Oxidative Stress-Mediated Thombospondin-2 Upregulation Contributes to Diabetic Endothelial Progenitor Cell Dysfunction via Matrix Metalloproteinase-9 Activation

Abstract

Endothelial progenitor cells (EPCs) play an essential role in angiogenesis but are dysfunctional in diabetes featuring excessive oxidative stress. Thrombospondin-2 (TSP-2) and matrix metalloproteinase-9 (MMP-9) possess potent anti-angiogenic properties, but their roles on EPC dysfunction in diabetes remain unknown. We tested the hypothesis that increased oxidative stress up-regulates TSP-2 and contributes to diabetic EPC dysfunction through MMP-9 activation in type 2 diabetes. Bone marrow-derived EPCs from adult male type 2 diabetic db/db mice (C57BLKS/J, 9 –13 weeks) and control db/+ mice on the same genetic background were used (blood glucose level, 451.0±8.2 mg/dL for db/db, n=38, vs. 141.3±3.7 mg/dL for db/+, n=28, p<0.01). TSP-2 mRNA and protein were both up-regulated in diabetic EPCs (3.43±0.41 folds of mRNA by qRT-PCR, n=6, p<0.01; 2.35±0.16 folds of protein by Western blot analysis, n= 5, p<0.01, vs. db/+). Silencing TSP-2 by its siRNA in diabetic EPCs improved their angiogenesis in vitro (Matrigel tube formation assay, n=4, p<0.05 vs. scrambled RNA). The increase of TSP-2 in diabetic EPCs was reversed by adenoviral vector-mediated overexpression of dominant-negative Rac1 (68±9%, n=5, p<0.05 vs. β-galactosidase reporter gene), which retards endogenous NADPH oxidase subunit Rac1 activity. Furthermore, both MMP-9 mRNA and enzymatic activity were significantly increased in diabetic EPCs (6.42±2.46 folds of mRNA by qRT-PCR, n=6, p<0.01; 1.37±0.08 folds activity by gelatin zymography, n=3, p<0.01, vs. db/+). The increased MMP-9 activity was significantly inhibited by silencing TSP-2 by its siRNA in diabetic EPCs (47±15%, n=5, p<0.05 vs. scrambled RNA). Our results suggest that up-regulation of TSP-2 mediated by increased oxidative stress contributes to dysfunction of EPC angiogenesis through MMP-9 activation in type 2 diabetes. These findings may provide a basis of targeting oxidative stress/TSP-2/MMP-9 pathway for restoring EPC angiogenic function and cell therapy of diabetic vascular complications.