RESUMEN
Highly active anti-retroviral therapy has proved successful in reducing morbidity and mortality associated with HIV infection though it has been linked to increased risk of cardiovascular disease. To date, the direct effects of the anti-retroviral drugs Efavirenz, Tenofovir and Emtricitabine on the vasculature relaxant response have not been elucidated, which impaired may predispose individuals to cardiovascular disease. Increased cellular oxidative stress and overactivation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) have been identified as central mediators of vascular dysfunction. The aim of this study was to investigate whether exposure to Efavirenz, Tenofovir or Emtricitabine directly causes endothelial cell dysfunction via overactivation of PARP. Exposure of ex vivo male rat aortic rings or in vitro endothelial cells to Efavirenz but not Tenofovir or Emtricitabine impaired the acetylcholine-mediated relaxant response, increased cellular oxidative stress and PARP activity, decreased cell viability and increased apoptosis and necrosis. Pharmacological inhibition of PARP protected against the Efavirenz-mediated impairment of vascular relaxation and endothelial cell dysfunction. Oestrogen exposure also protected against the Efavirenz-mediated inhibition of the vascular relaxant response, cell dysfunction and increased PARP activation. In conclusion, Efavirenz directly impairs endothelial cell function, which may account for the increased risk of developing cardiovascular complications with anti-retroviral therapy.
Asunto(s)
Antirretrovirales/farmacología , Benzoxazinas/farmacología , Emtricitabina/farmacología , Endotelio Vascular/enzimología , Poli(ADP-Ribosa) Polimerasas/fisiología , Tenofovir/farmacología , Alquinos , Animales , Antirretrovirales/toxicidad , Aorta Torácica/efectos de los fármacos , Aorta Torácica/enzimología , Benzoxazinas/toxicidad , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Ciclopropanos , Relación Dosis-Respuesta a Droga , Emtricitabina/toxicidad , Endotelio Vascular/efectos de los fármacos , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Tenofovir/toxicidadRESUMEN
Hyperlipidemia in the general population has been linked to the development of chronic kidney disease with both oxidative and endoplasmic reticulum stress implicated. Physiological levels (50-300 micromol/L) of saturated fatty acids such as palmitic acid (PA) cause cytotoxicity in vitro. We investigated cell type- and stimulus-specific signaling pathways induced by PA in renal proximal tubular cells and whether oxidative stress leads to ER stress or vice versa and which pathways predominate in signaling for PA-induced apoptosis and necrosis. NRK-52E cells were incubated with PA or hydrogen peroxide (H(2)O(2)) combined with SP600125 which blocks c-Jun N-terminal kinase (JNK) activation; salubrinal, which maintains eukaryotic initiation factor 2 alpha in its phosphorylated state and the antioxidant EUK-134 - a superoxide dismutase mimetic with catalase activity. We found that (i) PA causes both oxidative and ER stress leading to apoptosis which is mediated by phosphorylated JNK; (ii) oxidant-induced apoptosis generated by H(2)O(2) involves ER stress signaling and CHOP expression; (iii) the ER stress mediated by PA is largely independent of oxidative stress; (iv) in contrast, the apoptosis produced by PA is mediated partly via oxidative stress. PA-mediated cell signaling in renal NRK-52E cells therefore differs from that identified in neuronal, hepatic and pancreatic beta cells.
Asunto(s)
Retículo Endoplásmico/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Estrés Oxidativo/fisiología , Ácido Palmítico/toxicidad , Transducción de Señal/fisiología , Estrés Fisiológico/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Western Blotting , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Ratas , Transducción de Señal/efectos de los fármacos , Factor de Transcripción CHOP/metabolismoRESUMEN
Unsaturated fatty acids may counteract the lipotoxicity associated with saturated fatty acids. Palmitic acid induced endoplasmic reticulum (ER) stress and caused apoptotic and necrotic cell death in the renal proximal tubular cell line, NRK-52E. We investigated whether alpha-linolenic acid, an unsaturated fatty acid, protected against ER stress and cell death induced by palmitic acid or by other non-nutrient ER stress generators. Incubation of NRK-52E cells for 24h with palmitic acid produced a significant increase in apoptosis and necrosis. Palmitic acid also increased levels of three indicators of ER stress - the phosphorylated form of the eukaryotic initiation factor 2alpha (eIF2alpha), C/EBP homologous protein (CHOP), and glucose regulated protein 78 (GRP78). alpha-Linolenic acid dramatically reduced cell death and levels of all three indicators of ER stress brought about by palmitic acid. Tunicamycin, which induces ER stress by glycosylation of proteins, produced similar effects to those obtained using palmitic acid; its effects were partially reversed by alpha-linolenic acid. Salubrinal (a phosphatase inhibitor) causes increased levels of the phosphorylated form of eIF2alpha - this effect was partially reversed by alpha-linolenic acid. Palmitoleate, a monosaturated fatty acid, had similar effects to those of alpha-linolenic acid. These results suggest that part of the mechanism of protection of the kidney by unsaturated fatty acids is through inhibition of ER stress, eIF2alpha phosphorylation and consequential reduction of CHOP protein expression and apoptotic renal cell death.
Asunto(s)
Retículo Endoplásmico/metabolismo , Túbulos Renales Proximales/metabolismo , Ácido Palmítico/toxicidad , Estrés Fisiológico , Ácido alfa-Linolénico/metabolismo , Animales , Muerte Celular , Línea Celular , Supervivencia Celular , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas de Choque Térmico/metabolismo , Fosforilación , Ratas , Factor de Transcripción CHOP/metabolismoRESUMEN
Polychlorinated biphenyls (PCBs) are persistent environmental pollutants implicated in the development of pro-inflammatory events critical in the pathology of atherosclerosis and cardiovascular disease. PCB exposure of endothelial cells results in increased cellular oxidative stress, activation of stress and inflammatory pathways leading to increased expression of cytokines and adhesion molecules and ultimately cell death, all of which can lead to development of atherosclerosis. To date no studies have been performed to examine the direct effects of PCB exposure on the vasculature relaxant response which if impaired may predispose individuals to hypertension, an additional risk factor for atherosclerosis. Overactivation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) following oxidative/nitrosative stress in endothelial cells and subsequent depletion of NADPH has been identified as a central mediator of cellular dysfunction. The aim therefore was to investigate whether 2,2',4,6,6'-pentachlorobiphenyl (PCB 104) directly causes endothelial cell dysfunction via increased oxidative stress and subsequent overactivation of PARP. Exposure of ex vivo rat aortic rings to PCB 104 impaired the acetylcholine-mediated relaxant response, an effect that was dependent on both concentration and exposure time. In vitro exposure of mouse endothelial cells to PCB 104 resulted in increased cellular oxidative stress through activation of the cytochrome p450 enzyme CYP1A1 with subsequent overactivation of PARP and NADPH depletion. Pharmacological inhibition of CYP1A1 or PARP protected against the PCB 104-mediated endothelial cell dysfunction. In conclusion, the environmental contaminants, PCBs, can activate PARP directly impairing endothelial cell function that may predispose exposed individuals to development of hypertension and cardiovascular disease.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Contaminantes Ambientales/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Bifenilos Policlorados/farmacología , Acetilcolina/farmacología , Animales , Aorta Torácica/citología , Aorta Torácica/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/metabolismo , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Contaminantes Ambientales/química , Indicadores y Reactivos/metabolismo , Masculino , Ratones , Estructura Molecular , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , NADP/análisis , NADP/metabolismo , Óxido Nítrico/biosíntesis , Nitroazul de Tetrazolio/metabolismo , Oxidantes/efectos adversos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Bifenilos Policlorados/química , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Vasodilatación/efectos de los fármacosRESUMEN
Exposure to ricin, either by accident through ingestion of castor oil plant seeds or intentionally through its use as a bioweapon, invariably leads to multiple organ damage and death. Currently there is only a vaccine in advanced development to ricin, but no other antidote. Ricin causes systemic inflammation with increased proinflammatory cytokine release and subsequent multiple organ failure, particularly kidney and liver dysfunction. Activation of the cholinergic antiinflammatory pathway, specifically through the alpha7 nicotinic acetylcholine receptor (either indirectly through vagus nerve stimulation or directly through nicotine treatment) reduces proinflammatory gene expression. This activation also increases release of proinflammatory chemokines and cytokines, and has proven effective in a variety of inflammatory diseases. The aim of this study was to investigate whether nicotine treatment protected against ricin toxicity in mice. Male Balb/c mice exposed to ricin had increased serum levels of the inflammatory cytokine tumor necrosis factor-alpha and markers of both kidney (blood urea nitrogen, creatine) and liver (alanine tranaminase) dysfunction, with a subsequent increase in mortality. Nicotine administration 2 h after ricin injection significantly delayed and reduced ricin-induced mortality, an effect coupled with reduced serum levels of tumor necrosis factor-alpha and markers of kidney and liver dysfunction. Both the kidney and liver had markedly increased cellular oxidative stress following ricin exposure, an effect attenuated by nicotine administration. In conclusion, these data demonstrate that in cases of ricin poisoning, activation of the cholinergic antiinflammatory pathway may prove beneficial by reducing organ damage, delaying mortality, and allowing for a greater chance of survival.
Asunto(s)
Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Insuficiencia Multiorgánica/inducido químicamente , Insuficiencia Multiorgánica/tratamiento farmacológico , Nicotina/uso terapéutico , Ricina/toxicidad , Alanina Transaminasa/sangre , Animales , Nitrógeno de la Urea Sanguínea , Inflamación/metabolismo , Inflamación/mortalidad , Masculino , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos BALB C , Insuficiencia Multiorgánica/metabolismo , Insuficiencia Multiorgánica/mortalidad , Nicotina/administración & dosificación , Agonistas Nicotínicos/uso terapéutico , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Endogenous purines, including inosine, have been shown to exert immunomodulatory and anti-inflammatory effects in a variety of disease models. The dosage of inosine required for these effects has been shown to be between 200 and 600 mg kg(-1) because of the rapid metabolism of inosine in vivo. The aim of this study was to determine whether a metabolic resistant purine analog, INO-2002, exerts anti-inflammatory effects in an animal model of acute respiratory distress syndrome. Mice challenged with intratracheal LPS (50 microg) were treated with INO-2002 (30 or 100 mg kg(-1), i.p.) in divided doses at either 1 and 12 h or at 5 and 16 h. After 24 h, bronchoalveolar lavage fluid was obtained to measure leukocyte infiltration by myeloperoxidase levels, lung edema by protein levels, and proinflammatory chemokine (macrophage inflammatory protein 1alpha) and cytokine (TNF-alpha, IL-1, and IL-6) levels. INO-2002 (30 and 100 mg kg(-1)) reduced the LPS-mediated infiltration of leukocytes and edema as evidenced by bronchoalveolar lavage fluid reduction in levels of myeloperoxidase and protein. INO-2002 also downregulated expression of the proinflammatory mediators macrophage inflammatory protein 1alpha, TNF-alpha, IL-1, and IL-6. Delaying the start of treatment by 5 h after LPS administration affected the potency of INO-2002 protective effects, with 100 but not 30 mg kg(-1) having anti-inflammatory effects. The inosine analog INO-2002 largely suppressed LPS-induced inflammation in vivo at doses lower than those needed for the naturally occurring purine inosine. These data support the proposal that purine analogs, resistant to metabolic breakdown, may represent a useful addition to the therapy of acute respiratory distress syndrome.
Asunto(s)
Lesión Pulmonar Aguda/patología , Antiinflamatorios/farmacología , Inosina/farmacología , Pulmón/efectos de los fármacos , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Quimiocina CCL3/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Inosina/análogos & derivados , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Peroxidasa/metabolismo , Síndrome de Dificultad Respiratoria/inducido químicamente , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/metabolismo , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
In this study, we investigated the effect of the xanthine oxidase (XO) inhibitor, allopurinol (ALP), on cardiac dysfunction, oxidative-nitrosative stress, apoptosis, poly(ADP-ribose) polymerase (PARP) activity and fibrosis associated with diabetic cardiomyopathy in mice. Diabetes was induced in C57/BL6 mice by injection of streptozotocin. Control and diabetic animals were treated with ALP or placebo. Left ventricular systolic and diastolic functions were measured by pressure-volume system 10 weeks after established diabetes. Myocardial XO, p22(phox), p40(phox), p47(phox), gp91(phox), iNOS, eNOS mRNA and/or protein levels, ROS and nitrotyrosine (NT) formation, caspase3/7 and PARP activity, chromatin fragmentation and various markers of fibrosis (collagen-1, TGF-beta, CTGF, fibronectin) were measured using molecular biology and biochemistry methods or immunohistochemistry. Diabetes was characterized by increased myocardial, liver and serum XO activity (but not expression), increased myocardial ROS generation, p22(phox), p40(phox), p47(phox), p91(phox) mRNA expression, iNOS (but not eNOS) expression, NT generation, caspase 3/7 and PARP activity/expression, chromatin fragmentation and fibrosis (enhanced accumulation of collagen, TGF-beta, CTGF and fibronectin), and declined systolic and diastolic myocardial performance. ALP attenuated the diabetes-induced increased myocardial, liver and serum XO activity, myocardial ROS, NT generation, iNOS expression, apoptosis, PARP activity and fibrosis, which were accompanied by improved systolic (measured by the evaluation of both load-dependent and independent indices of myocardial contractility) and diastolic performance of the hearts of treated diabetic animals. Thus, XO inhibition with ALP improves type 1 diabetes-induced cardiac dysfunction by decreasing oxidative/nitrosative stress and fibrosis, which may have important clinical implications for the treatment and prevention of diabetic cardiomyopathy and vascular dysfunction.
Asunto(s)
Alopurinol/farmacología , Complicaciones de la Diabetes/prevención & control , Inhibidores Enzimáticos/farmacología , Cardiopatías/prevención & control , Xantina Oxidasa/antagonistas & inhibidores , Secuencia de Bases , Cartilla de ADN , Cardiopatías/complicaciones , HumanosRESUMEN
Endogenous purines including inosine have been shown to exert immunomodulatory and anti-inflammatory effects in a variety of disease models. The dosage of inosine required for protection is very high because of the rapid metabolism of inosine in vivo. The aim of this study was to determine whether a metabolic-resistant purine analogue, INO-2002, exerts anti-inflammatory effects in two animal models of type I diabetes. Type I diabetes was induced chemically with streptozotocin or genetically using the non-obese diabetic (NOD) female mouse model. Mice were treated with INO-2002 or inosine as required at 30, 100, or 200 mg/kg per day, while blood glucose and diabetes incidence were monitored. The effect of INO-2002 on the pancreatic cytokine profile was also determined. INO-2002 reduced both the hyperglycaemia and incidence of diabetes in both streptozotocin-induced and spontaneous diabetes in NOD mice. INO-2002 proved to be more effective in protecting against diabetes than the naturally occurring purine, inosine, when administered at the same dose. INO-2002 treatment decreased pancreatic levels of interleukin (IL)-12 and tumour necrosis factor-alpha, while increasing levels of IL-4 and IL-10. INO-2002 also reduced pancreatic levels of the chemokine MIP-1 alpha. The inosine analogue, INO-2002, was protected more effectively than the naturally occurring purine, inosine, against development of diabetes in two separate animal models. INO-2002 exerts protective effects by changing the pancreatic cytokine expression from a destructive Th1 to a protective Th2 profile. The use of analogues of inosine such as INO-2002 should be considered as a potential preventative therapy in individuals susceptible to developing type I diabetes.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 1/prevención & control , Inosina/análogos & derivados , Inosina/farmacología , Páncreas/efectos de los fármacos , Estreptozocina/farmacología , 5'-Nucleotidasa/metabolismo , Animales , Citocinas/sangre , Citocinas/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Tipo 1/inducido químicamente , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Hiperglucemia/inducido químicamente , Hiperglucemia/prevención & control , Inosina/metabolismo , Inosina Monofosfato/metabolismo , Insulina/sangre , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Interleucina-12/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Obesos , Páncreas/metabolismo , Pentosiltransferasa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Vitamin D is well characterized for its role in mineral homeostasis and maintenance of normal skeletal architecture. Vitamin D has been demonstrated to exert anti-inflammatory effects in a variety of disease states including diabetes, arthritis and inflammatory bowel disease. In these diseases poly[adenosine diphosphate (ADP)-ribose] polymerase (PARP) inhibitors have also proved effective as anti-inflammatory agents. Here we present data demonstrating that the active metabolite of vitamin D, 1alpha,25-dihydroxy-vitamin D3, is a PARP inhibitor. UV irradiation-mediated PARP activation in human keratinocytes can be inhibited by treatment with vitamin D, 7-dehydrocholesterol or 1alpha,25-dihydroxyvitamin D3. Inhibition of cytochrome P450 reversed the PARP inhibitory action of vitamin D and 7-dehydrocholesterol, indicating that conversion to 1alpha,25-dihydroxyvitamin D3 mediates their PARP inhibitory action. Vitamin D may protect keratinocytes against over-activation of PARP resulting from exposure to sunlight. PARP inhibition may contribute to the pharmacological and anti-inflammatory effects of vitamin D.
Asunto(s)
Calcitriol/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Poli(ADP-Ribosa) Polimerasas/metabolismo , Vitamina D/análogos & derivados , Vitamina D/farmacologíaRESUMEN
Growing evidence indicates that adenosine receptors could be promising therapeutic targets in autoimmune diseases. Here we studied the role of adenosine receptors in controlling the course of type 1 diabetes. Diabetes in CD-1 mice was induced by multiple-low-dose-streptozotocin (MLDS) treatment and in nonobese diabetic (NOD) mice by cyclophosphamide injection. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) prevented diabetes development in both MLDS-challenged mice and in cyclophosphamide-treated NOD mice. The effect of NECA was reversed by the selective A2B receptor antagonist N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1754). The selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were less efficacious in ameliorating the course of diabetes. NECA inhibited diabetes in A2A receptor KO mice and the selective A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethyl-carboxamidoadenosine (CGS21680) had no effect in normal mice, indicating a lack of role of A2A receptors. NECA failed to prevent cytokine-induced beta-cell death in vitro, but NECA strongly suppressed expression of the proinflammatory cytokines TNF-alpha, MIP-1alpha, IL-12, and IFN-gamma in pancreata, endotoxin, or anti-CD3-stimulated splenic cells, and T helper 1 lymphocytes, indicating that the beneficial effect of NECA was due to immunomodulation. These results demonstrate that adenosine receptor ligands are potential candidates for the treatment of type 1 diabetes.
Asunto(s)
Adenosina-5'-(N-etilcarboxamida)/uso terapéutico , Diabetes Mellitus Tipo 1/prevención & control , Diabetes Mellitus Tipo 1/fisiopatología , Agonistas del Receptor Purinérgico P1 , Receptores Purinérgicos P1/fisiología , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Células Cultivadas/efectos de los fármacos , Células Cultivadas/fisiología , Citocinas/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 1/patología , Amplificación de Genes , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Páncreas/patología , Receptores Purinérgicos P1/genética , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Overactivation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) contributes to the development of cell dysfunction and tissue injury in various pathophysiological conditions associated with oxidative and nitrosative stress, including myocardial reperfusion injury, heart transplantation, diabetic cardiomyopathy and chronic heart failure. In recent studies, we have demonstrated the beneficial effects of a novel ultrapotent PARP inhibitor, INO-1001, on cardiac and endothelial dysfunction and remodeling in rat model of advanced aging-associated chronic heart failure and in a mouse model of heart failure induced by aortic banding. In the current study, we have investigated the effect of INO-1001 on the development of heart failure induced by permanent ligation of the left anterior descending coronary artery, heart failure induced by doxorubicin and acute myocardial dysfunction induced by bacterial endotoxin. In the coronary ligation model, a significantly depressed left ventricular performance and impaired vascular relaxation of aortic rings were found, and PARP inhibition significantly improved both cardiac function and vascular relaxation. In the doxorubicin model, a single injection of doxorubicin induced high mortality and a significant decrease in left ventricular systolic pressure, +dP/dt, -dP/dt, stroke volume, stroke work, ejection fraction and cardiac output. Treatment with the PARP inhibitor reduced doxorubicin-induced mortality and markedly improved cardiac function. PARP inhibition did not interfere with doxorubicin's antitumor effect. In the endotoxin model of cardiac dysfunction, PARP inhibition attenuated the suppression of myocardial contractility elicited by endotoxin. The current data strengthen the view that PARP inhibition may represent an effective approach for the experimental therapy of various forms of acute and chronic heart failure.
Asunto(s)
Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Cardiopatías/tratamiento farmacológico , Cardiopatías/enzimología , Indoles/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Doxorrubicina/farmacología , Cardiopatías/inducido químicamente , Masculino , Ratones , Ratones Endogámicos BALB C , Poli(ADP-Ribosa) Polimerasas/metabolismo , Tasa de SupervivenciaRESUMEN
Novel indeno[1,2-c]isoquinolinone derivatives were synthesized and evaluated as inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These potent nonmutagenic PARP-1 inhibitors possess an additional five-membered ring between the B and C rings of 6(5H)-phenanthridinone. The most potent PARP-1 inhibitors were obtained from the substitution of the D ring at the C-9 position, in particular sulfonamide and N-acyl analogues (6 and 11). The 9-sulfonamide analogues 11a and 12a exhibited IC(50) values of 1 and 10 nM, respectively.
Asunto(s)
Indenos/síntesis química , Isoquinolinas/síntesis química , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/química , Indenos/química , Isoquinolinas/química , Relación Estructura-ActividadRESUMEN
Activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of various cardiovascular and inflammatory diseases. Here, we report that the gender-specific inflammatory response is preferentially down-regulated by PARP in male animals. Female mice produce less tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha in response to systemic inflammation induced by endotoxin than male mice and are resistant to endotoxin-induced mortality. Pharmacological inhibition of PARP is effective in reducing inflammatory mediator production and mortality in male, but not in female, mice. Ovariectomy partially reverses the protection seen in female mice. Endotoxin-induced PARP activation in circulating leukocytes is reduced in male, but not female, animals by pharmacological PARP inhibition, as shown by flow cytometry. Pretreatment of male mice with 17-beta-estradiol prevents endotoxin-induced hepatic injury and reduces poly(ADP-ribosyl)ation in vivo. In male, but not female, animals, endotoxin induces an impairment of the endothelium-dependent relaxant responses, which is prevented by PARP inhibition. In vitro oxidant-induced PARP activation is reduced in cultured cells placed in female rat serum compared with male serum. Estrogen does not directly inhibit the enzymatic activity of PARP in vitro. However, PARP and estrogen receptor alpha form a complex, which binds to DNA in vitro, and the DNA binding of this complex is enhanced by estrogen. Thus, estrogen may anchor PARP to estrogen receptor alpha and to the DNA and prevent its recognition of DNA strand breaks and hence its activation. In conclusion, the gender difference in the inflammatory response shows preferential modulation by PARP in male animals.
Asunto(s)
Vasos Sanguíneos/efectos de los fármacos , Endotoxinas/toxicidad , Inflamación/inducido químicamente , Poli(ADP-Ribosa) Polimerasas/metabolismo , Angiotensina II/metabolismo , Animales , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática/efectos de los fármacos , Estradiol/farmacología , Receptor alfa de Estrógeno/efectos de los fármacos , Femenino , Citometría de Flujo , Glucosa/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Ratones , Ovariectomía , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Caracteres Sexuales , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Complications of diabetes rather than the primary disease itself pose the most challenging aspects of diabetic patient management. Diabetic vascular dysfunction represents a problem of great clinical importance underlying the development of many of the complications including retinopathy, neuropathy and the increased risk of stroke, hypertension and myocardial infarction. Hyperglycaemia stimulates many cellular pathways, which result in oxidative stress, including increased production of advanced glycosylated end products, protein kinase C activation, and polyol pathway flux. Endothelial cells produce nitric oxide constitutively to regulate normal vascular tone; the combination of this nitric oxide with the hyperglycaemia-induced superoxide formation results in the production of reactive nitrogen species such as peroxynitrite. This nitrosative stress results in many damaging cellular effects, but it is these effects on DNA, which are the most damaging to the cell function; nitrosative stress induces DNA single stand breaks and leads to over-activation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP). PARP activation contributes to endothelial cell dysfunction and appears to be the central mediator in all the mechanisms by which hyperglycaemia-induces diabetic vascular dysfunction. This review focuses on the mechanism by which hyperglycaemia induces nitrosative stress and the role PARP activation plays in diabetic vascular dysfunction.
Asunto(s)
Complicaciones de la Diabetes/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Animales , Angiopatías Diabéticas/etiología , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Activación Enzimática , Humanos , Hiperglucemia/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Macro- and microvascular disease are the most common causes of morbidity and mortality in patients with diabetes mellitus. Diabetic cardiovascular dysfunction represents a problem of great clinical importance underlying the development of various severe complications including retinopathy, nephropathy, neuropathy and increase the risk of stroke, hypertension and myocardial infarction. Hyperglycemic episodes, which complicate even well-controlled cases of diabetes, are closely associated with increased oxidative and nitrosative stress, which can trigger the development of diabetic complications. Hyperglycemia stimulates the production of advanced glycosylated end products, activates protein kinase C, and enhances the polyol pathway leading to increased superoxide anion formation. Superoxide anion interacts with nitric oxide, forming the potent cytotoxin peroxynitrite, which attacks various biomolecules in the vascular endothelium, vascular smooth muscle and myocardium, leading to cardiovascular dysfunction. The pathogenetic role of nitrosative stress and peroxynitrite, and downstream mechanisms including poly(ADP-ribose) polymerase (PARP) activation, is not limited to the diabetes-induced cardiovascular dysfunction, but also contributes to the development and progression of diabetic nephropathy, retinopathy and neuropathy. Accordingly, neutralization of peroxynitrite or pharmacological inhibition of PARP is a promising new approach in the therapy and prevention of diabetic complications. This review focuses on the role of nitrosative stress and downstream mechanisms including activation of PARP in diabetic complications and on novel emerging therapeutical strategies offered by neutralization of peroxynitrite and inhibition of PARP.
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Complicaciones de la Diabetes/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Tirosina/análogos & derivados , Animales , Cardiomiopatías/etiología , Cardiomiopatías/metabolismo , Complicaciones de la Diabetes/etiología , Angiopatías Diabéticas/etiología , Nefropatías Diabéticas/etiología , Neuropatías Diabéticas/etiología , Retinopatía Diabética/etiología , Endotelio Vascular/metabolismo , Humanos , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Superóxidos/metabolismo , Tirosina/metabolismoRESUMEN
OGG-1 DNA glycosylase (OGG-1) is an enzyme involved in DNA repair. It excises 7,8-dihydro-8-oxoguanine, which is formed by oxidative damage of guanine. We have investigated the role of OGG-1 in inflammation using three models of inflammation: endotoxic shock, diabetes, and contact hypersensitivity. We found that OGG-1(-/-) mice are resistant to endotoxin (lipopolysaccharide, LPS)-induced organ dysfunction, neutrophil infiltration and oxidative stress, when compared with the response seen in wild-type controls (OGG(+/+)). Furthermore, the deletion of the OGG-1 gene was associated with decreased serum cytokine and chemokine levels and prolonged survival after LPS treatment. Type I diabetes was induced by multiple low-dose streptozotocin treatment. OGG-1(-/-) mice were found to have significantly lower blood glucose levels and incidence of diabetes as compared with OGG-1(+/+) mice. Biochemical analysis of the pancreas showed that OGG-1(-/-) mice had greater insulin content, indicative of a greater beta-cell mass coupled with lower levels of the chemokine MIP-1alpha and Th1 cytokines IL-12 and TNF-alpha. Levels of protective Th2 cytokines, IL-4 and IL-10 were significantly higher in the pancreata of OGG-1(-/-) mice as compared with the levels measured in wild-type mice. In the contact hypersensitivity induced by oxazolone, the OGG-1(-/-) mice showed reduced neutrophil accumulation, chemokine, and Th1 and Th2 cytokine levels in the ear tissue. The current studies unveil a role for OGG-1 in the regulation of inflammation.
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ADN Glicosilasas/fisiología , Animales , ADN Glicosilasas/deficiencia , ADN Glicosilasas/genética , Dermatitis por Contacto/enzimología , Dermatitis por Contacto/genética , Dermatitis por Contacto/metabolismo , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Endotoxinas/efectos adversos , Endotoxinas/metabolismo , Femenino , Inflamación/inducido químicamente , Inflamación/enzimología , Inflamación/genética , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Genéticos , Choque Séptico/enzimología , Choque Séptico/genética , Choque Séptico/metabolismo , EstreptozocinaRESUMEN
Nitrosative stress, that is, enhanced peroxynitrite formation, has been documented in both experimental and clinical diabetic neuropathy (DN), but its pathogenetic role remains unexplored. This study evaluated the role for nitrosative stress in two animal models of type 1 diabetes: streptozotocin-diabetic mice and diabetic NOD mice. Control (C) and streptozotocin-diabetic (D) mice were treated with and without the potent peroxynitrite decomposition catalyst FP15 (5 mg kg(-1) d(-1)) for 1 wk after 8 wk without treatment. Sciatic nerve nitrotyrosine (a marker of peroxynitrite-induced injury) and poly(ADP-ribose) immunoreactivities were present in D and absent in C and D+FP15. FP15 treatment corrected sciatic motor and hind-limb digital sensory nerve conduction deficits and sciatic nerve energy state in D, without affecting those variables in C. Nerve glucose and sorbitol pathway intermediate concentrations were similarly elevated in D and D+FP15 vs C. In diabetic NOD mice, a 7-day treatment with either 1 or 3 mg kg(-1) d(-1) FP15 reversed increased tail-flick latency (a sign of reduced pain sensitivity); the effect of the higher dose was significant as early as 3 days after beginning of the treatment. In conclusion, nitrosative stress plays a major role in DN in, at least, type 1 diabetes. This provides the rationale for development of agents counteracting peroxynitrite formation and promoting peroxynitrite decomposition, and their evaluation in DN.
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Neuropatías Diabéticas/fisiopatología , Metaloporfirinas/farmacología , Estrés Oxidativo/fisiología , Ácido Peroxinitroso/metabolismo , Especies de Nitrógeno Reactivo/fisiología , Animales , Glucemia/análisis , Creatina/análisis , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/metabolismo , Neuropatías Diabéticas/etiología , Ratones , Ratones Endogámicos NOD , Conducción Nerviosa/efectos de los fármacos , Neuronas Aferentes/fisiología , Fosfocreatina/análisis , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/análisis , Nervio Ciático/química , Nervio Ciático/fisiopatología , Tirosina/análogos & derivados , Tirosina/análisis , Aumento de Peso/efectos de los fármacosRESUMEN
Angiotensin II (AII) contributes to the pathogenesis of many cardiovascular disorders. Oxidant-mediated activation of poly(adenosine diphosphate-ribose) polymerase (PARP) plays a role in the development of endothelial dysfunction and the pathogenesis of various cardiovascular diseases. We have investigated whether activation of the nuclear enzyme PARP contributes to the development of AII-induced endothelial dysfunction. AII in cultured endothelial cells induced DNA single-strand breakage and dose-dependently activated PARP, which was inhibited by the AII subtype 1 receptor antagonist, losartan; the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, apocynin; and the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester. Infusion of sub-pressor doses of AII to rats for 7 to 14 d induced the development of endothelial dysfunction ex vivo. The PARP inhibitors PJ34 or INO-1001 prevented the development of the endothelial dysfunction and restored normal endothelial function. Similarly, PARP-deficient mice infused with AII for 7 d were found resistant to the AII-induced development of endothelial dysfunction, as opposed to the wild-type controls. In spontaneously hypertensive rats there was marked PARP activation in the aorta, heart, and kidney. The endothelial dysfunction, the cardiovascular alterations and the activation of PARP were prevented by the angiotensin-converting enzyme inhibitor enalapril. We conclude that AII, via AII receptor subtype 1 activation and reactive oxygen and nitrogen species generation, triggers DNA breakage, which activates PARP in the vascular endothelium, leading to the development of endothelial dysfunction in hypertension.
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Angiotensina II , Endotelio Vascular/fisiopatología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Enfermedades Vasculares/fisiopatología , Acetilcolina , Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Antihipertensivos/farmacología , Células Cultivadas , Enalapril/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Activación Enzimática/efectos de los fármacos , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Relajación Muscular/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/deficiencia , Poli(ADP-Ribosa) Polimerasas/genética , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Enfermedades Vasculares/inducido químicamente , Enfermedades Vasculares/prevención & controlRESUMEN
Aging is associated with profound changes in the structure and function of the heart. A fundamental understanding of these processes, using relevant animal models, is required for effective prevention and treatment of cardiovascular disease in the elderly. Here, we studied cardiac performance in 4- to 5-mo-old (young) and 24- to 26-mo-old (old) Fischer 344 male rats using the Millar pressure-volume (P-V) conductance catheter system. We evaluated systolic and diastolic function in vivo at different preloads, including preload recruitable stroke work (PRSW), maximal slope of the systolic pressure increment (+dP/dt), and its relation to end-diastolic volume (+dP/dt-EDV) as well as the time constant of left ventricular pressure decay, as an index of relaxation. The slope of the end-diastolic P-V relation (EDPVR), an index of left ventricular stiffness, was also calculated. Aging was associated with decrease in left ventricular systolic pressure, +dP/dt, maximal slope of the diastolic pressure decrement, +dP/dt-EDV, PRSW, ejection fraction, stroke volume, cardiac and stroke work indexes, and efficiency. In contrast, total peripheral resistance, left ventricular end-diastolic volume, left ventricular end-diastolic pressure, and EDPVR were greater in aging than in young animals. Taken together, these data suggest that advanced aging is characterized by decreased systolic performance accompanied by delayed relaxation and increased diastolic stiffness of the heart in male Fischer 344 rats. P-V analysis is a sensitive method to determine cardiac function in rats.
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Envejecimiento , Gasto Cardíaco Bajo/etiología , Gasto Cardíaco Bajo/fisiopatología , Volumen Sistólico , Función Ventricular Izquierda , Presión Ventricular , Animales , Presión Sanguínea , Diástole , Corazón/fisiopatología , Masculino , Ratas , Ratas Endogámicas F344 , Sístole , Remodelación VentricularRESUMEN
Increased production of reactive oxygen and nitrogen species has recently been implicated in the pathogenesis of cardiac and endothelial dysfunction associated with atherosclerosis, hypertension, and aging. Oxidant-induced cell injury triggers the activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP), which in turn contributes to cardiac and vascular dysfunction in various pathophysiological conditions including diabetes, reperfusion injury, circulatory shock, and aging. Here, we investigated the effect of a new PARP inhibitor, INO-1001, on cardiac and endothelial dysfunction associated with advanced aging using Millar's new Aria pressure-volume conductance system and isolated aortic rings. Young adult (3 months old) and aging (24 months old) Fischer rats were treated for 2 months with vehicle, or the potent PARP inhibitor INO-1001. In the vehicle-treated aging animals, there was a marked reduction of both systolic and diastolic cardiac function and loss of endothelial relaxant responsiveness of aortic rings to acetylcholine. Treatment with INO-1001 improved cardiac performance in aging animals and also acetylcholine-induced, nitric oxide-mediated vascular relaxation. Thus, pharmacological inhibition of PARP may represent a novel approach to improve cardiac and vascular dysfunction associated with aging.