Incidence of complications and mortality in a type 2 diabetes patient cohort study followed up from diagnosis in a primary healthcare centre.

AIMS: To determine the microvascular and macrovascular complications and mortality incidence rates and to identify the related factors in patients recently diagnosed with type 2 diabetes between 1991 and 2000 and followed until 2006. METHODS: Retrospective longitudinal study in a primary healthcare center. Patients without any measure of glycaemia in the 3 years previous to diabetes diagnosis were excluded. Annual incidence rates for microvascular and macrovascular complications and mortality were estimated. Analysis of KaplanMeier survival curves and Cox proportional risk models by gender were done. RESULTS: Of 469 patients [mean age: 60.4 (SD 10.7) years, 53.9% women], 80 died principally of tumoral (38.7%) and cardiovascular (30%) causes. The mean follow-up period was 8.81 years. (SD 3.21). The complication rates per 1000 patients/year (95% CI) were: microvascular complications 29.11 (22.97-36.38), macrovascular complications 24.10 (19.05-30.08) and mortality 19.23 (15.25-23.93), all of those being significantly greater in males except for cerebrovascular disease. Complications and mortality were associated with age, HbA1c, HDL-cholesterol, blood pressure and smoking with a different significance for each gender. HbA1c was related to microvascular complications in both sexes and to macrovascular complications only in women. CONCLUSION: The annual rates for death and complications in a Mediterranean type 2 diabetic patient cohort followed from diagnosis were lower than those published in Anglo-Saxon countries. Males showed higher death and complication rates except in terms of cerebrovascular disease. Predictors of complication and death were different depending on gender. In terms of mortality, unlike in other studies, only one-third of the deaths were for cardiovascular causes.

Menopausia y enfermedad cardiovascular / Menopause and cardiovascular disease

Varios estudios epidemiológicos han demostrado que las mujeres premenopáusicas están protegidas frente a las enfermedades cardiovasculares si las comparamos con los hombres de edad similar. Después de la menopausia el riesgo cardiovascular aumenta progresivamente. El tratamiento de reposición hormonal en las mujeres postmenopáusicas reduce de modo acusado la morbilidad y la mortalidad cardiovascular. Los efectos ateroprotectores de los estrógenos se han atribuido al efecto hormonal sobre el espectro lipídico. No obstante, el análisis de regresión múltiple sugiere que sólo el 25 por ciento de la reducción de los accidentes cardiovasculares es atribuible al efecto hipolipidemiante del tratamiento de reposición de estrógenos. Disponemos de numerosos datos que indican que la acción de los estrógenos sobre los vasos sanguíneos contribuye a los efectos protectores sobre el sistema cardiovascular. Los estrógenos inducen un mecanismo de vasodilatación e inhiben la respuesta de los vasos sanguíneos al insulto y del desarrollo de las lesiones aterosclerósicas. Los estrógenos no sólo alteran las concentraciones de los lípidos séricos, sino que modulan el sistema fibrinolítico y antioxidante e induce la producción de otras moléculas vasoactivas, como el NO o las prostaglandinas. Los efectos rápidos de los estrógenos sobre los vasos sanguíneos se producen por la activación de la sintetasa del óxido nítrico sin transcripción génica, mientras que los efectos a largo plazo implican cambios en la expresión génica (AU)

Expression of inducible nitric oxide synthase in the liver of bile duct-ligated Wistar rats with modulation by lymphomononuclear cells.

BACKGROUND: The current study evaluated whether biliary tract obstruction stimulates inducible nitric oxide synthase (iNOS) protein expression in the liver and analyzed the implication of lymphomononuclear cells and interleukin-4 (IL-4). METHODS: Male Wistar rats were used. Bile flow interruption was achieved by a complete division of the extrapancreatic common bile duct. iNOS expression was determined by both the Western blot technique and immunohistochemistry. RESULTS: iNOS protein was markedly expressed in the liver 7 days after bile duct obstruction. Treatment with thymostimulin (TP-1), a partially purified thymic extract, reduced the intensity of the expression of iNOS protein in the liver after bile duct ligation. Recent data have suggested that IL-4 attenuates iNOS protein expression. We then analyzed the involvement of this anti-inflammatory cytokine on the modulation of iNOS expression in the liver. The liver from rats that underwent bile duct ligation (BDL) showed a lower content of IL-4 than that of sham-operated (SO) rats. TP-1 treatment increased the content of IL-4 in the liver. Liver slices incubated in vitro with Escherichia coli lipopolysaccharide (LPS, 10 microg/mL) stimulated the expression of iNOS protein. The level of LPS-induced iNOS expression was reduced by lymphomononuclear cells obtained from sham-operated animals. However, lymphomononuclear cells isolated from BDL rats potentiated the induction of iNOS expression by LPS-stimulated liver. However, lymphomononuclear cells from TP-1-treated BDL rats failed to modify LPS-stimulated iNOS expression. The different effect of lymphomononuclear cells on the modulation of iNOS expression in the liver was associated with their ability to generate IL-4. CONCLUSIONS: The liver of jaundiced rats markedly expressed iNOS protein, which was associated to modifications in the content of IL-4 in the liver. Furthermore, lymphomononuclear cells modulate iNOS protein expression in the liver by a mechanism in which IL-4 is involved.

[The effect of triflusal on human platelet aggregation and secretion: the role of nitric oxide]. / Efecto del triflusal sobre la agregación y secreción de las plaquetas humanas: papel del óxido nítrico.

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.

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.

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.

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.

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.

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.

Comparison of in vitro effects of triflusal and acetysalicylic acid on nitric oxide synthesis by human neutrophils.

Recent studies have suggested that the protective anti-ischemic effects of acetylsalicylic acid are stronger than the inhibition of platelet thromboxane A2 synthesis. Since ischemic events still occur in acetylsalicylic acid-treated patients, the development of new drugs with more powerful protective effects is needed. We compared the effects of a new platelet antiaggregating drug, 2-acetoxy-4-trifluoromethyl-benzoic acid (triflusal) and of acetylsalicylic acid on the interaction between human neutrophils and platelets, examining the capability of neutrophils to generate nitric oxide (NO). Triflusal, in the presence of neutrophils, showed a greater antiplatelet potency than acetylsalicylic acid to inhibit thrombin-induced platelet activation. Significant stimulation of NO-mediated mechanisms in the presence of acetylsalicylic acid or triflusal was demonstrated by the following findings: (1) increased metabolism of arginine to citrulline, (2) increase of cGMP in the platelet/neutrophil system and (3) the inhibitory action of the L-arginine (L-Arg) competitive analogue, NG-nitro-L-arginine-methyl ester (L-NAME), which was reversed by L-Arg. Triflusal increased the stimulation of NO synthesis by neutrophils more than did of acetylsalicylic acid. The main metabolite of triflusal, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB), alone or in combination with acetylsalicylic acid, did not modify NO production by neutrophils. Therefore, the whole molecule of triflusal is needed to stimulate NO production by neutrophils. Our results show that, in the presence of neutrophils, triflusal exerts an antiplatelet effect greater than that of acetylsalicylic acid, demonstrating a more powerful stimulation of the NO/cGMP system. The present results indicate that it is possible to develop new and more potent acetylsalicylic acid-related antiplatelet drugs for the prevention of the myocardial ischemic/reperfusion processes.

Endogenous angiotensin II produced by endothelium regulates interleukin-1beta-stimulated nitric oxide generation in rat isolated vessels.

The endothelium is a source of several factors that regulate vascular functions. Angiotensin II is one of the main active factors released by the endothelium. The aim of the present work was to analyze the role of angiotensin II released by the endothelium in the regulation of the inducible nitric oxide synthase expression in rat isolated aortic vessels. Interleukin-1beta (0.03 U/L) stimulated nitrite release by the aortic vessels. The nitrite released was less in vessels with endothelium than in deendothelialized aortic segments. This effect was accompanied by a reduced expression of the inducible nitric oxide synthase in the aortic rings with endothelium. Exogenous angiotensin II inhibited IL-1beta-stimulated inducible nitric oxide synthase protein expression in both deendothelialized vessels and those with endothelium, although with reduced ability on the aortic segments with endothelium by a nitric oxide-independent mechanism. In the aortic rings with endothelium, either inhibition of the AT-1 receptor with losartan or blocking of angiotensin II generation with fosinopril enhanced interleukin-1beta-stimulated inducible nitric oxide synthase protein expression. In conclusion, the endothelium decreases inducible nitric oxide synthase expression in the vascular wall. Angiotensin II released from endothelial cells is a main mediator responsible for this inhibition through an AT-1-type receptor-dependent mechanism.