Fluvastatin upregulates inducible nitric oxide synthase expression in cytokine-stimulated vascular smooth muscle cells

Nitric oxide (NO) production by inducible NO synthase (iNOS) may play an important role in the pathogenesis of atherosclerosis. Although fluvastatin has been shown to reduce progression of atherosclerosis, it is not known whether it regulates iNOS expression. We investigated the effects of fluvastatin on iNOS expression and subsequent NO synthesis in vascular smooth muscle cells (VSMCs) and the mechanism by which fluvastatin exerts its effects. Fluvastatin significantly increased interleukin-1ss (IL-1ss)-induced nitrite production by VSMCs in a time-dependent (0 to 24 hours) and dose-dependent (10(-)(8) to 10(-)(5) mol/L) manner. Increased nitrite production by fluvastatin was accompanied by increased iNOS mRNA and protein accumulation. IL-1ss induced nuclear factor-kappaB activation in VSMCs, which was not affected by fluvastatin. Exogenous mevalonate significantly prevented the stimulatory effect of fluvastatin on nitrite production. Cotreatment with geranylgeranyl-pyrophosphate also reversed the effect of fluvastatin. Furthermore, both Rho inhibitor C3 exoenzyme and Rho kinase inhibitor Y-27632 significantly increased IL-1ss-induced nitrite accumulation in VSMCs. These results demonstrated that fluvastatin upregulates iNOS expression and subsequent NO formation in rat VSMCs through inhibition of Rho.     

beta-very low density lipoprotein enhances inducible nitric oxide synthase expression in cytokine-stimulated vascular smooth muscle cells

beta-very low-density lipoprotein (beta-VLDL), a collective term for VLDL and chylomicron remnants, has recently shown to potently promote the development of atherosclerosis. However, the effects of beta-VLDL on the accumulation of nitric oxide (NO) and the expression of inducible NO synthase (iNOS) in vascular smooth muscle cells (VSMC) have not been determined. In this study, we measured the accumulation of nitrite, stable metabolite of NO and examined the expression of iNOS protein and mRNA using Western blotting and RT-PCR, respectively, in VSMC. NF-kappaB activation in VSMC was examined by gel retardation assay. Incubation of cell cultures with interleukin-1beta (IL-1beta) for 24 h caused a significant increase in nitrite accumulation. Although beta-VLDL alone did not increase nitrite accumulation in unstimulated VSMC, beta-VLDL significantly enhanced nitrite accumulation in IL-1beta-stimulated VSMC in a time- and dose-dependent manner. beta-VLDL-induced nitrite accumulation in IL-1beta-stimulated VSMC was accompanied by an increase in iNOS protein and mRNA expression. In addition, IL-1beta induced NF-kappaB activation in VSMC, an effect that was increased by the addition of beta-VLDL. Use of specific tyrosine kinase inhibitor herbimycin A, genistein, or PP2 (Src family kinase inhibitor) indicated that tyrosine kinases are required for IL-1beta-stimulated and beta-VLDL-enhanced nitrite accumulation, while specific inhibition of ERK1/2 or p38-MAP kinase had no effects. Our results suggest that beta-VLDL enhances iNOS expression and nitrite accumulation in IL-1beta-stimulated VSMC through tyrosine kinase(s)-dependent mechanisms.     

高血压患者血清抗AT1-受体抗体对血管平滑肌细胞p44/p42丝裂原活化蛋白激酶表达的影响

目的已有研究表明抗血管紧张素Ⅱ受体1型(AT1-受体)自身抗体具有提高心肌细胞收缩频率的受体激动剂样作用,本研究观察该抗体对血管平滑肌细胞p44/p42丝裂原活化蛋白(MAP)激酶的影响,探讨高血压患者血清中抗AT1-受体自身抗体的生物学活性及其作用机制.方法我们从16例ELISA检测抗AT1-受体阳性的高血压患者血清中提取纯化了免疫球蛋白,用Western等方法检测抗体对培养大鼠血管平滑肌细胞的作用.结果发现抗AT1抗体可诱导平滑肌细胞p44/p42丝裂原活化蛋白(MAP)激酶的表达,与血管紧张素Ⅱ相似,并且这一效应可被AT1受体阻断剂氯沙坦抑制.结论P44/p42 MAP激酶是平滑肌细胞增殖肥厚的重要信号分子,抗AT1-受体抗体通过受体介导血管平滑肌细胞p44/p42 MAP激酶的表达,提示抗AT1-受体抗体具有引起血管平滑肌细胞肥厚增殖的作用.     

HMG-CoA reductase inhibitor enhances inducible nitric oxide synthase expression in rat vascular smooth muscle cells; involvement of the Rho/Rho kinase pathway

Little is known about the mechanism by which HMG-CoA reductase inhibitors affect inducible nitric oxide synthase (iNOS) expression. We investigated the effect of HMG-CoA reductase inhibitor cerivastatin on iNOS expression in cultured rat vascular smooth muscle cells (VSMCs). Quiescent VSMCs were incubated with or without various concentrations of drugs as follows: cerivastatin, C3 exoenzyme or Y-27632. Then, pretreated VSMCs were stimulated by a vehicle or interleukin (IL)-1beta (10 ng/ml). Treatment of VSMCs with cerivastatin (10(-7)-10(-5) mol/l), which inhibits isoprenylation of Rho and other small G proteins, significantly increased nitrite/nitrate (NOx) production and upregulated the expression of iNOS mRNA in IL-1beta-stimulated VSMCs. This effect of cerivastatin was abolished by cotreatment with mevalonate (2x10(-4) mol/l) or geranylgeranyl-pyrophosphate (GGPP) (10(-5) mol/l), but not by farnesyl-pyrophosphate (10(-5) mol/l). Furthermore, C3 exoenzyme (50 microg/ml), an inactivator of Rho protein, and Rho kinase inhibitor Y-27632 (10(-5) mol/l) also enhanced NOx production and the expression of iNOS mRNA in IL-1beta-stimulated VSMCs. Immunocytochemical study revealed that cerivastatin, C3 exoenzyme and Y-27632 did not affect the nuclear translocation of nuclear factor-kappaB in IL-1beta-stimulated VSMCs. Our study suggests that cerivastatin stimulates iNOS expression in IL-1beta treated VSMCs by its inhibitory effect on Rho/Rho kinase pathway. In addition, this effect of cerivastatin, by enhancing iNOS expression, may contribute to the prevention of restenosis after percutaneous coronary intervention and protect against atherothrombosis.     

Lipoxygenase products regulate nitric oxide and inducible nitric oxide synthase production in interleukin-1beta stimulated vascular smooth muscle cells.

In cultured vascular smooth muscle cells (VSMCs), interleukin-1beta (IL-1beta) stimulates inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. IL-1beta also activates phospholipase A2 (PLA2), and induces lipoxygenase (LOX) and cyclooxygenase-2 (COX-2). The present study investigated whether these metabolites are involved in the regulation of IL-1beta-induced NO production and iNOS expression. Pretreatment with ONO-RS-082, the secretory PLA2 (sPLA2) inhibitor, at 1 to 10 micromol/l reduced IL-1beta-stimulated nitrite production and iNOS expression. Nordihydroguaiaretic acid (NDGA, 1 to 10 micromol/l), the LOX inhibitor, also reduced IL-1beta (10 ng/ml)-stimulated nitrite production and iNOS expression in a dose-dependent manner. Exogenous 12(S)-hydroxyeicosatetraenoic acids (HETE) enhanced the IL-1beta-stimulated nitrite production and iNOS expression. On the other hand, the COX inhibitors, indomethacin and NS-398, had little effect on nitrite production or iNOS expression. These results suggest that LOX products play important roles in the regulation of stimulus-induced NO production in VSMCs.     

An anti-ulcer drug, geranylgeranylacetone, suppresses inducible nitric oxide synthase in cultured vascular smooth muscle cells

OBJECTIVE: Geranylgeranylacetone (GGA) is commonly used as an anti-ulcer drug. If GGA affects inducible nitric oxide synthase (iNOS) in the vascular tissue, it could influence disease progression in coronary arteries. We investigated the effects of the anti-ulcer drug GGA on iNOS activity in vascular smooth muscle cells. METHODS: We measured the production of nitrite, a stable metabolite of nitric oxide, in cultured rat vascular smooth muscle cells with the Griess reagent. iNOS protein and mRNA expressions were assayed by western blotting and northern blotting, respectively. The levels of nuclear factor (NF)-kappaB proteins in nuclear extracts were analyzed by gel retardation assay. Heat shock protein 70, a cytoprotective molecule, was evaluated by western blotting. RESULTS: Incubation of cultures with interleukin-1beta for 24 h caused a significant increase in nitrite generation. Interleukin-1beta-induced nitrite production by vascular smooth muscle cells was significantly suppressed by GGA in a dose-dependent manner. GGA-suppressed nitrite production was accompanied by decreased iNOS mRNA and protein accumulations. GGA by itself did not modulate the basal level of nitrite production. Interleukin-1beta induced NF-kappaB activation in vascular smooth muscle cells, and the addition of GGA further inhibited this NF-kappaB activation. GGA itself induced heat shock protein 70 expression in a dose-dependent manner. CONCLUSION: These findings demonstrated that GGA suppresses iNOS expression in cytokine-stimulated cultured vascular smooth muscle cells partially through the suppression of NF-kappaB activation, suggesting that GGA may modulate the pathophysiology of cardiovascular diseases including atherosclerosis. In addition, this effect may be associated with heat shock protein 70 production by GGA.     

Role of p42/p44 mitogen-activated-protein kinase and p21waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide

The purpose of this study was to determine the involvement of the p42/p44 mitogen-activated protein kinase (MAPK) pathway and induction of p21(waf1/cip1) in the antiproliferative effects of nitric oxide (NO) on rat aortic smooth muscle cells (RASMC). NO, like alpha-difluoromethylornithine (DFMO), interferes with cell proliferation by inhibiting ornithine decarboxylase (ODC) and, therefore, polyamine synthesis. S-nitroso-N-acetylpenicillamine or (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate inhibited RASMC growth at concentrations as low as 3 microM, and DFMO elicited effects at concentrations of 100 microM or greater. The cytostatic effect of NO and DFMO was prevented by the MAPK kinase 1/2 inhibitors PD 098,059 or U0126. This finding suggests that the p42/p44 MAPK pathway is involved in the inhibition of RASMC proliferation by NO. Western blot analysis revealed that treatment of RASMC with NO or DFMO leads to activation of p42/p44 MAPK and induction of p21(waf1/cip1). This effect was prevented by MAPK kinase 1/2 inhibitors, suggesting that induction of p21(waf1/cip1) depended on activation of p42/p44. Moreover, activation of p42/p44 and induction of p21(waf1/cip1) were prevented by exogenous putrescine but not ornithine, suggesting this effect was due to the inhibition of ODC by NO or DFMO. Finally, activation of p42/p44 MAPK and induction of p21(waf1/cip1) were cGMP-independent. Neither 1H-(1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one nor zaprinast influenced the cytostatic effect of NO or DFMO or their ability to activate these signal transduction pathways. These observations suggest that inhibition of ODC and accompanying putrescine production are the underlying mechanisms by which NO and DFMO activate the MAPK pathway to promote induction of p21(waf1/cip1) and consequent inhibition of cell proliferation.     

p38 mitogen-activated protein (MAP) kinase but not p44/p42 MAP kinase is involved in prostaglandin E1-induced vascular endothelial growth factor synthesis in osteoblasts

We investigated the mechanism underlying vascular endothelial growth factor (VEGF) synthesis stimulated by prostaglandin E1 (PGE1) in osteoblast-like MC3T3-E1 cells. PGE1 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. SB203580, a specific inhibitor of p38 MAP kinase, inhibited the PGE1-stimulated VEGF synthesis as well as PGE1-induced phosphorylation of p38 MAP kinase. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, which reduced the PGE1-induced phosphorylation of p44/p42 MAP kinase, had little effect on the VEGF synthesis stimulated by PGE1. AH-6809, an antagonist of the subtypes of the PGE receptor, EP1 and EP2, or SC-19220, an antagonist of EP1 receptor, did not inhibit the PGE1-induced VEGF synthesis. H-89, an inhibitor of cAMP-dependent protein kinase, and SQ22536, an inhibitor of adenylate cyclase, reduced the VEGF synthesis induced by PGE1. Cholera toxin, an activator of G(s), and forskolin, an activator of adenylate cyclase, induced VEGF synthesis. SB203580 and PD169316, another specific inhibitor of p38 MAP kinase, reduced the cholera toxin-, forskolin- or 8bromo-cAMP-stimulated VEGF synthesis. However, PD98059 failed to affect the VEGF synthesis stimulated by cholera toxin, forskolin or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP). SB203580 reduced the phosphorylation of p38 MAP kinase induced by forskolin or 8bromo-cAMP. These results strongly suggest that p44/p42 MAP kinase activation is not involved in the PGE1-stimulated VEGF synthesis in osteoblasts but that p38 MAP kinase activation is involved.     

Expression of inducible nitric oxide synthase in cultured smooth muscle cells from rat mesenteric lymphatic vessels

OBJECTIVE: The objective was to devise a method for establishing cultures of rat mesenteric lymphatic vessel smooth muscle cells (LSMC) and to investigate if inducible nitric oxide synthase (iNOS) expression could be activated in LSMC treated with bacterial lipopolysaccharide (LPS). METHODS: LSMC were successfully grown from explanted rat lymphatic microvessels and maintained by subculture. Treatment of LSMC for 24 h with LPS (1-100 microg/mL) activated iNOS protein induction, associated with (1) assay of increased nitrite concentrations in the medium representing cellular nitric oxide synthesis, and (2) demonstration of iNOS in cell extracts by Western blotting. RESULTS: The protein synthesis inhibitor cycloheximide (10 microM) blocked both LPS-induced nitrite formation and iNOS protein expression in LSMC. 1400 W (1 microM), a selective iNOS inhibitor, prevented LPS-induced nitrite formation but not iNOS expression. As well as induction of iNOS by LPS, "constitutive" iNOS was present in some cultures, producing nitrite in amounts that were also subsequently reduced after cell treatment with 1400 W. CONCLUSION: Rat mesenteric LSMC produce nitrite and express iNOS in response to bacterial LPS. Cultured LSMC may provide a useful model for studying mechanisms of iNOS induction in relation to possible influences of iNOS upon lymphatic vessel function.     

NAD(P)H oxidase activation by angiotensin II is dependent on p42/44 ERK-MAPK pathway activation in rat's vascular smooth muscle cells

OBJECTIVE: To determine whether the activation of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase and the increase of superoxide anion production by angiotensin II is dependent upon the activation of the ERK-MAPK pathway. METHODS: Hypertension was induced in Sprague-Dawley rats by infusing angiotensin II (200 ng/kg per min) through osmotic pumps for 12 days. The effects of treatments including an angiotensin II type 1 (AT(1)) blocker losartan (20 mg/kg per day), a tyrosine kinase inhibitor genistein (1.6 microg/kg per min), a specific ERK-MAPK inhibitor, PD98059 (2 mg/kg per day) and an antioxidant alpha-lipoic acid (500 mg/kg of chow) were evaluated during angiotensin infusion. The aortic superoxide anion production, the ERK-MAPK pathway activity and the systolic blood pressure (SBP), were measured following those treatments. RESULTS: Increases in the concentration of the superoxide anion (1622 to 3719 cpm), in NAD(P)H activity (107%) and in the ERK-MAPK activity (3.6-fold) in the aorta as well as a rise in the arterial pressure (136 to 184 mmHg) were observed 12 days after initiating the treatments (P < 0.05). When the angiotensin-treated rats were treated either with losartan, genistein, PD98059 or alpha-lipoic acid, increases in superoxide anion production, in NAD(P)H oxidase activity, in ERK-MAPK activity and in blood pressure were attenuated. A correlation between the superoxide anion production and the ERK-MAPK activity was also observed. CONCLUSIONS: The present study suggests that the NAD(P)H-dependent increase of the superoxide anion production in the vascular tissue following a treatment with angiotensin II is dependent on the activation of the ERK-MAPK pathway.     

17Beta-estradiol decreases nitric oxide synthase II synthesis in vascular smooth muscle cells

Several studies have provided evidence for a direct effect of 17beta-estradiol on vessel wall via interaction with the constitutively expressed nitric oxide synthase (NOS) by endothelium. The aim of the present study was to investigate the effect of 17beta-estradiol on inducible NOS (NOS II) in primary culture of smooth muscle cells (SMC) from rat aorta. We here prove that 17beta-estradiol decreases the content and activity of NOS II in SMC. This effect appears to be the consequence of ER activation, because: 1) ER alpha and ER beta are expressed in rat aorta SMC grown in culture; 2) low concentrations of hormone modulate NOS II activity; 3) the specific ER alpha antagonist ICI182,780 completely blocks 17beta-estradiol effect. On the other hand, progesterone is deprived of any effect on NOS II content or activity, proving the specificity of 17beta-estradiol effect. In addition, we show that 17beta-estradiol can counteract the increase in NOS II activity following cytokine treatment. The observation could indicate a novel mechanism for the protective effects exerted by these hormones in cardiovascular diseases and atherosclerosis in particular.