Regulation of RGS2 and second messenger signaling in vascular smooth muscle cells by cGMP-dependent protein kinase

RGS2, a GTPase-activating protein (GAP) for G(q)alpha, regulates vascular relaxation and blood pressure. RGS2 can be phosphorylated by type Ialpha cGMP-dependent protein kinase (cGKIalpha), increasing its GAP activity. To understand how RGS2 and cGKIalpha regulate vascular smooth muscle signaling and function, we identified signaling pathways that are controlled by cGMP in an RGS2-dependent manner and discovered new mechanisms whereby cGK activity regulates RGS2. We show that RGS2 regulates vasoconstrictor-stimulated Ca(2+) store release, capacitative Ca(2+) entry, and noncapacitative Ca(2+) entry and that RGS2 is required for cGMP-mediated inhibition of vasoconstrictor-elicited phospholipase Cbeta activation, Ca(2+) store release, and capacitative Ca(2+) entry. RGS2 is degraded in vascular smooth muscle cells via the proteasome. Inhibition of cGK activity blunts RGS2 degradation. However, inactivation of the cGKIalpha phosphorylation sites in RGS2 does not stabilize the protein, suggesting that cGK activity regulates RGS2 degradation by other mechanisms. cGK activation promotes association of RGS2 with the plasma membrane by a mechanism requiring its cGKIalpha phosphorylation sites. By regulating GAP activity, plasma membrane association, and degradation, cGKIalpha therefore may control a cycle of RGS2 activation and inactivation. By diminishing cGK activity, endothelial dysfunction may impair RGS2 activation, thereby blunting vascular relaxation and contributing to hypertension.     

Active Rho kinase (ROK-alpha ) associates with insulin receptor substrate-1 and inhibits insulin signaling in vascular smooth muscle cells.

Recent studies from our laboratory have shown that insulin stimulates myosin-bound phosphatase (MBP) in vascular smooth muscle cells (VSMCs) by decreasing site-specific phosphorylation of the myosin-bound subunit (MBS) of MBP via nitric oxide/cGMP-mediated Rho/Rho kinase inactivation. Here we tested potential interactions between Rho kinase and insulin signaling pathways. In control VSMCs, insulin inactivates ROK-alpha, the major Rho kinase isoform in VSMCs, and inhibits thrombin-induced increase in ROK-alpha association with the insulin receptor substrate-1 (IRS-1). Hypertension (in spontaneous hypertensive rats) or expression of an active RhoA(V14) up-regulates Rho kinase activity and increases ROK-alpha/IRS-1 association resulting in IRS-1 serine phosphorylation that leads to inhibition of both insulin-induced IRS-1 tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activation. In contrast, expression of dominant negative RhoA or cGMP-dependent protein kinase type I alpha inactivates Rho kinase, abolishes ROK-alpha/IRS-1 association, and potentiates insulin-induced tyrosine phosphorylation and PI3-kinase activation leading to decreased MBS(T695) phosphorylation and decreased MBP inhibition. Collectively, these results suggest a novel function for ROK-alpha in insulin signal transduction at the level of IRS-1 and potential cross-talk between cGMP-dependent protein kinase type I alpha, Rho/Rho kinase signaling, and insulin signaling at the level of IRS-1/PI3-kinase.     

Modulation of ANP-C receptor signaling by arginine-vasopressin in A-10 vascular smooth muscle cells: role of protein kinase C

We have previously shown that pretreatment of A-10 vascular smooth muscle cells (VSMC) with angiotensin II (Ang II) attenuated atrial natriuretic peptide receptor-C (ANP-C)-mediated inhibition of adenylyl cyclase without altering [125I]ANP binding. In the present studies, we have investigated the modulation of ANP-C receptor signaling by arginine-vasopressin (AVP). Pretreatment of A-10 VSMC with AVP for 24h resulted in a reduction in ANP receptor binding activity by about 50% (B(max); control cells, 22.9+/-2.5 fmol/mg protein, AVP-treated cells, 11.4+/-1.2 fmol/mg protein). In addition, the expression of ANP-C receptor as determined by immunoblotting was also decreased by about 50% by AVP treatment, which was prevented by GF109203X, an inhibitor of protein kinase C (PKC). The decreased expression of ANP-C receptor was reflected in an attenuation of ANP-C receptor-mediated inhibition of adenylyl cyclase. C-ANP(4-23) [des(Gln(18),Ser(19),Gln(20),Leu(21),Gly(22))ANP(4-23)-NH(2)], a ring deleted peptide of ANP that interacts specifically with ANP-C receptor, inhibited adenylyl cyclase activity by about 30% in control cells, which was completely attenuated in AVP-treated cells. This attenuated inhibition was significantly restored by GF 109203X. In addition, AVP treatment augmented the levels of Gialpha-2 and Gialpha-3 proteins; however, the Gi functions were completely attenuated. The increased expression of Gialpha proteins induced by AVP was inhibited by GF109203X as well as by actinomycin D treatments. In addition, AVP treatment also enhanced the expression of Gsalpha protein and Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, N-ethylcarboxamide adenosine (NECA), and forskolin (FSK), whereas the levels of Gbeta were not altered by AVP treatment. These results indicate that AVP-induced PKC signaling may be responsible for the down-regulation of ANP-C receptor that results in the attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase activity, and suggest a cross-talk between vasopressin V(1) and ANP-C receptor-mediated signaling pathways.     

Redox-dependent MAP kinase signaling by Ang II in vascular smooth muscle cells: role of receptor tyrosine kinase transactivation

We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 micromol/L) fluorescence and lucigenin (5 micromol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10(-5) mol/L), an intracellular scavenger, and diphenylene iodinium (DPI; 10(-6) mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.     

Activation of mitogen-activated protein kinase pathways by cyclic GMP and cyclic GMP-dependent protein kinase in contractile vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.     

Regulation of cGMP-dependent protein kinase expression by soluble guanylyl cyclase in vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the alpha1 and beta1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro.     

Impaired insulin-stimulated myosin phosphatase Rho-interacting protein signaling in diabetic Goto-Kakizaki vascular smooth muscle cells

Insulin resistance associated with Type 2 diabetes contributes to impaired vasorelaxation and therefore contributes to the enhanced incidence of hypertension observed in diabetes. In this study, we examined the role of insulin on the association of the myosin-binding subunit of myosin phosphatase (MYPT1) to myosin phosphatase Rho-interacting protein (MRIP), a relatively novel member of the myosin phosphatase complex that directly binds RhoA in vascular smooth muscle cells (VSMCs). Through a series of molecular and cellular studies, we investigated whether insulin stimulates the binding of MRIP to MYPT1 and compared the results generated from VSMCs isolated from both Wistar-Kyoto (WKY) control and Goto-Kakizaki (GK) diabetic rats. We demonstrate for the first time that insulin stimulates the binding of MRIP to MYPT1 in a dose- and time-dependent manner, as determined by immunoprecipitation, implying a regulatory role for MRIP in insulin-induced vasodilation signaling via MYPT1 interaction. VSMCs from GK model of Type 2 diabetes had impaired insulin-induced MRIP/MYPT1 binding as well as reduced MRIP expression. Adenovirus-mediated overexpression of MRIP in GK VSMCs led to significantly improved insulin-stimulated MRIP/MYPT1 binding. Finally, insulin-stimulated MRIP translocation out of stress fibers, which was observed in control VSMCs, was impaired in GK VSMCs. We believe the impaired expression of MRIP, and therefore decreased insulin-stimulated MRIP/MYPT1 association, in the GK diabetic model may contribute to the impaired insulin-mediated vasodilation observed in the diabetic vasculature and provides a novel therapeutic strategy for the treatment of Type 2 diabetes.     

Hydrogen peroxide inhibits insulin signaling in vascular smooth muscle cells

Both insulin resistance and reactive oxygen species (ROS) have been reported to play essential pathophysiological roles in cardiovascular diseases, such as hypertension and atherosclerosis. However, the mechanistic link between ROS, such as H2O2 and insulin resistance in the vasculature, remains undetermined. Akt, a Ser/Thr kinase, mediates various biological responses induced by insulin. In this study, we examined the effects of H2O2 on Akt activation in the insulin-signaling pathway in vascular smooth muscle cells (VSMCs). In VSMCs, insulin stimulates Akt phosphorylation at Ser473. Pretreatment with H2O2 concentration- and time-dependently inhibited insulin-induced Akt phosphorylation with significant inhibition observed at 50 microM for 10 min. A ROS inducer, diamide, also inhibited insulin-induced Akt phosphorylation. In addition, H2O2 inhibited insulin receptor binding partially and inhibited insulin receptor autophosphorylation almost completely. However, pretreatment with a protein kinase C inhibitor, GF109203X (2 microM), for 30 min did not block the inhibitory effects of H2O2 on insulin-induced Akt phosphorylation, suggesting that protein kinase C is not involved in the inhibition by H2O2. We conclude that ROS inhibit a critical insulin signal transduction component required for Akt activation in VSMCs, suggesting potential cellular mechanisms of insulin resistance, which would require verification in vivo.     

Regulation of ERK5 by insulin and angiotensin-II in vascular smooth muscle cells

ERK5 is involved in proliferation of vascular smooth muscle cells (VSMC). The proliferative actions of insulin and angiotensin-II (A-II) in VSMC are mediated in part by ERK1/2. We hypothesized that insulin and A-II also regulate ERK5 activity in VSMC. Acute treatment (<60min) with insulin or A-II increased phosphorylation of ERK1/2 at 15min and ERK5 at 5min. Chronic treatment (< or = 8h) with insulin increased ERK1/2 phosphorylation by 4h and ERK5 by 8h. A-II-stimulated phosphorylation of ERK1/2 by 8h and ERK5 by 4h. The EC(50) for insulin treatment effecting ERK1/2 and ERK5 phosphorylation was 1.5 and 0.1nM, whereas the EC(50) for A-II was 2nM, each. Insulin plus A-II induced an additive effect only on ERK5 phosphorylation. Inhibition of insulin- and A-II-stimulated phosphorylation of ERK5 and ERK1/2 by PD98059 and Wortmannin exhibited differential and time-dependent effects. Taken together, these data indicate that insulin and A-II regulate the activity of ERK5, but different from that seen for ERK1/2.     

Potential role of insulin signaling on vascular smooth muscle cell migration, proliferation, and inflammation pathways

To investigate the role of insulin signaling pathways in migration, proliferation, and inflammation of vascular smooth muscle cells (VSMCs), we examined the expression of active components of the phosphatidyl inositol 3 (PI-3) kinase (p-Akt) and mitogen-activated protein kinase (MAPK) (p-Erk) in primary cultures of VSMCs from human coronary arteries. VSMCs were treated in a dose-response manner with insulin (0, 1, 10, and 100 nM) for 20 min, and Akt and Erk phosphorylation were measured by Western blot analysis. In separate experiments, we evaluated the effect of 200 μM palmitate, in the presence and absence of 8 μM pioglitazone, on insulin-stimulated (100 nM for 20 min) Akt and Erk phosphorylation. The phosphorylation of Akt and Erk in VSMCs exhibited a dose dependency with a three- to fourfold increase, respectively, at the highest dose (100 nM). In the presence of palmitate, insulin-induced Akt phosphorylation was completely abolished, and there was a threefold increase in p-Erk. With addition of pioglitazone, the phosphorylation of Akt by insulin remained unchanged, whereas insulin-stimulated Erk phosphorylation was reduced by pioglitazone. These data in VSMCs indicate that high palmitate decreases insulin-stimulated Akt phosphorylation and stimulates MAPK, whereas preexposure peroxisome proliferator-activated receptor-γ agonist pioglitazone preserves Akt phosphorylation and simultaneously attenuates MAPK signaling. Our results suggest that metabolic and mitogenic insulin signals have different sensitivity, are independently regulated, and may play a role in arterial smooth muscle cells migration, proliferation, and inflammation in conditions of acute hyperinsulinemia.     

Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells

In smooth muscle, a Rho-regulated systemof myosin phosphatase exists; however, it has yet to be establishedwhether Rho kinase, one of the downstream effectors of Rho, mediatesthe regulation of myosin phosphatase activity in vivo. In the presentstudy, we demonstrate in permeabilized vascular smooth muscle cells(SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dosedependently inhibits Rho-mediated enhancement ofCa²⁺-induced 20-kDa myosin lightchain (MLC₂₀) phosphorylationdue to abrogating Rho-mediated inhibition ofMLC₂₀ dephosphorylation. By animmune complex phosphatase assay, we found that guanosine 5'-O-(3-thiotriphosphate)(GTPS) stimulation of permeabilized SMCs caused a decrease in myosinphosphatase activity with an increase in the extent of phosphorylationof the 130-kDa myosin-binding regulatory subunit (MBS) of myosinphosphatase in a Rho-dependent manner. HA-1077 abolished both of theRho-mediated events. Moreover, we observed that the pleckstrinhomology/cystein-rich domain protein of Rho kinase, a dominant negativeinhibitor of Rho kinase, inhibited GTPS-induced phosphorylation ofMBS. These results provide direct in vivo evidence that Rho kinasemediates inhibition of myosin phosphatase activity with resultantenhancement of MLC₂₀phosphorylation in smooth muscle and reveal the usefulness of HA-1077as a Rho kinase inhibitor.

     

Rho expression and activation in vascular smooth muscle cells

Phenotypic modulation of smooth muscle cells (SMC) involves dramatic changes in expression and organization of contractile and cytoskeletal proteins, but little is known of how this process is regulated. The present study used a cell culture model to investigate the possible involvement of RhoA, a known regulator of the actin cytoskeleton. In rabbit aortic SMC seeded into primary culture at moderate density, Rho activation was high at two functionally distinct time-points, first as cells modulated to the "synthetic" phenotype, and again upon confluence and return to the "contractile" phenotype. Rho expression increased with time, such that maximal expression occurred upon return to the contractile state. Transient transfection of synthetic state cells with constitutively active RhoA (Val14RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile phenotype. Actin and myosin filaments were tightly packed and highly organised while vimentin localised to the perinuclear region; focal adhesions were enlarged and concentrated at the cell periphery. Conversely, inhibition of endogenous Rho by C3 exoenzyme resulted in complete loss of contractile filaments without affecting vimentin distribution; focal adhesions were reduced in size and number. Treatment of synthetic state SMC with known regulators of SMC phenotype, heparin and thrombin, caused a modest increase in Rho activation. Long-term confluence and serum deprivation induced cells to return to a more contractile phenotype and this was augmented by heparin and thrombin. The results implicate RhoA for a role in regulating SMC phenotype and further show that activation of Rho by heparin and thrombin correlates with the ability of these factors to promote the contractile phenotype.     

Interactive role of tyrosine kinase,protein kinase C,and Rho/Rho kinase systems in the mechanotransduction of vascular smooth muscles

Blood vessels are always subjected to hemodynamic stresses including blood pressure and blood flow. The cerebral artery is particularly sensitive to hemodynamic stresses such as pressure and stretch, and shows contractions that are myogenic in nature; i.e., the mechanical response is generated by the vascular smooth muscle itself. The artery constricts in response to an increase in intraluminal pressure, and dilates in response to a decrease in the intraluminal pressure. We provide herein some insights into the mechanotransduction of vascular tissue; i.e., we discuss how the tissue is receptive to mechanical force and how the latter induces the specific signals leading to myogenic contraction in terms of mechanosensor action and subsequent intracellular signaling. The interactive role of tyrosine kinase, protein kinase C, and Rho/Rho-kinase systems in the mechanotransduction process is discussed, which systems also seem to play an important role in the development of experimental cerebral vasospasm. The study of the mechanotransduction in vascular tissue may aid in clarifying the mechanisms underlying vasospastic episodes and pathologic remodeling in cardiovascular diseases, and may potentially have therapeutic consequences.     

The role of Src kinase in insulin-like growth factor-dependent mitogenic signaling in vascular smooth muscle cells

Activation of the MAPK pathway mediates insulin-like growth factor-I (IGF-I)-dependent proliferation in vascular smooth muscle cells (SMC). Our previous studies have shown that IGF-I-induced Shc phosphorylation is necessary for sustained activation of MAPK and increased cell proliferation of SMCs, and both Shc and the tyrosine phosphatase SHP-2 must be recruited to the membrane protein SHPS-1 in order for Shc to be phosphorylated. These studies were undertaken to determine whether Src kinase activity is required to phosphorylate Shc in response to IGF-I in SMC and because SHP-2 binds to Src whether their interaction was also required for IGF-I-stimulated mitogenesis. Our results show that IGF-I induces activation of Src kinase and that is required for Shc phosphorylation and for optimal MAPK activation. We tested whether Shc is a substrate of c-Src in SMC by disrupting Src/Shc association using a peptide containing a YXXL (Tyr328) motif sequence derived from Src. The peptide blocked the binding of Src and Shc in vitro and in vivo. Cells expressing a mutant Src (Src-FF) that had Tyr328/Tyr358 substituted with phenylalanines (Src-FF) showed defective Src/Shc binding, impaired IGF-I-dependent Shc phorylation, and impaired mitogenesis. This supports the conclusion that Src phosphorylates Shc. IGF-I induced both Src/SHP-2 and Src/SHPS-1 association. SMCs expressing an SHP-2 mutant that had the polyproline-rich region of SH2 deleted (SHP-2Delta10) had disrupted SHP-2/Src association, impaired IGF-I-dependent Shc phosphorylation, and an attenuated mitogenic response. IGF-I-induced association of Src and SHPS-1 was also impaired in SHP-2Delata10-expressing cells, although SHP-2/SHPS-1 association was unaffected. Upon IGF-I stimulation, a complex assembles on SHPS-1 that contains SHP-2, c-Src, and Shc wherein Src phosphorylates Shc, a signaling step that is necessary for an optimal mitogenic response.     

Excitation-contraction coupling in pulmonary vascular smooth muscle involves tyrosine kinase and Rho kinase

We investigated the mechanisms that underlie the responses to norepinephrine (NE) and thromboxane (Tx) A(2) (TxA2) in the canine pulmonary vasculature with fura 2 fluorimetric, intracellular microelectrode, and force transduction techniques. KCl, caffeine, and cyclopiazonic acid elevated intracellular Ca2+ concentration levels and tone, indicating that Ca2+ mobilization is sufficient to produce contraction. However, contractions evoked by NE or the TxA2 mimetic U-46619 were unaffected by nifedipine or by omitting external Ca2+ and were reduced only partially by depleting the internal Ca2+ store; furthermore, NE-evoked depolarization was subthreshold for voltage-dependent Ca2+ currents. Agonist-evoked contractions were insensitive to inhibitors of protein kinase C (calphostin C and chelerythrine), mitogen-activated protein kinase kinase (PD-98059), and p38 kinase (SB-203580) but were abolished by the tyrosine kinase inhibitor genistein and the Rho kinase inhibitor Y-27632. We conclude that, although Ca2+ influx and Ca2+ release are sufficient for contraction, they are not necessary for adrenergic or TxA2 contractions. Instead, excitation-contraction coupling involves the activation of tyrosine kinase and Rho kinase, leading to enhanced Ca2+ sensitivity of the contractile apparatus.     

Regulation of vascular smooth muscle cells by micropatterning

Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.     

Inhibition of Rho protein stimulates iNOS expression in rat vascular smooth muscle cells

Inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMCs) is upregulated in arterial injury and plays a role in regulating VSMC proliferation and restenosis. Inflammatory cytokines [e.g., interleukin-1beta (IL-1beta)] released during vascular injury induce iNOS. Small GTP-binding proteins of the Ras superfamily play a major role in IL-1beta-dependent signaling pathways. In this study, we examined the role of Rho GTPases in regulating iNOS expression in VSMCs. Treatment of VSMCs with mevastatin, which inhibits isoprenylation of Rho and other small GTP-binding proteins, produced significantly higher amounts of IL-1beta-evoked NO and iNOS protein compared with control. Similarly, bacterial toxins [Toxin B from Clostridium difficile and C3 ADP-ribosyl transferase (C3) toxin from Clostridium botulinium] that specifically inactivate Rho proteins increased NOS products (NO and citrulline) and iNOS expression. Toxin B increased the activity of iNOS promoter-reporter construct in VSMCs. Both toxins enhanced IL-1beta-stimulated iNOS expression and NO production. These data demonstrate for the first time that inhibition of Rho induces iNOS and suggest a role for Rho protein in IL-1beta-stimulated NO production in VSMCs.