Hz) enhanced TFPI synthesis and secretion by VSMCs in a time-independent manner (1 to 48 hours) without changes in protein level of smooth muscle myosin heavy chain. Application of 5% static stretch had no effect. In postmenopausal women, free TFPI increases as vascular wall function deteriorates and PP increases. These findings are supported by the increase in TFPI synthesized by VSMCs in response to cyclic stress in vitro. They suggest that VSMCs require pulsatility to interfere with the coagulation process and highlight the relevance of plasma free TFPI levels to cardiovascular diseases.

Effect of Tensile Stress on Smooth Muscle Cells

Cellular responses to mechanical stresses play an important role in the physiology of many cell types in healthy and diseased states. In vascular smooth muscle cells (VSMC), as in any other cells anchored to extracellular matrix, external mechanical stresses are imposed on a preexisting force equilibrium generated by the cytoskeletal tension. The ability to measure real-time mechanosensitive events at sub-cellular level in response to discrete and physiologically relevant mechanical stimulation is the critical component in understanding mechanically-induced cellular remodeling. Mechanical perturbation of a VSMC by the atomic force microscope (AFM) tip mimics the tensile stress in the vessel wall. By integrating tensile stress stimulation with simultaneous optical imaging using total internal reflection fluorescence and fast spinning-disk confocal microscopy, we have the unique opportunity of investigating in real-time the mechanically-induced cross-talk between apical and basal cell surface. Thus, we were able to measure vinculin and actin recruitment at focal adhesions upon tensile stress stimulation, and stress fibers bundling and reassembly. Understanding the real-time contractile and adhesion events associated with live VSMC response to force, provides fundamental new information regarding the coordinated cellular responses involved in VSMC adaptation to the local extracellular environment in the vessel wall.

Cyclic strain upregulates VEGF and attenuates proliferation of vascular smooth muscle cells

ObjectiveVascular smooth muscle cell (VSMC) hypertrophy and proliferation occur in response to strain-induced local and systemic inflammatory cytokines and growth factors which may contribute to hypertension, atherosclerosis, and restenosis. We hypothesize VSMC strain, modeling normotensive arterial pressure waveforms in vitro, results in attenuated proliferative and increased hypertrophic responses 48 hrs post-strain.MethodsUsing Flexcell Bioflex Systems we determined the morphological, hyperplastic and hypertrophic responses of non-strained and biomechanically strained cultured rat A7R5 VSMC. We measured secretion of nitric oxide, key cytokine/growth factors and intracellular mediators involved in VSMC proliferation via fluorescence spectroscopy and protein microarrays. We also investigated the potential roles of VEGF on VSMC strain-induced proliferation.ResultsProtein microarrays revealed significant increases in VEGF secretion in response to 18 hours mechanical strain, a result that ELISA data corroborated. Apoptosis-inducing nitric oxide (NO) levels also increased 43% 48 hrs post-strain. Non-strained cells incubated with exogenous VEGF did not reproduce the antimitogenic effect. However, anti-VEGF reversed the antimitogenic effect of mechanical strain. Antibody microarrays of strained VSMC lysates revealed MEK1, MEK2, phospo-MEK1T385, T291, T298, phospho-Erk1/2T202+Y204/T185+T187, and PKC isoforms expression were universally increased, suggesting a proliferative/inflammatory signaling state. Conversely, VSMC strain decreased expression levels of Cdk1, Cdk2, Cdk4, and Cdk6 by 25-50% suggesting a partially inhibited proliferative signaling cascade.ConclusionsSubjecting VSMC to cyclic biomechanical strain in vitro promotes cell hypertrophy while attenuating cellular proliferation. We also report an upregulation of MEK and ERK activation suggestive of a proliferative phenotype. Hhowever, the proliferative response appears to be aborogated by enhanced antimitogenic cytokine VEGF, NO secretion and downregulation of Cdk expression. Although exogenous VEGF alone is not sufficient to promote the quiescent VSMC phenotype, we provide evidence suggesting that strain is a necessary component to induce VSMC response to the antimitogenic effects of VEGF. Taken together these data indicate that VEGF plays a critical role in mechanical strain-induced VSMC proliferation and vessel wall remodeling. Whether VEGF and/or NO inhibit signaling distal to Erk 1/2 is currently under investigation.

Cyclic stretch stimulates vascular smooth muscle cell alignment by redox-dependent activation of Notch3

Mice deficient in Notch3 have defects in arterial vascular smooth muscle cell (VSMC) mechanosensitivity, including impaired myogenic responses and autoregulation, and inappropriate VMSC orientation. Experiments were performed to determine if Notch3 is activated by mechanical stimulation and contributes to mechanosensitive responses of VSMCs, including cell realignment. Cyclic, uniaxial stretch (10%, 1 Hz) of human VSMCs caused Notch3 activation, demonstrated by a stretch-induced increase in hairy and enhancer of split 1/hairy-related transcription factor-1 expression, translocation of Notch3 to the nucleus, and a decrease in the Notch3 extracellular domain. These effects were prevented by inhibiting the expression [small interfering (si)RNA] or proteolytic activation of Notch3 {N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alanine-2-aminoethyl amide (TAPI-1; 50 μmol/l) to inhibit TNF-α-converting enzyme (TACE) or N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT; 20 μmol/l) to inhibit γ-secretase}. Stretch increased the activity of ROS within VSMCs, determined using dichlorodihydrofluorescein fluorescence. Catalase (1,200 U/ml), which degrades H₂O₂, inhibited the stretch-induced activation of Notch3, whereas in nonstretched cells, increasing H₂O₂ activity [H₂O₂ or manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin] caused activation of Notch3. Stretch increased the activity of TACE, which was prevented by catalase. Stretch-induced activation of p38 MAPK in VSMCs was inhibited either by catalase or by inhibiting Notch3 expression (siRNA). Stretch caused VSMCs to realign perpendicular to the direction of the mechanical stimulus, which was significantly inhibited by catalase or by inhibiting the expression (siRNA) or activation of Notch3 (TAPI-1 or DAPT). Therefore, cyclic uniaxial stretch activates Notch3 signaling through a ROS-mediated mechanism, and the presence of Notch3 is necessary for proper stretch-induced cell alignment in VSMCs. This mechanism may contribute to the physiological role of Notch3 in mediating developmental maturation of VSMCs.

Suppression of vascular smooth muscle cell responses induced by TNF-α in GM3 synthase gene transfected cells

The natural accumulation of ganglioside GM3 (N-glycolylneuraminic acid) on atherosclerotic lesions is a common theory. The present study is the first to examine the effects of the GM3 synthase gene on the responses of vascular smooth muscle cells (VSMC) to tumor necrosis factor-α (TNF-α). We found that overexpression of the GM3 synthase gene inhibited DNA synthesis and ERK1/2 activity induced by TNF-α in VSMC, whereas the basal levels of DNA synthesis and ERK1/2 activity remained unchanged. In addition, GM3 synthase gene transfectants significantly reduced the migration and invasion of VSMC following TNF-α treatment, compared with empty vector transfectants. Furthermore, TNF-α-induced matrix metalloproteinase-9 (MMP-9) expression and promoter activity were also decreased in GM3 synthase gene transfectants. GM3 synthase gene expression markedly suppressed the TNF-α-stimulated transcriptional activity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB), which are the controlling factors of MMP-9 expression. Consistent with these results, the addition of anti-GM3 antibody into the GM3 synthase gene transfectants blocked inhibition of DNA synthesis, ERK1/2 activity, migration and invasion. Finally, GM3 synthase gene transfectants treated with anti-GM3 antibody reversed the suppression of MMP-9 expression by reducing AP-1 and NF-κB binding activity. These results suggest regulatory roles for the GM3 synthase gene in VSMC proliferation and migration during the formation of atherosclerotic lesions.

Thrombospondin-1 inhibition of vascular smooth muscle cell responses occurs via modulation of both cAMP and cGMP

Nitric oxide (NO) drives pro-survival responses in vascular cells and limits platelet adhesion, enhancing blood flow and minimizing thrombosis. The matricellular protein thrombospondin-1 (TSP1), through interaction with its receptor CD47, inhibits soluble guanylyl cyclase (sGC) activation by NO in vascular cells. In vascular smooth muscle cells (VSMCs) both intracellular cGMP and cAMP regulate adhesion, contractility, proliferation, and migration. cGMP can regulate cAMP through feedback control of hydrolysis. Inhibition of the cAMP phosphodiesterase-4 selectively interfered with the ability of exogenous TSP1 to block NO-driven VSMC adhesion but not cGMP accumulation, suggesting that cAMP also contributes to VSMC regulation by TSP1. Inhibition of phosphodiesterase-4 was sufficient to elevate cAMP levels, and inhibiting guanylyl cyclase or phosphodiesterase-3, or adding exogenous TSP1 reversed this increase in cAMP. Thus, TSP1 regulates VSMC cAMP levels in part via cGMP-dependent inhibition of phosphodiesterase-3. Additionally basal cAMP levels were consistently elevated in both VSMCs and skeletal muscle from TSP1 null mice, and treating null cells with exogenous TSP1 suppressed cAMP levels to those of wild type cells. TSP1 inhibited both forskolin and isoproterenol stimulated increases in cAMP in VSMCs. TSP1 also abrogated forskolin and isoproterenol stimulated vasodilation. Consistent with its ability to directly limit adenylyl cyclase-activated vasodilation, TSP1 also limited cAMP-induced dephosphorylation of myosin light chain-2. These findings demonstrate that TSP1 limits both cGMP and cAMP signaling pathways and functional responses in VSMCs and arteries, by both phosphodiesterase-dependent cross talk between these second messengers and by inhibition of adenylyl cyclase activation.

ID family protein expression and regulation in hypoxic pulmonary hypertension

Bone morphogenetic protein (BMP) signaling has been linked to the development of pulmonary hypertension (PH). Inhibitors of differentiation (ID) proteins (ID1-4) are a family of basic helix-loop-helix transcription factors that are downstream targets of the BMP signaling pathway, but the role that ID proteins play in the development of PH is unknown. To address this, we evaluated pulmonary expression of ID proteins in a mouse model of hypoxia-induced PH. There is selective induction of ID1 and ID3 expression in hypoxic pulmonary vascular smooth muscle cells (VSMCs) in vivo, and ID1 and ID3 expression are increased by hypoxia in cultured pulmonary VSMCs in a BMP-dependent fashion. ID4 protein is barely detectable in the mouse lung, and while ID2 is induced in hypoxic peripheral VSMCs in vivo, it is not increased by hypoxia or BMP signaling in cultured pulmonary VSMCs. In addition, the PH response to chronic hypoxia is indistinguishable between wild type and Id1 null mice. This is associated with a compensatory increase in ID3 but not ID2 expression in pulmonary VSMCs of Id1 null mice. These findings indicate that ID1 is dispensable for mounting a normal pulmonary vascular response to hypoxia, but suggest that ID3 may compensate for loss of ID1 expression in pulmonary VSMCs. Taken together, these findings indicate that ID1 and ID3 expression are regulated in a BMP-dependent fashion in hypoxic pulmonary VSMCs, and that ID1 and ID3 may play a cooperative role in regulating BMP-dependent VSMC responses to chronic hypoxia.

The use of micropatterning to control smooth muscle myosin heavy chain expression and limit the response to transforming growth factor β1 in vascular smooth muscle cells

In the healthy artery, contractile vascular smooth muscle cells (VSMCs) have an elongated shape and are highly aligned but transition to a synthetic phenotype in culture, while additionally becoming well spread and randomly organized. Thus, controlling VSMC phenotype is a challenge in tissue engineering. In this study, we investigated the effects of micropatterning on contractile protein expression in VSMCs at low and high passage and in the presence of transforming growth factor beta 1 (TGFβ1). Micropatterning led to significantly decreased cell area, increased elongation, and increased alignment compared to non-patterned VSMCs independent of passage number. In the presence of serum, micropatterning led to increased smooth muscle myosin heavy chain (SM-MHC) and α-actin expression in low passage VSMCs, but had no effect on high passage VSMCs. Micropatterning was as effective as TGFβ1 in up-regulating SM-MHC at low passage; however, micropatterning limited VSMC response to TGFβ1 at both low and high passage. Investigation of TGFβ receptor 1 revealed higher expression in non-patterned VSMCs compared to patterned at high passage. Our studies demonstrate that micropatterning is an important regulator of SM-MHC expression in contractile VSMCs and that it may provide a mechanism for phenotype stabilization in the presence of growth factors.

Can microRNAs control vascular smooth muscle phenotypic modulation and the response to injury?

Vascular smooth muscle cell (VSMC) migration and proliferation are critical events in vascular proliferative diseases. Recent studies have established microRNAs (miRNAs) as important mediators for the modulation of VSMC phenotype by targeting transcription factors and the cytoskeleton, which act as molecular switches for VSMC differentiation. The importance of miRNAs for VSMC development, differentiation, and function is evident by the fact that loss of the miRNA processing enzyme Dicer in VSMCs results in embryonic lethality due to severe vascular abnormalities. Similar abnormalities are observed in adult miR-143/145 knockout mice, indicating that these miRNAs are important for VSMC differentiation and function. However, since miR-143/145 knockout is not embryonically lethal, additional miRNA must be required during embryonic development of VSMCs. In addition, specific miRNAs such as miR-145, miR-21, and miR-221 have been found to regulate neointimal hyperplasia following vascular injury, which provides interesting possibilities for future therapeutical targets against vascular disease. Herein, we summarize recent advances regarding the role of miRNAs in VSMC phenotype modulation and response to injury.

Protein tyrosine phosphatase PTPεM negatively regulates PDGF β-receptor signaling induced by high glucose and PDGF in vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation and migration and vascular endothelial cell (VEC) dysfunction are closely associated with the development of atherosclerosis. We previously demonstrated that protein tyrosine phosphatase ε M (PTPεM) promotes VEC survival and migration. The present study investigates the biological functions of PTPεM in VSMCs and determines whether PTPεM is implicated in diabetes-accelerated atherosclerosis. We overexpressed wild-type and inactive PTPεM and an small interfering RNA (siRNA) of PTPεM by using an adenovirus vector to investigate the effects of PTPεM upon platelet-derived growth factor (PDGF)- and high glucose (HG)-induced responses of rat VSMCs in vitro. We found that PTPεM decreased PDGF-induced DNA synthesis and migration by reducing the phosphorylation level of the PDGF β-receptor (PDGFRβ) with subsequently suppressed H(2)O(2) generation. The HG content in the medium generated H(2)O(2), upregulated PDGFRβ expression and its tyrosine-phosphorylation, and elevated NADPH oxidase 1 (Nox1) expression even without exogenous PDGF, all of which were downregulated by PTPεM. The PDGFR inhibitor AG1296 also blocked HG-induced Nox1 expression and H(2)O(2) production. Moreover, HG suppressed PTPεM expression itself, which was blocked by the antioxidant N-acetyl-l-cysteine. The effects of PTPεM siRNA were the opposite of those of wild-type PTPεM. Therefore, PTPεM negatively regulates PDGFRβ-mediated signaling pathways that are crucial for the pathogenesis of atherosclerosis, and PTPεM may be involved in diabetes-accelerated atherosclerosis.