Antiinflammatory and Antiarteriosclerotic Actions of HMG-CoA Reductase Inhibitors in a Rat Model of Chronic Inhibition of Nitric Oxide Synthesis
Recent studies suggest that some of the beneficial effects of 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) may be due to their cholesterol-lowering independent effects on the blood vessels. Chronic inhibition of endothelial nitric oxide (NO) synthesis by oral administration of Nω-nitro-l-arginine methyl ester (L-NAME) to rats induces early vascular inflammation as well as subsequent arteriosclerosis. The aim of the study is to test whether treatment with statins attenuates such arteriosclerotic changes through their cholesterol-lowering independent effects. We investigated the effect of statins (pravastatin and cerivastatin) on the arteriosclerotic changes in the rat model. We found that treatment with statins did not affect serum lipid levels but markedly inhibited the L-NAME–induced vascular inflammation and arteriosclerosis. Treatment with statins augmented endothelial NO synthase activity in L-NAME–treated rats. We also found the L-NAME induced increase in Rho membrane translocation in hearts and its prevention by statins. Such vasculoprotective effects of statins were suppressed by the higher dose of L-NAME. In summary, in this study, we found that statins such as pravastatin and cerivastatin inhibited vascular inflammation and arteriosclerosis through their lipid-lowering independent actions in this model. Such antiarteriosclerotic effects may involve the increase in endothelial NO synthase activity and the inhibition of Rho activity.
- endothelium-derived factors
- monocyte chemoattractant protein-1
- nitric oxide synthase
Recent studies suggest that 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) reduce the incidence of myocardial infarctions and ischemic strokes.1,2 The beneficial effects of statins are likely to result from stabilization of unstable atheroma prone to rupture, because cholesterol-lowering therapy with statins increases markers of plaque stability.3,4 Although such effects of statins are predominantly attributed to their lipid-lowering effects, subgroup analysis of the data from clinical trials suggests that there may be beneficial effects independent of their cholesterol-lowering effects.1,2,5 Endothelium-dependent nitric oxide (NO)-mediated vasodilatation has been demonstrated to improve at the early phase of statin treatment in humans.6–8 It is recently reported that addition of statins to cultured human endothelial cells increases endothelial-type NO synthase expression and activity.9 Thus, one may hypothesize that some of the beneficial effects of statins may be due to their cholesterol-lowering independent effects on endothelial cells. However, it is unclear whether statins attenuate cardiovascular inflammation and arteriosclerosis in vivo through their cholesterol-lowering independent effects.
Endothelium-derived NO has been recognized to be an antiinflammatory and antiarteriosclerotic molecule. Mice lacking endothelial-type NO synthase exhibit hypertension and an enhanced vascular remodeling in response to injury.10 We recently reported that chronic inhibition of NO synthesis by administration of Nω-nitro-l-arginine methyl ester (L-NAME) induces early inflammation (monocyte infiltration into the coronary vessels associated with induction of monocyte chemoattractant protein-1 [MCP-1] expression) and subsequent arteriosclerosis (medial thickening and perivascular fibrosis) in the rats.11,12 The importance of our observation is supported by the well-recognized concept that the adhesion of mononuclear cells to, and their infiltration into, the blood vessel wall have been assumed to be crucial early events of arteriosclerosis.13
Such rat model may be useful to investigate lipid-lowering independent antiarteriosclerotic effects of statins, because administration of statins even at high doses to normal rats does not change serum cholesterol levels.14 Accordingly, in the present study, we used this rat model to investigate the antiarteriosclerotic actions of statins.
Materials and Methods
Animals and Experimental Protocol
The present study protocol was reviewed and approved by the Committee on Ethics on Animal Experiments, Kyushu University Faculty of Medicine, and the experiments were conducted according to the Guidelines for Animal Experiments of Kyushu University Faculty of Medicine. Twenty-week-old male Wistar-Kyoto rats were obtained from an established colony at the Animal Research Institution of Kyushu University Faculty of Medicine.
Several groups of 20-week-old male Wistar-Kyoto rats were studied. The control group received untreated chow and drinking water. L group received L-NAME in drinking water (1 mg/mL). Lx+Py group received L-NAME in drinking water (1.0 or 1.8 mg/mL) and pravastatin sodium (50, 100, or 250 mg/kg per day, dissolved in 5% carboxymethyl cellulose sodium solution and given by gavage). Lx+Cy group received L-NAME in drinking water and cerivastatin sodium intraperitoneally (1, 2, or 10 mg/kg per day). Treatment with statins was started 2 days before L-NAME administration was begun. On day 3 of L-NAME administration, systolic blood pressure (the tail-cuff method) was measured. Then the rats were anesthetized and euthanized for morphometric, immunohistological, and biological analyses. Furthermore, some rats in the control, L-NAME, L1.0+P100, and L1.0+C2 groups were euthanized on day 28 for morphometric analysis. Pravastatin sodium and cerivastatin sodium were generously provided by Sankyo Co, Ltd (Tokyo) and Bayer Yakuhin, Ltd (Osaka), respectively.
Serum total cholesterol, triacylglycerol, and HDL cholesterol levels were determined with commercially available kits from Wako Pure Chemicals. Serum levels of L-NAME and Nω-nitro-l-arginine (L-NNA) was measured by postcolumn fluorometric detection system for liquid chromatography.15 Serum NOx (NO2 plus NO3) concentration was measured with use of a fluorometric assay kit (Wako). Serum statin concentrations were measured with use of high-performance liquid chromatography/mass spectrometry.16 The angiotensin-converting enzyme (ACE) activity was measured in cardiac tissues by fluorometric assay as described.11,17 Aortic NOx-generating capacity was measured by the Greiss method as described.12
Histopathology and Immunohistochemistry
Histopathology and Immunohistochemistry were performed as described.11 Some sections were subjected to immunostaining using antibodies against rat macrophage/monocytes (ED-1, Serotec, Oxford, UK), proliferating cell nuclear antigen (PCNA) (Dako A/S, Glostrup, Denmark), endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) (Transduction Laboratories), or nonimmune IgG (Zymed Laboratory Inc). Cell enumeration was performed as described.11,12 Evaluation of the medial thickness and perivascular fibrosis of coronary arteries on day 28 was performed as previously described.11
Northern Blot Analysis
Northern blot hybridization was performed as described.12 A rat MCP-1 cDNA probe,12 rat transforming growth factor-β1 (TGF-β1) probe,18,19 and a mouse GAPDH cDNA (American Type Culture Collection, Manassas, Va) probe were used.
Western Blot Analysis
Proteins were prepared from the aorta and heart and separated on SDS-PAGE as described.20 Immunoblotting was performed using monoclonal antibodies to eNOS, iNOS (Transduction Laboratories), and to RhoA (Santa Cruz Biotechnology Inc). Immunodetection was accomplished using appropriate horseradish peroxidase-linked secondary antibody and the enhanced chemiluminescence (ECL) kit (Amersham Corp). Membrane and cytosolic proteins in cardiac tissue were isolated as described previously.20 Immunoblotting for RhoA in membrane and cytosolic fraction was performed as described above.
Data are expressed as mean±SE. Statistical analysis of differences was compared by ANOVA and Bonferroni’s multiple comparison test. A level of P<0.05 was considered statistically significant.
Systolic Arterial Pressure and Serum Lipids and L-NA
The L group showed a progressive rise in systolic arterial pressure throughout the study (Table 1). This increase in systolic blood pressure showed no significant change in all groups with statin treatment on day 3, whereas it was normalized on days 7, 14, 21, and 28 of treatment in the L1.0+P100 and L1.0+C2 groups.
Serum total, HDL cholesterol, and triacylglycerol concentrations in the statin-treated rats were not statistically different from those seen in control or L-NAME–treated rats (Table 2). Serum L-NAME and L-NNA concentrations were measured on day 3 of L-NAME treatment. L-NNA was not detected in the serum of control rats. In the L group, L-NNA concentration was 111±15 mmol/L, which was not significantly different from those in the L1.0+P100 or L1.0+C2 groups (Table 2). Serum L-NAME was below detection level (<0.02 μmol/L) in all groups (data not shown), indicating conversion of L-NAME to L-NNA after absorption.21
Effect of Statins on Local ACE Activity
Cardiac ACE activity was increased in the L group on day 3. This increased ACE activity was not influenced by treatment with statins (Table 2).
Effects of Statins on Inflammatory and Proliferative Changes
We found no evidence of inflammation in the control rats (Figure 1A). In contrast, on day 3, attachment of mononuclear leukocytes to the endothelium of coronary vessels and their infiltration into the vessel wall were noted in the L group (Figure 1A). A marked mononuclear leukocyte infiltration into the perivascular area and the myocardial interstitial area was also observed in this group. The majority of these cells were found to be ED-1–positive monocytes (Figure 1A). Nuclear staining with PCNA antibody was observed in some endothelial cells, vascular smooth muscle cells in the media, monocytes, and myofibroblast-like cells (Figure 1A).
When ED-1–positive monocytes or PCNA-positive cells were counted by a single observer who was blind to the treatment protocols, the number of immunopositive cells per section was significantly greater in the L group than in the control group (Figure 2). The increases in ED-1–positive cells and PCNA positive cells were significantly reduced by treatment with statins in a dose-dependent manner (Figures 2A through 2D).
Effects of Statins on MCP-1 and TGF-β1 Gene Expression
When cardiac MCP-1 and TGF-β1 mRNA levels were examined by Northern blot analysis in the control, L-NAME, L1.0+P100, and L1.0+C2 groups on day 3, both MCP-1 and TGF-β1 mRNA levels were significantly increased in L group. Such increased gene expressions were significantly reduced in the L1.0+P100 and L1.0+C2 groups (Figure 3).
Effect of Statins on NO Synthase Expression and Activity
NOx-generating capacity from the aortic segments was examined in the control, L, L1.0+P100, and L1.0+C2 group. NOx-generating capacity in the thoracic aorta was markedly decreased in the L group. The blunted NOx-generating capacity by L-NAME was significantly restored by statin treatment (Figure 4A). To provide a direct evidence suggesting restoration of NO production in vivo, serum NOx was measured by a fluorometric assay. Serum NOx concentration was significantly decreased in the L group, treatment of statins markedly restored serum NOx level in the L1.0+P100 and L1.0+C2 groups (Figure 4B).
eNOS and iNOS protein expression was examined by Western blot analysis. Compared with the control group, aortic eNOS protein expression did not change in the L group (Figure 4C). In contrast, aortic eNOS protein level was significantly higher in the L1.0+P100 and L1.0+C2 groups than in the control or L groups (Figure 4C). Aortic iNOS protein expression could not be detected in any groups (data not shown). In the heart, there was no difference in eNOS protein expression between the control and L groups, Statins increased eNOS protein level in L-NAME–treated rats (Figure 4D). iNOS protein could not be detected in the heart of any groups (data not shown).
Immunohistochemical study showed strong iNOS inductions mainly in lesional inflammatory cells in the L group (Figure 1C). Statins markedly reduced such iNOS protein level as assessed by immunohistochemistry (Figure 1C). In contrast, endothelial NOS immunoreactivity in coronary arteries was similar in control and L group. Statins markedly increased eNOS immunoreactivity in the endothelial layer of coronary arteries (Figure 1C).
Effects of Statins on RhoA Membrane Translocation
Immunoblot for RhoA (a major isoform of Rho) in the membrane and cytosolic fraction of cardiac tissue exhibited that L-NAME significantly increased membrane translocation of RhoA on day 3. Cotreatment with statins significantly decreased membrane translocation of RhoA with a concomitant increase of RhoA in the cytosol (Figure 4E).
Effects of Statins on Vascular Remodeling on Day 28
The increases in medial thickening (the wall-to-lumen ratio) and perivascular fibrosis of coronary arteries seen in the L group was inhibited in the L1.0+P100 and L1.0+C2 groups (Figures 1B and 5⇓). In addition, cardiac fibrosis was also inhibited in the L1.0+P100 and L1.0+C2 groups (data not shown).
Serum Statin Concentrations
Serum pravastatin concentration was 7.4±0.8 ng/mL in the L1.0+P100 group. Serum maximum concentrations of pravastatin after oral administration at 20 mg in healthy human subjects are 26.1 to 38.4 ng/mL.22 Serum cerivastatin concentration was 2.1±3.1 ng/mL in the L1.0+C2 group. Serum maximum concentrations of cerivastatin after oral administration at 0.3 or 0.4 mg in healthy human subjects are 3.7 and 6.3 ng/mL, respectively.23
Effects of High-Dose L-NAME on the Vasculoprotective Effects of Statins
To examine if the beneficial effects of statins are due to restoration of eNOS activity, we determined if the higher dose of L-NAME could block the vasculoprotective effects of statins. The higher dose of L-NAME suppressed statin-induced restoration of NO production (Figure 4B), statin-induced inhibition of vascular inflammatory/proliferative changes (Figures 2E and 2F), and statin-induced inhibition of RhoA translocation to membrane (data not shown) on day 3 of L-NAME administration. The high dose of L-NAME did not affect eNOS expression in both the heart and aorta (data not shown). Also, the high dose of L-NAME attenuated statin-induced inhibition of vascular remodeling on day 28 (data not shown).
We have demonstrated herein that (1) treatment with statins such as pravastatin and cerivastatin inhibits early vascular inflammation and (2) statins also inhibit proliferative changes and the development of cardiovascular remodeling in a rat model of chronic inhibition of NO synthesis. Importantly, such antiinflammatory and antiarteriosclerotic effects of statins cannot be attributed to their lipid-lowering effects.
We have recently reported that MCP-1 mediates early vascular inflammation and subsequent vascular medial thickening18 and that TGF-β1 mediates perivascular and myocardial fibrosis19 in our model. Our present observations suggest that treatment with statins reduced such pathological disorders by inhibiting gene expression and biological activity of MCP-1 and TGF-β1. Because restoration of endothelial NO-mediated vasodilation is an early event after cholesterol-lowering therapy with statins,6–8 we hypothesized that the beneficial effects of statins seen in this study involved the increase in NO synthase expression or activity. We found that treatment with statins augmented endothelial-type NO synthase protein expression and restored NO-generating capacity and serum NOx concentration. Such augmented endothelial NO synthase expression and activity are in agreement with prior in vitro studies that statins increased NO synthesis from the endothelial cells by increasing NO synthase mRNA stability.24 Therefore, statins are likely to restore the L-NAME–induced inhibition of endothelial NO synthase activity by augmenting NO synthase protein expression and thereby elicited antiinflammatory and antiarteriosclerotic actions in this model. Another several lines of evidence in our study may also support our hypothesis. We have demonstrated that the antiinflammatory and antiproliferative effects of statins in regular-dose L-NAME (1 mg/mL)–treated rats completely disappeared in rats treated with high-dose L-NAME (1.8 mg/mL) (Figures 2E and 2F). We have noticed that disappearance of the vasculoprotective effects of statins in high-dose L-NAME–treated rats corresponded to failure of restoration of NO production (Figure 4B), inhibition of vascular inflammatory/proliferative changes (Figures 2E and 2F), and inhibition of RhoA membrane translocation. These data substantially support the idea that the beneficial effects of statins observed in this model may be mediated at least in part by their effect on eNOS activity. Other possible mechanisms for vasculoprotective effects of statins, which were not investigated in the present study, include inhibition of oxidative stress, inflammatory cytokine activity, or matrix metalloproteinase activity.
Our present finding is also consistent with the recently clarified role of Rho GTPase in mediating cardiovascular disease.25 For example, Rho promotes cell-cycle progression and proliferation in vascular smooth muscle cells,26 which are central events in the pathogenesis of several vascular lesions; Rho also mediates the activation of the proinflammatory transcription factor nuclear factor-κB (NF-κB) in response to cytokines.27 Furthermore, Rho proteins may also be involved in mediating increases in oxidative stress.28
We next hypothesized that the increased NO synthase activity by statins might result from inhibition of small GTPase protein Rho activity. We could demonstrate in the present study that statins prevented the L-NAME–induced increase in Rho activation. Since angiotensin II has been shown to induce Rho activation in several cell types in vitro,29,30 the L-NAME–induced increase in Rho activation may be mediated by increased angiotensin II activity. Evidence suggests that the Rho pathway controls multiple cell functions such as adhesion, proliferation, migration, and the calcium sensitivity of the contractile proteins,31 and that blockade of the Rho pathway with C3 toxin may increase NO synthase protein expression in vitro24 and in vivo.32 Accordingly, these results suggest that treatment with statins augment endothelial type NO synthase activity at least in part by inhibiting Rho activation.
It is reported that treatment with statins lower blood pressure in hypertensive rats.33 Therefore, normalization of the L-NAME–induced hypertension by statins at the late stage seen in the present study (Table 1) may not be an extremely surprising finding. Because we previously demonstrated that normalization of L-NAME–induced hypertension by hydralazine treatment did not inhibit such pathological changes,11 it is unlikely that the antihypertensive effect of statins may contribute largely to the prevention of cardiovascular pathological changes. However, we cannot rule out the possibility that some of the beneficial effects of statins might have been due to normalization of blood pressure.
We previously demonstrated that the increased angiotensin II activity by overexpression of ACE mediates the cardiovascular pathological changes in this model. There was no significant difference in the enzyme activity between hearts from the L-NAME and L-NAME plus statin groups. Thus, it is likely that statins did not affect cardiac angiotensin II activity but inhibited inflammatory and proliferative disorders induced by increased angiotensin II activity in our experiments.
Although very high doses of statins (pravastatin at 100 mg/kg per day and cerivastatin at 2 mg/kg per day) were needed to inhibit early inflammation and late arteriosclerosis, serum concentrations of statins in our rat model were equivalent to those reported in humans.22,23 This discrepancy may be explained by more rapid metabolism of statins from circulation in rats.34,35 No serious side effects were observed by administration of such high doses of statins to rats. Thus, it is unlikely that vasculoprotective effects of statins seen in the present study resulted from clinically irrelevant serum concentrations.
In conclusion, this study has demonstrated direct antiinflammatory and antiarteriosclerotic effects of statins in a rat model of chronic inhibition of NO synthesis. Such vasculoprotective effects of statins are not likely to be mediated by their lipid-lowering actions but seem to be mediated by augmentation of endothelial-type NO synthase expression and activity by the inhibition of Rho activity. The observed vasculoprotective effects of statins are not related to hydrophilic versus lipophilic properties of statins, because both hydrophilic statin (pravastatin) and lipophilic statins (cerivastatin) exhibited similar effects. Recent clinical trials showed that reduction of ischemic cardiovascular events in patients after myocardial infarction by pravastatin therapy was related closely with the decrease in blood C-reactive protein levels.36 Pravastatin therapy reduced monocyte infiltration into atherosclerotic plaque and preserved endothelial functions in atherosclerotic monkeys.37 Such antiinflammatory effects of pravastatin are suggested to be a cholesterol-lowering independent action. The present study may provide a new aspect of how statins reduce cardiovascular ischemic events.
This study was supported by Grants-in-Aid for Scientific Research (11470164, 11158216, 11557056, 10307019, and 10177226) from the Ministry of Education, Science and Culture, Tokyo, Japan.
Original received May 9, 2001; revision received July 25, 2001; accepted July 25, 2001.
Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, MacFarlane PW, McKillop JH, Packard CJ. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. West of Scotland Coronary Prevention Study Group. N Engl J Med. 1995; 333: 1301–1307.
Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford JD, Cole TG, Brown L, Warnica JW, Arnold JM, Wun CC, Davis BR, Braunwald E. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events Trial investigators. N Engl J Med. 1996; 335: 1001–1009.
Libby P, Schoenbeck U, Mach F, Selwyn AP, Ganz P. Current concepts in cardiovascular pathology: the role of LDL cholesterol in plaque rupture and stabilization. Am J Med. 1998; 104: 14S–18S.
Shiomi M, Ito T, Tsukada T, Yata T, Watanabe Y, Tsujita Y, Fukami M, Fukushige J, Hosokawa T, Tamura A. Reduction of serum cholesterol levels alters lesional composition of atherosclerotic plaques: effect of pravastatin sodium on atherosclerosis in mature WHHL rabbits. Arterioscler Thromb Vasc Biol. 1995; 15: 1938–1944.
Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. N Engl J Med. 1998;339:1349–1357.
Anderson TJ, Meredith IT, Yeung AC, Frei B, Selwyn AP, Ganz P. The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. N Engl J Med. 1995; 332: 488–493.
Egashira K, Hirooka Y, Kai H, Sugimachi M, Suzuki S, Inou T, Takeshita A. Reduction in serum cholesterol with pravastatin improves endothelium-dependent coronary vasomotion in patients with hypercholesterolemia. Circulation. 1994; 89: 2519–2524.
Treasure CB, Klein JL, Weintraub WS, Talley JD, Stillabower ME, Kosinski AS, Zhang J, Boccuzzi SJ, Cedarholm JC, Alexander RW. Beneficial effects of cholesterol-lowering therapy on the coronary endothelium in patients with coronary artery disease. N Engl J Med. 1995; 332: 481–487.
Laufs U, La Fata V, Plutzky J, Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation. 1998; 97: 1129–1135.
Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, Huang PL. Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. J Clin Invest. 1998; 101: 1225–1232.
Takemoto M, Egashira K, Usui M, Numaguchi K, Tomita H, Tsutsui H, Shimokawa H, Sueishi K, Takeshita A. Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats. J Clin Invest. 1997; 99: 278–287.
Tomita H, Egashira K, Kubo-Inoue M, Usui M, Koyanagi M, Shimokawa H, Takeya M, Yoshimura T, Takeshita A. Inhibition of NO synthesis induces inflammatory changes and monocyte chemoattractant protein-1 expression in rat hearts and vessels. Arterioscler Thromb Vasc Biol. 1998; 18: 1456–1464.
Ross R , Atherosclerosis—an inflammatory disease. N Engl J Med. 1999; 340: 115–126.
Fujioka T, Nara F, Tsujita Y, Fukushige J, Fukami M, Kuroda M. The mechanism of lack of hypocholesterolemic effects of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in rats. Biochim Biophys Acta. 1995; 1254: 7–12.
Fujiwara M, Ishida Y, Nimura N, Toyama A, Kinoshita T. Postcolumn fluorometric detection system for liquid chromatographic analysis of amino and imino acids using o-phthalaldehyde/N-acetyl-l-cysteine reagent. Anal Biochem. 1987; 166: 72–78.
Kawabata K, Matsushima N, Sasahara K. An automated method for the simultaneous determination of pravastatin and its main metabolite in human plasma by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. Biomed Chromatogr. 1998; 12: 271–275.
Takemoto M, Egashira K, Tomita H, Usui M, Okamoto H, Kitabatake A, Shimokawa H, Sueishi K, Takeshita A. Chronic angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade: effects on cardiovascular remodeling in rats induced by the long-term blockade of nitric oxide synthesis. Hypertension. 1997; 30: 1621–1627.
Koyanagi M, Egashira K, Kitamoto S, Ni W, Shimokawa H, Takeya M, Yoshimura T, Takeshita A. Role of monocyte chemoattractant protein-1 in cardiovascular remodeling induced by chronic blockade of nitric oxide synthesis. Circulation. 2000; 102: 2243–2248.
Tomita H, Egashira K, Ohara Y, Takemoto M, Koyanagi M, Katoh M, Yamamoto H, Tamaki K, Shimokawa H, Takeshita A. Early induction of transforming growth factor-β via angiotensin II type 1 receptors contributes to cardiac fibrosis induced by long-term blockade of nitric oxide synthesis in rats. Hypertension. 1998; 32: 273–279.
Liao JK, Homcy CJ. Specific receptor-guanine nucleotide binding protein interaction mediates the release of endothelium-derived relaxing factor. Circ Res. 1992; 70: 1018–1026.
Krejcy K, Schwarzacher S, Raberger G. Distribution and metabolism of NG-nitro-l-arginine and NG-nitro-l-arginine methylester in canine blood in vitro. Naunyn Schmiedebergs Arch Pharmacol. 1993; 347: 342–345.
Pan HY, DeVault AR, Swites BJ, Whigan D, Ivashkiv E, Willard DA, Brescia D. Pharmacokinetics and pharmacodynamics of pravastatin alone and with cholestyramine in hypercholesterolemia. Clin Pharmacol Ther. 1990; 48: 201–207.
Muck W, Unger S, Kawano K, Ahr G. Inter-ethnic comparisons of the pharmacokinetics of the HMG-CoA reductase inhibitor cerivastatin. Br J Clin Pharmacol. 1998; 45: 583–590.
Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem. 1998; 273: 24266–24271.
Laufs U, Liao JK. Targeting Rho in cardiovascular disease. Circ Res. 2000; 87: 526–528.
Laufs U, Marra D, Node K, Liao JK. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of p27(Kip1). J Biol Chem. 1999; 274: 21926–21931.
Pruefer D, Scalia R, Lefer AM. Simvastatin inhibits leukocyte-endothelial cell interactions and protects against inflammatory processes in normocholesterolemic rats. Arterioscler Thromb Vasc Biol. 1999; 19: 2894–2900.
Wagner AH, Kohler T, Ruckschloss U, Just I, Hecker M. Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation. Arterioscler Thromb Vasc Biol. 2000; 20: 61–69.
Aoki H, Izumo S, Sadoshima J. Angiotensin II activates RhoA in cardiac myocytes: a critical role of RhoA in angiotensin II-induced premyofibril formation. Circ Res. 1998; 82: 666–676.
Yamakawa T, Tanaka S, Numaguchi K, Yamakawa Y, Motley ED, Ichihara S, Inagami T. Involvement of Rho-kinase in angiotensin II-induced hypertrophy of rat vascular smooth muscle cells. Hypertension. 2000; 35: 313–318.
Hall A. The cellular functions of small GTP-binding proteins. Science. 1990; 249: 635–640.
Laufs U, Endres M, Stagliano N, Amin-Hanjani S, Chui D-S, Yang S-X. Neuroprotection mediated by changes in the endothelial actin cytoskeleton. J Clin Invest. 2000; 106: 15–24.
Park JK, Muller DN, Mervaala EM, Dechend R, Fiebeler A, Schmidt F, Bieringer M, Schafer O, Lindschau C, Schneider W, Ganten D, Luft FC, Haller H. Cerivastatin prevents angiotensin II-induced renal injury independent of blood pressure- and cholesterol-lowering effects. Kidney Int. 2000; 58: 1420–1430.
Boberg M, Angerbauer R, Kanhai WK, Karl W, Kern A, Radtke M, Steinke W. Biotransformation of cerivastatin in mice, rats, and dogs in vivo. Drug Metab Dispos. 1998; 26: 640–652.
Komai T, Kawai K, Tokui T, Tokui Y, Kuroiwa C, Shigehara E, Tanaka M. Disposition and metabolism of pravastatin sodium in rats, dogs and monkeys. Eur J Drug Metab Pharmacokinet. 1992; 17: 103–113.
Ridker PM, Rifai N, Pfeffer MA, Sacks F, Braunwald E. Long-term effects of pravastatin on plasma concentration of C-reactive protein. The Cholesterol and Recurrent Events (CARE) Investigators. Circulation. 1999; 100: 230–235.
Williams JK, Sukhova GK, Herrington DM, Libby P. Pravastatin has cholesterol-lowering independent effects on the artery wall of atherosclerotic monkeys. J Am Coll Cardiol. 1998; 31: 684–691.