High serum TNF-alpha level in Type 2 diabetic patients with microangiopathy is associated with eNOS down-regulation and apoptosis in endothelial cells

A high dose of tumor necrosis factor (TNF)-alpha induces endothelial dysfunction and enhances apoptosis in vitro. The present study was conducted to examine whether incubating human umbilical vein endothelial cells (HUVECs) with serum from Type 2 diabetic patients complicated with retinopathy and/or microalbuminemia demonstrate endothelial dysfunction. Serum levels of TNF-alpha and vascular endothelial growth factor (VEGF) were elevated in diabetic patients. Plasma levels of TNF-alpha, two soluble TNF-alpha receptors (sTNFR), and VEGF were assessed in diabetic patients (CD, n=21) complicated with retinopathy and/or nephropathy, uncomplicated diabetic patients (UD, n=18), and in healthy normal participants (NS, n=16). In HUVECs incubated with patient's serum, endothelial constitutive nitric oxide synthase (eNOS) protein expressions were measured by Western blot analysis. Apoptosis in HUVECs was determined by optical microscopy, DNA fragmentation, and CPP32-like protease activity. Serum TNF-alpha, sTNFR-I, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS, in CD were significantly higher than in UD or NS. While, serum sTNFR-I and VEGF levels were significantly increased in the both diabetic patients, compared with those of NS, no difference was observed in the serum TNF-alpha, sTNFR-II, and ADMA levels between UD and NS. eNOS down-regulation and apoptosis were seen in HUVECs incubated with serum from CD for 24 h, but those observations were completely counteracted in the incubation by the addition of the antihuman TNF-alpha antibody. These results imply that eNOS down-regulation in CD is associated with high serum TNF-alpha levels despite of high serum of VEGF levels. Therefore, endothelial dysfunction in diabetic patients complicated with microangiopathy may, in part, be attributed to high serum TNF-alpha levels.