Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells.

Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors.

Involvement of Rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells.

Endothelial dysfunction is characterized by an impaired vasodilatory response to endothelial agonists as well as by alterations in adhesion and coagulation processes. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) have been shown to be useful in the reversal of endothelial dysfunction, an effect that may be independent of the reduction in cholesterol levels. Both the L-arginine-nitric oxide-cGMP and endothelin pathways are involved in the regulation of vascular tone. Here, we show that the basal transcription rate of the preproendothelin-1 gene was decreased by simvastatin (10 micromol/L) in bovine aortic endothelial cells. Transfection studies with the preproendothelin-1 gene promoter showed that mevalonate (100 micromol/L) was able to prevent the inhibitory effect mediated by simvastatin. Protein geranylgeranylation, but not farnesylation, proved to be crucial for a correct expression of the preproendothelin-1 gene. The C3 exotoxin from Clostridium botulinum that selectively inactivates Rho GTPases, the processing of which involves geranylgeranylation, reproduced the inhibitory effect of simvastatin on the expression of preproendothelin-1. Overexpression of dominant-negative mutants of RhoA and RhoB led to a significant reduction in the preproendothelin-1 promoter activity, whereas the expression of wild-type and constitutively active forms of these proteins resulted in an increase, in support that Rho proteins are required for the basal expression of the preproendothelin-1 gene. Finally, we show that the Rho-dependent activation of the preproendothelin-1 gene transcription was inhibited by simvastatin. Thus, the control of vascular tone and proliferative response mediated by endothelin-1 is regulated at multiple levels, among which the Rho proteins play an essential role.

Association of angiotensinogen M235T and A(-6)G gene polymorphisms with coronary heart disease with independence of essential hypertension: the PROCAGENE study. Prospective Cardiac Gene.

OBJECTIVES: We examined the relationship between the angiotensinogen (AGT) gene M235T polymorphism, the variant promoter of the AGT gene A(-6)G and the angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and coronary heart disease (CHD) in native Gran Canaria Island habitants, who have the highest rates of CHD in Spain. BACKGROUND: Some studies subject that the ACE (I/D) polymorphism could be associated with CHD, while AGT (M235T) has been related to essential hypertension. METHODS: We studied 304 subjects with angiographic evidence of coronary artery disease and a clinical diagnosis of myocardial infarction or unstable angina and 315 age- and gender-matched controls. Blood was drawn and DNA extracted. Angiotensin-converting enzyme (I/D) gene polymorphism was analyzed by polymerase chain reaction (PCR) and AGT gene polymorphisms by restriction fragment length polymorphism-PCR and mutagenically-separated PCR. RESULTS: The ACE (I/D) polymorphism showed no association with CHD, whereas the frequency distribution of AGT (M235T) genotypes among patients and controls (235T: 29.1% and 19.0%; M235T: 48.5% and 50.2%; M235: 22.4% and 30.8%, respectively) was statistically different (p = 0.005) and not related to the presence of essential hypertension. Similar results were observed with the AGT A(-6)G polymorphism. In multiple logistic regression analysis, CHD odds ratio associated with 235T and M235 homozygotes were 1.7 (1.1 to 2.6) and 0.54 (0.36 to 0.82), respectively. CONCLUSIONS: This study shows that genetic variation of the AGT (M235T), but not the ACE (I/D), genotypes contributes to the presence of CHD independently of blood pressure profile in a subset of the Spanish population with a high prevalence of cardiovascular disease.

Presence of nitric oxide synthase activity in roots and nodules of Lupinus albus.

NO is a widespread messenger molecule in physiology. We were interested in investigating whether an NO-generating system could be present in plants. NO and L-[14C]citrulline were synthesized by roots and nodules of Lupinus albus in an L-arginine-dependent manner. L-[14C]Citrulline production was inhibited by N(G)-monomethyl-L-arginine, a nitric oxide synthase antagonist, in a competitive way. NADPH-diaphorase activity was localized in the vascular bundles in root and nodules, and also in the nodule infected zone. This staining was significantly reduced in the presence of N(G)-monomethyl-L-arginine. These results indicate the presence of a putative nitric oxide synthase in plants.

Role of reactive oxygen species in the signalling cascade of cyclosporine A-mediated up-regulation of eNOS in vascular endothelial cells.

1. Cyclosporine A (CsA) increases eNOS mRNA expression in bovine cultured aortic endothelial cells (BAEC). As some effects of CsA may be mediated by reactive oxygen species (ROS), present experiments were devoted to test the hypothesis that the CsA-induced eNOS up-regulation could be dependent on an increased synthesis of ROS. 2. CsA induced a dose-dependent increase of ROS synthesis, with the two fluorescent probes used, DHR123 (CsA 1 microM: 305+/-7% over control) and H2DCFDA (CsA 1 microM: 178+/-6% over control). 3. Two ROS generating systems, xanthine plus xanthine oxidase (XXO) and glucose oxidase (GO), increased the expression of eNOS mRNA in BAEC, an effect which was maximal after 8 h of incubation (XXO: 168+/-21% of control values. GO: 208+/-18% of control values). The ROS-dependent increased eNOS mRNA expression was followed by an increase in eNOS activity. 4. The effect of CsA on eNOS mRNA expression was abrogated by catalase, and superoxide dismutase (SOD). In contrast, the antioxidant PDTC augmented eNOS mRNA expression, both in basal conditions and in the presence of CsA. 5. The potential participation of the transcription factor AP-1 was explored. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. 6. The present results support a role for ROS, particularly superoxide anion and hydrogen peroxide, as mediators of the CsA-induced eNOS mRNA up-regulation. Furthermore, they situate ROS as potential regulators of gene expression in endothelial cells, both in physiological and pathophysiological situations.

CsA and FK506 up-regulate eNOS expression: role of reactive oxygen species and AP-1.

Cyclosporine A (CsA) and FK506 increase endothelial nitric oxide synthase (eNOS) mRNA expression in cultured bovine aortic endothelial cells (BAEC). CsA appears to increase eNOS mRNA levels mainly by increasing the rate of transcription, although a small contribution of mRNA stabilization could not be ruled out. CsA and FK506 induced an increase of ROS synthesis with the fluorescent probe used, DHR123. The ROS generating system glucose oxidase (GO) increased the expression of eNOS mRNA in BAEC. This upregulation of eNOS mRNA by CsA or GO was abrogated by catalase. As AP-1 is a redox-sensitive transcription factor and the bovine eNOS promoter has an AP-1 consensus sequence, a role of this factor in the up-regulation of eNOS mRNA was studied. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. The potential participation of ROS and the transcription factor AP-1 in the regulation of eNOS gene expression is suggested.

Effects of the angiotensinogen gene M235T and A(-6)G variants on blood pressure and other vascular risk factors in a Spanish population.

Angiotensinogen (AGT) gene polymorphism has shown significant differences in the allelic frequencies between hypertensive and normotensive subjects. This allele frequency varies among ethnic groups. There are still some controversies related to the 235T-variant as a marker for essential hypertension. As part of an extensive case-control study carried out in a Spanish population, we selected the 237 subjects with a diagnosis of essential hypertension according to the established criteria. A group of 242 normotensives matched for age and gender was used as control. Smoking habits, a previous diabetes and hypertension medical history, body mass index (BMI) and blood pressure (BP) values were recorded. Glucose, plasma creatinine, lipid profile with Lp(a), homocysteine and microalbuminuria were measured. Angiotensinogen M235T-gene polymorphism was determined by polymerase chain reaction (PCR) from genomic DNA. A(-6)G polymorphism was determined by mutagenically separated PCR (MS-PCR). BP values, BMI and microalbuminuria were significantly higher in hypertensive subjects; 31.6% of hypertensives and 40.1% normotensives were active smokers. M235T-genotype frequencies were not different in the hypertensive and normotensive population. Similarly, homocigotic AA predominate in the hypertensives but without statistical significance. The association of 235T-genotype or the changes in the promoter activity due to A(-6) substitution with essential hypertension was not confirmed in the multivariate regression analyses. Only a previous family history of hypertension and BMI were significantly associated with hypertension. Journal of Human Hypertension (2000) 14, 789-793

[Genes and kidney disease. Candidate genes]. / Genes y enfermedad renal. Generalidades. Genes candidatos.

Molecular biology techniques have provided important advances in the search for causal relationships in complex diseases supporting traditional epidemiologic studies. Genetic epidemiology allows us to detect genetic variants that could be related to the onset and progression of different diseases. In cardiovascular and renal diseases, this approach linking traditional risk factors to new described ones and those allelic variants, which contribute to the development of these manifestations permits a better understanding of individual disease susceptibility. This is usually afforded through case-control studies evaluating allelic variants of candidate genes previously associated with the disease. Even in this candidate gene search, association-based methods are more powerful than linkage studies in complex traits if we assume that some of the typed polymorphisms are causative although with subtle phenotypic effects. Some brief examples may illustrate the progress in the understanding of renal and cardiovascular diseases.

Phosphorylation of calmodulin by the epidermal-growth-factor-receptor tyrosine kinase.

An epidermal-growth-factor(EGF)-receptor preparation isolated by calmodulin-affinity chromatography from rat liver plasma membranes is able to phosphorylate calmodulin. Calmodulin phosphorylation was enhanced 3-8-fold by EGF, was dependent on the presence of a polycation or basic protein and was inhibited by micromolar concentrations of Ca2+. Phosphate incorporation into calmodulin occurs predominantly on tyrosine residues. Partial proteolysis of phosphocalmodulin by thrombin identifies Tyr99, located in the third calcium-binding domain of calmodulin, as the phosphorylated residue. Stoichiometric measurements show a 32P/calmodulin molar ratio of approximately 1 when optimal phosphorylation conditions are used.

Involvement of transcriptional mechanisms in the inhibition of NOS2 expression by dexamethasone in rat mesangial cells.

In previous studies we reported that stimulation of rat mesangial cells (RMC) with lipopolysaccharide (LPS) + tumor necrosis factor alpha (TNF-alpha) (L/T) elicits inducible nitric oxide synthase (NOS2) mRNA expression, which is inhibited by dexamethasone (DX). We have now analyzed the mechanisms responsible for this inhibitory effect. Dexamethasone had no destabilizing effect on NOS2 mRNA. Transfection of RMC with several luciferase reporter constructs from the 5' flanking regulatory region of the rat NOS2 gene established the importance of the NF-kappa B site in the transcriptional activation of the NOS2 gene. DNA mobility shift assays showed activation by L/T of the NF-kappa B complex in a time-dependent manner. Dexamethasone specifically inhibited this activation in a process dependent on the glucocorticoid receptor and with a markedly greater effect when it was added prior to L/T. Dexamethasone increased the expression of the I kappa B-alpha transcript and protein in the cytoplasm. While treatment of RMC with L/T induced the transient decrement of cytoplasmic p65 levels and its appearance in the nucleus, preincubation with DX prevented this effect. Co-immunoprecipitation and immunocytochemical studies demonstrated that I kappa B-alpha is associated with p65 in the cytoplasm of RMC after treatment with DX and L/T. These results prove that inhibition of NF-kappa B-mediated transcription is a crucial mechanism by which DX inhibits NOS2 expression, and that this occurs by increasing cytoplasmic I kappa B-alpha levels and sequestering the activating subunits of NF-kappa B in the cytoplasm. The need for previous induction of I kappa B-alpha could provide a molecular explanation for the limited efficacy of these agents in the therapy of septic shock.