Biomedical subjects

F González-Fernández

Publications and source records attributed to F González-Fernández.

13 recordsLinked to original sources

PubMed2001

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.

Animals↗

PubMed2000

[The effect of triflusal on human platelet aggregation and secretion: the role of nitric oxide].

INTRODUCTION AND AIMS: The thrombotic process is a multicellular phenomenon in which not only platelets are involved but also neutrophils are involved. Recent in vitro studies performed in our laboratory have demonstrated that triflusal reduced platelet aggregation by stimulating nitric oxide (NO) production by neutrophils. The aim of the present study was to evaluate whether the in vivo treatment with triflusal could also modify the ability of neutrophils to produce NO. Furthermore, the role of NO released by neutrophils on platelet aggregation and secretion was also tested. METHODS: The study was performed in 12 healthy volunteers of 32 +/- 6 years of age. The volunteers were treated with triflusal (600 mg/day) for 5 days and platelets and neutrophils were isolated before and after treatment. The ability of neutrophils to produce NO and the capacity of inhibiting platelet aggregation and secretion of transforming growth factor-beta (TGF-beta) were assessed. RESULTS: After the treatment with triflusal we obtained the following results: a) an increase in NO production by neutrophils; b) potentiation of the inhibition of platelet aggregation by neutrophils, an effect that was reverted by incubating neutrophils with an L-arginine antagonist, L-NAME, and c) the presence of neutrophils reduced the release of TGF-beta by platelets measured as index of platelet secretion by a NO-independent mechanism. CONCLUSIONS: Triflusal (600 mg/day/5 days) stimulated NO production by neutrophils. After the treatment with triflusal, neutrophils inhibited both platelet aggregation and secretion. The antiaggregating effect of neutrophils was an NO-dependent mechanism while the inhibition of platelet secretion mediated by neutrophils after the treatment with triflusal was an NO-independent mechanism.

Adult↗

PubMed2000

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.

Animals↗

PubMed1999

Estrogen stimulates neuronal nitric oxide synthase protein expression in human neutrophils.

Recent studies have postulated the contribution of nitric oxide (NO) released by the endothelium to the beneficial effects of estrogen. Despite a neuronal-type NO synthase (nNOS) described in neutrophils, less is known about the effect of estrogen in these cells. The aim of the present study was to analyze the expression of nNOS protein in human neutrophils under different estrogenic conditions. We first analyzed nNOS expression in neutrophils obtained from premenopausal women. During the first 2 days of the follicular phase (low circulating estrogen concentrations), nNOS expression in neutrophils was reduced with respect to that found in neutrophils obtained from the same donors during the ovulatory phase (high circulating estrogen concentrations). Moreover, the expression of nNOS protein in neutrophils obtained from postmenopausal women after transdermal estrogen therapy was markedly enhanced with respect to that observed before the treatment. In vitro incubation of neutrophils derived from men for 6 hours with 17beta-estradiol (10(-10) to 10(-8) mol/L) upregulated the expression of nNOS protein. The 17beta-estradiol receptor antagonists, tamoxifen (10(-8) mol/L) and ICI 182780 (10(-8) mol/L), inhibited the upregulation of nNOS protein induced by 17beta-estradiol. The putative functional implication was denoted by a reduced expression of the CD18 antigen on the surface of 17beta-estradiol-incubated neutrophils, which was accompanied by a decreased adhesive capacity. Both effects were prevented by an NO antagonist. In conclusion, the in vivo levels of circulating estrogen concentrations seem to be associated with the level of nNOS protein expression in neutrophils from women. Moreover, low doses of 17beta-estradiol upregulate nNOS protein expression in neutrophils from men. The increased ability of 17beta-estradiol-incubated neutrophils derived from men to produce NO reduced their adhesive properties.

Adult↗

PubMed1999

[Expression of inducible nitric oxide synthase in the carotid of rats after endothelial skinning: the effects of platelets and treatment with 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.

Abciximab↗

PubMed1999

Aspirin inhibits inducible nitric oxide synthase expression and tumour necrosis factor-alpha release by cultured smooth muscle cells.

BACKGROUND: Inflammatory related cardiovascular disease, i.e. cardiac allograft rejection, myocarditis, septic shock, are accompanied by cytokine production, which stimulates the expression of inducible nitric oxide (iNOS). MATERIALS AND METHODS: The aim of the present study was to examine whether anti-inflammatory doses of acetylsalicylic acid (aspirin) could regulate iNOS protein expression in bovine vascular smooth muscle cells (BVSMCs) in culture. RESULTS: Interleukin 1 beta (IL-1 beta, 0.03 U mL-1) induced nitric oxide release by BVSMCs. Aspirin inhibited nitric oxide release from IL-1 beta-stimulated BVSMCs in a dose-dependent manner. In addition, aspirin significantly inhibited iNOS protein expression in BVSMCs and reduced the translocation of the nuclear factor-kappa B (NF-kappa B). Furthermore, aspirin and the blockade of NO generation by BVSMCs reduced the production of tumour necrosis factor alpha (TNF-alpha) by these cells. CONCLUSION: High doses of aspirin inhibited iNOS protein expression in BVSMCs and decreased NF-kappa B mobilization. The inhibition of iNOS expression by aspirin was further associated with a reduced ability of BVSMCs to produce TNF-alpha. This study could provide new mechanisms of action for aspirin in the treatment of the inflammation-related cardiovascular diseases.

6-Ketoprostaglandin F1 alpha↗

PubMed1999

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.

Adult↗

PubMed1999

NO from smooth muscle cells decreases NOS expression in endothelial cells: role of TNF-alpha.

Despite the evidence that cytokines stimulate nitric oxide (NO) production by inducible nitric oxide synthase (iNOS), several reports recently demonstrated that the hypotensive response related to endothelial nitric oxide synthase (eNOS) activity could be inhibited by the same cytokines. The aim of the present work was to analyze whether NO generated by vascular smooth muscle cells (VSMC) could modify eNOS protein expression in endothelial cells. Bovine aortic endothelial cells (BAEC) and bovine VSMC (BVSMC) in coculture were used for the study. Interleukin-1beta (IL-1beta, 10 ng/ml)-treated BVSMC, which expressed iNOS protein, decreased eNOS protein expression in BAEC. The presence of NO antagonists N(omega)-nitro-L-arginine methyl ester (10(-3) mol/l) or N(G)-monomethyl-L-arginine (10(-3) mol/l) prevented the decrease in eNOS protein expression induced by IL-1beta-treated BVSMC. Surprisingly, two different NO donors, 3-morpholinosydnonimine (10(-4) mol/l) and S-nitroso-N-acetyl-D,L-penicillamine (10(-4) mol/l), failed to modify eNOS expression in BAEC, suggesting the existence of a diffusible mediator released from IL-1beta-treated BVSMC that acts on endothelial cells by reducing eNOS expression. The presence of NO antagonists reduced tumor necrosis factor-alpha (TNF-alpha) production by IL-1beta-stimulated BVSMC. This effect was also produced in the presence of a protein kinase G inhibitor, guanosine-5'-O-(2-thiodiphosphate) trilithium salt. A polyclonal antibody against TNF-alpha prevented eNOS expression in the BAEC-BVSMC coculture. In conclusion, NO by itself failed to modify eNOS protein expression in endothelial cells but increased TNF-alpha generation by IL-1beta-stimulated BVSMC and, in this way, reduced eNOS expression in the endothelium.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Acetylcholine↗

PubMed1998

[Endothelial dysfunction: a global response].

The endothelium is a dynamic organ involved in the genesis and development of the cardiovascular diseases. Nitric oxide (NO) is one of the factors released from endothelium. NO is generated by endothelial cells through the activity of a constitutive nitric oxide synthase (cNOS). Smooth muscle cells generate NO by an inducible NOS isoform (iNOS). NO regulates vascular tone, different mechanisms involved in the interaction of blood cells to the vascular wall, the growth of smooth muscle cells and the matrix protein synthesis. The lack of an endothelium-dependent vasodilatory response has been defined as endothelial dysfunction. It has been demonstrated a reduced endothelium-dependent vasodilation response in hypertension, aging, atherosclerosis ... and in patients without evident coronary disease. Although the cNOS has been initially described as constitutive, in recent years it has been demonstrated that several pathophysiological stimuli such as hypoxia, chronic exercise, cytokines regulate its level of expression. Our laboratory has demonstrated that an endothelial cytosolic protein regulates the half-lives of eNOS mRNA. This endothelial cytosolic protein could be a target for specific drugs to prevent endothelial dysfunction.

Endothelium, Vascular↗

PubMed1989

Sucralfate in the treatment of reflux esophagitis in children. Preliminary results.

In order to evaluate the use of sucralfate in the treatment of children with reflux esophagitis, we studied 66 children aged from 4 months to 12 years (mean 5.9 years, SD 3.5) diagnosed to have gastroesophageal reflux by means of esophageal isotopic examination and radiology. An endoscopic examination was carried out in all cases. None of the patients suffered from kidney disease or had taken antacids, cimetidine, sucralfate or antirheumatic drugs in the two weeks prior to the study. Patients were divided into three groups matched according to age, grade of esophagitis, sex, nutritional state and semiology and treated with sucralfate in tablets, cimetidine, or sucralfate in suspension; no dietetic or postural measures were used. On days 14, 28, 42 and 56, clinical control was carried out and endoscopy was done on day 28, this being repeated on day 56 if the course was not satisfactory. From the statistical analysis of the results we deduce that there are no differences between the three groups. Therefore sucralfate appears to be a useful drug for the treatment of children with esophagitis due to GER.

Child↗

PubMedDate not supplied

Evidence that an endothelial cytosolic protein binds to the 3'-untranslated region of endothelial nitric oxide synthase mRNA.

Changes in the endothelial nitric oxide synthase (eNOS) expression could be involved in the endothelium-dependent vasorelaxing dysfunction associated with cardiovascular diseases. We have recently demonstrated the existence of endothelial cytosolic proteins that bind to the 3'-untranslated region (3'-UTR) of eNOS mRNA and could be involved in eNOS mRNA stabilization. In the present work, we have characterized the cytosolic proteins that bind to 3'-UTR eNOS mRNA. An endothelial cytosolic protein (MW 60-kD) specifically bound to 3'-UTR eNOS mRNA as determined by a cross-linking assay followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The endothelial cytosolic protein recognized a cytidine (C)-rich region within 3'-UTR eNOS mRNA. Furthermore, tumor necrosis factor-alpha (TNF-alpha) increased the level of the 60-kD endothelial cytosolic protein. In addition, TNF-alpha reduced eNOS mRNA levels and this was prevented by coincubation with cycloheximide. Cycloheximide also prevented the binding activity of the endothelial cytosolic protein to 3'-UTR eNOS mRNA. In summary, these data suggest that a 60-kD endothelial cytosolic protein binds to 3'-UTR eNOS mRNA. TNF-alpha increased the 60-kD protein levels. Cycloheximide prevented the binding activity of the cytosolic protein to 3'-UTR eNOS mRNA related to TNF-alpha; this effect was associated with greater eNOS mRNA levels. Further specific studies are needed to determine the involvement of this 60-kD endothelial cytosolic protein in the regulation of eNOS mRNA stabilization and in the endothelial dysfunction associated with cardiovascular diseases.

3' Untranslated Regions↗