RESUMEN
Recent studies on the pathogenesis of diabetic retinopathy have focused on correcting adverse biochemical alterations, but there have been fewer efforts to enhance prosurvival pathways. Bcl-2 is the archetypal member of a group of antiapoptotic proteins. In this study, we investigated the ability of overexpressing Bcl-2 in vascular endothelium to protect against early stages of diabetic retinopathy. Transgenic mice overexpressing Bcl-2 regulated by the pre-proendothelin promoter were generated, resulting in increased endothelial Bcl-2. Diabetes was induced with streptozotocin, and mice were sacrificed at 2 months of study to measure superoxide generation, leukostasis, and immunohistochemistry, and at 7 months to assess retinal histopathology. Diabetes of 2 months duration caused a significant decrease in expression of Bcl-2 in retina, upregulation of Bax in whole retina and isolated retinal microvessels, and increased generation of retinal superoxide and leukostasis. Seven months of diabetes caused a significant increase in the number of degenerate (acellular) capillaries in diabetic animals. Furthermore, overexpression of Bcl-2 in the vascular endothelium inhibited the diabetes-induced degeneration of retinal capillaries and aberrant superoxide generation, but had no effect on Bax expression or leukostasis. Therefore, overexpression of Bcl-2 in endothelial cells inhibits the capillary degeneration that is characteristic of the early stages of diabetic retinopathy, and this effect seems likely to involve inhibition of oxidative stress.
Asunto(s)
Capilares/metabolismo , Retinopatía Diabética/metabolismo , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis , Endotelio Vascular/embriología , Inmunohistoquímica/métodos , Leucostasis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microcirculación , Estrés Oxidativo , Superóxidos/metabolismoRESUMEN
Pharmacologic treatment of diabetic retinopathy via eyedrops could have advantages but has not been successful to date. We explored the effect of topical Nepafenac, an anti-inflammatory drug known to reach the retina when administered via eyedrops, on the development of early stages of diabetic retinopathy and on metabolic and physiologic abnormalities that contribute to the retinal disease. Streptozotocin-induced diabetic rats were assigned to three groups (0.3% Nepafenac eyedrops, vehicle eyedrops, and untreated control) for comparison to age-matched nondiabetic control animals. Eyedrops were administered in both eyes four times per day for 2 and 9 months. At 2 months of diabetes, insulin-deficient diabetic control rats exhibited significant increases in retinal prostaglandin E(2), superoxide, vascular endothelial growth factor (VEGF), nitric oxide (NO), cyclooxygenase-2, and leukostasis within retinal microvessels. All of these abnormalities except NO and VEGF were significantly inhibited by Nepafenac. At 9 months of diabetes, a significant increase in the number of transferase-mediated dUTP nick-end labeling-positive capillary cells, acellular capillaries, and pericyte ghosts were measured in control diabetic rats versus nondiabetic controls, and topical Nepafenac significantly inhibited all of these abnormalities (all P < 0.05). Diabetes-induced activation of caspase-3 and -6 in retina was partially inhibited by Nepafenac (all P < 0.05). Oscillatory potential latency was the only abnormality of retinal function reproducibly detected in these diabetic animals, and Nepafenac significantly inhibited this defect (P < 0.05). Nepafenac did not have a significant effect on diabetes-induced loss of cells in the ganglion cell layer or in corneal protease activity. Topical ocular administration of Nepafenac achieved sufficient drug delivery to the retina and diabetes-induced alterations in retinal vascular metabolism, function, and morphology were inhibited. In contrast, little or no effect was observed on diabetes-induced alterations in retinal ganglion cell survival. Local inhibition of inflammatory pathways in the eye offers a novel therapeutic approach toward inhibiting the development of lesions of diabetic retinopathy.
Asunto(s)
Antiinflamatorios/uso terapéutico , Bencenoacetamidas/uso terapéutico , Retinopatía Diabética/tratamiento farmacológico , Fenilacetatos/uso terapéutico , Retina/efectos de los fármacos , Administración Tópica , Animales , Antiinflamatorios/farmacología , Bencenoacetamidas/farmacología , Caspasa 3/metabolismo , Caspasa 6/metabolismo , Ciclooxigenasa 2/metabolismo , Diabetes Mellitus Experimental/complicaciones , Retinopatía Diabética/metabolismo , Dinoprostona/metabolismo , Potenciales Evocados Visuales/efectos de los fármacos , Inflamación/tratamiento farmacológico , Inflamación/patología , Masculino , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Óxido Nítrico/metabolismo , Soluciones Oftálmicas/uso terapéutico , Fenilacetatos/farmacología , Distribución Aleatoria , Ratas , Ratas Endogámicas Lew , Retina/metabolismo , Retina/patología , Vasos Retinianos/efectos de los fármacos , Estreptozocina , Superóxidos/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Oxidative stress is believed to play a significant role in the development of diabetic retinopathy. In this study, we have investigated the effects of elevated glucose concentration on the production of superoxide anion by retina and retinal cells, the cellular source of the superoxide, the effect of therapies that are known to inhibit diabetic retinopathy on the superoxide production, and the role of the superoxide in cell death in elevated glucose concentration. Superoxide release was measured from retinas collected from streptozotocin-diabetic rats (2 months) treated with or without aminoguanidine, aspirin, or vitamin E, and from transformed retinal Müller cells (rMC-1) and bovine retinal endothelial cells (BREC) incubated in normal (5 mM) and high (25 mM) glucose. Diabetes (retina) or incubation in elevated glucose concentration (rMC-1 and BREC cells) significantly increased superoxide production, primarily from mitochondria, because an inhibitor of mitochondrial electron transport chain complex II normalized superoxide production. Inhibition of reduced nicotinamine adenine dinucleotide phosphate (NADPH) oxidase or nitric oxide synthase had little or no effect on the glucose-induced increase in superoxide. Treatment of diabetic animals with aminoguanidine, aspirin, or vitamin E for 2 months significantly inhibited the diabetes-induced increase in production of superoxide in the retinas. Despite the increased production of superoxide, no increase in protein carbonyls was detected in retinal proteins from animals diabetic for 2-6 months or rMC-1 cells incubated in 25 mM glucose for 5 d unless the activities of calpain or the proteosome were inhibited. Addition of copper/zinc-containing superoxide dismutase to the media of rMC-1 and BREC cells inhibited the apoptotic death caused by elevated glucose. Diabetes-like glucose concentration increases superoxide production in retinal cells, and the superoxide contributes to impaired viability and increased cell death under those circumstances. Three therapies that inhibit the development of diabetic retinopathy all inhibit superoxide production, raising a possibility that these therapies inhibit retinopathy in part by inhibiting a hyperglycemia-induced increase in superoxide production.
Asunto(s)
Hiperglucemia , Mitocondrias/metabolismo , Retina/citología , Retina/patología , Superóxidos , Actinas/química , Animales , Apoptosis , Bovinos , Muerte Celular , Línea Celular Transformada , Supervivencia Celular , Cobre/química , Retinopatía Diabética/patología , Transporte de Electrón , Glucosa/química , Glucosa/metabolismo , Inmunohistoquímica , Masculino , Óxido Nítrico Sintasa/metabolismo , Estrés Oxidativo , Oxígeno/metabolismo , Ratas , Ratas Endogámicas Lew , Retina/metabolismo , Superóxidos/metabolismo , Zinc/químicaRESUMEN
Aminoguanidine inhibits the development of retinopathy in diabetic animals, but the mechanism remains unclear. Inasmuch as aminoguanidine is a relatively selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), we have investigated the effects of hyperglycemia on the retinal nitric oxide (NO) pathway in the presence and absence of aminoguanidine. In vivo studies utilized retinas from experimentally diabetic rats treated or without aminoguanidine for 2 months, and in vitro studies used bovine retinal endothelial cells and a transformed retinal glial cell line (rMC-1) incubated in 5 mm and 25 mm glucose with and without aminoguanidine (100 microg/mL). NO was detected as nitrite and nitrate, and nitrotyrosine and iNOS were detected using immunochemical methods. Retinal homogenates from diabetic animals had greater than normal levels of NO and iNOS (p < 0.05), and nitrotyrosine was greater than normal, especially in one band immunoprecipitated from retinal homogenates. Oral aminoguanidine significantly inhibited all of these increases. Nitrotyrosine was detected immunohistochemically only in the retinal vasculature of non-diabetic and diabetic animals. Retinal endothelial and rMC-1 cells cultured in high glucose increased NO and NT, and aminoguanidine inhibited both increases in rMC-1 cells, but only NT in endothelial cells. Hyperglycemia increases NO production in retinal cells, and aminoguanidine can inhibit this abnormality. Inhibition of diabetic retinopathy by aminoguanidine might be mediated in part by inhibition of sequelae of NO production.
Asunto(s)
Diabetes Mellitus/metabolismo , Retinopatía Diabética/prevención & control , Guanidinas/farmacología , Óxido Nítrico/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Tirosina/análogos & derivados , Animales , Bovinos , Células Cultivadas , GMP Cíclico/metabolismo , Complicaciones de la Diabetes , Diabetes Mellitus/inducido químicamente , Retinopatía Diabética/etiología , Retinopatía Diabética/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Hiperglucemia/inducido químicamente , Hiperglucemia/metabolismo , Inmunohistoquímica , Masculino , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Estrés Oxidativo/efectos de los fármacos , Ácido Peroxinitroso/metabolismo , Ratas , Ratas Sprague-Dawley , Retina/citología , Retina/patología , Estreptozocina , Tirosina/metabolismoRESUMEN
OBJECTIVES: To investigate, in an initial study, the use of microarray analysis (MA) to develop an information base for urolithiasis. MA enables the screening of thousands of genes simultaneously making it the technique of choice for situations where the results are known, but the underlying mechanisms are not. Little is known about the pathological changes occurring in the kidney during urolithiasis and this has severely hampered efforts to develop effective therapeutics. MATERIALS AND METHODS: Male rats were treated with 0.75% ethylene glycol for 2, 4 or 8 weeks; after death the kidneys were processed for RNA isolation and MA, conducted using a rat-based chip (one kidney/chip) and the results confirmed by reverse transcription-polymerase chain reaction (RT-PCR, 21 probe sets; control, four rats; treated, five rats). Targets were defined as different by the software if the fold change (FC) was >or= 2, and sorted into functional categories using a data-mining tool. The repeatability of MA was investigated by subjecting the 4-week samples to MA in two independent runs. RESULTS: The results for targets with a FC of >or= 2 were plotted (y = 1.01x - 0.75; r(2) 0.84). Comparing the results obtained by RT-PCR and MA showed a good qualitative correlation for those targets having a FC of >or= 5 as determined by MA. Changes in the expression of genes associated with tubule function and regulation, oxidative damage, and inflammation were the most common in the functional categories. Changes in the expression of tubule-specific markers indicated that there was damage to the proximal (gamma-adducin, organic anion and cation transporters, sodium-hydrogen exchange protein-isoform 3) and distal tubules (gamma-adducin, kallikrein) at 2 and 4 weeks. Increased expression of mitochondrial uncoupling protein indicated that there were changes to the mitochondria and oxidative stress at 2 and 4 weeks. CONCLUSION: This study shows the power of MA as an exploratory technique, and changes in the expression of several physiologically important genes whose expression has not previously been reported to be affected by hyperoxaluria or calcium oxalate crystalluria.