Department of Ophthalmology, The First People's Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China.
Eye (Lond). 2010 Jan;24(1):145-51. doi: 10.1038/eye.2009.157. Epub 2009 Jun 26.
To investigate the effect of high glucose on insulin-induced vascular endothelial growth factor (VEGF) expression in bovine retinal microvascular endothelial cells (BRECs) and to probe into related mechanisms.
BRECs were isolated as primary cultures and identified by immunostaining. Passage cells were initially exposed to normal (5 mM) or high glucose (30 mM) for 3 days, and equimolar L-glucose was supplemented for osmotic equation. BRECs were then treated with 100 nM insulin for 24 h or not, and cells were prepared for the determination of VEGF mRNA expression by real-time PCR. VEGF protein was determined by human umbilical vein endothelial cell proliferation assay, immunofluorescence, and ELISA. BRECs were treated with 5 or 30 mM glucose for 3 days and then cells cultured with 5 mM glucose were exposed to the PI3-K inhibitor wortmannin (100 nM), the P42/44 mitogen-activated protein kinase (MAPK) inhibitor U0126 (50 microM), or to the protein kinase C (PKC) inhibitor GF109203X (2 microM) 1 h before addition of 100 nM insulin. Twenty-four hours after incubation with insulin, the cells were subjected to real-time PCR and ELISA analyses.
Insulin or high glucose alone markedly increased VEGF mRNA and protein levels in BRECs (P<0.05, two-way ANOVA). However, the combination of insulin and high glucose displayed a weaker effect in promoting VEGF expression than did insulin alone (P<0.05, t-test). Pretreatment of cells with PI3-K inhibitor significantly (P<0.05, one-way ANOVA) suppressed the insulin-induced VEGF expression; neither pretreatment with the PKC inhibitor nor with the P42/p44 MAPK inhibitor showed an effect on the expression of VEGF at the mRNA or protein level (P>0.05, one-way ANOVA).
Both insulin and high glucose can markedly increase VEGF expression in BRECs at the mRNA and protein level. We propose that insulin may upregulate VEGF expression through the PI3-K signalling pathway in BRECs, and high glucose may attenuate insulin-induced VEGF expression by impairing PI3-K signalling pathways.
研究高葡萄糖对牛视网膜微血管内皮细胞（BRECs）胰岛素诱导的血管内皮生长因子（VEGF）表达的影响，并探讨相关机制。
原代培养 BRECs 并通过免疫染色进行鉴定。将传代细胞最初暴露于正常（5 mM）或高葡萄糖（30 mM）中 3 天，并补充等摩尔 L-葡萄糖以进行渗透压平衡。然后，将 BRECs 用 100 nM 胰岛素处理 24 小时或不处理，并通过实时 PCR 测定 VEGF mRNA 表达。通过人脐静脉内皮细胞增殖测定、免疫荧光和 ELISA 测定 VEGF 蛋白。将 BRECs 用 5 或 30 mM 葡萄糖处理 3 天，然后用 5 mM 葡萄糖培养细胞，并用 PI3-K 抑制剂wortmannin（100 nM）、P42/44 丝裂原激活蛋白激酶（MAPK）抑制剂 U0126（50 microM）或蛋白激酶 C（PKC）抑制剂 GF109203X（2 microM）预处理 1 小时，然后加入 100 nM 胰岛素。用胰岛素孵育 24 小时后，进行实时 PCR 和 ELISA 分析。
胰岛素或高葡萄糖单独处理可显著增加 BRECs 中的 VEGF mRNA 和蛋白水平（P<0.05，双因素方差分析）。然而，与胰岛素单独处理相比，胰岛素和高葡萄糖联合处理对 VEGF 表达的促进作用较弱（P<0.05，t 检验）。细胞用 PI3-K 抑制剂预处理可显著抑制胰岛素诱导的 VEGF 表达（P<0.05，单因素方差分析）；用 PKC 抑制剂或 P42/p44 MAPK 抑制剂预处理对 VEGF 的 mRNA 或蛋白水平均无影响（P>0.05，单因素方差分析）。
胰岛素和高葡萄糖均可显著增加 BRECs 中 VEGF 的 mRNA 和蛋白水平。我们提出，胰岛素可能通过 BRECs 中的 PI3-K 信号通路上调 VEGF 表达，而高葡萄糖可能通过损害 PI3-K 信号通路来减弱胰岛素诱导的 VEGF 表达。
