Modeling of diseases of retinal ischemia in vitro: Possible participation of autocrine vascular endothelial growth factor signaling

Purpose: To establish and evaluate a novel in vitro model of retinal ischemia, and to determine whether an autocrine pathway of retinal microvascular endothelial cells (RMVECs) by vascular endothelial growth factor (VEGF) signaling plays a role based on this model. Methods: Primary RMVECs were isolated from the retinas of C57/BL6J rats and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the biobag at the time of 12, 24, 48 and 72 h, and evaluated with a blood-gas analyzer. The control groups were incubated under normoxic conditions for the same length of time. Cell proliferation was evaluated by the CCK-8 method. Apoptosis was assayed using a flow cytometry method. RNA and protein expressions of VEGF-A, VEGFR-2 and iNOs were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Results: The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 h, the pO₂ was below 4.5 kPa, pCO₂ and pH shifted slightly. Real-time RT-PCR revealed that the expressions of VEGF-A, VEGFR-2 and iNOs mRNA in hypoxic groups increased in comparison to those in the normoxia groups (p < 0.01) and the expression of mRNA increased significantly in a time-dependent fashion in the hypoxic groups (p < 0.01), peaking at 48 h, and then decreasing. Western blot analysis revealed that the expression of relative proteins ranked in this order. CCK-8 analysis revealed that the proliferative capacity of RMVECs in the hypoxic groups was significantly higher than those in the normoxic groups at each time point (p < 0.05). At 48 h, the proliferative capacity was highest in the hypoxia groups (p < 0.05). Data acquisition from flow cytometry showed that cell survival rates in the hypoxic groups were higher than those in the normoxic groups and apoptosis rates dropped accordingly. The survival rate was highest at 48 h. Conclusion: These findings suggested that a novel in vitro model of retinal ischemia using the biobag had a good authenticity. According to the well-established in vitro hypoxia model by the biobag, RMVECs include the requisite elements for an autocrine pathway that may serve to amplify the angiogenic effects of VEGF.