Walter Gonzalez

Angiotensin II Stimulates Endothelial Vascular Cell Adhesion Molecule-1 via Nuclear Factor-κB Activation Induced by Intracellular Oxidative Stress

The recruitment of monocytes via the endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) is a key step in the formation of the initial lesion in atherosclerosis. Because angiotensin (Ang) II may be involved in this process, we investigated its role on the signaling cascade leading to VCAM-1 expression in endothelial cells. Ang II stimulates mRNA and protein expression of VCAM-1 in these cells via the AT(1) receptor. This effect was enhanced by N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, and blocked by pyrrolidinedithiocarbamate, an antioxidant molecule. Ang II activated the redox-sensitive transcription factor nuclear factor-kappaB and stimulated the degradation of both inhibitor of kappaB (IkappaB)alpha and IkappaBbeta with different kinetics. The degradation of IkappaBs induced by Ang II was not modified by incubation with exogenous superoxide dismutase and catalase, suggesting that this effect was not mediated by the extracellular production of O(2)(-). In contrast, rotenone and antimycin, 2 inhibitors of the mitochondrial respiratory chain, inhibited the Ang II-induced IkappaB degradation, showing that generation of reactive oxygen species in the mitochondria is involved on Ang II action. BXT-51702, a glutathione peroxidase mimic, inhibited the effect of Ang II, and aminotriazole, an inhibitor of catalase, enhanced it, suggesting a role for H(2)O(2) in IkappaB degradation. This is confirmed by experiments showing that Ang II stimulates the intracellular production of H(2)O(2) in endothelial cells. These results demonstrate that Ang II induced an intracellular oxidative stress in endothelial cells, which stimulates IkappaB degradation and nuclear factor-kappaB activation. This activation enhances the expression of VCAM-1 and probably other genes involved in the early stages of atherosclerosis.

Molecular Plasticity of Vascular Wall During NG-Nitro-l-Arginine Methyl Ester–Induced Hypertension : Modulation of Proinflammatory Signals

It has previously been reported that hypertension induced by the chronic blockade of NO production is characterized by a proinflammatory phenotype of the arterial wall associated with a periarterial accumulation of inflammatory cells. In the present study, the cellular and molecular mechanisms involved in the luminal and perivascular accumulation of inflammatory cells were evaluated in the aortas of N(G)-nitro-L-arginine methyl ester (L-NAME)-treated rats. Because the medial layer remains intact, putative markers of the resistance of the vascular wall to cell migration and to oxidative stress were also explored. For this purpose, monocyte adhesion, cytokine expression, superoxide anion production, and nuclear factor-kappa B (NF-kappa B) activation were assessed in the aortas of L-NAME-treated rats. Expressions of tissue inhibitor of metalloproteinases-1 (TIMP-1) and heme oxygenase-1 (HO-1) in the aortic wall were also studied as possible markers of such resistance. Chronic blockade of NO production increased ex vivo monocyte adhesion to the endothelium, increased the production of superoxide anions, and activated the NF-kappa B system. In concert with this modification of the redox state of the vascular wall in L-NAME-treated rats, the expression of proinflammatory cytokines interleukin-6, monocyte chemoattractant protein-1, and macrophage colony-stimulating factor was increased. In parallel, expressions of both TIMP-1 and HO-1 were increased. All these changes were prevented by treatment with an angiotensin-converting enzyme inhibitor (Zofenopril). Hypertension associated with a proinflammatory phenotype of the vascular wall induced by blockade of NO production could be due to an increase in oxidative stress, which, in turn, activates the NF-kappa B system and increases gene expression. In parallel, the arterial wall overexpresses factors such as TIMP-1 and HO-1, which could participate in the resistance to cell migration and oxidative stress.

Endothelin antagonists in salt-dependent hypertension associated with renal insufficiency

Bosentan is a nonspecific antagonist for endothelin (ET) receptors, and BQ123 is a specific inhibitor for ET-A receptors. We compared the effects of bosentan (10 mg/kg intravenously, i.v.) and BQ123 (10 mg/kg/h i.v.) on blood pressure and renal function in deoxycorticosterone acetate (DOCA)-salt rats, Dahl salt-sensitive (Dahl-S) rats, and normotensive Wistar rats. In normotensive Wistar rats, bosentan and BQ123 decreased blood pressure. Only BQ123 decreased glomerular filtration rate (GFR) and filtration fraction. These results indicate that ET-A receptors play a role in glomerular function. In DOCA-salt rats, bosentan and BQ123 caused a decrease in blood pressure to normal range and a decrease in renal vascular resistances. Bosentan decreased filtration fraction. Paradoxically, BQ123 caused a decrease in GFR. In Dahl-S rats, bosentan and BQ123 decreased blood pressure, but blood pressure did not reach normal ranges. Bosentan did not modify renal function, but BQ123 caused a decrease in the GFR and filtration fraction. Our results confirm the importance of specific and nonspecific ET antagonists in decreasing blood pressure in models of salt-dependent hypertension. However nonspecific inhibition of ET action did not improve renal function and specific inhibition of ET-A receptors by BQ123 temporarily worsened renal function.

Characterization of neutral endopeptidase in vascular cells, modulation of vasoactive peptide levels

We characterized neutral endopeptidase activity and protein in the three aortic layers and in corresponding cultured primary cells. Neutral endopeptidase was expressed in all three layers of rat aorta with higher protein level and activity in the adventitia than in the media and intimal endothelium. Neutral endopeptidase was also found in primary cultured fibroblasts, smooth muscle and endothelial cells derived from the corresponding layers. Neutral endopeptidase activity and protein were higher in the fibroblasts and smooth muscle cells than in endothelial cells. Neutral endopeptidase inhibition prevented atrial natriuretic peptide (ANP) degradation in endothelial and smooth muscle cells. It potentiated ANP-stimulated cyclic GMP production in these cells. Neutral endopeptidase inhibition also reduced bradykinin degradation and potentiated bradykinin-stimulated release of arachidonic acid in fibroblasts and endothelial cells. Our data demonstrate the presence and functional activity of neutral endopeptidase in all three cell layers of rat aorta as well as in primary cells of the vessel. The data suggest that local concentrations of vasoactive peptides in the vessel wall might be regulated by the neutral endopeptidase cleavage pathway in the immediate vicinity of their target cells.

Insulin secretion in rats with chronic nitric oxide synthase blockade

SummaryNitric oxide, which is produced from l-arginine by a nitric oxide-synthase enzyme, has been shown to be a ubiquitous messenger molecule. Recently, it has been suggested that nitric oxide might influence insulin secretion by activating the soluble guanylate cyclase and generating cyclic guanosine monophosphate (cGMP). We have investigated the role of the nitric oxide pathway in insulin secretion by evaluating the insulin response to several secretagogues in rats in which nitric oxide-synthase was chronically inhibited by oral administration of the l-arginine analogue, NG-nitro-l-arginine methyl ester (l-NAME). Blood pressure and aortic wall cGMP content were used as indices of nitric oxide-synthase blockade. Insulin secretion was evaluated after an intravenous bolus of d-glucose, l-arginine or d-arginine. Chronic l-NAME administration induced a 30% increase in blood pressure and a seven-fold drop in arterial cGMP content. Body weight, fasting plasma glucose and insulin were not influenced by l-NAME administration. First-phase insulin secretion (1+3 min) in response to glucose was not significantly different in l-NAME and control rats. The areas under the insulin curve were similar in both groups. Insulin secretion in response to d-arginine or l-arginine in l-NAME-treated and control rats were also similar. In conclusion, chronic nitric oxide-synthase blockade increases blood pressure and decreases aortic cGMP content, but does not alter insulin secretion in response to several secretagogues. Chronic oral administration of l-NAME in the rat provides an adequate animal model for studying the l-arginine nitric oxide-pathway.

Transforming Growth Factor-β Regulation of Endothelin Expression in Rat Vascular Cell and Organ Cultures

Transforming growth factor (TGF)-β increases the production of the vasoactive peptide endothelin (ET) in cultures of vascular endothelial cells (EC) and vascular smooth muscle cells (VSMC), but the physiologic or pathologic significance of this regulation has not been determined. The present studies test the hypothesis that when EC and VSMC are in direct contact or close proximity, ET expression is, at least in part, dependent on TGF-β. The effects of TGF-β on ET-1 mRNA (Northern analysis and reverse transcription polymerase chain reaction) and peptide (radioimmunoassay) levels were assessed in rat EC and VSMC and vascular organ cultures. TGF-β2 (1 ng/ml) increased ET-1 mRNA in VSMC and EC plus VSMC cultures and increased ET-1 peptide in EC, VSMC, and EC plus VSMC cultures. TGF-β2 also increased ET-1 mRNA and peptide in vascular organ cultures. Antibodies that neutralized the activities of TGF-β1 and TGF-β2 decreased ET-1 mRNA in EC plus VSMC cultures and in vascular organ cultures. These data indicate that when EC and VSMC are in direct contact or close proximity, TGF-β increases ET expression and active TGF-β is present and promotes ET expression. These data suggest that TGF-β is a determinant of vascular ET expression in vivo, and that TGF-β regulation of ET expression would affect cardiovascular function in health and disease.

Effects of angiotensin-converting enzyme and neutral endopeptidase inhibitors: influence of bradykinin

These experiments compare the effects of a neutral endopeptidase inhibitor, retrothiorphan, 1-[(1-mercaptomethyl-2-phenyl)ethyl]amino-1-oxopropanoic acid, a converting enzyme inhibitor, enalaprilat, and the combination of the two inhibitors on changes in blood pressure and renal function induced by exogenous and endogenous bradykinin in deoxycorticosterone acetate (DOCA)-salt rats. Enalaprilat potentiated the exogenous bradykinin-induced hypotensive responses while retrothiorphan potentiated the effects on urinary cyclic-GMP (cGMP) and bradykinin. The combination potentiated the exogenous bradykinin-induced hypotensive effects and the bradykinin-induced urinary excretion of cGMP, bradykinin and prostaglandin. The bradykinin B2 receptor antagonist, Hoe 140, had no effect on the enalaprilat- and retrothiorphan-induced changes in blood pressure and renal function. In conclusion, while angiotensin-converting enzyme and neutral endopeptidase are involved in the vascular and renal catabolism of exogenous bradykinin, the effects of the peptidase inhibitors do not appear to depend on the protection of endogenous bradykinin under acute conditions in DOCA-salt rats.

Endothelium-independent conversion of angiotensin I by vascular smooth muscle cells

Abstract. The conversion of angiotensin I (AT-I) to angiotensin II (AT-II) by angiotensin I-converting enzyme (ACE) is a key step in the action of angiotensins. ACE is constitutively expressed in endothelial cells, but can also be detected at low levels in smooth muscle cells (SMC). Furthermore, in rats the ACE activity can be induced in SMC in vivo by experimental hypertension or vascular injury and in vivo by corticoid treatment. This study was therefore undertaken to evaluate the conversion of AT-I and its subsequent effects in SMC in basal conditions and after stimulation by dexamethasone. Using rat and human SMC, showed that dexamethasone induced ACE expression and that this enzyme was functional, leading to AT-II-dependent intracellular signaling. A fourfold increase in phospholipase C activity in response to AT-I was observed in dexamethasone-activated SMC compared with quiescent SMC. This effect of dexamethasone on signal transduction is dependent on ACE activity, whereas AT-II receptor parameters remain unchanged. The action of AT-I was blocked by an AT1 receptor antagonist, suggesting that it was mediated by AT-II. Similarly, dexamethasone-induced ACE expression was present in human SMC, and calcium signaling was mobilized in response to AT-I in activated human cells. Experiments performed with cocultures of endothelial cells and SMC in a Transwell system showed that the response to AT-I was limited to the compartment where AT-I was localized, suggesting that AT-I does not pass through the endothelial cell barrier to interact with underlying SMC. Our data suggest that in rat, as in human SMC, the conversion of AT-I into AT-II and the signal transduction in response to AT-I are ACE expression-dependent. In addition, the present findings show that this SMC response to AT-I is endothelium-independent, supporting the idea of a local generation of AT-II in the vascular wall.

Diastereoselective synthesis of mixanpril, an orally active dual inhibitor of neutral endopeptidase and angiotensin converting enzyme

Abstract A new diastereoselective synthesis of mixanpril, N-[(2S,3R)-2-benzoylthiomethyl-3-phenylbutanoyl]-L-alanine, a dual inhibitor of neutral endopeptidase and angiotensin converting enzyme, which could be used in the treatment of chronic hypertension and cardiac failure has been developed in order to obtain large quantities of this compound necessary for preclinical screening. As expected we showed that a complete inhibition of both enzymes was obtained after oral administration of mixanpril in anaesthesized rats.

Pulmonary endothelium as a site of synthesis and storage of interleukin-6 in experimental congestive heart failure.

Pulmonary endothelium is an early upstream hemodynamic target of left ventricular dysfunction. Interleukin 6 (IL‐6) is a pro‐inflammatory cytokine reported to increase in congestive heart failure (CHF) patients.