Xia Ling, Wang Hong, Munk Snezana, Frecker Helena, Goldberg Howard J, Fantus I George, Whiteside Catharine I
University Health Network, University of Toronto, Toronto, ON, Canada.
Am J Physiol Endocrinol Metab. 2007 Nov;293(5):E1280-8. doi: 10.1152/ajpendo.00223.2007. Epub 2007 Aug 21.
Vascular endothelial growth factor (VEGF) is implicated in the development of proteinuria in diabetic nephropathy. High ambient glucose present in diabetes stimulates VEGF expression in several cell types, but the molecular mechanisms are incompletely understood. Here primary cultured rat mesangial cells served as a model to investigate the signal transduction pathways involved in high-glucose-induced VEGF expression. Exposure to high glucose (25 mM) significantly increased VEGF mRNA evaluated by real-time PCR by 3 h, VEGF cellular protein content assessed by immunoblotting or immunofluorescence within 24 h, and VEGF secretion by 24 h. High-glucose-induced VEGF expression was blocked by an antioxidant, Tempol, and antisense oligonucleotides directed against p22(phox), a NADPH oxidase subunit. Inhibition of protein kinase C (PKC)-beta(1) with the specific pharmacological inhibitor LY-333531 or inhibition of PKC-zeta with a cell permeable specific pseudosubstrate peptide also prevented enhanced VEGF expression in high glucose. Enhanced VEGF secretion in high glucose was prevented by Tempol, PKC-beta(1), or PKC-zeta inhibition. In normal glucose (5.6 mM), overexpression of p22(phox) or constitutively active PKC-zeta enhanced VEGF expression. Hypoxia inducible factor-1alpha protein was significantly increased in high glucose only by 24 h, suggesting a possible contribution to high-glucose-stimulated VEGF expression at later time points. Thus reactive oxygen species generated by NADPH oxidase, and both PKC-beta(1) and -zeta, play important roles in high-glucose-stimulated VEGF expression and secretion by mesangial cells.
血管内皮生长因子（VEGF）与糖尿病肾病蛋白尿的发生有关。糖尿病中存在的高环境葡萄糖刺激多种细胞类型中的VEGF表达，但其分子机制尚未完全阐明。在此，原代培养的大鼠系膜细胞作为模型，以研究高糖诱导VEGF表达所涉及的信号转导途径。通过实时PCR评估，暴露于高糖（25 mM）3小时后VEGF mRNA显著增加，通过免疫印迹或免疫荧光评估，24小时内VEGF细胞蛋白含量增加，24小时时VEGF分泌增加。抗氧化剂Tempol和针对NADPH氧化酶亚基p22(phox)的反义寡核苷酸可阻断高糖诱导的VEGF表达。用特异性药理抑制剂LY-333531抑制蛋白激酶C（PKC）-β(1)或用细胞可渗透的特异性假底物肽抑制PKC-ζ也可防止高糖条件下VEGF表达增强。Tempol、PKC-β(1)或PKC-ζ抑制可防止高糖条件下VEGF分泌增加。在正常葡萄糖（5.6 mM）条件下，p22(phox)过表达或组成型活性PKC-ζ可增强VEGF表达。仅在24小时时，高糖条件下缺氧诱导因子-1α蛋白显著增加，提示其可能在后期对高糖刺激的VEGF表达有贡献。因此，NADPH氧化酶产生的活性氧以及PKC-β(1)和PKC-ζ在高糖刺激系膜细胞VEGF表达和分泌中起重要作用。
