Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide.
BACKGROUND
The vascular endothelial growth factor (VEGF) is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 (ICAM-1) is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells (ECs) is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs.


METHODS
Bovine retinal ECs (BRECs) were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF (100 ng/ml) and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase (eNOS) were detected using Western blotting. Griess reaction was used to detect nitric oxide (NO).


RESULTS
Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C (PKC) altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase (PI3K) or reactive oxygen species (ROS) significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased.


CONCLUSIONS
ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.
Zhang
Xiao-ling
XL
Department of Ophthalmology, First Affiliated Hospital, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, China. zhangxle@mail.xjtu.edu.cn
Wen
Liang
L
Chen
Yan-jiong
YJ
Zhu
Yi
Y
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012160:Retina / Q000166:cytology*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; D018799:Intercellular Adhesion Molecule-1; D009569:Nitric Oxide