Inhibitory Effect And Its Mechanism Of PF4 On Retinal Neovascularization

Aim Retina neovascularization(RNV)is newly formed abnormal blood vessels on the retina or optic disk that extend along the retina surface or into the vitreous cavity,it is an important cause of irreversible blindness in several age people.The aim of this study is to construct a homogeneous and stable classical retinal neovascularization model,oxygen-induced retinopathy(OIR)mice model,and to identify platelet factor 4(PF4),an important cytokine in the occurrence and development of RNV,by protein array analysis,to further explore the effect of PF4 on the formation of RNV and the proliferation,migration and tube formation of retinal vascular endothelial cells,and its downstream molecular mechanisms.To further our understanding of the occurrence and development mechanism of RNV and provide new clues and idea for the treatment of RNV-related diseases.Methods 1.The neonatal mice on postmatal day 7(P7)were exposed to 75% oxygen for 5 days to P12,and then fed in normal oxygen for 5 days to P17 to construct the OIR model.Isolectin B4(IB4)staining of whole mount retinal and HE staining of paraffin section of retina were used to detect the formation of retinal avascular areas and neovascularization,thus confirming the success of the establishment of OIR model in young mice.Then,the protein expression difference between the OIR retinas and normal retinas was analyzed by protein array analysis.PF4,an important cytokine in the occurrence and development of RNV,was found.The results of protein array were further confirmed by western blot(WB)and enzyme-linked immunosorbent assay(ELISA).2.In vivo,the inhibition effect of PF4 on RNV was confirmed by injecting PF4 into the vitreous of OIR mice and further detecting retinal avascular areas and neovascularization by retinal whole mount isolectin B4(IB4)staining.Vascular endothelial growth factor(VEGF)and Tumor necrosis factor alpha(TNF-?)were administered to stimulate angiogenesis and mimic pro-angeogenesis environment in vitro.After PF4 intervention,the inhibition effect of PF4 on RF/6A cells was confirmed by detecting the proliferation,migration and tube formation function of RF/6A cells.3.The differences of protein expression among normal retina,OIR retinas and the retinas of OIR mice injected with PF4 were detected and analyzed by protein array.PRAS40 was found to be the target of PF4 in the progress of RNV.The lentivirus expressing PRAS40 and its mutant version with deficient phosphorylation sites were constructed and injected into eyes of OIR mice.Retina whole mount IB4 staining was performed to detect the formation of retinal avascular areas and neovascularization,thus confirming the counteracting effect of PRAS40 overexpression on the inhibition effect of RNV induced by PF4 in vivo.The plasmid expressing PRAS40 was constructed and transfected into RF/6A cells.The ability of proliferation,migration and tube formation of RF/6A cells after injection was tested in vitro to confirm the counteraction of PRAS40 overexpression on the inhibition effect of PF4 inhibiting the function of RF/6A cells.To clarify the mechanism of PF4 in inhibiting RNV and retinal vascular endothelial cell function.Results 1.The neonatal mice were exposed to 75% oxygen environment since P7 for 5 days to P12,and then kept in normal oxygen environment for another 5 days to P17.The OIR mice model was successfully constructed.Retina whole mount IB4 staining showed that the OIR retinas have a large area of avascular zone and their retinal neovascularization were significantly more than that of the normal mice.Retinal paraffin section of HE staining showed that the number of pre-retinal nuclei of the OIR retinas was significantly more than that of their normal counterpart.The results of protein array analysis showed that PF4 was the cytokine with most significant change between OIR retinas and normal retinas.ELISA further confirmed that PF4 in OIR retinas was significantly more than that of normal retinas.2.Retina whole mount IB4 staining showed that the avascular area and neovascularization of OIR retinas were significantly more than that of normal retinas,while the avascular area and neovascularization of PF4 injected OIR retinas were significantly less than that of OIR retinas.The proliferation,migration and tube formation ability of RF/6A cells in the group with VEGF and TNF-? were significantly higher than that of normal group,while the proliferation,migration and tuve formation ability of RF/6A cells treated with PF4,VEGF and TNF-? were significantly lower than that of cells only with VEGF and TNF-?.3.Retina whole mount IB4 staining showed that the avascular area and neovascularization of PF4-injected OIR retinas were significantly reduced when compared with that of OIR retinas,while the avascular area and neovascularization in PRAS40 lentivirus-injected OIR mice were significantly increased when compared with that of only PF4-injected OIR retinas,however,the avascular area and neovascularization in mutant PRAS40 lentivirus-injected OIR mice were similar with that of only PF4-injected OIR retinas.The proliferation,migration and tube formation ability of RF/6A cells in PF4-treated group were significantly stronger than that of VEGF and TNF-alpha-stimulated group.The proliferation,migration and tube formation ability of RF/6A cells with transfection of PRAS40 plasmid were significantly stronger than that of RF/6A cells which were only treated with PF4.Conclusion PF4 can effectively inhibit vessel obliteration and neovascularization in OIR retina and suppress proliferation,migration and tube formation of retinal vascular endothelial cells.PF4 exhibit its inhibititory effect on RNV and angiogenesis of vascular endothelial cells through by downregulating the phosphorylation of PRAS40.