Felodipine inhibits nuclear translocation of p42/44 mitogen-activated protein kinase and human smooth muscle cell growth.

OBJECTIVE: Smooth muscle cell (SMC) proliferation contributes to vascular structural changes in cardiovascular disease. Ca(2+) antagonists exert antiproliferative effects and may also be clinically beneficial in the patients. However, the underlying mechanisms of action remain elusive. Activation of mitogen-activated protein kinases (MAPK), in particular p42/44mapk plays a central role in cell proliferation. We hypothesise that Ca(2+) antagonists inhibit cell proliferation by interfering with the p42/44mapk pathway in human SMC. METHODS: SMC were cultured from human aorta. Cell proliferation was analysed by [3H]thymidine incorporation. Activation of p42/44mapk and the nuclear target protein Elk-1 was analysed by phosphorylation and p42/44mapk nuclear translocation by confocal microscope. RESULTS: PDGF-BB (10 ng/ml) stimulated [3H]thymidine incorporation, phosphorylated p42/44mapk, caused nuclear translocation of the enzymes and phosphorylated the nuclear target protein Elk-1. Felodipine (10(-7) to 10(-5) mol/l) inhibited [3H]thymidine incorporation to PDGF-BB, had no effect on p42/44mapk phosphorylation. However, p42/44mapk nuclear translocation and Elk-1 activation stimulated by PDGF-BB were prevented by the Ca(2+) antagonist. CONCLUSION: Activation of p42/44mapk, subsequent nuclear translocation and activation of Elk-1 are essentially associated with human SMC proliferation. The Ca(2+) antagonist felodipine prevents p42/44mapk nuclear translocation (but not its activation) associated with inhibition of human SMC growth.     

Low-dose cerivastatin inhibits spontaneous atherogenesis in heterozygous watanabe hyper lipidemic rabbits

The aim of this study was to investigate whether cerivastatin (BAYw6228), a new potent 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, was able to prevent atherogenesis in heterozygous Watanabe heritable-hyperlipidemic (WHHL) rabbits, a model never tested before using this HMG-CoA reductase inhibitor. The heterozygous WHHL rabbits of our breeding developed mild hypercholesterolemia along with focal atherosclerotic lesions in the thoracic aorta. A 9-week treatment with cerivastatin at doses comparable to those used in humans (50 microg/kg/day) reduced serum total cholesterol levels (from 94.4 +/- 10.9 to 43.6 +/- 10.5 mg/dl, p < 0.005) and prevented aortic lesion development (intima/media ratio: 0.058 +/- 0.032 vs 0.946 +/- 0.282 in the placebo group, p < 0.0005). Using a panel of monoclonal antibodies specific to macrophages and able to recognize different smooth muscle cell (SMC) phenotypes, we observed that cerivastatin treatment affected the differentiation properties of SMCs and drastically reduced SMC and macrophage accumulation in the intima of the thoracic aorta. These data show that in the presence of moderate atherosclerotic lesions, such as those of heterozygous WHHL rabbits, low doses of cerivastatin exert an antiatherogenic effect.     

Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model

OBJECTIVE: Angiogenesis is a complex multistep process that involves endothelial cell (EC) migration, proliferation and differentiation into vascular tubes. NO has been reported to be a downstream mediator in the angiogenic response to a variety of growth factors, but the mechanisms by which NO promotes neovessel formation is not clear. We hypothesized that NO directly contributes to EC migration and capillary tube formation. METHODS: Since previous studies have noted important biological differences between NO produced pharmacologically by NO-donor compounds compared to that from NO synthase (NOS), we used a cell-based gene transfer approach to increase NO production in a co-culture model of in vitro angiogenesis. Rat smooth muscle cells (SMCs) were transfected with plasmids containing VEGF(121), VEGF(165) (SMC(VEGF)), endothelial NOS (SMC(eNOS)) or the empty vector (SMC(Cont)). Expression of the eNOS in SMC(eNOS) was confirmed by Northern analysis, NADPH-diaphorase activity, and nitrite/nitrate levels, whereas VEGF production was confirmed using ELISA. Calf pulmonary artery ECs (CPAECs) were cultured on the fibrin matrix with (co-culture) or without underlying SMCs (monoculture). RESULTS: Co-culture of ECs with SMC(Cont) had no effect on EC differentiation compared with EC in monoculture (differentiation index, DI=2.8+/-3.4 vs. 2.1+/-2.8, respectively, NS). In contrast, co-culture with SMC(eNOS) resulted in the formation of extensive capillary-like structures within 48 h (DI=17.2+/-5.9, P<0.001 versus SMC(Cont)), which was significantly inhibited using a NOS inhibitor, L-NAME (3 mM) (DI=4.5+/-3.04, P<0.001 versus SMC(eNOS)). Similarly, SMC(VEGF121) induced an angiogenic response (DI=14.2+/-3.8), which was also significantly inhibited by L-NAME (DI=5.9+/-1.8, P<0.05). In using the Boyden chamber model, SMC(eNOS), but not SMC(Cont) increased EC migration to a similar extent as SMC(VEGF121), and both were significantly inhibited with L-NAME. CONCLUSIONS: These data support an important paracrine role for endogenously produced NO in EC migration and differentiation in vitro, and suggest that the cell-based eNOS gene transfer may be a useful approach to increase new blood vessel formation in vivo.     

An HMG-CoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro

BACKGROUND: Unstable atherosclerotic plaques that cause acute coronary events usually contain abundant macrophages expressing matrix metalloproteinases (MMPs) and tissue factor (TF), molecules that probably contribute to plaque rupture and subsequent thrombus formation. Lipid lowering with HMG-CoA reductase inhibitors reduces acute coronary events. METHODS AND RESULTS: To test whether lipid lowering with an HMG-CoA reductase inhibitor retards macrophage accumulation in rabbit atheroma, we administered cerivastatin to immature Watanabe heritable hyperlipidemic rabbits (cerivastatin group, n=10, cerivastatin 0.6 mg x kg(-1) x d(-1); control group, n=9, saline 0.6 mL x kg(-1) x d(-1)) for 32 weeks and measured macrophage accumulation and expression of MMPs and TF. Serum cholesterol levels after 32 weeks were 809+/-40 mg/dL (control group) and 481+/-24 mg/dL (treated group). Cerivastatin diminished accumulation of macrophages in aortic atheroma. Macrophage expression of MMP-1, MMP-3, MMP-9, and TF also decreased with cerivastatin treatment. Cerivastatin reduced the number of macrophages expressing histone mRNA (a sensitive marker of cell proliferation) detected by in situ hybridization but did not alter macrophages bearing a marker of death (TUNEL staining). Cerivastatin treatment (>or=0.01 micromol/L) also reduced growth, proteolytic activity due to MMP-9, and TF expression in cultured human monocyte/macrophages. CONCLUSIONS: These results suggest that lipid lowering with HMG-CoA reductase inhibitors alters plaque biology by reducing proliferation and activation of macrophages, prominent sources of molecules responsible for plaque instability and thrombogenicity.     

Pharmacodynamics, safety, tolerability, and pharmacokinetics of the 0.8-mg dose of cerivastatin in patients with primary hypercholesterolemia.

Cerivastatin is a third generation hydroxy-methyl-glutaryl-Co-enzyme A (HMG-CoA) reductase inhibitor proven to lower low-density lipoprotein (LDL) cholesterol 28% to 31% in patients with primary hypercholesterolemia when given at 0.3 mg/day. This study evaluates the safety, tolerability, pharmacodynamics, and pharmacokinetics of cerivastatin 0.8 mg once daily for 4 weeks. In this randomized, double-blind, placebo-controlled parallel group trial conducted at 2 study centers, 41 patients (63% women) with primary hypercholesterolemia were placed on an American Heart Association Step 1 diet for 4 weeks. Single-blind placebo was administered for the final 2 weeks, before randomization. Patients received cerivastatin 0.8 mg (n = 28) or placebo (n = 13) once each evening for 28 days. Cerivastatin at 0.8 mg daily was well tolerated. No discontinuations occurred during the study. Adverse events were mild and transient. One cerivastatin-treated patient experienced asymptomatic creatinine kinase, 8x the upper limit of normal (ULN) elevation on the last day of the study, which resolved 6 days after the completion of the study. Cerivastatin 0.8 mg daily significantly reduced LDL cholesterol compared with placebo (-44.0 +/- 2.0% vs 2.2 +/- 2.8%, p <0.0001); total cholesterol (-30.8 +/- 1.4% vs 2.6 +/- 2.1%, p <0.0001), triglycerides (-11.2 +/- 5.9% vs 15.9 +/- 8.6%, p <0.02), but did not significantly alter high-density lipoprotein (HDL) cholesterol (3.2 +/- 2.1% vs -1.2 +/- 3.1%, p = NS). The pharmacokinetics of the 0.8-mg dose revealed dose proportional elevations in the 24-hour area under the curve and maximum plasma concentration relative to 0.3- and 0.4-mg doses with no change in time to maximum concentration or the elimination half-life in plasma. The increased efficacy and lack of clinically significant laboratory abnormalities or adverse events demonstrates a need for a large long-term study to confirm the safety and efficacy of this dose of cerivastatin.     

Cerivastatin prevents tumor necrosis factor-alpha-induced downregulation of endothelial nitric oxide synthase: role of endothelial cytosolic proteins

Cardiovascular disease is accompanied by an impaired endothelium-dependent vasodilatory response. Loss of endothelial nitric oxide synthase (eNOS) expression may contribute to endothelial dysfunction. The aim of the present study was to analyze the effect of cerivastatin, a novel HMG CoA reductase inhibitor, on tumor necrosis factor-alpha (TNF-alpha)-induced downregulation of eNOS protein expression in bovine aortic endothelial cells (BAEC). TNF-alpha (10 ng/ml)- incubated BAEC showed a reduced expression of eNOS protein and decreased eNOS mRNA stabilization. This effect was associated with an increased binding activity of BAEC cytosolic proteins to the 3'-untranslated region (3'UTR) of eNOS mRNA. Cerivastatin prevented TNF-alpha-induced downregulation of eNOS protein expression in a concentration-dependent manner (10(-8) to 10(-5) M). Cerivastatin also prevented the binding of the cytosolic proteins to 3'-UTR of eNOS mRNA and was associated with eNOS mRNA stabilization. The reduced expression of eNOS protein by TNF-alpha was also prevented by coincubation with cycloheximide. In addition cycloheximide inhibited the binding activity of the cytosolic proteins to 3'-UTR of eNOS mRNA, suggesting the inducible character of the mentioned-cytosolic proteins. TNF-alpha stimulated the translocation of nuclear factor-kappaB (NF-kappaB), an effect that was not modified by cerivastatin. Furthermore, an inhibitor of NF-kappaB translocation, pyrrolidine dithiocarbamate failed to modify both the downregulation of eNOS expression and the increased binding activity of the cytosolic proteins to 3'-UTR of eNOS mRNA by TNF-alpha. The effect of cerivastatin on eNOS expression and the binding activity of the cytosolic proteins were reversed by coincubation with L-mevalonate. In conclusion, cerivastatin stabilized eNOS mRNA and upregulated eNOS expression in the endothelium, and this was associated with a decreased binding activity of cytosolic proteins to 3'-UTR of eNOS mRNA. The effect of cerivastatin on the regulation of eNOS expression was independent of NF-kappaB mobilization by TNF-alpha. These findings suggest that cerivastatin may have beneficial effects on the endothelial dysfunction associated with cardiovascular diseases beyond its effect on lowering cholesterol.     

Effects of cerivastatin on forearm vascular responses, blood pressure responsiveness and ambulatory blood pressure in type 2 diabetic men

OBJECTIVE: The objective of the study was to investigate the effects of cerivastatin therapy on forearm endothelial dependent acetylcholine (ACH) and independent (nitroprusside) vasodilator responses, blood pressure (BP) responses to intravenous infusions of angiotensin II (AII) and noradrenaline (NA) and on 24-h ambulatory BP recordings in type 2 diabetic men. DESIGN: Eleven type 2 diabetic men aged 59 +/- 9 years with total cholesterol levels of 5.0 +/- 1.26 mmol/l, triglycerides of 2.23 mmol/l and high-density lipoprotein cholesterol levels of 1.24 mmol/l completed a double-blind, randomized, crossover trial comparing 8 weeks of cerivastatin therapy (800 microg of nocte) with placebo. Forearm vascular resistance (FVR) responses to intrabrachial-arterial infusions of ACH (3-24 microg/min), nitroprusside (2-16 microg/min), the nitric oxide(NO) synthase inhibitor l-nitro-mono-methyl arginine (l-nmma) (8 micromol/min), ACH during l-NMMA infusion and BP responses to intravenous infusions of AII (12.5-50 ng/min) and NA (20-400 ng/min) were measured at the end of each treatment period. Twenty-four-hour ambulatory BP recordings were also performed. RESULTS: FVR responses to ACH during l-NMMA infusion were significantly (p = 0.026) greater during cerivastatin than during placebo therapy. In contrast, FVR responses to ACH in the absence of NO synthase inhibition did not differ significantly between cerivastatin and placebo therapies (p = 0.81). FVR increased by 31.4 +/- 57.3% in response to l-NMMA infusion during cerivastatin therapy compared with 6.1 +/- 41.2% during placebo therapy (p = 0.20). FVR responses to nitroprusside did not differ between cerivastatin and placebo therapies (p = 0.28), nor did BP responses to AII (systolic BP, p = 0.99; diastolic BP, p = 0.98) or NA (systolic BP, p = 0.21; diastolic BP, p = 0.48). Mean 24-h BP was similar during cerivastatin (123 +/- 10 or 70 +/- 7 mmHg) and placebo therapies (129 +/- 11 or 74 +/- 7 mmHg) (systolic BP, p = 0.26; diastolic BP, p = 0.41). CONCLUSION: Cerivastatin increases FVR responses to ACH in type 2 diabetic men with mild dyslipidaemia but only following NO synthase inhibition. This may indicate an improvement in endothelium-derived hyperpolarizing factor-mediated responses.     

Statins prevent pulsatile stretch-induced proliferation of human saphenous vein smooth muscle cells via inhibition of Rho/Rho-kinase pathway

OBJECTIVE: Pulsatile forces regulate vascular remodeling and trigger vascular diseases such as saphenous vein graft disease. The saphenous vein is exposed to high pressure and pulsatility only after implantation. Statins have been proved to reduce the incidence of vein graft failure. Thus, we investigated the molecular mechanisms of pulsatile stretch-induced saphenous vein smooth muscle cell (SMC) proliferation and potential beneficial effects of statins. METHODS AND RESULTS: Human saphenous vein SMCs were subjected to cyclic stretch (60 cycles/min) in Flex I plates. Cerivastatin and simvastatin significantly prevented stretch-induced increase in SMC proliferation. Stretch induced the membrane accumulation of Rho A and Rho kinase inhibitors (Y-27632 and hydroxyfasudil) and dominant negative Rho A mutant significantly prevented stretch-induced SMC proliferation. In addition, stretch increased the levels of both p44/42 mitogen-activated protein (MAP) kinase and Akt phosphorylation. MAP kinase kinase (MEK)1/2 inhibitor U0126, phosphatidylinositol (PI) 3-kinase inhibitors (wortmannin and LY294002), and dominant negative Akt mutant significantly prevented stretch-induced SMC proliferation. Cerivastatin significantly prevented stretch-induced membrane accumulation of Rho A. On the other hand, stretch-induced phosphorylation of p44/42 MAP kinase and Akt was not prevented by cerivastatin. Mevalonate restored the preventive effect of cerivasatain on stretch-induced Rho A membrane accumulation. Stretch induced hyperphosphorylation of retinoblastoma protein (pRb), which was prevented by cerivastatin and the Rho kinase inhibitors. CONCLUSION: Statins prevent stretch-induced saphenous vein SMC proliferation via inhibition of the Rho/Rho-kinase pathway. This may explain the beneficial effects of this class of drug, especially for patients after coronary artery bypass grafting.     

Effect of platelet factors on migration of cultured bovine aortic endothelial and smooth muscle cells

Endothelial cell (EC) injury and the response of EC and smooth muscle cells (SMCs) to injury contribute to the pathophysiology in patients with vascular disease and atherosclerosis. Since platelets have been suggested to play an important role in modulating vascular injury, the present study was undertaken to examine the influence and mechanism of action of individual platelet factors on bovine aortic EC and SMC migration using an in vitro wound assay system. Serotonin decreased EC proliferation and reduced EC migration 21 +/- 1% (p less than 0.005), which was attenuated by imipramine. Transforming growth factor-beta reduced EC proliferation and decreased EC migration 52 +/- 3% (p less than 0.005). Norepinephrine increased EC proliferation but decreased EC migration 26 +/- 2% (p less than 0.005), which was abolished by phenoxybenzamine. Histamine increased EC proliferation but reduced EC migration 29 +/- 2% (p less than 0.005), which was attenuated by diphenhydramine. Platelet-derived growth factor decreased EC proliferation and decreased EC migration 40 +/- 2% (p less than 0.005). In contrast, serotonin increased SMC proliferation and increased SMC migration 31 +/- 2% (p less than 0.005), which was abolished by ketanserin. Transforming growth factor-beta increased SMC migration 35 +/- 5% (p less than 0.005). Norepinephrine increased SMC proliferation and increased SMC migration 43 +/- 4% (p less than 0.005), which was abolished by propranolol. Histamine increased SMC proliferation and increased SMC migration 38 +/- 3% (p less than 0.005), which was abolished by cimetidine. Platelet-derived growth factor increased SMC proliferation and increased SMC migration 40 +/- 3% (p less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of p27(Kip1) in human intestinal cell differentiation

BACKGROUND & AIMS: Growth arrest and differentiation are generally considered to be temporally and functionally linked phenomena in the intestinal epithelium. METHODS: To delineate the mechanism(s) responsible for the loss of proliferative potential as committed intestinal cells start to differentiate, we have analyzed the regulation of G(1)-phase regulatory proteins in relation to differentiation in the intact epithelium as well as in well-established intestinal cell models that allow the recapitulation of the crypt-villus axis in vitro. RESULTS: With intestinal cell differentiation, we have observed an induction of the cell cycle inhibitors p21(Cip), p27(Kip1), and p57(Kip2) expression with an increased association of p27(Kip1) and p57(Kip2) with cyclin-dependent kinase 2 (Cdk2). At the same time, there was an accumulation of the hypophosphorylated form of the pRb proteins and a strong decline in Cdk2 activity. Stable expression of a p27(Kip1) antisense complementary DNA in Caco-2/15 cells did not prevent growth arrest induced by confluence, but repressed villin, sucrase-isomaltase, and alkaline phosphatase expression. CONCLUSIONS: Our results indicate that the growth arrest that precedes differentiation involves the activation of Rb proteins and the inhibition of Cdk2. Furthermore, intestinal cell differentiation apparently requires a function of p27(Kip1) other than that which leads to inhibition of Cdks.     

Reduction of Plasma Cholesterol Levels and Induction of Hepatic LDL Receptor by Cerivastatin Sodium (CAS 143201-11-0, BAY w 6228), a New Inhibitor of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase, in Dogs

The effects of cerivastatin sodium (BAY w 6228), a new type of inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, on plasma cholesterol concentrations and the induction of hepatic LDL receptors were investigated with beagle dogs and Hep G2 cells. Oral administration of cerivastatin (0.01, 0.03, and 0.1 mg/kg per day) for 3 weeks reduced plasma total and very low-density lipoprotein plus low-density lipoprotein (VLDL + LDL) cholesterol concentrations and increased hepatic LDL receptor binding activity in dogs. Scatchard plot analysis revealed a 1.9-fold increase in the maximum binding capacity of hepatic LDL receptors in cerivastatin-treated animals. Similar results were obtained by administration of pravastatin (1.0 and 5.0 mg/kg/day) for 3 weeks. Binding activity of the LDL receptor, as well as receptor mRNA and protein concentrations, were increased in a dose-dependent manner (0.01–1.0 μM) by exposure of Hep G2 cells to cerivastatin. The results suggest that cerivastatin reduces plasma cholesterol concentrations by increasing hepatic LDL receptor expression. The mechanism of lowering cholesterol concentration by cerivastatin was the same as with the other previously examined HMG-CoA reductase inhibitors, but the effects with cerivastatin were apparent at doses much lower than the effective doses of the other drugs. Cerivastatin, therefore, shows potential for clinical use as a potent and efficacious plasma cholesterol-lowering drug. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Inhibition of HMG-CoA reductase with cerivastatin lowers dense low density lipoproteins in patients with elevated fasting glucose, impaired glucose tolerance and type 2 diabetes mellitus.

OBJECTIVE: While 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors effectively decrease LDL cholesterol, it remains controversial whether these agents also lower dense LDL, which are considered particularly atherogenic. METHODS: We examined the effects of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor cerivastatin on lipids, lipoproteins, and apolipoproteins in 69 patients with elevated fasting glucose, impaired glucose tolerance, or type 2 diabetes, combined hyperlipoproteinemia and increased concentrations of dense LDL (apo B in LDL-5 plus LDL-6 > 25 mg/dl). The study was a multicenter, double-blind, randomized, parallel-group comparison of cerivastatin at 0.4 mg daily for 12 weeks (n = 34) and placebo (n = 35). RESULTS: Cerivastatin significantly reduced cholesterol (- 20 %, p < 0.001), IDL cholesterol - 37 %, p < 0.001), LDL cholesterol (- 26 %, p < 0.001), apolipoprotein B (- 25 %, p < 0.001), triglycerides (- 12 %, p < 0.05), and raised HDL cholesterol (+ 7.5 %, p < 0.05) and apolipoprotein AI (+ 7.2 %, p < 0.05). Cerivastatin signficantly lowered apolipoprotein B in all LDL subfractions (- 21 to - 28 %, p < 0.05). Absolute changes were greatest in dense LDL and the change in dense LDL made the largest contribution to the change of total LDL. The change of dense LDL was highly correlated with baseline values. There was no consistent relationship between the effect of cerivastatin on triglycerides and the decrease of dense LDL. CONCLUSIONS: The HMG CoA reductase inhibitor cerivastatin lowers total and LDL cholesterol and the concentration of dense LDL in patients with elevated fasting glucose, impaired glucose tolerance or type 2 diabetes.     

Contribution of Rho A and Rho kinase to platelet-derived growth factor-BB-induced proliferation of vascular smooth muscle cells

In order to identify small G protein (s) which contributes to the proliferation of vascular smooth muscle cells (VSMCs), we examined the effect of an HMG-CoA reductase inhibitor (cerivastatin), a farnesyltransferase inhibitor (FTI-277), a geranyl geranyl transferase inhibitor (GGTI-286) and a Rho kinase inhibitor (Y-27632) on the proliferation of cultured rat VSMCs stimulated with 20ng/ml platelet-derived growth factor (PDGF)-BB. Cerivastatin and GGTI-286, but not FTI-277, suppressed the PDGF-BB-induced activation of extracellular signal related kinase (ERK1/2). The inhibitory effect of cerivastatin on the PDGF-BB-induced activation of ERK1/2 was fully recovered by the addition of geranylgeranyl pyrophosphate (GGPP), but not farnesyl pyrophosphate (FPP). Cerivastatin and GGTI-286, but not FTI-277, suppressed the PDGF-BB-induced [3H] thymidine incorporation and activation of ornitine decarboxylase (ODC), both of which were fully recovered by the addition of GGPP, but not FPP. These data indicate that the PDGF-BB-induced activation of ERK1/2 and proliferation of VSMCs depend upon geranylgeranylated small G protein. Immunoblotting analysis revealed the upregulation of Rho A protein in the membrane fractions of VSMCs stimulated by PDGF-BB. Furthermore, Y-27632 suppressed the PDGF-BB-induced activation of ERK1/2 and proliferation of VSMCs. On the basis of these data, we conclude that PDGF-BB stimulates the proliferation of VSMCs via the activation of Rho A. Rho kinase plays an important role in this process as an effector of Rho A.     

Inhibition of MCF-7 breast cancer cell proliferation by 5alpha-dihydrotestosterone; a role for p21(Cip1/Waf1)

Androgens inhibit the growth of breast cancer cells in vitro and in vivo by mechanisms that remain poorly defined. In this study, treatment of asynchronously growing MCF-7 breast cancer cells with the androgen, 5alpha-dihydrotestosterone (DHT), was shown to inhibit cell proliferation and induce moderate increases in the proportion of G1 phase cells. Consistent with targeting the G1-S phase transition, DHT pretreatment of MCF-7 cultures impeded the serum-induced progression of G1-arrested cells into S phase and reduced the kinase activities of cyclin-dependent kinase (Cdk)4 and Cdk2 to less than 50% of controls within 3 days. DHT treatment was associated with greater than twofold increases in the levels of the Cdk inhibitor, p27(Kip1), while p21(Cip1/Waf1) protein levels remained unchanged. During the first 24 h of DHT treatment, levels of Cdk4-associated p21(Cip1/Waf1) and p27(Kip1) were reduced coinciding with decreased levels of Cdk4-associated cyclin D3. In contrast, DHT treatment caused increased accumulation of Cdk2-associated p21(Cip1/Waf1), with no significant alterations in levels of p27(Kip1) bound to Cdk2 complexes. These findings suggest that DHT reverses the Cdk4-mediated titration of p21(Cip1/Waf1) and p27(Kip1) away from Cdk2 complexes, and that the increased association of p21(Cip1/Waf1) with Cdk2 complexes in part mediates the androgen-induced growth inhibition of breast cancer cells.     

Avenanthramide, a polyphenol from oats, inhibits vascular smooth muscle cell proliferation and enhances nitric oxide production

The proliferation of vascular smooth muscle cells (SMC) and impaired nitric oxide (NO) production are both crucial pathophysiological processes in the initiation and development of atherosclerosis. Epidemiological data have indicated that diets rich in whole grain foods are associated with a reduced risk of developing atherosclerosis. Avenanthramides are polyphenols found exclusively in oats (Avena sativa L.). The present study was conducted to examine the effect of synthetically prepared avenanthramide-2c on the proliferation of SMC and NO production by SMC and human aortic endothelial cells (HAEC). Avenanthramide-2c significantly inhibited serum-induced SMC proliferation. At a concentration of 120 microM, avenanthramide-2c inhibited more than 50% of SMC proliferation, as measured by [3H] thymidine incorporation, and increased the doubling time of rat SMC line (A10) from 28 to 48 h. Treatment of human SMC with 40, 80, and 120 microM avenanthramide-2c inhibited cell number increase by 41, 62, and 73%, respectively. In addition, avenanthramide-2c treatment significantly and dose-dependently increased NO production in both SMC and HAEC. At a concentration of 120 microM, avenanthramide-2c increased NO production by three-fold in SMC, and by nine-fold in HAEC. These increases were in parallel with the up-regulation of mRNA expression for endothelial NO synthase (eNOS) in both vascular SMC and HAEC. These results suggest that the avenanthramides of oats may contribute to the prevention of atherosclerosis through inhibition of SMC proliferation and increasing NO production.     

Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption

Our objective was to determine the precise role of endothelial nitric oxide synthase (eNOS) as a modulator of cardiac O2 consumption and to further examine the role of nitric oxide (NO) in the control of mitochondrial respiration. Left ventricle O2 consumption in mice with defects in the expression of eNOS [eNOS (-/-)] and inducible NOS [iNOS (-/-)] was measured with a Clark-type O2 electrode. The rate of decreases in O2 concentration was expressed as a percentage of the baseline. Baseline O2 consumption was not significantly different between groups of mice. Bradykinin (10(-4) mol/L) induced significant decreases in O2 consumption in tissues taken from iNOS (-/-) (-28+/-4%), wild-type eNOS (+/+) (-22+/-4%), and heterozygous eNOS(+/-) (-22+/-5%) but not homozygous eNOS (-/-) (-3+/-4%) mice. Responses to bradykinin in iNOS (-/-) and both wild-type and heterozygous eNOS mice were attenuated after NOS blockade with N-nitro-L-arginine methyl ester (L-NAME) (-2+/-5%, -3+/-2%, and -6+/-5%, respectively, P<0.05). In contrast, S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4) mol/L), which releases NO spontaneously, induced decreases in myocardial O2 consumption in all groups of mice, and such responses were not affected by L-NAME. In addition, pretreatment with bacterial endotoxin elicited a reduction in basal O2 consumption in tissues taken from normal but not iNOS (-/-)-deficient mice. Our results indicate that the pivotal role of eNOS in the control of myocardial O2 consumption and modulation of mitochondrial respiration by NO may have an important role in pathological conditions such as endotoxemia in which the production of NO is altered.     

Short-term effects of statin therapy in patients with hyperlipoproteinemia after liver transplantation: results of a randomized cross-over trial

BACKGROUND/AIMS: Hyperlipoproteinemia is frequent following liver transplantation and may lead to atherosclerosis. Lipid-lowering agents may be useful, but could interfere with the function of the transplanted organ and with immunosuppression. We therefore evaluated in a prospective, randomized, open-labeled cross-over trial the effect of two frequently used 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (pravastatin 10 mg d(-1) and cerivastatin 0.1 mg d(-1)) in hyperlipoproteinemic patients after liver transplantation. METHODS: Sixteen patients (6.3 +/- 2.0 years post-transplantation, cyclosporine n = 11, tacrolimus n = 5) with hyperlipoproteinemia (cholesterol 246 +/- 42, triglycerides 191 +/- 87, low-density lipoprotein (LDL)-cholesterol 161 +/- 35, high-density lipoprotein (HDL)-cholesterol 44 +/- 11 mg d(-1)) were included. Treatment periods of 6 weeks were separated by a 4-week washout period. RESULTS: Both medications were tolerated well, no effects on serum concentrations of liver enzymes or immunosuppressive agents were observed. Cerivastatin and pravastatin decreased (P < 0.001) cholesterol by 21 +/- 10% and 15 +/- 10%, LDL-cholesterol by 27 +/- 14% and 17 +/- 15%, respectively, while triglyceride and HDL-cholesterol concentrations did not change significantly. LDL/HDL-cholesterol markedly improved (P < 0.001) by 29 +/- 16% (cerivastatin) and 16 +/- 16% (pravastatin). Cerivastatin was more potent than pravastatin in patients receiving cyclosporine A, while there was no significant difference in patients receiving tacrolimus. CONCLUSIONS: Low-dose cerivastatin and pravastatin significantly improve lipid profiles following liver transplantation without affecting liver function or immunosuppression.     

Expression and function of recombinant endothelial nitric oxide synthase in human endothelial cells

Endothelial dysfunction is frequently involved in the pathogenesis of vascular disease. While nitric oxide (NO) inhibits smooth muscle cell proliferation, its effect on endothelial cell proliferation is unclear. The aim of this study was to determine if adenoviral-mediated gene transfer of endothelial NO synthase (eNOS) to human umbilical vein endothelial cells (HUVECs) would result in increased generation of NO and affect endothelial cell proliferation. HUVECs were transduced with adenoviral vectors encoding eNOS (AdeNOS) or beta-galactosidase (Ad beta gal) or exposed to diluent (control). AdeNOS-transduced cells showed increased eNOS expression as detected by Western blot analysis, and increased concentrations of cGMP (control 0.7 +/- 0.1; Ad beta gal 0.9 +/- 0.2; AdeNOS 3.1 +/- 0.5 pmol/mg protein; p < 0.001) and nitrite (control 11.8 +/- 1.2; Ad beta gal 13.3 +/- 1.7; AdeNOS 21.1 +/- 2.2 nmol/mg protein/hour; p < 0.01). DNA synthesis as assessed by [(3)H]thymidine incorporation and cell counts were significantly reduced (by approximately 30%) in AdeNOS-transduced HUVECs. Expression of mitogen-activated protein kinase was also decreased in AdeNOS-transduced cells. This study shows that adenoviral-mediated gene transfer of eNOS to HUVECs inhibits endothelial cell proliferation.