Prostaglandin E₂ Promotes Pathological Retinal Neovascularization Via EP4R-EGFR-Gab1 Signalling Pathway

Background:A considerable proportion of retinal diseases in ophthalmology are caused by retinal vascular abnormalities,such as DR,ROP and RVO.Retinal neovascularization results in retinal hemorrhage,vitreous hematocele and retinal proliferation.Therefore,the generation and development of retinal neovascularization are closely related to the severity of the disease.In order to find a better method to treat retinal neovascularization,it is very urgent for us to study the mechanism of retinal neovascularization.Prostaglandin E₂（PGE₂）,biosynthesised from arachidonic acid by the cyclooxygenase（COX）enzyme,is a crucial growth-factor inducer and a potent proangiogenic mediator.PGE₂ traditionally stimulates its G-protein-coupled plasma membrane receptors（E-prostanoid1–4 receptors[EP1–4Rs]）,activating multiple signal transduction pathways that lead to downstream responses.Studies have shown that PGE₂ is involved in cell proliferation,angiogenesis and migration through EP4R,but the specific mechanism of PGE2 promoting retinal angiogenesis is still unclear.Research Object:Oxygen-induced retinopathy（OIR）is the most widely used animal model for forms of pathological angiogenesis such as retinopathy of prematurity and proliferative diabetic retinopathy（PDR）.PGE₂,the major product of cyclooxygenases,is implicated in cellular proliferation,angiogenesis and migration via prostanoid receptor EP4.The aim of this study was to investigate the role of PGE₂ and its EP4receptor（EP4R）in the promotion of retinal neovascularization.Research Contents:1.The role of the PGE₂/EP4R signaling pathway in the process of OIR.2.The PGE₂/EP4R signaling pathway mediates the retinal neovascularization.3.The PGE₂/EP4R signalling pathway activates the AKT through non G-protein coupling to promote the proliferation and migration of hRMECs.Research Methods:1.The OIR was established by incubating fetal rats from high-oxygen environment to relatively anoxic environment.2.PGE₂,Cay10598 and AH23848 were injected into the vitreous of OIR rats,according to the experimental design.3.Fundus photography and OCT were performed on the OIR rats to investigate the morphological changes of the retina.4.Evens Blue and IB4 staining were used to observe the retinal vascularized and neovascularized areas of the ratina.5.The hRMECs were cultured by the conventional methods.6.Western Blot was used to detect the expression of related target proteins in the PGE₂/EP4R signaling pathway.7.WST-1 assay was used to detect the proliferation activity of hRMECs cells stimulated by various drugs.8.Ki67 immunofluorescence assay was used to detect the proliferation capacity of hRMECs cells after drug stimulation.9.Transwell assay was performed to detect the proliferation and migration of hRMECs stimulated by PGE₂ and EP4R agonist treatment.10.CO-IP experiment was used to detect the association between EGFR and Gab1,and the association between EGFR and EP4R.Research Results:1、PGE₂/EP4R signalling influenced morphological changes in OIR rats.PGE₂-and Cay10598-treated rats showed significantly increased retinal NV while there was fewer pathological neovascular tufts in the retinas of the AH23848-treated rats.2、PGE₂/EP4R signalling specifically promoted the formation of new vascular sprouts during retinal angiogenesis.3、PGE₂/EP4R signalling promoted hRMEC proliferation and migration.PGE₂/EP4R-induced proliferation and migration of hRMECs were independent of AC/cAMP/PKA signalling pathways.4、PGE₂/EP4R signalling induced hRMEC proliferation and migration via Akt activation.hRMECs pretreated with LY294002,a PI3K inhibitor,effectively abolished the proliferation and migration induced by PGE₂ and Cay10598 exposure.5、PGE₂ recruited EGFR to the EP4R and enhanced cellular proliferation via Akt activation.Pretreatment of the hRMECs with AG1478,an EGFR inhibitor,dramatically inhibited PGE₂-induced proliferation and migration of hRMECs.Phosphorylation of Akt at Thr308,and Ser473 induced by PGE₂ was diminished in the cells pretreated with AG1478.6、EGFR recruits Gab1 and results in the upregulation of activated Akt（p-Akt）.We observed an augmented formation of the Gab1-EGFR complex and an increased phosphorylation of Gab1 in PGE₂-or Cay10598-treated hRMECs.Conclusion:These results indicate that PGE₂/EP4R mediated hRMECs proliferation by promoting the activation of the EGFR/Gab1/Akt signalling pathway and that this network is thus a potential therapeutic target for pathological intraocular angiogenesis.