VEGF was reported to be increased in both the vitreous and the ocular fluids of patients with diabetic retinopathy.18 VEGF is currently viewed as a major effector for retinal neovascularization in all proliferative retinopathies.19 VEGF may also be involved in the genesis of retinal vascular leakage and nonperfusion, two major complications of diabetes. In diabetic rats, the rates of retinal endothelial barrier breakdown and VEGF activity were increased in proportion to the duration of the diabetes.20 VEGF can trigger many of the retinal vascular changes caused by diabetes, including ICAM-1 up-regulation, leukocyte adhesion to endothelial cells, vascular permeability, and capillary nonperfusion.3,4 To study the role of VEGF in the induction of retinal endothelial ICAM-1 and the underlying mechanism, we first isolated and cultured BRECs and then treated the cells with VEGF. The results showed that VEGF increased ICAM-1 expression and induced eNOS phosphorylation and NO generation in BRECs. Previous studies found that the up-regulation of ICAM-1 and VEGF coincides in experimental diabetes and that the inhibition of VEGF suppresses ICAM-1 expression and leukostasis.21 ICAM-1 participates in different types of cell-cell interactions and transendothelial migration; the process leads to blood-retinal barrier breakdown and capillary nonperfusion.22 Thus, the up-regulation of ICAM-1 seems to play a crucial role in the development of diabetic retinopathy.23 Endothelial NO is known to play a role in the regulation of retinal vascular functions and is postulated to contribute to the pathophysiology of retinopathy.23 VEGF has been shown to stimulate endothelial NO production by activating eNOS, 24,25 which may mediate the angiogenic and inflammatory activities of VEGF.7,26,27 We showed that VEGF increased eNOS activation and NO generation, as well as ICAM-1 production in microvascular ECs, which differs from the effects of other pro-inflammatory stimuli such as LPS.
Our results revealed that although VEGF increased NO production and ICAM-1 expression, the up-regulation of ICAM-1 was not directly mediated by NO. In order to examine whether NO is involved in the signal transduction of ICAM-1 induction by VEGF, we used L-Arginine and its analogy L-NAME to block NO production. It is significant to note that in accordance with our findings, Murohara and colleagues28 found that L-NAME did not alter the expression of multiple angiogenesis-related cell adhesion molecules, including ICAM-1. ECs contain at least two high affinity receptors for VEGF or for Flk-1/KDR and Flt-1; both belong to the family of receptor-tyrosine kinases. Autophosphorylation of these receptors leads to the activation of PI3K and the subsequent phosphorylation of PKC.29,30 In our experiment, we used Calphostin C to block PKC and revealed that neither of them altered ICAM-1 expression or eNOS phosphorylation, indicating that PKC did not participate in the up-regulation of ICAM-1 by VEGF. Interestingly, blocking PI3K with LY294002 could significantly decrease protein expression of ICAM-1 and phosphorylation of eNOS. Furthermore, LY294002 also attenuated NO production. Thus, PI3K, but not PKC, is involved in the up-regulation of ICAM-1 by VEGF.
In summary, retinal ischemia can induce that the expression of VEGF in retinal ECs in diabetes mellitus. VEGF activates its downstream effector PI3K, then through the VEGF/PI3K/AKT/eNOS pathway, promotes NO production. However, VEGF can also increase ROS production and eNOS uncoupling; ROS induces ICAM-1 expression by activating the transcriptional factors NF-κB and AP-1, and O2- combines with NO to form peroxynitrite, an effect that is stronger than the addition of O2-, to promote ICAM-1 expression.
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