Cultured Rat Vascular Smooth Muscle Research Articles

Insulin activates p38 mitogen-activated protein (MAP) kinase via a MAP kinase kinase (MKK) 3/MKK 6 pathway in vascular smooth muscle cells.

Induction of stress-activated mitogen-activated protein (MAP) kinases such as p38 could be important for the development of cardiovascular diseases since p38 MAP kinase activation stimulates apoptosis, cell growth, prostanoid formation and other cellular dysfunctions when induced by oxidants, hyperosmolarity, or pro-inflammatory cytokines. On the other hand, insulin resistance is one of the most important factors promoting atherogenesis, including cardiovascular diseases, but it is not clear how these different factors transmit their signals intracellularly at the cytosolic and nuclear levels. In this study, we investigated the effect of insulin on p38 mitogen-activated protein (MAP) kinase activation in cultured rat vascular smooth muscle cells (VSMC). VSMC were obtained from the aortas of male Wistar rats by the media explant technique. After being stimulated by insulin with SB-203580, PD-98059, or GF109203X, the cells were solubilized and the expressions of MAP kinases, MAP kinase kinases and p70 S6 kinase were examined by immunoblot analysis. The amount of DNA synthesis was measured by [3H]thymidine incorporation. Insulin activated p38 MAP kinase phosphorylation in a dose-dependent manner, and the phosphorylation was specifically inhibited by SB-203580, a p38 MAP kinase-specific inhibitor, but not by PD-98059, a specific inhibitor of upstream kinase (MEK), of extracellular signal-regulated kinase (ERK), or GF209203X, a protein kinase C-specific inhibitor. Insulin also activated MAP kinase kinase (MKK) 3/MKK 6 phosphorylation, the upstream kinase of p38 MAP kinase, but neither stress-activated protein kinase (SAPK)/ERK kinase (SEK1/MKK4) nor SAPK/c-jun NH2-terminal protein kinase. Surprisingly, phosphorylation of p70 S6 kinase and an increase of DNA synthesis by insulin were suppressed dose dependently by SB-203580. These results have established that insulin activates the p38 MAP kinase cascade via an MKK 3/6 pathway in rat VSMC, independently of a MEK-ERK cascade, and partly regulates cell growth.

Peroxisome proliferator-activated receptor-gamma ligands inhibit nitric oxide synthesis in vascular smooth muscle cells.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a key player in glucose metabolism. If PPARgamma ligands modulate nitric oxide (NO) synthesis in the vascular tissue, they may affect the process of plaque formation and postangioplasty restenosis. We investigated the effects of PPARgamma ligands on NO synthesis in vascular smooth muscle cells. Incubation of cultures with interleukin-1beta (10 ng/mL) for 24 hours caused a significant increase in the production of nitrite, a stable metabolite of NO, in cultured rat vascular smooth muscle cells. The PPARgamma agonists troglitazone and 15-deoxy-triangle up(12,14)-prostaglandin J(2) (15d-PG J(2)) dose-dependently inhibited nitrite production by interleukin-1beta-stimulated vascular smooth muscle cells. Decreased interleukin-1beta-induced nitrite production by the PPARgamma agonists was accompanied by decreased inducible NO synthase mRNA and protein accumulation. Interleukin-1beta induced nuclear factor-kappaB activation in vascular smooth muscle cells, and both troglitazone and 15d-PG J(2) markedly suppressed this nuclear factor-kappaB activation. PPARgamma ligands inhibit NO synthesis in cytokine-stimulated vascular smooth muscle cells, suggesting that these agonists may act directly on the vascular smooth muscle and influence the process of atherosclerosis and restenosis.

Suppression by eicosapentaenoic acid of oxidized low-density lipoprotein and lysophosphatidylcholine-induced migration in cultured rat vascular smooth muscle cells

The migration of medial smooth muscle cells into the intima is proposed to be an initial process of intimal thickening in atherosclerotic lesions. The present study was designed to determine whether pretreatment with the antiatherogenic agent eicosapentaenoic acid (EPA) inhibits the migration induced by oxidized low-density lipoprotein (LDL) and its major phospholipid component, lysophosphatidylcholine (lyso-PC), in cultured rat vascular smooth muscle cells (VSMCs) using Boyden's chamber method. The effects of EPA pretreatment on angiotensin II (Ang II)- and platelet-derived growth factor BB (PDGF BB)-induced migration were also examined in these cells. Oxidized LDL and lyso-PC induced migration in a concentration-dependent manner. EPA pretreatment clearly suppressed oxidized LDL (200 microg/mL)- and lyso-PC (10(-5) mol/L)-induced migration between 40 and 160 micromol/L. EPA pretreatment also suppressed Ang 11 (10(-7) mol/L)- and PDGF BB (5 ng/mL)-induced migration at a concentration of 80 and 160 micromol/L. However, in a trypan blue exclusion test, dead cells stained with trypan blue were not found 24 hours after treatment with EPA. These results suggest that EPA suppresses VSMC migration induced by oxidized LDL and lyso-PC, as well as Ang II and PDGF BB. These preliminary data concerning the effects of EPA may partly explain the antiatherosclerotic effects of this agent.

Inhibitors of Na-K-ATPase in human urine: effects of ouabain-like factors and of vanadium-diascorbate on calcium mobilization in rat vascular smooth muscle cells

Endogenous ouabain-like factors (OLF) may play a role in the pathogenesis of volume-dependent hypertension by raising intracellular free calcium ([Ca 2 + ] i) as a consequence of inhibition of the sodium pump. In previous studies we described the presence of two low molecular (Mr ≈ 400) inhibitors of Na-K-ATPase in human urine, ie, a more polar OLF-1 and a more apolar OLF-2. We subsequently identified the active compound in OLF-2 as vanadium (V IV)-diascorbate (Mr 416). OLF-1, OLF-2, and V-diascorbate inhibited dose-dependently porcine Na-K-ATPase in vitro. In the present study we investigated the effects of urinary OLF-1, OLF-2, and V-diascorbate on calcium mobilization, ie, on [Ca 2 + ] i in cultured rat vascular smooth muscle (VSM) cells in comparison to the effects of ouabain, angiotensin II (A II), and arginine-vasopressin (AVP). [Ca 2 + ] i was determined by the fura-2 method. OLF-1 and OLF-2 (each ≈ 10 −4 mol/L), obtained as single spots by thin-layer chromatography, produced a rise in [Ca 2 + ] i in VSM cells from 45 ± 7 to 99 ± 22 and from 48 ± 9 to 92 ± 2 nmol/L (each n = 5; P < .05), respectively, after 3 min. V-diascorbate also increased [Ca 2+] i slowly and dose-dependently, eg, from 56 ± 14 to 102 ± 15 nmol/L at a concentration of 10 −6 mol/L (n = 5; P < .05) after 3 min. A similar slow rise in [Ca 2 + ] i from 53 ± 10 to 185 ± 3 nM (n = 5; P < .05) after 3 min was found with ouabain (10 −6 mol/L). As standard vasoconstrictor, All (10 −8 mol/L) rapidly increased [Ca 2 + ] i from 23 ± 4 to 846 ± 50 nmol/L (n = 7; P < .01) within 30 sec. This effect was enhanced to 1389 ± 161 nM (n = 7; P < .01) when VSM cells were preincubated with V-diascorbate (10 −6 mol/L) for 10 min. AVP (10 −7 mol/L) also rapidly increased [Ca 2 + ] i to 418 ± 11 nmol/L within 30 sec (n = 7; P < .01). This effect was enhanced in the presence of OLF-2 ( ≈10 −4 mol/L) or ouabain (10 −6 mol/L) to 523 ± 14 and 560 ± 19 nmol/L, respectively (each n = 7); P < .01). The calcium channel blocker verapamil, the intracellular calcium release blocker TMB-8, and the unselective cation channel blocker Ni 2 + partly blunted the A II- or AVP-induced rise in [Ca 2 + ] i and prevented the OLF-2- and V-diascorbate–induced increase in [Ca 2 + ] i. Thus, OLF-1, OLF-2 and V-diascorbate, the active component of OLF-2, reveal effects similar to those of ouabain on [Ca 2 + ] i in VSM cells, ie, they produce a slow rise in [Ca 2 + ] i subsequent to inhibition of the sodium pump. The physiologic and pathologic roles of these and additional OLF in body fluid and blood pressure regulation and in hypertension have yet to be evaluated.

Mechanical stretch stimulates growth of vascular smooth muscle cells via epidermal growth factor receptor.

We have studied whether activation of epidermal growth factor receptor (EGFR) is involved in stretch-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation and protein synthesis in cultured rat vascular smooth muscle cells (VSMC). Cyclic stretch (1 Hz) induced a rapid (within 5 min) phosphorylation of ERK1/2, an effect that was time and strength dependent and inhibited by an EGFR kinase inhibitor (AG-1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG-1296). The stretch rapidly (within 2 min) induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR as revealed by blockade with AG-1478 as well as immunoprecipitation with anti-EGFR antibody coupled with immunoblotting with anti-phosphotyrosine antibody. The stretch rapidly (within 2 min) induced association of tyrosine-phosphorylated EGFR with adaptor proteins (Shc/Grb2) as revealed by coprecipitation with glutathione-S-transferase-Grb2 fusion protein coupled with immunoblotting with anti-phosphotyrosine, anti-EGFR, and anti-Shc antibodies. Transfection of a dominant-negative mutant of H-Ras also inhibited stretch-induced ERK1/2 activation. Treatment with a stretch-activated ion channel blocker (Gd(3+)) and an intracellular Ca(2+) antagonist (TMB-8) inhibited stretch-induced phosphorylation of EGFR and ERK1/2. Treatment with AG-1478 and a mitogen-activated protein kinase kinase inhibitor (PD-98059), but not AG-1296, blocked [(3)H]leucine uptake stimulated by a high level of stretch. These data suggest that ERK1/2 activation by mechanical stretch requires Ca(2+)-sensitive EGFR activation mainly via stretch-activated ion channels, thereby leading to VSMC growth.

Induction of cyclooxygenase-2 by angiotensin II in cultured rat vascular smooth muscle cells.

Angiotensin II (Ang II) stimulates the release of prostaglandins (PGs) in various cells and tissues. Recently, cyclooxygenase-2 (COX-2) emerged as a new key regulator for PG synthesis. In the present study, we investigated whether Ang II regulates COX-2 expression in cultured rat vascular smooth muscle cells (VSMCs). Ang II markedly increased the expression of COX-2 mRNA in a time- and dose-dependent manner. This effect was completely blocked by the Ang II type 1 receptor antagonist losartan but not by the Ang II type 2 receptor antagonist PD123319. The p42/44 mitogen-activated protein kinase (MAPK) kinase-1 inhibitor PD98059 and the p38 MAPK inhibitor SB203580 significantly suppressed Ang II-induced COX-2 mRNA and protein expression. Ang II did not increase transcription of the COX-2 gene, as examined with a COX-2 promoter/luciferase chimeric plasmid construct. Instead, it suppressed the degradation of COX-2 mRNA. PD98059 and SB203580 markedly enhanced the decay of COX-2 mRNA induced by Ang II, implying that p42/44 and p38 MAPK activated by Ang II play a role in the regulation of COX-2 through stabilization of its mRNA. The COX-2-specific inhibitor NS-398 attenuated Ang II-stimulated DNA and protein synthesis, as well as PGE(2) production by VSMCs. These results suggest that Ang II regulates COX-2 expression and PG production and modulates cell proliferation through MAPK-mediated signaling pathways in rat VSMCs.

Inhibition of Bradykinin-Induced Phosphoinositide Hydrolysis and Ca 2+ Mobilisation by Phorbol Ester in Rat Cultured Vascular Smooth Muscle Cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca 2+ concentration ([Ca 2+] i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca 2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC 50) and maximal inhibition of BK induced an increase in [Ca 2+] i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca 2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-α, βI, βII, δ, ϵ, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K D and B max of the BK receptor for binding (control: K D = 1.7 ± 0.2 nM; B max = 47.3 ± 4.4 fmol mg protein ), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-α, βI, βII, δ, ϵ, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca 2+ mobilisation in VSMCs.

Annexin VII and Annexin XI Are Tyrosine Phosphorylated in Peroxovanadate-treated Dogs and in Platelet-derived Growth Factor-treated Rat Vascular Smooth Muscle Cells

The intraperitoneal administration of peroxovanadate results in the rapid accumulation of many tyrosine-phosphorylated proteins in the liver and kidney of treated animals. The availability of large pools of tyrosine-phosphorylated proteins derived from normal tissues facilitates the purification and identification of previously unknown targets for cellular tyrosine kinases. Using this procedure, we have thus far identified four proteins in the liver and kidney of peroxovanadate-treated dogs. Two of these, annexin VII and annexin XI, were novel and had not been previously reported to be substrates of tyrosine kinases while the remaining two, ezrin and clathrin, have been reported to be tyrosine phosphorylated in some cell culture systems. In the present study, isolated proteins were identified both by sequence analysis and immunological methods. Annexin VII and annexin XI are present in cultured rat vascular smooth muscle cells and both were tyrosine phosphorylated in response to a physiological ligand, platelet-derived growth factor-BB (PDGF-BB). Furthermore, the extent of tyrosine phosphorylation in response to PDGF-BB was augmented by the co-addition of peroxovanadate to cell cultures. In vitro phosphorylation assays showed that PDGF receptor, calcium-dependent tyrosine kinase (CADTK/Pyk-2), Src kinase, and epidermal growth factor receptor all were able to phosphorylate purified annexin VII and XI on tyrosine residues. These findings confirm the usefulness of phosphatase inhibition by peroxovanadate as a tool for identifying previously unknown physiological targets for cellular protein tyrosine kinases.

Endothelin-mediated vascular growth requires p42/p44 mitogen-activated protein kinase and p70 S6 kinase cascades via transactivation of epidermal growth factor receptor.

Endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor peptide, exerts a growth-promoting effect on vascular smooth muscle cells, implicating its pathogenic role in vascular remodeling. To gain insight into the cellular and molecular mechanism whereby ET-1 induces vascular growth, we studied whether transactivation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor, are required for activation of p42/p44 mitogen-activated protein (MAP) kinase and p70 S6 kinase (p70S6K), and subsequent growth-promotion by ET-1 in cultured rat vascular smooth muscle cells. Immunoblotting with antiphosphotyrosine antibody revealed that ET-1 rapidly (within 2 min) and transiently induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR. ET-1 rapidly increased association of EGFR and Shc with glutathione-S-transferase-Grb2 fusion protein. The ET-1-induced activation of MAP kinase was reduced by an EGFR kinase inhibitor (AG1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG1296). AG1478 dose-dependently decreased ET-1-stimulated MAP kinase activity as well as [3H]leucine and [3H]thymidine uptake. The ET-1-induced tyrosine phosphorylation of EGFR, as well as MAP kinase activation, was inhibited by an ETA receptor antagonist and intracellular Ca2+ antagonists but not by an ETB receptor antagonist, pertussis toxin, or protein kinase C inhibitors. In addition, dominant negative mutant of H-Ras and a MAP kinase kinase (MEK-1) inhibitor (PD98059) completely blocked ET-1-induced MAP kinase activation as well as [3H]leucine and [3H]thymidine uptake. Both AG1478 and PD98059 inhibited ET-1-induced phosphorylation and activation of p70S6K. Furthermore, rapamycin, a selective inhibitor of mammalian target of rapamycin, completely blocked ET-1-stimulated [3H]leucine and [3H]thymidine uptake. These results suggest that ETA receptor-mediated vascular growth by ET-1 requires both MAP kinase and p70S6K cascades mediated partly via Ca2+-dependent EGFR transactivation.

The effect of erythropoietin on interleukin-1beta mediated increase in nitric oxide synthesis in vascular smooth muscle cells.

Recently, we observed that recombinant human erythropoietin (rHuEPO) inhibits the interleukin (IL)-1beta induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMC). The mechanisms of these inhibitory effects of rHuEPO were evaluated. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify a specific erythropoietin receptor (EpoR). Tyrosine phosphorylation of phospholipase C (PLC) was analyzed by combination of immunoprecipitation and Western blotting. Protein kinase C (PKC) activities were analyzed by phosphorylation assay of myelin basic protein (MBP4-14). VSMC were incubated with test agents for 24 h and nitrite as a stable NO metabolite was measured. iNOS mRNA and protein expression was analyzed by Northern and Western blotting, respectively. RT-PCR analysis revealed that EpoR m-RNA was expressed; furthermore, it might be alternatively spliced in VSMC. rHuEPO induced tyrosine phosphorylation of PLC-gamma1 and activation of PKC. rHuEPO inhibited not only IL-1beta induced nitrite production, but also the expression of iNOS mRNA and protein. These inhibitory effects of rHuEPO were reversed in the presence of PKC inhibitors, calphostin C (1 pmol/l) or staurosporine (10 nmol/l). PKC activation by phorbol myristate acetate inhibited nitrite production. The inhibitory effect of rHuEPO on IL-1beta induced nitrite production was also eliminated in PKC depleted cells or in the existence of anti-EpoR antibody. rHuEPO inhibits IL-1beta induced NO production by suppressing iNOS mRNA and protein expressions through EpoR, and the PLC-gamma1 and PKC pathway may be involved.

Failure of cdc2 promoter activation and G(2)/M transition by ANG II and AVP in vascular smooth muscle cells.

The physiological role of the vasoconstrictive hormones arginine vasopressin (AVP) and angiotensin II (ANG II) in the development of vascular hyperplasia is still unclear. We examined the effects of these hormones on cell cycle regulation of cultured rat vascular smooth muscle cells (VSMC). AVP and ANG II were able to induce G(1)/S transition and DNA synthesis in serum-starved quiescent VSMC but failed to promote further progression into G(2)/M phases. AVP and ANG II enhanced the expression and activity of cdk2, cyclin E, and proliferating cell nuclear antigen but did not induce expression of cdc2/cyclin B complex, a critical regulator of G(2)/M transition. The failure of cdc2 mRNA induction was found to be caused by a defect in cdc2 promoter activation. Binding of free E2F-1 to the cdc2 promoter did not occur in hormone-treated VSMC, which may account for the defective induction of cdc2. The absence of cdc2 promoter activation and G(2)/M transition may be important for the prevention of hyperplasia under physiological conditions but underlies the hypertrophy of VSMC.

Effects of lovastatin on expression of cell cycle regulatory proteins in vascular smooth muscle cells

The sequential appearance of cyclins D and E is thought to initiate subsequent DNA synthesis in proliferating cells. Previous studies have reported that DNA synthesis in cultured rat vascular smooth muscle cells (VSMCs) was suppressed by the HMG-CoA reductase inhibitor lovastatin. The effects of lovastatin on cell cycle regulatory proteins in proliferating VSMCs, however, are largely unknown. Thus, we investigated the sequential expression of cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 4, CDK2, and p27Kip1 in cultured rat VSMCs stimulated by platelet-derived growth factor (PDGF)-BB in the presence or absence of lovastatin. Quiescent VSMCs, with and without lovastatin (20 microM) pretreatment for nine hours, were stimulated by PDGF-BB (25 ng/ml). The incorporation of tritiated thymidine was done to assess DNA synthesis. VSMC lysates were obtained every 6 hours for up to 36 hours after stimulation and were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using relevant polyclonal antibodies. Autoradiograms were analyzed using a densitometer. The peak expression of cyclins D1 and E occurred at 18 and 30 hours of PDGF stimulation, respectively. Concomitant expression of CDK4 and CDK2 was also observed. The expression of p27Kip1, by contrast, was reduced in association with DNA synthesis. Lovastatin suppressed DNA synthesis and reduced the expression of cyclin D1 and cyclin E, whereas p27Kip1 expression was strongly induced by lovastatin pretreatment. CDK4 and CDK2 expression was unaffected by lovastatin treatment. PDGF-BB induces cyclins D1 and E prior to the onset of DNA synthesis in VSMCs. Lovastatin may suppress DNA synthesis in VSMCs by inducing p27Kip1 and reducing expression of cyclins D1 and E.

Role of Glutathione S-transferase 8-8 in Allylamine Resistance of Vascular Smooth Muscle Cells in Vitro

Allylamine (AA) is a cardiovascular toxin that causes lesions resembling atherosclerosis in several mammalian species. AA's toxic effects are thought to be exerted through its conversion to acrolein (AC), a potent electrophilic alkylating agent and atherogen. Semicarbazide sensitive amine oxidase (SSAO) catalyzes the oxidation of AA to AC. Glutathione S-transferases (GST) can catalyze the first step of detoxification of AC to mercapturic acid. Our previous studies suggest that the isozyme rGST8-8 is a principal defense against electrophilic stress exerted by α,β-unsaturated carbonyls such as AC. In the present studies, we use cultured rat vascular smooth muscle cells (VSMC) to examine the relative roles of SSAO and rGST8-8 in the cytotoxic effects of the atherogens, AA and AC. Exposure derived AA-resistant cells (VSMC-AA) were 3.5-fold more resistant to AA when compared to VSMC and 1.8-fold more resistant to acrolein. SSAO activity was 2-fold higher in VSMC-AA than in VSMC. Consistent with the role of SSAO in biotransformation of AA, the SSAO inhibitor semicarbazide (SC; 100 μM) provided nearly complete protection from AA to both VSMC-AA and VSMC. As expected, SC did not affect the cytotoxicity of AC. Pretreatment with 100 μM sulfasalazine (SS), a GST inhibitor, potentiated AA and AC toxicity in both VSMC-AA and VSMC, indicating a protective role of GST. Catalytic efficiency (Kcat/Km) of GSTs was higher toward 4-hydroxynonenal (4-HNE) (0.65 mM−1 s−1) than toward 1-chloro-2,4-dinitrobenzene (CDNB) (0.14 mM−1 s−1) for VSMC. In VSMC-AA, Kcat/Km was increased 4.1-fold toward CDNB (0.58 mM−1 s−1) and 6-fold toward 4HNE (3.9 mM−1 s−1) when compared to VSMC, indicating a preferential increase in VSMC-AA of GST isozymes which utilize α,β-unsaturated carbonyls. Western blots confirmed induction of rGST8-8 in VSMC-AA. Expression of recombinant mGSTA4 (the mouse homolog of rGST8-8) in VSMC caused a 1.6-fold increase in resistance to AA and AC. This resistance was fully reversed by 50 μM SS. Our results demonstrate that GSTs are an important defense against electrophilic atherogens and that isozymes with high activity toward α,β-unsaturated carbonyls are particularly important in the vascular wall.

Alteration of microtubule polymerization modulates arteriolar vasomotor tone.

Microtubules are important cytoskeletal elements that have been shown to play a major role in many cellular processes because of their mechanical properties and/or their participation in various cell signaling pathways. We tested the hypothesis that depolymerization of microtubules would alter vascular smooth muscle (VSM) tone and hence contractile function. In our studies, isolated cremaster arterioles exhibited significant vasoconstriction that developed over a 20- to 40-min period when they were treated with microtubule depolymerizing drugs colchicine (10 microM), nocodazole (10 microM), or demecolcine (10 microM). Immunofluorescent labeling of microtubules in cultured rat VSM revealed that both colchicine and nocodazole caused microtubule depolymerization over a similar time course. The vasoconstriction was maintained over a wide range of intraluminal pressures (30-170 cmH(2)O). The increased tone was not affected by endothelial denudation, suggesting that it was due to an effect on VSM. Microtubule depolymerization with demecolcine or colchicine had no effect on VSM intracellular Ca(2+) concentration ([Ca(2+)](i)). These data indicate that microtubules significantly interact with processes leading to the expression of vasomotor tone. The mechanism responsible for the effect of microtubules on vasomotor tone appears to be independent of both the endothelium and an increase in VSM [Ca(2+)](i).

Differential inhibitory actions by glucocorticoid and aspirin on cytokine-induced nitric oxide production in vascular smooth muscle cells.

Glucocorticoids and nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory and immune diseases. Nitric oxide (NO) has a diversity of physiological functions, but its excess production has been implicated in the inflammatory process. The present study was designed to elucidate the mechanisms by which glucocorticoids and NSAIDs affect inducible nitric oxide synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMCs). Both interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha potently stimulated nitrite/nitrate (NOx) production with a concomitant expression of iNOS mRNA and protein as demonstrated by Northern and Western blot analysis, respectively. Both IL-1beta and TNF-alpha activated nuclear factor (NF)-kappaB as demonstrated by electrophoretic mobility shift assay. Dexamethasone, salicylate and aspirin, but not indomethacin, dose dependently inhibited cytokine-stimulated NOx production and iNOS protein expression. Dexamethasone decreased cytokine-induced NF-kappaB activation and iNOS mRNA expression, but neither salicylate nor aspirin affected NF-kappaB activation or iNOS mRNA expression. IL-1beta caused a rapid increase in phosphorylated IkappaB-alpha levels and subsequent transient decrease in IkappaB-alpha levels, an inhibitor of NF-kappaB, as revealed by Western blot analysis using specific antibodies for phosphorylated and nonphosphorylated IkappaB-alpha. These effects were blocked by pretreatment with dexamethasone. Aspirin dose dependently inhibited iNOS enzymatic activity, whereas salicylate and dexamethasone had limited effect. The present study demonstrates that 1) inhibitory effect of dexamethasone on cytokine-induced iNOS expression and NO production in rat VSMCs, although potentially acting at multiple levels, is partly mediated by inhibition of NF-kappaB activation resulting from decreased phosphorylation and degradation of IkappaB-alpha, 2) both salicylate and aspirin inhibit cytokine-stimulated NO production at translational and/or posttranslational levels without affecting NF-kappaB- mediated iNOS gene expression, and 3) aspirin directly inhibits iNOS enzyme activity. These data suggest the differential inhibitory mechanisms of iNOS-mediated NO synthesis by glucocorticoids and NSAIDs in the vasculature.

A possible mechanism of TPA-mediated downregulation of neurotrophin-3 gene expression in rat cultured vascular smooth muscle cells

We have previously reported that in cultured rat vascular smooth muscle cells (VSMCs), neurotrophin-3 (NT-3) gene expression was suppressed by TPA (12- O-tetradecanoyl phorbol-13-acetate), which induces an AP-1 transcription factor. In the present study, to clarify the mechanism for TPA-mediated downregulation of NT-3 gene expression, effects of cycloheximide and dexamethasone (Dex) on the TPA-mediated downregulation were examined in VSMCs. Pretreatment with cycloheximide, an inhibitor of protein synthesis, or simultaneous treatment with Dex, an inhibitor of AP-1, suppressed the TPA-mediated downregulation of NT-3 gene expression. Furthermore, co-transfection of c- fos and c- jun expression vectors into VSMCs resulted in decrease in the NT-3 gene expression. The present findings suggest that TPA-induced AP-1 de novo synthesis causes the downregulation of NT-3 gene expression in VSMCs.

Bradykinin modulates arginine vasopressin-induced calcium influx in vascular myocytes

We investigated direct, endothelium-independent effects of bradykinin on arginine vasopressin-induced calcium influx in vascular smooth muscle cells. We studied cultured rat vascular smooth muscle cells by using the whole-cell voltage-clamp and calcium fluorescence imaging methods. Exposing cultured vascular smooth muscle cells (A7r5 cell line) to arginine vasopressin (100 nM) produced a transient increase in [Ca 2+] i, followed by a sustained increase in [Ca 2+] i. This was readily reversible ( n=28). At a holding potential of −40 to −60 mV, arginine vasopressin induced a sustained inward current correlated with a sustained increase in [Ca 2+] i. Bradykinin (30 nM to 30 μM) had no effect on arginine vasopressin-induced [Ca 2+] i transients. However, during the sustained phase of increased [Ca 2+] i, bradykinin reversibly attenuated relative fluorescence and inward current in the presence of arginine vasopressin ( n=14). This was concentration dependent and inhibited by [ d-Phe 7]-bradykinin (30 μM), a kinin receptor antagonist. Also, sustained arginine vasopressin-mediated increases in [Ca 2+] i and inward current were attenuated by Ca 2+-free or La 3+-supplemented perfusate but not by nifedipine ( n=5). Conclusions: (1) Bradykinin can attenuate arginine vasopressin-induced and sustained Ca 2+ influx and sustained inward current through a novel endothelium-independent process. (2) The direct effect of bradykinin on arginine vasopressin-induced increases in [Ca 2+] i sustained Ca 2+ influx in vascular smooth muscle cells is concentration dependent and kinin-receptor mediated. (3) Arginine vasopressin-induced sustained [Ca 2+] i elevation correlates with the activation of a dihydropyridine-insensitive, Ca 2+-conducting inward current.

Troglitazone upregulates nitric oxide synthesis in vascular smooth muscle cells.

We investigated the effects of troglitazone on cytokine-stimulated nitric oxide (NO) production in cultured rat vascular smooth muscle cells (VSMC). The increase in NO formation caused by interleukin-1alpha (IL-1) was enhanced by troglitazone in a concentration-dependent manner. Bacterial lipopolysaccharide-stimulated NO synthesis was also increased by troglitazone. The combinations of IL-1, tumor necrosis factor-alpha, or lipopolysaccharide with interferon-gamma (IFN) were strong stimuli for induction of NO synthesis in VSMC, which were further potentiated by the presence of troglitazone. When troglitazone was added at increasing intervals after the stimulation of VSMC with IL-1, the enhancement in NO production decreased as the interval lengthened, suggesting that troglitazone alters NO synthase (NOS) expression by VSMC rather than having a direct affect on VSMC NOS activity. Troglitazone had no effect on IL-1-elicited or IL-1/IFN-elicited nuclear factor-kappaB activity in VSMC. Troglitazone inhibited the degradation of cytokine-induced NOS mRNA. Thus troglitazone appears to enhance IL-1-induced NOS mRNA levels by prolonging its half-life rather than activating its transcription, which is nuclear factor -kappaB-dependent. No expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) was detected in VSMC, and 15-deoxy-D12,14 prostaglandin J2, the natural ligand for the PPARgamma, did not resemble the effect of troglitazone on IL-1-induced NO synthesis. These results indicate that troglitazone upregulates cytokine-stimulated NO synthesis in VSMC through PPARgamma-independent mechanisms. Considering its inhibitory effects on the action of numerous growth factors on VSMC, the direct vascular effects of troglitazone shown in this study may have important implications for prevention of restenosis and possibly atherosclerosis.

Targeting Gbeta gamma signaling in arterial vascular smooth muscle proliferation: a novel strategy to limit restenosis.

Restenosis continues to be a major problem limiting the effectiveness of revascularization procedures. To date, the roles of heterotrimeric G proteins in the triggering of pathological vascular smooth muscle (VSM) cell proliferation have not been elucidated. betagamma subunits of heterotrimeric G proteins (Gbetagamma) are known to activate mitogen-activated protein (MAP) kinases after stimulation of certain G protein-coupled receptors; however, their relevance in VSM mitogenesis in vitro or in vivo is not known. Using adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of Gbetagamma signaling (betaARKct), we evaluated the role of Gbetagamma in MAP kinase activation and proliferation in response to several mitogens, including serum, in cultured rat VSM cells. Our results include the striking finding that serum-induced proliferation of VSM cells in vitro is mediated largely via Gbetagamma. Furthermore, we studied the effects of in vivo adenoviral-mediated betaARKct gene transfer on VSM intimal hyperplasia in a rat carotid artery restenosis model. Our in vivo results demonstrated that the presence of the betaARKct in injured rat carotid arteries significantly reduced VSM intimal hyperplasia by 70%. Thus, Gbetagamma plays a critical role in physiological VSM proliferation, and targeted Gbetagamma inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.

Homocysteine increases nitric oxide synthesis in cytokine-stimulated vascular smooth muscle cells.

Elevated plasma homocysteine levels have been reported to be an independent risk factor for vascular disease. However, there have been no reports concerning the effects of homocysteine on the production of nitric oxide (NO), another modulator of vascular function and proliferation, by the vascular smooth muscle. We investigated the effects of homocysteine on NO synthesis by measuring the production of nitrite, a stable metabolite of NO, in cultured rat vascular smooth muscle cells (VSMCs). Incubation of cultures with interleukin (IL)-1beta 10 ng/mL for 24 hours caused a significant increase in nitrite generation. The IL-1beta-induced nitrite production by VSMCs was significantly increased by homocysteine in a dose-dependent manner. This effect of homocysteine was significantly inhibited in the presence of NG-monomethyl-L-arginine or actinomycin D. The homocysteine-induced nitrite production was accompanied by increased inducible NO synthase mRNA and protein accumulation. Cysteine, glutathione, or hydrogen peroxide also increased nitrite accumulation in IL-1beta-stimulated VSMCs. Coincubation with the radical scavenger catalase or superoxide dismutase markedly reduced homocysteine-induced nitrite accumulation. Homocysteine enhances NO synthesis in IL-1beta-stimulated VSMCs, and oxidative products are involved in the effect of homocysteine.