Juan A. Rodríguez-Feo

Expression of Constitutive and Inducible Nitric Oxide Synthases in the Vascular Wall of Young and Aging Rats

Two NO synthase (NOS) isoforms have been described in vessels, an endothelial constitutive NOS (eNOS) and an inducible NOS (iNOS). The purpose of the present study was to examine the endothelium-dependent and endothelium-independent hypotensive response in aging rats, analyzing the ability of their vessels to produce NO. The studies were performed in 2 groups of euvolemic, conscious, male Wistar rats: aging rats (n=20, 18 months old) and young rats (n=20, 5 months old). The hypotensive responses to acetylcholine, bradykinin, and sodium nitroprusside were determined. Furthermore, the expression of the NOS isoforms by Western blot and the eNOS and iNOS activities, defined as Ca2+-dependent and Ca2+-independent conversion of [14C]L-arginine into [14C]L-citrulline, respectively, were also determined. In the aging rats, we found an impaired hypotensive response to acetylcholine and bradykinin (2 NO- and endothelium-dependent hypotensive agents) that was accompanied by a preserved hypotensive response to sodium nitroprusside. Aging rats also demonstrated an enhanced sensitivity response to the pressor effect of the L-arginine antagonist L-Nomega-nitro-L-arginine and a reduced vasoconstrictor response to angiotensin II. The inhibition of NO synthesis normalized the pressor effect of angiotensin II in the aging animals. Nitrite plus nitrate plasma levels were increased in aging rats. Furthermore, cGMP content was also higher in the aging vessels. In the aging aortas, the expression of both eNOS and iNOS isoforms was enhanced. However, in aging rats, the activity of the eNOS isoform was markedly reduced, a finding that was accompanied by the presence of iNOS activity. The vessel wall of aging rats showed an enhanced expression of eNOS and iNOS isoforms. However, eNOS activity was reduced in the aging animals. These findings could explain the impaired endothelium-dependent hypotensive response associated with aging.

Effect of losartan on human platelet activation.

ObjectivePrevious studies have demonstrated that losartan can block the thromboxane A2 receptor on the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation.MethodsPlatelets were obtained from 15 healthy men, aged 26–40 years. Platelet activati

ROLE OF NITRIC OXIDE IN THE CONTROL OF APOPTOSIS IN THE MICROVASCULATURE

Cell death occurs by either apoptosis or necrosis. Apoptosis is a cellular event in which a sequence of biochemical and morphological changes conclude in the death of the cell. Apoptosis is an important mechanism to control the number of cells and maintain tissue architecture. Nitric oxide (NO) is a multifunctional molecule that is synthesized by a family of enzymes, namely nitric oxide synthases (NOS). NO is implicated in several physiological functions within the microvascular environment, i.e. regulation of vascular tone, antiplatelet and antileukocyte properties and modulation of cell growth. Several investigations have demonstrated effects of NO on gene transcription. In this regard, NO has been also implicated in the apoptotic processes. The goal of the present review is to summarize the current knowledge about the relationship between NO and different genes involved in the apoptotic phenomena with focus in the cells of the microvascular environment, i.e. monocytes/macrophages, endothelium and vascular smooth muscle cells. Different studies have revealed that stimulation and inhibition of different genes are required to stimulate apoptosis. NO modulates the expression of bcl-2 family members, p53, interleukin-1 beta-converting enzyme family proteases and the cytokine receptor Fas. Therefore, NO generated from NO donors or synthesized by NOS induces cell death via apoptosis in a variety of different cell types. On the other hand, in the endothelial cells NO seems to have a relevant role in the maintenance of the confluent endothelial monolayer inhibiting apoptotic-related mechanisms. Furthermore, the redox states of the cells play an important role in the effects of NO as promotor of apoptosis. There have been exciting advances in the understanding of the molecular relationship between apoptosis and NO. Therefore, NO could be an important mediator to consider in the context of future therapeutic applications particularly considering apoptosis as a mechanism to maintain vascular architecture.

Reduction of the soluble cyclic GMP vasorelaxing system in the vascular wall of stroke-prone spontaneously hypertensive rats: effect of the α1-receptor blocker doxazosin

Objective The aim of the present study was to analyse the nitric oxide (NO)/cyclic GMP (cGMP) relaxing system in spontaneously hypertensive rats of the stroke-prone substrain (SHRSP). Design The study was performed in 20-week-old SHRSP rats. A group of normotensive Wistar–Kyoto (WKY) rats was used as control. Results The endothelium-dependent relaxation to acetylcholine was reduced in SHRSP rats (n = 15). No modifications in the expression of the endothelial nitric oxide synthase were found in the vascular wall of WKY rats (n = 15) and SHRSP rats. SHRSP rats demonstrated an impaired relaxing response to the NO-donor sodium nitroprusside that was accompanied by a reduction in the level of the main second messenger of NO, cyclic GMP. The expression of the soluble guanylate cyclase (sGC) β1-subunit was markedly reduced in the vascular wall of SHRSP rats. In the experimental model of SHRSP, an increased concentration of catecholamines has been reported. Therefore, we evaluated the effect of an α1-receptor blocker, doxazosin, on the NO/cGMP system. Doxazosin [10 mg/kg body weight (bw) per day for 15 days, n = 15] reduced mean arterial pressure (MAP) in SHRSP rats. Treatment with doxazosin preserved the endothelium-independent relaxation response to sodium nitroprusside in aortic segments from SHRSP rats which was associated with an increased expression of the sGC β1-subunit. A dose of doxazosin (1 mg/kg bw per day, n = 15) that did not modify MAP partially prevented sGC protein expression in the vascular wall. Conclusions Independently of the endothelial NO-generating system, impaired vasorelaxation could also result from vascular smooth muscle cell layer dysfunction. Doxazosin treatment improved the endothelial-independent relaxation and preserved the cGMP generating system in the vascular wall of SHRSP rats.

Expression of Inducible Nitric Oxide Synthase After Endothelial Denudation of the Rat Carotid Artery Role of Platelets

There is functional evidence suggesting that endothelial denudation stimulates inducible nitric oxide synthase (iNOS) activity in the vascular wall. In vitro studies have shown that iNOS expression in smooth muscle cells is reduced by endothelial cells. In the present study we have analyzed the time course of iNOS protein expression in the arterial wall after in vivo deendothelialization. Endothelial denudation was performed in the left carotid artery of Wistar rats, and the right carotid artery was used as control. Whereas iNOS protein was weakly expressed 6, 24, and 48 hours after endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after vascular damage. Because platelet adhesion and aggregation occur early after endothelial damage, we studied the role of activated platelets in the negative modulation of iNOS protein expression during the first 2 days after endothelial denudation. Early after in vivo endothelial injury, platelet-depleted rats showed a marked iNOS protein expression in the vascular wall. Similar results were obtained by blocking the platelet glycoprotein (GP) IIb/IIIa. Although iNOS protein is present in the arterial wall several days after endothelial denudation, early after arterial wall injury iNOS protein is weakly expressed. Platelets play a crucial role in preventing iNOS protein expression early after endothelial damage, an effect that can be avoided with GP IIb/IIIa blockers. Although iNOS protein was weakly expressed in vivo in the rat carotid artery wall 6, 24, and 48 hours after balloon endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after arterial damage. iNOS expression could be increased early after endothelial injury by removing circulating platelets and by an antibody against the GP IIb/IIIa. In conclusion, platelets prevent iNOS protein expression early after endothelial balloon damage, an effect that can be avoided with GP IIb/IIIa blocking agents.

Doxazosin modifies Bcl-2 and Bax protein expression in the left ventricle of spontaneously hypertensive rats.

BACKGROUND: Increased apoptosis has recently been reported in the heart of spontaneously hypertensive rats (SHRs). OBJECTIVE: To investigate the molecular basis of apoptosis in the left ventricle of SHRs in terms of the expression of Bcl-2 protein (which protects from apoptosis) and Bax protein (which acts as an apoptotic promoter). In addition, we analysed the involvement of alpha -adrenergic receptors in the left ventricular apoptosis of SHRs. METHODS: The study was performed in untreated SHRs (n=16) and SHRs that were orally treated with doxazosin (10 mg/kg body weight per day, for 15 days), a selective alpha1-receptor blocker (n=16). A group of Wistar-Kyoto (WKY) rats (n=16) was used as the control. RESULTS: The left ventricles of untreated SHRs showed a significant increase in Bcl-2 protein expression and a reduced presence of Bax protein. The ratio of Bcl-2:Bax in SHRs was higher than in WKY rats, suggesting an anti-apoptotic state. Paradoxically, both the number of apoptotic cardiac cells and the cleavage of an 85-kDa fragment of the poly (ADP-ribose) polymerase (PARP), a marker of caspase-3 activity, were higher in the left ventricle of SHRs than in WKY rats, suggesting an apoptotic situation. Bax promotes cell apoptosis when it is bound to Bcl-2. We then determined the abundance of Bax-Bcl-2 complexes in the left ventricle of the two groups of animals. Bax-Bcl-2 complexes were more abundant in SHRs than WKY rats. In a second set of experiments, we analysed the role of alpha1-adrenergic blockade by doxazosin in the above-described mechanisms. Doxazosin treatment reduced the formation of Bax-Bcl-2 complexes in the left ventricle of SHRs, and this was accompanied by a decrease in the levels of 85kDa PARP and a reduction in apoptotic left ventricular cells. CONCLUSIONS: The present work suggests that the presence of Bax-Bcl-2 complexes in the left ventricle could be a more reliable marker of the apoptotic state than the determination of the absolute expression of Bcl-2 and Bax proteins. Moreover, the inhibition of alpha1 -adrenergic receptors by doxazosin decreased the abundance of BaxBcl-2 complexes and promoted a reduction of apoptosis in the left ventricle of SHRs.

Efecto del losartán sobre la activación de plaquetas humanas por tromboxano A2

Introduccion y objetivo Estudios previos han demostrado que el losartan, antagonista de los receptores de tipo AT-1 de la angiotensina II (Ang II) podria bloquear al receptor del tromboxano A2 (TXA2) en la pared vascular. El objetivo del trabajo fue estudiar el efecto del losartan sobre la activacion de plaquetas humanas. Materiales y metodos Las plaquetas fueron obtenidas de 15 voluntarios sanos con edades comprendidas entre los 26 y 40 anos. La activacion plaquetaria fue medida por cambios en la transmision de luz del plasma rico en plaquetas estimuladas por un analogo sintetico del TXA2, el U46619 (5 × 10 –6 mol/l). Resultados El U46619 estimulo la agregacion de las plaquetas, siendo significativamente inhibida por el losartan de manera dosis dependiente. Solo dosis altas del EXP 3174, el metabolito hepatico principal del losartan, consiguieron inhibir la activacion plaquetaria inducida por el U46619. Captopril, inhibidor de la enzima convertidora de angiotensina, no fue efectivo en modificar la agregacion plaquetaria inducida por el analogo del TXA2. A pesar de que las plaquetas expresan receptores de tipo AT-1 de la Ang II, la Ang II exogena no modifico la agregacion plaquetaria inducida por U46619. La union del U46619 a las plaquetas fue competitivamente inhibida por el losartan en forma dependiente de la dosis. Sin embargo, solo dosis altas de EXP 3174 redujeron la union del U46619. Captopril no modifico la union del U46619 a las plaquetas. Conclusiones Losartan disminuyo la agregacion plaquetaria por un mecanismo dependiente de TXA2. El EXP 3174 demostro una menor potencia que losartan en reducir la activacion plaquetaria por TXA2. El captopril y la Ang II exogena no tuvieron efecto sobre la activacion de plaquetas humanas. Estos resultados sugieren que el losartan redujo la activacion plaquetaria inducida por el TXA2 por un mecanismo independiente del bloqueo de los receptores de tipo AT-1.

Doxazosina y guanilato ciclasa soluble en un modelo de ratas hipertensas

Introduccion Aunque existen diferentes estudios sobre la capacidad del endotelio de generar oxido nitrico en la disfuncion endotelial asociada a la hipertension, el sistema de la guanilato ciclasa soluble (GCs) ha sido menos estudiado. Objetivo Analizar en ratas espontaneamente hipertensas que desarrollan accidentes cerebrovasculares (SHRSP) el nivel de expresion de la GCs en la pared vascular. Tambien se estudio el efecto del tratamiento con doxazosina, antagonista a1-adrenergico, sobre la expresion de la GCs en la pared vascular de estos animales. Metodos Se utilizaron ratas SHRSP (edad, 20 semanas; n = 24). Un grupo de estas ratas fue tratado con doxazosina (10 mg/kg peso/dia; n = 12) durante 2 semanas, y se comparo con ratas Wistar-Kyoto (WKY) normotensas (n = 12). Resultados Segmentos aorticos aislados de ratas SHRSP presentaron una disminucion de la respuesta vasodilatadora al nitroprusiato sodico. La respuesta al nitroprusiato sodico mejoro en las ratas SHRSP tratadas con doxazosina respecto de las SHRSP sin tratamiento. La expresion de la b1-GCs determinada por Western blot e inmunohistoquimica estaba disminuida en las ratas SHRSP respecto a las WKY. La administracion de doxazosina aumento la expresion de GCs, particularmente en la capa media cuando se comparara con ratas SHRSP no tratadas. Conclusiones En las ratas SHRSP existe una disminucion de la expresion de b1-GCs en la pared vascular y una reduccion en la respuesta a nitroprusiato sodico que mejora tras la administracion de doxazosina cuando se compara con SHRSP no tratadas. Estos resultados sugieren la importancia que tambien puede tener el sistema de la GCs en el tratamiento global de la disfuncion endotelial.

Expresión de la óxido nítrico sintasa inducible en carótida de rata tras denudación endotelial: efecto de las plaquetas y del tratamiento con abciximab

BACKGROUND: Functional evidence suggests that endothelial denudation stimulates inducible nitric oxide synthase (iNOS) activity in the vascular wall. In vitro studies done in our laboratory have shown that iNOS expression in smooth muscle cells is reduced by endothelial cells. The object of this study was to analyze the iNOS protein expression in the arterial wall after in vivo deendothelialization, and the role of platelet activation abciximab in the expression of this protein. MATERIALS AND METHODS: Endothelial denudation was performed in the left carotid artery of Wistar rats. The right carotid artery was used as control. RESULTS: iNOS protein was only weakly expressed at 6, 24 and 48 hours after endothelial denudation. Since platelet adhesion and aggregation occur early after endothelial damage, we have analyzed the role of activated platelets in iNOS protein expression during the first two days after angioplasty. Early after in vivo endothelial injury, thrombocytopenic rats showed a marked iNOS protein expression. Similar results were obtained by blocking the platelet glycoprotein IIb/IIIa in rats treated with abciximab (Reopro). CONCLUSIONS: iNOS protein is weakly expressed in the arterial wall after endothelial denudation. Platelets play a crucial role preventing iNOS protein expression early after endothelial damage through a mechanism that depends on GP IIb/IIIa, an effect that can be avoided with glycoprotein IIb/IIIa, blockers, such as abciximab.

Effect of losartan on human platelet activation

OBJECTIVE Previous studies have demonstrated that losartan can block the thromboxane A2 receptor on the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. METHODS Platelets were obtained from 15 healthy men, aged 26-40 years. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by the thromboxane A2 analog U46619 (5 x 10(-6) mol/l) or ADP (10(-5) mol/l). RESULTS U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-dependent manner. Only a high dose of EXP 3174 (5 x 10(-5) mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I converting inhibitor, failed to modify U46619-induced platelet aggregation. Furthermore, the binding of [3H]-U46619 to platelets was competitively inhibited by losartan, whereas only a high dose of EXP 3174 reduced the binding of [3H]-U46619. Captopril failed to modify the binding of [3H]-U46619 to platelets. Losartan also reduced the platelet activation induced by ADP (10(-5) mol/l), a platelet agonist partially dependent on thromboxane A2. In addition, when thromboxane A2 generation was blocked by aspirin, ADP-induced platelet aggregation was inhibited to a similar degree to the inhibition induced by losartan. Exogenous angiotensin II did not elicit any modification of either U46619- or ADP-stimulated platelet aggregation. CONCLUSIONS Losartan decreased platelet aggregation by a thromboxane A2-dependent mechanism. EXP 3174 was less potent than losartan in reducing thromboxane A2-dependent platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced thromboxane A2-dependent platelet activation independently of its effect on angiotensin II.