RESUMO
Endothelial expression of E-selectin is enhanced in diabetic patients with retinopathy, however, the underlying mechanisms are unclear. Therefore, this study was aimed to determine if endothelial expression of E-selectin is stimulated with serum from type 2 diabetic patients with retinopathy, and whether this process is related to NAD(P)H oxidase-derived oxidative stress. Serum was obtained from type 2 diabetic patients with (T2DR) or without (T2DM) retinopathy, and age-matched non-diabetic healthy person (Control). Serum was added to in vitro-grown human coronary artery endothelial cells (HCAEC), after which E-selectin expression, reactive oxygen species (ROS) production, and NAD(P)H oxidase activity were measured. Serum from T2DR induced a significantly higher expression of E-selectin than serum from T2DM and control in association with an enhanced production of ROS in HCAEC. T2DR serum enhanced E-selectin expression in a ROS-dependent manner since this process was significantly attenuated not only by tiron (1 mM), a superoxide scavenger, but also by DPI (10 micromol/L) and apocynin (100 micromol/L), inhibitors of NAD(P)H oxidase. Furthermore, the activity of NADH oxidase was markedly increased by T2DR serum, and this was accompanied by the enhanced membrane translocation of p47phox, a cytosolic subunit of NAD(P)H oxidase. These findings suggest that serum from T2DR induced up-regulation of E-selectin expression in HCAEC, and this process might be dependent on activation of endothelial NADH oxidase via an enhanced membrane translocation of p47phox.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Retinopatia Diabética/sangue , Selectina E/metabolismo , Endotélio Vascular/metabolismo , NADPH Oxidases/metabolismo , Soro/fisiologia , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico/farmacologia , Acetofenonas/farmacologia , Antioxidantes/farmacologia , Células Cultivadas , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Diabetes Mellitus Tipo 2/complicações , Retinopatia Diabética/etiologia , Endotélio Vascular/citologia , Inibidores Enzimáticos/farmacologia , Feminino , Sequestradores de Radicais Livres/farmacologia , Humanos , Indicadores e Reagentes/farmacologia , Masculino , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismoRESUMO
The increased levels of cell adhesion molecules (CAM) have been identified in diabetic vasculatures, but the underlying mechanisms remain unclear. To determine the relationship among vascular production of superoxide, expression of CAM and diabetes, superoxide generation and expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E- and P-selectin in the aorta from control (C57BL/6J) and diabetic mice (ob/ob) were measured. In situ staining for superoxide using dihydroethidium showed an increased superoxide production in diabetic aorta in association with an enhanced NAD(P)H oxidase activity. Immunohistochemical analysis revealed that the endothelial expression of ICAM-1 (3.5 +/- 0.4) and VCAM-1 (3.8 +/- 0.3) in diabetic aorta was significantly higher than that in control aorta (0.9 +/- 0.5 and 1.6 +/- 0.3, respectively). Furthermore, there was a strong positive correlation (r = 0.89, p < 0.01 in ICAM-1 and r = 0.88, p < 0.01 in VCAM-1) between ICAM-1/VCAM-1 expression and vascular production of superoxide. The present data indicate that the increased production of superoxide via NAD(P)H oxidase may explain the enhanced expression of CAM in diabetic vasculatures.