The Role Of15-Lipoxygenase-1in Hypoxia-induced Retinal Neovascularization

Part One Establishment and Evaluation of a Novel Model of Hypoxia-induced Retinal Nevoscularization in VitroPurpose:To establish and evaluate a novel in vitro model of hypoxia-induced retinal nevoscularization.Methods:Primary retinal microvascular endothelial cells were isolated from the retinas of C57/BL6J rats and identified by an evaluation for FITC-marked CD31. Cultures were exposed to Bio-bags for0.5,1,2,3,12,24,48and72h. The pO2, pCO2, and pH values of the culture medium in the different hypoxia times were measured with a blood-gas analyzer.Results:Routine evaluation for FITC-marked CD31showed that cells were>95%pure. When the cells had been exposed to the Biobag for2h, the pO2was5.60Kpa, the pCO2was6.04Kpa, and the pH was7.07. When the cells had been exposed to the Biobag for more than2h, the pO2was4.5Kpa, the pCO2and the pH changed slightly.Conclusion:These findings suggested that a novel in vitro model of retinal nevoscularization using the Bio-bag had a good authenticity. Part Two A Hypoxia-driven VEGF Autocrine Loop Necessary for Proliferation of Retinal Microvascular Endothelial CellsPurpose:To determine whether an autocrine pathway of retinal microvascular endothelial cells (RMVECs) by vascular endothelial growth factor (VEGF) signaling plays a role in retinal novascularization.Methods:The hypoxia models were established with the Bio-bag at the time of12,24,48and72h, and evaluated with a blood-gas analyzer. The control groups were incubated in a normoxic condition for the same length of time. Cells proliferation was evaluated by the CCK-8method. 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:Real-time RT-PCR revealed that the expressions of VEGF-A, VEGF-R2and iNOs mRNA in hypoxic groups increased in comparison with those in normoxia groups (p<0.01) and the expressions of mRNA increased significantly in a time-dependent fashion in hypoxic groups during48h (p<0.01), peaking at48hours, then decreased. Western blot analysis revealed that the expressions of relative proteins ranked in that order. CCK-8analysis revealed that the proliferative capacity of RMVECs in hypoxic groups was significantly higher than those in normoxic groups at each time point (p<0.05). At48hours, the proliferative capacity was highest in hypoxia groups (p<0.05). Data acquisition from flow cytometry showed that cell survival rates in hypoxic groups were higher than those in normoxic groups and apoptosis rates dropped accordingly. The survival rate was highest at48hours.Conclusion:According to the well-established in vitro hypoxia model by the Bio-bag, RMVECs include the requisite elements for an autocrine pathway that may serve to amplify the angiogenic effects of VEGF. Part Three The Role of15-Lipoxygenase-1in Hypoxia-induced Retinal NeovascularizationPurpose:To investigate whether15-LOX-1plays a role in preventing hypoxia-induced proliferation of retinal microvascular endothelial cells(RMVECs) and the underlying mechanism.Methods:Experiments were performed using RMVECs treated with and without transfer Ad-15-LOX-1or Ad-GFP both under hypoxia and normoxia condition at12,24,48,72h. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8method. RNA and protein expressions of15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot.Results:We verified RMVECs could be infected with Ad-15-LOX-1or Ad-GFP via Fluorescence microscopy. CCK-8analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (p<0.05). In a hypoxic condition, the proliferative capacities of RMVECs in15-LOX-1group were significantly inhibited (p<0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2and eNOs mRNA increased in hypoxia group compared with normoxia group(p<0.01). However, the expressions of15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (p<0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2and eNOs mRNA decreased significantly in15-LOX-1group compared with hypoxia group(p<0.01). However,15-LOX-1and PPAR-r mRNA increased significantly in15-LOX-1group compared with hypoxia group(p<0.01). There is no significant difference of the mRNA expressions between GFP group and hypoxia group(p>0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order.Conclusions:Our results suggested that15-LOX-1and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of15-LOX-1overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization(RNV). Overexpression15-LOX-1on RMVECs of hypoxia-induced RNV blocked signaling cascades by preventing hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR2could be an additional mechanism whereby15-LOX-1prevented the hypoxia-induced RNV.