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

Kyoto Prefectural University of Medicine, Kioto, Kyōto, Japan
Atherosclerosis (Impact Factor: 3.99). 03/2003; 166(2):213-22. DOI: 10.1016/S0021-9150(02)00329-5
Source: PubMed


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.

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    • "In contrast, statins up-regulate IL-1-induced iNOS promoter activity in VSMCs (Chen et al., 2000; Muniyappa et al., 2000), airway epithelial cells (Kraynack et al., 2002), fibroblasts (Hausding et al., 2000), and cardiac myocytes (Ikeda et al., 2001). This stimulatory action might occur through the inhibition of the geranylgeranylation of Rho family proteins and downstream Rhoassociated kinase (ROCK) signaling (Chen et al., 2000; Muniyappa et al., 2000; Yamamoto et al., 2003). "
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    ABSTRACT: An optimal level of NO has protective effects in atherosclerosis, whereas large amounts contribute to septic shock. To study how statins, the potent inhibitors of cholesterol synthesis, regulate NO in the vascular wall, we determined their effects on interleukin-1beta (IL-1beta)- and lipopolysaccharide (LPS)-induced NO production in aortic vascular smooth muscle cells (VSMCs). Compared with the large amounts of NO and inducible NO synthase (iNOS) protein expression induced by LPS, the responses of IL-1beta were modest. Various statins were found to inhibit LPS-induced iNOS expression and NO production, although they potentiated IL-1beta responses. In addition, fluvastatin increased IL-1beta-induced p65 nuclear translocation and nuclear factor kappaB (NF-kappaB) activity, although it inhibited those induced by LPS. To address the role of small G proteins in statin's actions, farnesyl transferase inhibitors [alpha-hydroxyfarne-sylphosphonic acid and (2S)-2-[[(2S)-2-[(2S,3S)-2-[(2R)-2-amino-3-mercaptopropyl]amino]-3-methylpentyl]oxy]-1-oxo-3-phenylpropyl]amino]-4-(methylsulfonyl)-butanoic acid 1-methylethyl ester (L-744382)], Rac inhibitor (NSC23766), and Rho-associated kinase (ROCK) inhibitor [N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride (Y-27632)] were used. We found that Y-27632 potentiated IL-1beta-induced iNOS expression, p65 nuclear translocation, IkappaB kinase (IKK), and NF-kappaB activation, whereas it had minimal effects on LPS-induced responses. In contrast, farnesyl transferase inhibitors blocked iNOS protein expression induced by LPS and IL-1beta, whereas NSC23766 had no effect. Further studies showed that LPS down-regulated Rho and ROCK activity, whereas IL-1beta increased them, suggesting a negative role of Rho and ROCK signaling, which is regulated in contrary manners by IL-1beta and LPS, in IKK/NF-kappaB activation. Through abrogating this negative signaling, statins differentially regulate iNOS expression induced by LPS and IL-1beta in VSMCs. These differential actions of statins on iNOS gene regulation might provide an additional explanation for the pleiotropic beneficial effects of statins.
    Preview · Article · Apr 2006 · Molecular Pharmacology
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    • "In the last 10 years of drug development, the mevalonate pathway has become an important target for pharmacological research. This pathway seems to play a key role in cellular proliferation and transformation, providing cells with a number of essential products, including sterols, steroids, ubiquinone, isoprenoids, etc. Adjustment of this pathway has provided various efficient drugs, including treatments for cardiac disease with the statin family of drugs and cancer with farnesyl transferase inhibitors (Karp et al., 2001; Yamamoto et al., 2003). Until now, the drugs acting on this pathway had been first screened by enzymatic in vitro studies . "
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    ABSTRACT: We describe the development of a cell system for in vivo screening of inhibitors of the mevalonate pathway. To this aim, we have constructed a bicistronic mRNA, transcribed from a constitutive cytomegalovirus promoter, containing the Renilla reniformis luciferase RNA open reading frame sequence as first cistron and the Firefly luciferase RNA sequence as a second cistron. The intercistronic space is made of the R17 binding sequence of the bacteriophage R17 protein. A chimeric protein able to bind to a specific sequence in the hairpin and to induce internal ribosome entry in the RNA switches on translation of the second cistron. This chimeric protein is made up of the bacteriophage RNA binding domain (R17) fused to the ribosome recruitment core of the eIF-4G1 eukaryotic translation initiation factor and to the CAAX box of H-Ras addressing the protein to the plasma membrane where it is not efficient. Internal ribosome entry upstream of the Firefly cistron is therefore under the dependence of the mevalonate pathway inhibitors. Indeed, products that are able to inhibit protein farnesylation rescue the cytoplasmic location of the R17-eIF-4G-CAAX protein, which once more becomes a translation factor for the expression of the second cistron. To exemplify the system, the present work checks the ability of various antiestrogens to interfere with the mevalonate pathway. It seems that pure antiestrogen, able to selectively bind the estrogen receptor, is unable to switch on the second Firefly cistron although selective antiestrogen-binding-site ligands are able to do so.
    Preview · Article · Jul 2005 · Molecular Pharmacology
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    ABSTRACT: Numerous epidemiological studies indicate that flavonoid intake as part of a balanced diet confers beneficial health effects in man, including improved cardiovascular function, reduced incidence of cancer and amelioration of symptoms associated with inflammatory disorders (Boots et al., 2008). A recent area of interest that may be fruitful is the study of anti-inflammatory effects of flavonoids in combination with statins. Porcine coronary artery (PCA) segments were incubated overnight at 37°C in modified Krebs-Henseleit solution with or without 1µg/ml lipopolysaccharides (LPS), with either (0.1–10µM) quercetin, or 10µM quercetin 3′-suphate and 10µM quercetin-3-glucuronide, or with (0.01-10µM) epicatechins, 10µM catecchin and10µM epigallocatechin gallate. (0.03-3µM) simvastatins and 10µM pravastatin are also used in this study. In addition, since many quercetin-rich foods also contain significant amounts of myricetin, this flavonoid has also been examined. After 16 to 18 hours, segments were prepared for isometric tension recording in Krebs-Henseleit solution. The segments were then exposed to cumulatively increasing concentrations of KCl and then U46619. Responses are shown as milliNewton or calculated as the concentration causing 50% of the maximum effect (-log EC50). For nitrite measurement, segments of the PCA were incubated in DMEM at 37°C for 24 hours, with or without 1μg/mL LPS. The nitrite content (nmol) of the bathing medium was determined by spectrophotometry using the Griess reaction, while inducible nitric oxide synthase was identified immunohistochemically. Differences between mean values were assessed by ANOVA (post-hoc Dunnett test). Prolonged exposure to LPS caused hyporesponsiveness of the PCA associated with increase in nitrite production by a mechanism that appears to involve the induction of nitric oxide synthase. Nitrite content of the incubation medium increased 3 to 10-fold following exposure to LPS and inducible nitric oxide synthase was detected in the adventitia. The results indicated that all of the tested flavonoids and statins are able to suppress LPS-induced changes in vascular responses, nitrite production and expression of inducible nitric oxide synthase. While 10µM myricetin was inactive. In conclusion I have demonstrated that quercetin, and its principal human metabolites and catechins oppose pro-inflammatory events in both endothelial cells and vascular smooth muscle cells. Possibly through a mechanism involving inhibition of NFkB. Since pre-treatment of the PCA with statins reduced LPS-induced changes in vasoconstrictor responses, suppressed the induction of nitric oxide synthase caused by LPS and the associated increase in nitrite production. It is unlikely that the effect of the statin involves direct inhibition of NOS. These findings are consistent with clinical studies suggesting that prior use of statins may afford protection against bacterial sepsis.
    Full-text · Article · Jul 2011
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