RESUMEN
OBJECTIVE: Hypertension is a risk factor for arterial thrombosis. We investigated the effects of angiotensin II (ANG II)-dependent hypertension on the platelet proteome. METHODS AND RESULTS: Hypertension was induced in cyp1a1ren-2 transgenic rats by feeding indole-3-carbinol (n = 10) and in Fischer 344 rats by subcutaneously infusing ANG II (n = 7). After 14 days of hypertension (SBP 180 mmHg) and 10 days after normalization of blood pressure, changes in the platelet proteome were assessed by two-dimensional differential in-gel electrophoresis. In a subset of animals (n = 4), repeated blood withdrawals were performed. Of 1040 protein spots, 45 displayed hypertension-associated changes (>1.5-fold, P < 0.01) in both models (34 increased, 11 decreased). All were reversible within 10 days. Thirty-eight spots were identified by mass spectrometry and assigned to 20 distinct proteins. The majority of spots with increased intensity constituted protein fragments. Repeated blood withdrawals and stimulation of megakaryocytopoiesis by a thrombopoietin receptor agonist induced changes in the same protein spots but in the opposite direction to those induced by ANG II-dependent hypertension. CONCLUSION: ANG II-dependent hypertension is associated with enhanced protein degradation in platelets. As these changes are reversible, the proteins identified might be used to develop a biomarker for monitoring recent blood pressure history.
Asunto(s)
Angiotensina II/metabolismo , Plaquetas/metabolismo , Hipertensión/metabolismo , Proteoma/metabolismo , Aldosterona/metabolismo , Animales , Animales Modificados Genéticamente , Humanos , Indoles/farmacología , Masculino , Espectrometría de Masas/métodos , Proteómica/métodos , Ratas , Ratas Endogámicas F344 , Renina/metabolismo , Factores de RiesgoRESUMEN
Polyarteritis nodosa is a necrotizing vasculitis of medium-sized arteries of unknown origin. Hypertension is present in 30% of patients with polyarteritis nodosa. In those cases, high renin levels are thought to be secondary to renal involvement. The present study was performed to identify causal factors of polyarteritis nodosa. In cyp1a1ren-2 transgenic rats, vasculitis of medium-sized arteries resembling classical polyarteritis nodosa can be induced. In this model, oral administration of indole-3-carbinol (I3C) activates the liver-specific cyp1a1 promoter, leading to prorenin expression in a dose-dependent manner. After the first 6 weeks of chronic induction with 0.125% I3C, the mean arterial pressure reached a plateau of about 170 mmHg. Ten out of 11 I3C-treated rats, which were chronically instrumented with a telemetric device to measure blood pressure, developed polyarteritis nodosa within 10 weeks of I3C treatment. I3C alone or instrumentation alone did not cause polyarteritis nodosa. The angiotensin-converting enzyme inhibitor captopril completely prevented the development of polyarteritis nodosa, indicating that local angiotensin II generation is a pathogenetic factor in this model. The renin-angiotensin system can play a primary role in the development of polyarteritis nodosa in rats.
Asunto(s)
Poliarteritis Nudosa/fisiopatología , Sistema Renina-Angiotensina , Animales , Anticuerpos Anticitoplasma de Neutrófilos/metabolismo , Anticuerpos Antinucleares/metabolismo , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Complejo CD3/metabolismo , Captopril/farmacología , Movimiento Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Indoles/farmacología , Masculino , Poliarteritis Nudosa/enzimología , Poliarteritis Nudosa/patología , Ratas , Ratas Transgénicas , Renina/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Pérdida de Peso/efectos de los fármacosRESUMEN
OBJECTIVE: Prorenin has been associated with cardiovascular disease and the development of glomerulosclerosis via a renin/prorenin receptor. In cyp1a1ren-2 transgenic rats, prorenin levels and arterial pressure can be increased by oral administration of indole-3-carbinol (I3C). The transgenic strain has been used as a model of malignant hypertension. METHODS: The present study was designed to test the hypotheses that (i) low doses of I3C would result in dose-dependent sustained increases in arterial pressure without signs of malignancy, making cyp1a1ren-2 transgenic rats a useful model to study nonmalignant hypertension, and (ii) cyp1a1ren-2 transgenic rats would develop glomerulosclerosis when they were chronically exposed to 0.125% I3C in their diet. RESULTS: I3C treatment for 2 weeks resulted in increases of plasma prorenin concentrations and arterial pressure in a dose-dependent manner. Rats thrived well over a period of 12 weeks on dietary I3C concentrations (wt/wt) of 0.125%. Plasma prorenin concentration rose from 0.1 +/- 0.1 microg to 17.9 +/- 5.0 mug angiotensin I/ml per h (P < 0.01) and mean arterial pressure increased to a plateau of 170 +/- 5 mmHg (P < 0.001) between weeks 6 and 12. After 12 weeks of 0.125% I3C, rats exhibited moderate hypertensive renal vasculopathy, but no histological signs of glomerulosclerosis. CONCLUSIONS: The cyp1a1ren-2 transgenic rat model allows for chronic dose-dependent titration of arterial pressure by a simple and non-invasive intervention, making this strain a useful model to study malignant and nonmalignant arterial hypertension. High circulating prorenin levels per se do not cause glomerulosclerosis.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Glomeruloesclerosis Focal y Segmentaria/prevención & control , Indoles/administración & dosificación , Renina/sangre , Administración Oral , Aldosterona/sangre , Animales , Animales Modificados Genéticamente , Citocromo P-450 CYP1A1/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Glomeruloesclerosis Focal y Segmentaria/sangre , Glomeruloesclerosis Focal y Segmentaria/genética , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Ratones , Regiones Promotoras Genéticas/genética , Ratas , Ratas Endogámicas F344 , Renina/genética , Tiempo , VolumetríaRESUMEN
Allogeneic and autoimmune islet destruction limits the success of islet transplantation in autoimmune diabetic patients. This study was designed to investigate whether ex vivo gene transfer of viral interleukin-10 (vIL-10) protects BioBreeding (BB) rat islets from autoimmune destruction after transplantation into diabetic BB recipients. Islets were transduced with adenoviral constructs (Ad) expressing the enhanced green fluorescent protein (eGFP), alpha-1 antitrypsin (AAT) or vIL-10. Transduction efficiency was demonstrated by eGFP-positive cells and vIL-10 production. Islet function was determined in vitro by measuring insulin content and insulin secretion and in vivo by grafting AdvIL-10-transduced islets into syngeneic streptozotocin (SZ)-diabetic, congenic Lewis (LEW.1 W) rats. Finally, gene-modified BB rat islets were grafted into autoimmune diabetic BB rats. Ad-transduction efficiency of islets increased with virus titre and did not interfere with insulin content and insulin secretion. Ad-transduction did not induce Fas on islet cells. AdvIL-10-transduced LEW.1 W rat islets survived permanently in SZ-diabetic LEW.1 W rats. In diabetic BB rats AdvIL-10-transduced BB rat islets were rapidly destroyed. Prolongation of islet culture prior to transplantation improved the survival of gene-modified islets in BB rats. Several genes including those coding for chemokines and other peptides associated with inflammation were down-regulated in islets after prolonged culture, possibly contributing to improved islet graft function in vivo. Islets transduced ex vivo with vIL-10 are principally able to cure SZ-diabetic rats. Autoimmune islet destruction in diabetic BB rats is not prevented by ex vivo vIL-10 gene transfer to grafted islets. Graft survival in autoimmune diabetic rats may be enhanced by improvements in culture conditions prior to transplantation.
Asunto(s)
Diabetes Mellitus Experimental/patología , Herpesvirus Humano 4/metabolismo , Interleucina-10/genética , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Transducción Genética , Adenoviridae , Animales , Autoinmunidad/inmunología , Glucemia/análisis , Análisis por Conglomerados , Citometría de Flujo , Vectores Genéticos , Insulina/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Endogámicas BB , Ratas Endogámicas Lew , Estreptozocina , Receptor fas/metabolismoRESUMEN
The metabolic syndrome is associated with an increased risk for coronary heart disease. The underlying mechanisms are not well understood. The present study was designed to investigate coronary function in Wistar Ottawa Karlsburg W (WOKW) rats, a new animal model of the metabolic syndrome. The responses of coronary artery segments from WOKW and Dark Agouti (DA) control rats of different ages to several physiological vasoconstrictors and vasodilators were measured in a small vessel wire myograph, and potential mechanisms involved in the differential responses between the two strains were investigated. WOKW showed increased alpha(1)-adrenoceptor-mediated coronary constriction at 3 and 10 months of age, as well as seriously blunted beta-adrenoceptor-mediated coronary relaxation at 16 months of age. Responses to non-adrenergic agonists were not altered in WOKW compared to DA. The alpha(1)-adrenoceptor-mediated coronary constriction in WOKW was completely blocked by rho-kinase inhibition. Induced hyperinsulinemia did not cause increased alpha(1)-adrenoceptor-mediated coronary constriction or impaired beta-adrenoceptor-mediated coronary relaxation in DA. The association between blunted coronary beta-adrenoceptor responsiveness and the metabolic syndrome was confirmed in Zucker diabetic fatty rats. We conclude that the metabolic syndrome in WOKW rats is associated with impaired coronary function due to altered adrenoceptor sensitivity. The latter may contribute to inappropriately elevated coronary tone in insulin-resistant subjects, especially when sympathetic activity to the heart is increased.
Asunto(s)
Vasos Coronarios/fisiopatología , Síndrome Metabólico/fisiopatología , Envejecimiento/fisiología , Animales , Presión Sanguínea/fisiología , Calcio/metabolismo , Creatinina/metabolismo , Modelos Animales de Enfermedad , Implantes de Medicamentos , Espacio Extracelular/metabolismo , Hiperinsulinismo/metabolismo , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacología , Técnicas In Vitro , Insulina/administración & dosificación , Insulina/farmacología , Masculino , Músculo Liso Vascular/fisiología , Miografía , Norepinefrina/fisiología , Proteinuria/genética , Proteinuria/metabolismo , Ratas , Ratas Endogámicas , Ratas Zucker , Receptores Adrenérgicos alfa 1/fisiología , Receptores Adrenérgicos alfa 2/fisiología , Vasoconstricción/fisiología , Vasodilatación/fisiología , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Local excess of nitric oxide (NO) has been implicated in beta-cell damage, thus, a possible approach to the treatment of autoimmune IDDM is the selective inhibition of inducible nitric oxide synthase (iNOS). A series of variously substituted hexahydropyridazine-1-carbothioamides, -carbothioimidic acid esters and -carboximidamides was synthesized and dose-dependently evaluated as potential inhibitors of iNOS. The screening of the title compounds was performed with insulin-producing RIN-5AH cells and a combination of IL1-1 beta and IFN-gamma as inducers of cellular NO production. The structure-activity analysis revealed that the variation of substituents in the position 1 of the hexahydropyridazine strongly influences the inhibitory activity to iNOS as well as being critical for RIN cell survival. Among the compounds tested, the hexahydropyridazine-1-carbothioamides showed particularly significant inhibitory effects. However, for an efficient iNOS inhibition substitution at the nitrogen of the 1-carbothioamide group is important. Thus, the introduction of aliphatic chains such as propyl or butyl and of cyclic moieties such as cyclohexyl, 3-methoxyphenyl, and 4-methoxyphenyl (IC(50): 0.5-2.1 mM), respectively, provided compounds with similar inhibitory activity to aminoguanidine (IC(50): 0.3 mM), a common standard substance used for the selective inhibition of iNOS. However, the 1-carboximidamides, which represent more structurally related semicyclic derivatives of aminoguanidine, caused only incomplete iNOS inhibition. The hexahydropyridazine-1-carbothioimidic acid esters caused dose- and substituent-dependent damage of RIN-5AH cells. The toxicity of the synthesized compounds increased markedly if aliphatic substituents at the exocyclic N atom(s) were replaced by variously substituted aromatic rings.
Asunto(s)
Amidas/síntesis química , Amidas/farmacología , Imidoésteres/síntesis química , Imidoésteres/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Amidas/química , Línea Celular , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/patología , Humanos , Imidoésteres/química , Concentración 50 Inhibidora , Óxido Nítrico/análisis , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Relación Estructura-ActividadRESUMEN
Autoimmune beta-cell destruction occurs directly by cell-mediated cytotoxicity or indirectly by cytokines released from infiltrating lymphocytes. Cytokines (IL-1beta/IFN-gamma) modify or induce expression of MHC antigens and ICAM-1 on beta-cells which can lead to an improved binding of T-lymphocytes to beta-cells and finally to an enhanced cell-mediated cytotoxicity. Cytokines also induce Fas-expression and inducible nitric oxide synthase (iNOS) causing generation of nitric oxide (NO) which is toxic for beta-cells. The iNOS inhibitor aminoguanidine (AG) delays diabetes onset, but does not reduce diabetes incidence. We wanted to know whether AG inhibits cytokine-induced expression of Fas, MHC antigens and ICAM-1 on beta-cells of LEW.1W and BB/OK rat islets after culture with IL-1beta/IFN-gamma. NO was completely inhibited by 5.0 mmol/L AG while 0.5 mmol/L had no inhibitory effect. AG downregulated Fas-expression on the surface of beta-cells. Cytokine-induced/enhanced expression of MHC class-II and ICAM-1 was not affected by any AG concentration. AG syngergistically increased cytokine-induced enhancement of MHC class-I antigen density. AG possibly blocks the indirect pathway of beta-cell damage in vivo due to inhibition of Fas and iNOS and improves direct cell-mediated cytotoxicity due to drastic increased MHC class-I expression. Inhibition of only one pathway of beta-cell destruction is not sufficient to prevent diabetes.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Guanidinas/farmacología , Islotes Pancreáticos/efectos de los fármacos , Óxido Nítrico Sintasa/biosíntesis , Receptor fas/biosíntesis , Animales , Antígenos de Superficie/metabolismo , Citocinas/farmacología , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Insulina/metabolismo , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II , Ratas , Ratas Endogámicas LewRESUMEN
In the pathogenesis of type-1 diabetes insulin-producing beta-cells are destroyed by cellular autoimmune processes. The locality of beta-cell destruction is the inflamed pancreatic islet. During insulitis cytokines released from islet-infiltrating mononuclear cells affect beta-cells at several levels. We investigated whether cytokine-induced beta-cell destruction is associated with changes in the expression of the surface receptors intercellular adhesion molecule (ICAM)-1 and Fas. Islets from diabetes-prone and congenic diabetes-resistant BB rats were exposed to interleukin (IL)-1beta alone or in combination with interferon (IFN)-gamma plus tumour necrosis factor (TNF)-alpha. Cytokines decreased islet insulin content, suppressed glucose stimulated insulin secretion and generated enhanced amounts of nitric oxide and DNA-strand breaks. While no membrane alterations of IL-1beta treated islets cells were detectable, the cytokine combination caused damage of cell membranes. Independent of diabetes susceptibility IL-1beta treated islet beta-cells expressed a significantly increased amount of ICAM-1 on their surfaces which was not further increased by IFN-gamma+TNF-alpha. However, IL-1beta induced Fas expression was significantly enhanced only on beta-cells from diabetes-prone BB rats. From these results we suggest that IL-1beta mediates the major stimulus for ICAM-1 induction which is possibly a necessary but not sufficient step in the process of beta-cell destruction. Obviously, the additional enhancement of Fas expression on the surface of beta-cells is important for destruction. The combined action of all three cytokines induced the expression of Fas on the beta-cell surface independent of diabetes susceptibility, indicating that such a strong stimulus in vitro may induce processes different from the precise mechanisms of beta-cell destruction in vivo.