Effects Of PGE2 On Retinal Microvascular Endothelial Cell Injury And Angiogenesis In Diabetic Retinopathy

BackgroundAs a common and refractory ocular disease,diabetic retinopathy(DR)is the main cause of blindness in the adult population.Its mechanism remains unclear.Evidence showed that chronic inflammation might play key role in the pathogenesis of DR: first,inflammatory mediators such as interleukin-1 receptor antagonist,vascular endothelial growth factor(VEGF),prostaglandin E2(PGE2),and so on are detected in the eyes of diabetic patients;second,hyperglycemia and other stresses resulted in the increase of inflammatory mediators,which causes leukocyteendothelial interactions,retinal microvascular damage,pericyte loss and capillary occlusion and degeneration;third,the retina ischemia and hypoxia induced by capillary occlusion provokes the retinal neovascularization.Then micro-aneurysms,retinal and vitreous haemorrhage,macular edema,and eventually blindness develop.Inflammasome is the core of inflammation.Its activation contributed to the pathogenesis of DR.Its Nucleotide-binding domain and Leucine-rich Repeat receptor contains a Pyrin domain 3(NLRP3).In rat model and h RMECs,NLRP3 up-regulated caspase-1 cleavage,which stimulated IL-1? and IL-18 maturation and increased cell apoptosis and retinal vascular permeability.Prostaglandins(PGs)act as important inflammatory mediators.Studies showed that PGE2 up-regulated the expression of NLRP3 inflammasome in h RMECs.PGE2 induced VEGF-A up-regulation dose-dependently in müller cells.The level of PGE2 in ocular fluid showed significantly higher in the DR patients than in the non-DR patients.The hypothesis is that PGE2 plays a key role in the development of RMECs injury and intraocular neo-vascularazation by activating NLRP3 and inducing VEGFA expression.Purpose1.To identify the PGE2/EP2 R signaling pathway in RMECs injury.2.To clarify the role of PGE2/EP4 R pathway in RMECs proliferation and retinal neo-vascularization.MethodPart 1.AnimalA rat model of diabetes was established by intra-peritoneal injection of STZ(60mg/kg).Blood glucose remained >16.7 mmol/L.Diabetic rats were randomly to 4groups: dimethylsulfoxide(DMSO),PGE2,EP4 R agonist Cay10598 or EP4 R antagonist AH23848 were injected into the vitreous cavity.Additional blank control group in normal rats received injection of saline solution(154mmol/l Na Cl)in the vitreous cavity.Retinal tissue thickness,changes in vascular density,neovascularization,and NF-?B translocation from the cytoplasm to the nucleus were observed.Part 2.Human vitreous fluidThe vitreous PGE2,TXB2 concentrations were measured by ELISA methods in the PDR patients who required vitrectomy.Age-matched non-diabetic participants with other conditions requiring vitrectomy(epiretinal membrane or macular hole)served as a control group.Part 3.CellThe expression of cyclooxygenase(COX)2,four type EP receptors,cleaved caspase-1 in high glucose-stimulated h RMECs was detected by Western blot.The m RNA of EP receptors in h RMECs treated with 30 m M glucose and NLRP3,IL-1? in h RMECs treated with PGE2,lipopolysaccharide(LPS)were measured by q RT-PCR.HRMECs were given with PGE2,EPR agonist and antagonist.q RT-PCR was used to detect the expression of NLRP3,pro-IL-1?,and VEGF-A m RNA.Western blot was used to detect the level of cleaved-caspase-1,VEGF-A protein,and p65 in cytoplasm or nucleus.ELISA was used to detect the content of IL-1? in cell culture supernatant.LDH assay detected the cytotoxicity of PGE2,IL-1?,EP2 R,EP4R to h RMECs.The proliferation of h RMECs by PGE2,Cay10598 and AH23848 was detected by wound-healing assay.EMSA was used to detect the NF-?B binding activity.Results1.Activation of PGE2 and expression pattern of EP receptor were identified in hyperglycemia environment.1.1 The level of PGE2,TXB2 in vitreous humor of patients with PDR was significant high than that of non-DR patients.1.2 COX2 were dramatically increased in the retinal vascular of STZ-treated rats.1.3 COX2 was induced in a time-dependent manner in cultured h RMECs stimulated with 30 m M glucose.1.4 After 72 hours of stimulation with 30 m M glucose,four type EP receptor expression increased in cultured h RMECs at the protein and m RNA level.2.PGE2/EP2 R signaling pathway promoted h RMECs injury in DR.2.1 After stimulation of h RMECs with 30 m M glucose and LPS+ATP,cleaved caspase-1 level significantly increased.The m RNA expression of NLRP3 and IL-1?was elevated.After the stimulation concentration of PGE2 increased to 1 ?M,the expression levels of NLRP3 and IL-1? m RNA in h RMECs significantly increased.2.2 PGE2 and its EP receptor agonists promoted the expression of NLRP3,proIL-1? and IL-1? at m RNA level on h RMECs.After AH6809 administration,PGE2 activation was significantly reduced to 36.5%(1.60±0.11 vs 2.53±0.03,P<0.01).The promoted elevation of PGE2 to IL-1? could be attenuated by EP2 R and EP4 R antagonists.2.3 PGE2 and EP2 R agonist had significant stimulatory effects on the expression of caspase-1 in h RMECs with hyperglycaemia.EP2 R antagonist AH6809 could significantly inhibit the expression of caspase-1 in h RMECs induced by high glucose.2.4 After PGE2 and Butaprost were given to h RMECs,the expression of LDH was significantly higher than that of the control group.AH6809 could partially inhibit the damage of PGE2 and Butaprost to h RMECs.IL-1? caused h RMECs injury.The relative expression of LDH was significantly higher than that of the control group,and its expression increased with the prolongation of action time.3.The role of PGE2/EP4 R pathway in RMECs migration and retinal neovascularization.3.1 After intravitreal injection of PGE2,Cay10598 and AH23848 in STZinduced diabetic rats.a.histology: The retinal tissue showed that the retinal tissue was thicker than normal rat and the microvessel density was increased.PGE2 and Cay10598 further promoted the proliferation of retinal cells,which was characterized by thickening of retinal tissue and increased microvessel density,after the use of AH23848,the thickness of the retina becomes thinner and the vessel density decreased 78.1%(5.33±0.42 vs 1.17±0.31,P<0.01).b.Retinal tissues of STZ-induced diabetic rats(injected with PGE2 and Cay10598,AH23848)were stained with CD105/IB4/DAPI,and observed by fluorescence microscopy.AH23848 decreased 92.3% neovascularization in the rat retinainduced by PGE2 and Cay10598(4.33±0.67 vs 0.33±0.21,P<0.01).c.The serum content of VEGF-A in STZ-induced diabetic rats was significantly higher than that in normal rats,and this elevation was effectively alleviated after the administration of AH23848.3.2 PGE2 and Cay10598 could promote the migration of h RMECs and this effect was enhanced with the prolongation of stimulation time.After AH23848 pretreatment h RMECs,the proliferation and migration rate of endothelial cells were slow down.3.3 PGE2,Cay10598 promoted VEGF-A expression at protein and m RNA level in h RMECs,NF-?B banding activity and translocation from the cytoplasm to the nucleus,while AH23848 could inhibit the affection of PGE2.Conclusion1.The PGE2/EP2 R signaling pathway is mainly involved in the inflammatory process of DR,and mediated h RMECs injury by activating NLRP3 /caspase1-1/IL-1?.2.The PGE2/EP4 R signaling pathway play a role in cell migration and neovascularization in RMECs,It achieved this function by PGE2-induced VEGF-A up-regulation through promoted NF-?B banding activity and translocation from the cytoplasm to the nucleus.