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

Makino, Naoki; Maeda, Toyoki; Sugano, Masahiro; Satoh, Shinji; Watanabe, Reiko; Abe, Nobuyuki

doi:10.1016/j.jdiacomp.2005.04.002

« Previous Next »Journal of Diabetes and Its Complications
Volume 19, Issue 6 , Pages 347-355, November 2005

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

Naoki Makino
- Section of Molecular and Clinical Gerontology, Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Beppu 874-0838, Japan
- Corresponding author. Tel.: +81 977 27 1681; fax: +81 977 27 1682.
Toyoki Maeda
- Section of Molecular and Clinical Gerontology, Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Beppu 874-0838, Japan
Masahiro Sugano
- Section of Molecular and Clinical Gerontology, Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Beppu 874-0838, Japan
Shinji Satoh
- Section of Molecular and Clinical Gerontology, Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Beppu 874-0838, Japan
Reiko Watanabe
- Abe Medical Clinic, Beppu, Japan
Nobuyuki Abe
- Abe Medical Clinic, Beppu, Japan

Received 12 January 2005; received in revised form 15 March 2005; accepted 14 April 2005.

Abstract

A high dose of tumor necrosis factor (TNF)-α 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-α and vascular endothelial growth factor (VEGF) were elevated in diabetic patients. Plasma levels of TNF-α, two soluble TNF-α 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-α, 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-α, 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-α antibody. These results imply that eNOS down-regulation in CD is associated with high serum TNF-α 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-α levels.

Keywords: TNF-α, eNOS, HUVECs, Type 2 diabetes, VEGF, Microangiopathy