Response Of Vascular Smooth Muscle Cells Research Articles

PPARγ agonist, rosiglitazone, regulates angiotensin II-induced vascular inflammation through the TLR4-dependent signaling pathway

Atherosclerosis is increasingly recognized as a chronic inflammatory disease. Angiotensin II (Ang II) is a critical factor in inflammatory responses, so as to promote the pathogenesis of atherosclerosis. Toll-like receptor 4 (TLR4) activates signaling pathways leading to the expression of pro-inflammatory cytokines implicated in the etiology of atherosclerosis. Peroxisome proliferator-activated receptor γ (PPARγ) agonists are considered to be important in modulating vascular inflammation and atherosclerosis. Herein, we investigated the modulatory effects of rosiglitazone on Ang II-mediated inflammatory responses both in vivo and in vitro. We also examined whether TLR4-dependent signaling pathway was involved in the inhibitory effects of rosiglitazone on Ang II-induced pro-inflammatory responses in vascular smooth muscle cells (VSMCs). Male Sprague–Dawley rats received Ang II by subcutaneous infusion and/or rosiglitazone per os for 7 days. Systolic blood pressure rise in Ang II-infused rats was attenuated by rosiglitazone. Rosiglitazone also reduced Ang II-induced generation of pro-inflammatory mediators (TLR4, matrix metalloproteinase-9 and tumor necrosis factor-α), but enhanced production of anti-inflammatory mediators (PPARγ and 6-keto-PGF1α) both in vivo and in vitro. Furthermore, treatment of VSMCs with both the TLR4 inhibitor and TLR4 small-interfering RNA (siRNA) showed that the modulatory effects of rosiglitazone on Ang II-mediated inflammatory responses in VSMCs were related to TLR4. Treatment of the cells with rosiglitazone had little effect on Ang II receptors expression (AT1 and AT2), but downregulated AT1-dependent ERK1/2 activation. Then, treatment of VSMCs with TLR4 siRNA, interferon-gamma-inducible protein 10 (IP-10) siRNA and with the special protein kinase C (PKC) inhibitor further revealed that the signaling pathway (TLR4/IP-10/PKC/NF-κB) was involved in the inhibitory effects of rosiglitazone on Ang II-induced pro-inflammatory responses in VSMCs. In conclusion, TLR4 may be a drug target involved in the ameliorative effects of PPARγ agonist, rosiglitazone, on Ang II-mediated inflammatory responses in VSMCs. Moreover, rosiglitazone exerts its anti-inflammatory effect by interfering with the TLR4-dependent signaling pathway (ERK1/2/TLR4/IP-10/PKC/NF-κB) to prevent and treat atherosclerotic diseases.

The treatment of collagen fibrils by tissue transglutaminase to promote vascular smooth muscle cell contractile signaling

The enzyme tissue transglutaminase 2 (TG2) appears to play an important role in several physiological processes such as wound healing, the progression of cancer and of vascular disease. Additionally, TG2 has been proposed as a means of stabilizing collagen extracellular matrix (ECM) scaffolds for tissue engineering applications. In this report, we examined the effect of TG2 treatment on the mechanical properties of the ECM, and associated cell responses. Using a model ECM of fibrillar collagen, we quantitatively examined vascular smooth muscle cell (vSMC) response to untreated, or TG2 treated collagen. We show that cells respond to TG2 treated collagen with increased spreading, an increase in contractile response as indicated by elevated F-actin polymerization and myosin light chain phosphorylation, and increased proliferation, without apparent changes in integrin specificity or matrix topography. Comparative atomic force microscopy loading studies indicate that TG2 treated fibrils are 3 times more resistant to shearing force from an AFM tip than untreated fibrils. The data suggest that TG2 treatment of collagen increases matrix mechanical stiffness, which apparently alters the contractile and proliferative response of vSMC.

The signal transduction pathways of heat shock protein 27 phosphorylation in vascular smooth muscle cells

The objective of this study is to investigate the signal transduction pathways that regulate heat shock protein 27 (HSP27) phosphorylation and migration of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) induced by angiotensin II (AngII) and platelet derived growth factor-BB (PDGF-BB). The activity of HSP27 was evaluated by Western blot with specific phospho-HSP27 antibody. F-actin polymerization was detected by FITC-Phalloidine staining using confocal microscopy. Modified Boyden chamber technique was employed for VSMCs migration assessment. Within a given concentration, the phosphorylation of HSP27 induced by AngII and PDGF-BB was blocked by the specific P38MAPK inhibitor SB202190, the specific PI3K inhibitor LY294002 and the specific ERK1/2 inhibitor U0126 in a concentration-dependent manner, with a peak inhibition rate at 87.2%, 78.4% and 37.3%, respectively, induced by AngII (P < 0.01), with a peak inhibition rate at 85.0%, 55.3% and 41.0%, respectively, induced by PDGF-BB (P < 0.01).The migration of VSMCs induced by AngII and PDGF-BB was inhibited by 100 micromol/l SB202190, 30 micromol/l LY294002, and 30 micromol/l U0126, with a inhibition rate at 60.1%, 71.7% and 47.3%, respectively, provoked by AngII (P < 0.01), with a inhibition rate at 55.3%, 55.6% and 38.1%, respectively, provoked by PDGF-BB (P < 0.01). P38MAPK and PI3 K/Akt are important pathways that contribute to the phosphorylation of HSP27 and migration of VSMCs in response to AngII and PDGF-BB. ERK1/2 might be involved in HSP27 phosphorylation and migration of VSMCs provoked by AngII and PDGF-BB.

Differential Effects of Selective Cyclooxygenase-2 Inhibitors on Vascular Smooth Muscle Ion Channels May Account for Differences in Cardiovascular Risk Profiles

Celecoxib, rofecoxib, and diclofenac are clinically used cyclooxygenase-2 (COX-2) inhibitors, which have been under intense scrutiny because long-term rofecoxib (Vioxx; Merck, Whitehouse Station, NJ) treatment was found to increase the risk of adverse cardiovascular events. A differential risk profile for these drugs has emerged, but the underlying mechanisms have not been fully elucidated. We investigated the effects of celecoxib, rofecoxib, and diclofenac on ionic currents and calcium signaling in vascular smooth muscle cells (VSMCs) using patch-clamp techniques and fura-2 fluorescence and on arterial constriction using pressure myography. Celecoxib, but not rofecoxib or diclofenac, dramatically enhanced KCNQ (K(v)7) potassium currents and suppressed L-type voltage-sensitive calcium currents in A7r5 rat aortic smooth muscle cells (native KCNQ currents or overexpressed human KCNQ5 currents) and freshly isolated rat mesenteric artery myocytes. The effects of celecoxib were concentration-dependent within the therapeutic concentration range, and were reversed on washout. Celecoxib, but not rofecoxib, also inhibited calcium responses to vasopressin in A7r5 cells and dilated intact or endothelium-denuded rat mesenteric arteries. A celecoxib analog, 2,5-dimethyl-celecoxib, which does not inhibit COX-2, mimicked celecoxib in its enhancement of vascular KCNQ5 currents, suppression of L-type calcium currents, and vasodilation. We conclude that celecoxib inhibits calcium responses in VSMCs by enhancing KCNQ5 currents and suppressing L-type calcium currents, which ultimately reduces vascular tone. These effects are independent of its COX-2 inhibitory actions and may explain the differential risk of cardiovascular events in patients taking different drugs of this class.

PPARα activator fenofibrate modulates angiotensin II-induced inflammatory responses in vascular smooth muscle cells via the TLR4-dependent signaling pathway

Angiotensin II (Ang II) is a crucial contributor to inflammatory processes involved in development and progression of atherosclerotic lesion. Toll-like receptor 4 (TLR4) signaling responsible for the initiation of inflammation also participates in pathogenesis of atherosclerosis. The protective effect of peroxisome proliferator-activated receptor α (PPARα) activators on atherosclerosis may be due to their impact on vascular inflammation, plaque instability and thrombosis. However, mechanisms underlying the inhibitory effects of PPARα activators on Ang II-induced vascular inflammation and the TLR4-dependent signaling pathway involved in vascular smooth muscle cells (VSMCs) remain unclear. The present study demonstrated that PPARα activator fenofibrate decreased Ang II-induced generation of pro-inflammatory mediators such as TLR4, MMP-9 and TNF-α, but enhanced production of anti-inflammatory molecules like PPARα and 6-keto-PGF 1α both in vivo and in vitro. Meanwhile, treatment of VSMCs with the TLR4 inhibitor or TLR4 siRNA showed that the inhibitory effects of fenofibrate on Ang II-induced inflammatory responses in VSMCs were dependent on TLR4. Furthermore, fenofibrate depressed Ang II-induced inflammatory responses in VSMCs by intervening the downstream effector molecules of the TLR4-dependent signaling pathway, including interferon-gamma inducible protein 10 (IP-10), protein kinases C (PKC) and nuclear factor κB (NF-κB). Thus, these findings provide the evidence for beneficial effects of PPARα activator fenofibrate to counter-regulate vascular inflammation induced by Ang II. More importantly, anti-inflammatory action of fenofibrate via interfering with the TLR4-dependent signaling pathway (TLR4/IP-10/PKC/NF-κB) works in concert to protect against atherosclerosis.

Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells

Tyrosine phosphatase non-receptor type substrate-1 (SHPS-1), a transmembrane protein, plays a vital role in cell migration and proliferation. Our previous studies have shown that insulin-like growth factor-I (IGF-I) stimulates SHPS-1 phosphorylation, leading to recruitment of SHP-2, c-Src, Shc, and Grb2.p85 to phosphorylated SHPS-1. Assembly of this signaling complex is required for optimal stimulation of both mitogen-activated protein and phosphatidylinositol 3-kinase pathways. The main aim of the present study was to identify novel proteins that interacted with the cytoplasmic domain of SHPS-1 (SHPS-1/CD) in response to IGF-I stimulation and define the role of these interactions in mediating specific biological functions. We performed a functional proteomic screening to identify SHPS-1 binding partners using combination of mRNA display and the tandem affinity purification-tag methods. Screening identified a number of proteins not previously known to interact with phosphorylated SHPS-1/CD. These novel SHPS-1 binding partners represent several functional categories including heat shock proteins, protein kinases and phosphatases, and proteins that regulate transcription or translation. In Vivo and in vitro studies suggested that most of the proteins bound to SHPS-1 via binding to one of the four SH2 domain containing proteins, SHP-2, CTK, SUPT6H, and STAT1, that directly bound to SHPS-1. Although the binding of most of these proteins to SHPS-1 was positively regulated by IGF-I, a few were negatively regulated, suggesting differential regulation of protein complexes assembled on SHPS-1/CD in response to IGF-I. Further studies showed that truncation of SHPS-1/CD significantly impaired IGF-I-dependent AKT signal transduction and subsequent biological functions including cell survival, protein synthesis, protein aggregation, and prevention of apoptosis. The results emphasize the importance of formation of SHPS-1 signaling complex induced by IGF-I and provide novel insights into our knowledge of the role of this molecular scaffold in regulation of IGF-I-stimulated signal transduction and biological actions.

Abscisic Acid Released by Human Monocytes Activates Monocytes and Vascular Smooth Muscle Cell Responses Involved in Atherogenesis

Abscisic acid (ABA) is a phytohormone recently identified as a new endogenous pro-inflammatory hormone in human granulocytes. Here we report the functional activation of human monocytes and vascular smooth muscle cells by ABA. Incubation of monocytes with ABA evokes an intracellular Ca2+ rise through the second messenger cyclic ADP-ribose, leading to NF-kappaB activation and consequent increase of cyclooxygenase-2 expression and prostaglandin E2 production and enhanced release of MCP-1 (monocyte chemoattractant protein-1) and of metalloprotease-9, all events reportedly involved in atherogenesis. Moreover, monocytes release ABA when exposed to thrombin-activated platelets, a condition occurring at the injured vascular endothelium; monocyte-derived ABA behaves as an autocrine and paracrine pro-inflammatory hormone-stimulating monocyte migration and MCP-1 release, as well as vascular smooth muscle cells migration and proliferation. These results, and the presence of ABA in human arterial plaques at a 10-fold higher concentration compared with normal arterial tissue, identify ABA as a new signal molecule involved in the development of atherosclerosis and suggest a possible new target for anti-atherosclerotic therapy.

Evidence for Telomerase Activation in VSMCs Exposed to Hyperglycemic and Hyperhomocysteinemic Conditions

While diabetes and homocysteinemia have been demonstrated to be independent risk factors for progression of vascular disease, the activation of telomerase has not been linked to the potent effects of high levels of homocysteine (Hcy) and glucose on vascular smooth muscle cells (VSMC). We examined the proliferative response of VSMCs, resulting from hyperglycemia and hyperhomocysteinemia and their effects on telomerase activity. Primary cultures of VSMC from human aorta and arteries were used in this experiment from 3rd -5th passages. Hcy or/and glucose were added to the cell culture media in doses equivalent to plasma levels of Hcy in patients with moderate, high homocysteinemia, or physiologically high concentrations of glucose as seen in diabetics. The modified telomeric repeat amplification protocol was used for telomerase activity assay. Cytotoxicity, viability, proliferation, protein phosphorylation, were determined in cultures treated and not treated with Hcy and glucose. The mitogenic effect of Hcy and glucose on VSMC, independently and together, was observed at 48 hours after treatment. The viable cell numbers were significantly increased at doses comparable to plasma levels of Hcy in hyperhomocysteinemia, as compared to untreated cultures (p < 0.01). Cells exposed to high levels of glucose also exhibited an increased proliferation response (p = 0.01). Telomerase activity was detected in all sets of VSMC cultures exposed to high levels of glucose or/and Hcy (p < 0.01). However, a significant difference was not observed in telomerase activity, when high Hcy or high glucose was used alone or in concert. Significant dual phosphorylation of p38 MAPK was observed in treated cultures but it did not correlate with the telomerase activity detected. These data establish a link with telomerase activation and the mitogenic effect of hyperhomocysteinemia and hyperglycemia.

Effects of the cardiopulmonary bypass on cerebral vasomotor changes

Objective To study the cerebral vasomotor changes after different cardiopulmonary bypass time and modes. Methods Thirty SD rats were equally divided into 5 groups,which were subjec-ted to CPB for 60 min and 120 min,deep hypothermie low flow bypass 60 min, deep hypothermie circulato-ry arrest 60 min, and sham respectively. The right middle cerebral artery was harvested and prepared for assessment of the myogenic tone with different transmural pressure, the endothelial cell response induced by different concentration of acetylcholine, and the vascular smooth muscle cell response induced by the so-dium nitroprusside and serotonin. The hippocampus tissue was harvested to observe the ultramicrostructure of endothelial cells in capillary by electron microscope. Results The CPB was performed successfully in all the rats. The deep hypothermic bypass altered the results of myogenic tone test with different transmural pressure compared with the sham group [(34.62±2.36) % and (33.83±2.28) % vs (37.75± 1.92) %, P<0.05]. Acetylcholine caused a dose-dependent vasodilation in the sham group that was ab-sent in animals undergoing CPB [(24.26±1.90) % vs (8.59±1.48) %, P<0.05]. Significantly, this was apparent after only 60 min of CPB and the vasodilation response of endothelial cells was worse in DHLF and DHCA groups than in simple CPB. The vascular smooth muscle cell response induced by the so-dium nitroprusside and serotonin was intact in all the rats. Conclusion The specific loss of acetylcholine-induced vasodilation suggests endothelial cell dysfunction,which appeares early after the onset of CPB. The deep hypothermic bypass can cause the dysfunction of cerebrovascular autonomic regulation. And these cer-ebral vasomotor changes may contribute to the neurologic injuries after CPB. Key words: Cardiopulmonary bypass; Cerebrovascular; Brain injury

TNF-α regulates vascular smooth muscle cell responses in genetic hypertension

Cellular and molecular events in vascular smooth muscle cells (VSMC) from Wistar–Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were investigated. SHR-derived VSMC showed increased proliferative capacity and MAP kinase levels in comparison with WKY-derived VSMC. Flow cytometry analysis revealed that progression from G1 to S phase was faster in SHR-derived VSMC in response to tumor necrosis factor-α (TNF-α) as compared with cells from WKY. The G1 cell cycle-associated proteins such as cyclin D1, cyclin E, CDK2 and CDK4, and kinase activities associated with CDK2 and CDK4, were increased in SHR-derived VSMC. In addition, CDK inhibitor p21 was elevated in SHR-derived cells. Matrix metalloproteinase-9 (MMP-9) expression and migration were also increased in response to TNF-α in SHR-derived cells. This increase was characterized by the up-regulation of MMP-9, which was transcriptionally regulated at the AP-1 and NF-κB sites in the MMP-9 promoter. These results suggest that the hypertensive-associated increase in VSMC proliferative capacity, G1 to S-phase cell-cycle progress and MMP-9 expression may play a role in vascular remodeling in hypertension.

Prolyl hydroxylase 2 deficiency limits proliferation of vascular smooth muscle cells by hypoxia-inducible factor-1α-dependent mechanisms

Arterial O(2) levels are thought to modulate vascular smooth muscle cell (VSMC) proliferation and vascular remodeling, but the mechanisms involved are poorly understood. Here, we tested the hypothesis that PHD2, a prolyl hydroxylase domain (PHD)-containing O(2) sensor, modulates growth factor-induced proliferative responses of human pulmonary artery SMC (HPASMC). We found that both PHD1 and PHD2 were robustly expressed by HPASMC, and inhibiting prolyl hydroxylase activity pharmacologically by using the nonselective dioxygenase inhibitor dimethyloxalylglycine (DMOG) inhibited proliferation and cyclin A expression induced by PDGF-AB or FGF-2. Specific knockdown of PHD2 using small interfering RNAs had similar effects. The inhibitory effects of DMOG and PHD2 knockdown on proliferation and cyclin A expression were seen under both normoxic (20% O(2)) and moderately hypoxic (5% O(2)) conditions, and PHD2 expression was not affected by O(2) level nor by stimulation with PDGF or FGF-2, indicating that the proproliferative influence of PHD2 does not involve alterations of its expression. Knockdown of PHD2 increased hypoxia-inducible factor (HIF)-1alpha expression, as expected, but we also found that HIF-1alpha knockdown abolished the inhibitory effect of PHD2 knockdown on PDGF-induced cyclin A expression. Therefore, we conclude that PHD2 promotes growth factor-induced responses of human VSMC, acting by HIF-1alpha-dependent mechanisms. Given the role of PHD2 as an oxygen sensor in mammalian cells, these results raise the possibility that PHD2 links VSMC proliferation to O(2) availability.

The effects of RhoA on vascular reactivity following hemorrhagic shock in rats

Objective To observe the effects of RhoA on vascular reactivity following hemorrhagic shock （HS） in rats. Methods The superior mesenteric artery （SMA） from HS rats was adopted to assay the vascular reactivity via observing the contraction initiated with norepinephrine （NE） by isolated organ perfusion system. With transwell culture, the contractile response of Vascular smooth muscle cell （VSMC） to NE 10 or 90 rain after hypoxia was detected. Meanwhile, the effect of the U-46619 and C3enzyme, RhoA activity regulating agents, on vascular reactivity was observed. Results As compared with the control group, the cumulative dose-response curves of SMA to NE at early shock （ immediate after shock） shifted to the left, and the maximal contractions （Emax） of NE was （1. 684 ± 0. 101 ） g/mg tissue and increased significantly. The contractile response of VSMC to NE was increased at 10 min after hypoxia,RhoA agonist U-46619 （10 s mol/L） further increased the contractile response of SMA and VSMC to NE, and C3enzyme decreased reactivity at early shock or 10 min after hypoxia. The cumulative dose-re- sponse curves of SMA to NE at 2 h after shock shifted to the fight, Emax of NE was （0.608 ± 0.045 ） g/ mg tissue and decreased significantly, and the contractile response of VSMC to NE was decreased at 90 min after hypoxia. RhoA agonist U-46619 could increase the vascular reactivity in the late period of shock or at 90 min after hypoxia,and C3enzyme abolished U-46619-induced the increase of the contractile response of SMA and VSMC to NE. Conclusion Vascular reactivity is biphasic change following HS. RhoA may take part in the regulation of biphasic vascular reactivity regulation after HS. Key words: Shock,hemorrhagic ; RhoA ; Vascular

Nonesterified fatty acid exposure activates protective and mitogenic pathways in vascular smooth muscle cells by alternate signaling pathways

Vascular smooth muscle cells (VSMC) are dynamic cells exposed to fluctuating concentrations of nutrients on a daily basis. Nonesterified fatty acids (NEFA) have been indicted as potential mediators of atherosclerosis and exaggerated VSMC remodeling observed in diabetes, and in vitro data support a model of VSMC activation by NEFA. However, recent observations suggest that metabolic stressors such as oxidants and NEFA may also simultaneously induce cytoprotective events as part of a homeostatic “off switch.” Our group has established that the transcription factor cyclic adenosine monophosphate response element binding protein (CREB) is important for maintenance of VSMC quiescence, differentiation, and survival. We therefore examined whether acute physiologic NEFA exposure would regulate CREB in primary cultures of bovine aortic VSMC and explored the relationship between signaling to the cytoprotective CREB and the activating mitogen-activated protein kinase pathways. In vitro exposure of VSMC to 3 classes of unsaturated NEFA leads to significant acute, transient, dose-dependent, and repeatedly inducible CREB activation. As expected, extracellular signal–regulated kinase, P38 mitogen-activated protein kinase, Akt, Jun N-terminal kinase, and protein kinase C (PKC) pathways are also activated by NEFA. Using a battery of pharmacologic inhibitors and antioxidants, we demonstrate that CREB activation is mediated by a novel PKC isoform and is reactive oxygen species independent, whereas extracellular signal–regulated kinase activation, in contrast, is mediated by reactive oxygen species and is PKC independent. These data suggest parallel and mechanistically distinct stimulation of separate stabilizing and activating pathways in VSMC response to acute NEFA-mediated stress. Furthermore, the down-regulation of CREB in models of chronic metabolic stress reported in the literature would be expected to disrupt this homeostasis and shift the balance toward VSMC activation, consistent with emerging models of atherosclerosis.

Biomechanical stress induces novel arterial intima-enriched genes: implications for vascular adaptation to stress

The arterial vasculature is subjected to considerably greater biomechanical stress than the venous circulation. This is reflected in the difference in morphology between large arteries and veins, however little is known about the molecular differences that arise as a consequence of biomechanical stress. Previously, we identified a group of arterial intima-enriched (AIE) genes: sciellin, periplakin, SPRR3, envoplakin, galectin 7, and plakoglobin that are functionally related in that they contribute to the stress properties of stratified epithelium. We sought to test our hypothesis that these genes were regulated by biomechanical stress in vascular smooth muscle cells (VSMCs). Immunofluorescence was employed to determine the expression of the AIE genes in saphenous vein coronary artery bypass grafts. Furthermore, we used a model of cyclic stress to determine if the AIE genes were regulated by biomechanical stress in VSMCs in vitro. Sciellin and periplakin were upregulated in saphenous vein coronary artery bypass grafts after arterialization, but were absent in non-arterialized saphenous veins. Sciellin, SPRR3, and periplakin transcripts were all upregulated (4.67-, 4.95-, 2.77-fold, respectively) by prolonged exposure to cyclic strain (24-72 h), but not at earlier time points. These findings suggest a novel role for several human AIE genes in the VSMC response to arterialization and extended cyclic strain. Biomechanical stress has long been implicated in vascular pathologies. We report the novel finding of a group of genes, previously studied in stratified epithelium, that were regulated by prolonged cyclic stress in vascular smooth muscle cells. This may have important implications to vascular disease.

Mechanical Activity At Focal Adhesion Sites

We investigated whether mechanical force applied to extracellular matrix proteins (ECM)-integrin focal adhesion sites would induce mechanical activity characteristic of specific ECM type. We used atomic force microscopy (AFM) to apply forces to ECM adhesion sites on vascular smooth muscle cells (VSMC) isolated from resistance arterioles. The tip of the AFM probes were fused with a borosilicate bead (5 um diameter) coated with fibronectin (FN), collagen type-I (CNI), collagen type-IV (CNIV), laminin (LN) or vitronectin (VN). ECM-coated beads induced clustering of a5 and b3 integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward (z-axis) pulling force (800∼1600 pN) applied to the bead-cell contact site for FN specific focal adhesions induced a force-generating response from the cells resulting in a downward pull by the cell. Depolymerization of the actin cytoskeleton with cytochalasin D blocked whilst stabilization of the actin cytoskeleton with jasplakinolide enhanced this micromechanical event. Myosin light chain kinase inhibition (ML7) and an inhibitor of cSrc tyrosine kinase (PP2) also blocked the response. Furthermore, inhibitory antibodies to a5 and b3 integrins blocked the micromechanical cell event in a concentration-dependent manner. Similar experiments with CNI, CNIV, VN, or LN failed to induce micromechanical events. Our results demonstrate that mechanical force applied through FN at single focal adhesion sites induces a micromechanical event that is actin, myosin light chain kinase and tyrosine kinase dependent. Importantly, the data illustrate that there are different mechanical characteristics for focal adhesions formed by different ECM proteins. FN appears of particular relevance in its ability to induce a force-generating reaction from sites of focal adhesion in VSMC in response to applied forces.

Orai1 Channel And Ca2+-independent Phospholipase A2 Are The New Determinants Of Proliferation And Migration Of Vascular Smooth Muscle Cells

Ca2+ entry is known to play an important role in proliferation and migration of vascular smooth muscle cells (SMC), but the molecular mechanisms that mediate these processes are far from being understood. Here we introduce Ca2+-independent phospholipase A2β (iPLA2β) and Orai1-encoded plasma membrane channel (that mediate store-operated Ca2+ entry (SOCE) in non-excitable as well as excitable cells) as new molecular determinants of proliferation and migration of aortic SMC. Patch-clamp, Ca2+ imaging, immunocytochemistry and molecular approaches were used in this study. We demonstrated that molecular knock down of either iPLA2β or Orai1 (but not TRPC1) channel resulted in full inhibition of store-operated current and Ca2+ entry in primary aortic SMC. Transfection of SMC with siRNA to Orai1 impaired their proliferation: Orai1 knock down resulted in 69±4% decrease in the number of BrDU positive cells and 60±1% reduction in the rate of their proliferation. Similar effects were observed in iPLA2β-deficient cells (65±3% and 71±4% reductions, respectively). Interestingly, transfection of SMC with antisense to TRPC1 did not affect SOCE, but significantly reduced the rate of cell proliferation (by 48±4%). Orai1 and TRPC1 produced additive effects on proliferation, and knock down of both channels resulted in 76±8% reduction in the rate of SMC proliferation, consistent with different mechanisms for Orai1and TRPC1-mediated Ca2+ entry. Molecular knock down of iPLA2β, or Orai1 channel also impaired SMC ability to migrate in response to 20% FBS, high glucose, or SERCA inhibition. These results suggest that SOCE mediated by iPLA2β-dependent activation of Orai1 channel in SMC is required for their proliferation and migration, and may be involved in vascular SMC responses and angiogenesis triggered by different physiological and pathological conditions.

Role of V 1a Receptor in AVP-Induced Restoration of Vascular Hyporeactivity and Its Relationship to MLCP-MLC 20 Phosphorylation Pathway

Our previous study showed that small dosage of arginine vasopressin (AVP) had beneficial effect on shock by improving the shock-induced vascular hyporeactivity. But the mechanism is not clear. The objective of the present study is to investigate the role of V(1a) and V(2) receptors in AVP-mediated restoration of hemorrhage-induced vascular hyporesponsiveness and its relationship to protein kinase C (PKC), myosin light chain phosphatase (MLCP), and myosin light chain (MLC(20)) phosphorylation signal transduction pathway. We used isolated superior mesenteric artery (SMA) from hemorrhagic shock rats (40 mmHg for 2 hours) and 90 minutes hypoxia-treated vascular smooth muscle cell (VSMC) to study the effects of V(1a) and V(2) receptor inhibitors on AVP-mediated regulation of vascular reactivity and calcium sensitivity. Meanwhile the effects of AVP and V(1a) and V(2) receptor antagonists on the activity of MLCP and myosin light chain kinase (MLCK), and the phosphorylation of MLC(20), and the expression of PKC-alpha, delta, and epsilon isoforms were also studied. AVP significantly improved the reactivity of SMA to norepinephrine (NE) and Ca(2+) following hemorrhagic shock and increased the hypoxia-induced decrease of contractile response of VSMC to NE. The PKC-alpha and epsilon expression in particulate fractions of VSMC following hemorrhagic shock and hypoxia was also increased by treatment with AVP. V(1a) receptor inhibitor significantly antagonized these effects of AVP. AVP treatment resulted in a significant increase of MLC(20) phosphorylation in SMA and a significant decrease of MLCP activity in VSMC, which was also inhibited by V(1a) receptor inhibitor. V(1a) receptor, not V(2) receptor, played an important role in AVP-mediated regulation of vascular reactivity and calcium sensitivity following hemorrhagic shock. The mechanism is mainly through PKC-MLCP-MLC(20) phosphorylation calcium sensitivity pathway.

TRPC1 Associates With BK Ca Channel to Form a Signal Complex in Vascular Smooth Muscle Cells

TRPC1 (transient receptor potential canonical 1) is a Ca(2+)-permeable cation channel involved in diverse physiological function. TRPC1 may associate with other proteins to form a signaling complex, which is crucial for channel function. In the present study, we investigated the interaction between TRPC1 and large conductance Ca(2+)-sensitive K(+) channel (BK(Ca)). With the use of potentiometric fluorescence dye DiBAC(4)(3), we found that store-operated Ca(2+) influx resulted in membrane hyperpolarization of vascular smooth muscle cells (VSMCs). The hyperpolarization was inhibited by an anti-TRPC1 blocking antibody T1E3 and 2 BK(Ca) channel blockers, charybdotoxin and iberiotoxin. These data were confirmed by sharp microelectrode measurement of membrane potential in VSMCs of intact arteries. Furthermore, T1E3 treatment markedly enhanced the membrane depolarization and contraction of VSMCs in response to several contractile agonists including phenylephrine, endothelin-1, and U-46619. In coimmunoprecipitation experiments, an antibody against BK(Ca) alpha-subunit [BK(Ca)(alpha)] could pull down TRPC1, and moreover an anti-TRPC1 antibody could reciprocally pull down BK(Ca)(alpha). Double-labeling immunocytochemistry showed that TRPC1 and BK(Ca) were colocalized in the same subcellular regions, mainly on the plasma membrane, in VSMCs. These data suggest that, TRPC1 physically associates with BK(Ca) in VSMCs and that Ca(2+) influx through TRPC1 activates BK(Ca) to induce membrane hyperpolarization. The hyperpolarizing effect of TRPC1-BK(Ca) coupling could serve to reduce agonist-induced membrane depolarization, thereby preventing excessive contraction of VSMCs to contractile agonists.

Differential Interactions of Thrombospondin-1, -2, and -4 with CD47 and Effects on cGMP Signaling and Ischemic Injury Responses

Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPalpha (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2.

Essential Role of TGF-β/Smad Pathway on Statin Dependent Vascular Smooth Muscle Cell Regulation

BackgroundThe 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins) exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-β (TGF-β) in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-β/Smad pathway in atherosclerosis and vascular cells.MethodologyIn cultured vascular smooth muscle cells (VSMCs) statins enhanced Smad pathway activation caused by TGF-β. In addition, statins upregulated TGF-β receptor type II (TRII), and increased TGF-β synthesis and TGF-β/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-β induced apoptosis and increased TGF-β-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-β/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected.ConclusionsStatins enhance TGF-β/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-β/Smad pathway is essential for statins-dependent actions in VSMCs.