Inhibition of proliferation of retinal microvascular endothelial cells by pericytes through down-regulating KDR/Flk-1 in a co-culture system / 国际眼科杂志(Guoji Yanke Zazhi)

ABSTRACT

· AIM:To investigate the role of pericytes in growth of retinal microvascular endothelial cells with a co-culture system in order to understand some mechanism of angiogenesis in hypoxia induced retinal neovascular disorders.(RMECs) were isolated by a modified protocol using CD31 coated Dynabeads, and identified by immunocytochemical staining with anti-Factor Ⅷ and CD31 antibodies. Rat retinal pericytes were isolated and characterized by immunofluorescent staining with PDGFR-β; and desmin antibodies. Pericytes and RMECs were cultured in a contact co-culture system both under normoxia and hypoxia by Millicell chamber. RMECs proliferation was evaluated by MTT and cell cycle assay with flow cytometry. RT-PCR was used to detect the alteration of KDR/Flk-1 mRNA level in RMECs under normoxia or hypoxia in the co-culture system.harvested with the modified isolating method. The two cell types were identified by positive Factor Ⅷ, CD31 and PDGFR-β, desmin cytochemical staining respectively.RMECs proliferated significantly under hypoxia from 3 to 9d with a maximal rate on day 6 (24.9%, P ＜ 0.01) by MTT. In the co-culture system, the proliferation of RMECs was inhibited by pericytes. After 6d exposure to hypoxia,the fraction of S-phase RMECs number was greatly increased by 43.9% (P ＜ 0.01). In the co-culture system,RMECs proliferation was inhibited by pericytes through decreasing the fraction of S-phase cell number both under normoxia (3.6%, P＜0.05) and under hypoxia (15.1%,P＜0.01). KDR/Flk-1 mRNA level in single cultured RMECs was shown to increase approximately 1.3-fold when exposed to hypoxia. Compared with single cultured RMECs, co-culture with pericytes could decrease KDR/Flk-1 mRNA by 45.1% (P＜0.05) and 27.7% (P ＜ 0.05) under normoxia and hypoxia condition respectively.pericytes could inhibit proliferation of RMECs under both normoxia and hypoxia. The inhibition effects of pericytes maybe, at least in part, due to downregulation of KDR/Flk-1 of RMECs. These findings confirm that pericytes could be a potential inhibitor in the pathogenesis of retinal neovascularization.