Maria E. Pueyo

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.

Endothelium-derived nitric oxide and vascular physiology and pathology.

Abstract. In 1980, Furchgott and Zawadzki demonstrated that the relaxation of vascular smooth muscle cells in response to acetylcholine is dependent on the anatomical integrity of the endothelium. Endothelium-derived relaxing factor was identified 7 years later as the free radical gas nitric oxide (NO). In endothelium, the amino acid L-arginine is converted to L-citrulline and NO by one of the three NO synthases, the endothelial isoform (eNOS). Shear stress and cell proliferation appear to be, quantitatively, the two major regulatory factors of eNOS gene expression. However, eNOS seems to be mainly regulated by modulation of its activity. Stimulation of specific receptors to various agonists (e.g., bradykinin, serotonin, adenosine, ADP/ATP, histamine, thrombin) increases eNOS enzymatic activity at least in part through an increase in intracellular free Ca2+. However, the mechanical stimulus shear stress appears again to be the major stimulus of eNOS activity, although the precise mechanisms activating the enzyme remain to be elucidated. Phosphorylation and subcellular translocation (from plasmalemmal caveolae to the cytoskeleton or cytosol) are probably involved in these regulations. Although eNOS plays a major vasodilatory role in the control of vasomotion, it has not so far been demonstrated that a defect in endothelial NO production could be responsible for high blood pressure in humans. In contrast, a defect in endothelium-dependent vasodilation is known to be promoted by several risk factors (e.g., smoking, diabetes, hypercholesterolemia) and is also the consequence of atheroma (fatty streak infiltration of the neointima). Several mechanisms probably contribute to this decrease in NO bioavailability. Finally, a defect in NO generation contributes to the pathophysiology of pulmonary hypertension. Elucidation of the mechanisms of eNOS enzyme activity and NO bioavailability will contribute to our understanding the physiology of vasomotion and the pathophysiology of endothelial dysfunction, and could provide insights for new therapies, particularly in hypertension and atherosclerosis.

Chronic Blockade of NO Synthase Activity Induces a Proinflammatory Phenotype in the Arterial Wall Prevention by Angiotensin II Antagonism

Chronic blockade of NO production induces hypertension and early occlusive and fibrotic end-stage organ damage owing to vascular lesions in the brain, kidney, and heart. In this study, we evaluated the inflammatory phenotypic changes induced in the arterial wall by chronic N(G)-nitro-L-arginine methyl ester (L-NAME) administration and the effect of an angiotensin II receptor (AT1) antagonist, irbesartan, on these changes. For this purpose, 2 groups of rats received L-NAME in the drinking water (50 mg x kg(-1) x d(-1)) for 2 months. One group received no other treatment and the other was treated with irbesartan (10 mg x kg(-1) x d(-1)). A third group (controls) received neither L-NAME nor irbesartan. After 8 weeks, plasma, aortas, and left ventricles were sampled from all 3 groups. Expression of inducible NO synthase (iNOS) was evaluated at both the mRNA (quantitative reverse transcription-polymerase chain reaction) and the protein (Western blot and immunohistochemistry) level in the aorta. Expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was evaluated by reverse transcription-polymerase chain reaction, Western immunoblotting, and immunohistochemistry; inflammatory cell infiltration by immunohistochemistry; and fibrosis by Sirius red staining. Chronic L-NAME administration induced the expression of iNOS in the aorta, which was localized in smooth muscle cells as shown by immunohistochemistry and NADPH diaphorase activity. ICAM-1 and VCAM-1 expression was also increased in aortas of L-NAME-treated rats. These phenotypic changes of the vascular wall were associated with inflammatory cell infiltration and fibrosis in the heart. All of these pathological phenomena were prevented by the angiotensin II antagonist irbesartan. The proinflammatory phenotypic changes of the vascular wall induced by blockade of NOS activity could be involved in the interaction between endothelial dysfunction and the development of arteriosclerosis.

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.

Angiotensin II-elicited signal transduction via AT1 receptors in endothelial cells.

1 . Angiotensin II (AII) actions are mediated by two distinct types of receptors: AT1, which includes two subtypes, AT1A and AT1B, and AT2. AII produces vasoconstriction on the vascular wall acting directly on smooth muscle cells via AT1 receptors. AII receptors have recently been demonstrated on endothelial cells. But the pharmacological characteristics of these receptors and the intracellular signal pathways coupled to them remain unclear. 2 . The aim of this work was to characterize the AII receptor subtypes in rat aortic endothelial cells (RAEC) in primary culture and to evaluate the signal pathways coupled to these receptors by measuring the activation of phospholipase C (PLC) and phospholipase A2 (PLA2). 3 . Labelled AII bound to RAEC in a specific, saturable manner. Scatchard analysis showed a Kd of 1.87±0.49 nM and a Bmax of 50.2±10.9 × 103 sites per cell. AII was displaced by the AT1‐specific antagonist, DuP753 with a Ki of 17.37 ± 1.49 nM, but not by the AT2 receptor analogues CGP42771B or PD123177. These data were confirmed by the finding of AT1 mRNA in endothelial cells. Analysis of RNA expression by RT‐PCR showed the presence of both subtypes, AT1A and AT1B, in endothelial cells, whereas smooth muscle cells express only AT1A. 4 . The activation of PLC and PLA2 in response to AII was evaluated by measuring inositol phosphate production and arachidonic acid release, respectively. Both were enhanced by AII in a dose‐dependent manner, and inhibited by DuP753, but not by PD123177. 5 . We conclude that AT1 receptors are expressed by endothelial cells in primary culture and that phospholipase C and phospholipase A2 are activated via this receptor.

In vitro activation of human macrophages by alginate-polylysine microcapsules

Microencapsulated islets of Langerhans have been proposed as a bioartificial pancreas. However, foreign body reaction with fibrosis has been observed around implanted microcapsules. Since macrophages are present in this reaction and interleukin-1 (IL-1), a cytokine released by activated macrophages, may induce fibrosis, we tested the capacity of alginate-polylysine microcapsules to activate macrophages. Human monocytes were isolated from whole blood of healthy donors by a Ficoll density gradient and adherence to a plastic support. Monocytes were cultured for 24 h with: (1) alginate-polylysine microcapsules; (2) lipopolysaccharide (LPS) (positive control group); and (3) alone (negative control group). Monocyte activation was evaluated by measuring the secretion of IL-1 beta and the production of intracellular IL-1 alpha and IL-1 beta. Macrophages characterization was performed by immunocytological subtyping. IL-1 beta release and intracellular IL-1 beta and IL-1 alpha production were significantly higher when macrophages were cultured with alginate-polylysine microcapsules than when macrophages were cultured alone. In conclusion, macrophages are activated in vitro by alginate-polylysine microcapsules. This effect may be involved in the fibrosis observed in vivo around implanted microcapsules. In addition, interleukin-1, released during macrophage activation, may cross the microcapsule membrane and impair islet function.

Assessment of insulin sensitivity in glucokinase-deficient subjects

SummaryThe chronic hyperglycaemia of glucokinase-deficient diabetes results from a glucose-sensing defect in pancreatic beta cells and abnormal hepatic glucose phosphorylation. We have evaluated the contribution of insulin resistance to this form of chronic hyperglycaemia. Insulin sensitivity, assessed by the homeostasis model assessment (HOMA) method in 35 kindreds with glucokinase mutations, was found to be significantly decreased in 125 glucokinase-deficient subjects as compared to 141 unaffected first-degree relatives. Logistic regression analysis showed that in glucokinase-deficient subjects a decrease in insulin sensitivity was associated with deterioration of the glucose tolerance status. A euglycaemic hyperin-sulinaemic clamp was performed in 14 glucokinase-deficient subjects and 12 unrelated control subjects. In six patients and six control subjects the clamp was coupled to dideutero-glucose infusion to measure glucose turnover. Average glucose infusion rates (GIR) at 1 and 5 mU · kg body weight · min−1 insulin infusion rates were significantly lower in (the glucokinase-deficient) patients than in control subjects. Although heterogeneous results were observed, in 8 out of the 14 patients GIRs throughout the experiment were lower than 1 SD below the mean of the control subjects. Hepatic glucose production at 1 mU · kg body weight−1 · min−1 insulin-infusion rate was significantly higher in patients than in control subjects. In conclusion, insulin resistance correlates with the deterioration of glucose tolerance and contributes to the hyperglycaemia of glucokinase-deficient diabetes. Taken together, our results are most consistent with insulin resistance being considered secondary to the chronic hyperglycaemia and/or hypoinsulinaemia of glucokinase-deficiency. Insulin resistance might also result from interactions between the unbalanced glucose metabolism and susceptibility gene(s) to low insulin sensitivity likely to be present in this population.

Angiotensin II receptors in endothelial cells

1. Angiotensin II (Ang II), the main effector of the renin-angiotensin system, exerts its vasoconstrictory and trophic actions on smooth muscle cells via AT1 receptors. However, Ang II does not act only on smooth muscle cells, as Ang II receptors are also present in endothelial cells. 2. The receptor type on these cells differs depending on the origin of the endothelium and the species. The rat endothelial receptors are mostly of the AT1 type, but AT2 receptors have also been found. The pharmacological characteristics of the AT1 receptors on endothelial cells are similar to those of other cell types. 3. Ang II stimulates phospholipase C and phospholipase A2 activation via the AT1 receptor in endothelial cells. Ang II also stimulates the tyrosine phosphorylation of several proteins in these cells. 4. Some studies suggest that the AT1 receptor mediates the release of vasodilator molecules by endothelial cells and could modulate Ang II effect on smooth muscle cells. Ang II may also inhibit endothelial cell growth via the AT2 receptor. Finally, endothelial Ang II receptors may be implicated in the regulation of fibrinolysis.

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.

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.