Study On The Expression And Function Of Irisin In Mouse Retinal Vascular Development And Oxygen Induced Retinopathy

Background:The retina is highly metabolically active and consumes large amounts of oxygen and nutrients supplied by the blood vessels.The normal development of the vascular system is crucial for building a functional neuroretina.Formation of the retinal vascular network is finely regulated by the body,requiring interactions between different types of cells,among which astrocytes play a pivotal role.Vascular endothelial cells proliferate and form blood vessels under the stimulation of various factors and signal pathways,which is important under physiological and pathological conditions.Abnormal angiogenesis will destroy the delivery of oxygen and nutrition,resulting in metabolic imbalance and damage to neuroretinal function,and is associated with a variety of retinal neovascular diseases,such as retinopathy of prematurity(ROP)and proliferative diabetic retinopathy(PDR).Unlike normal vessels in the retina,these abnormal new vessels are more fragile and prone to bleeding and causing leakage.In this type of eye disease,vascular endothelial growth factor A(VEGFA)increases significantly.Previous studies have confirmed that VEGFA is a critical regulatory factor in the process of angiogenesis under both physiological and pathological conditions.Astrocytes are a primary source of VEGFA in physiological and pathological conditions in the retina.Intravitreal injection of drugs targeting VEGFA has become a standard method and first-line therapy in treating these diseases and has achieved beneficial therapeutic results.However,the economic pressure and severe complications caused by multiple injections and the poor responses in some patients cannot be ignored.Therefore,understanding the formation of vessels under physiological and pathological conditions and searching for better alternative treatment strategies for retinal neovascular diseases are still important basic and clinical topics in ophthalmic research.Irisin is a myokine found in muscle cells in 2012.It plays a role in bone and other tissues by binding with integrin receptors.Initially,irisin was found to induce browning in white adipose tissue and to regulate the metabolism of energy,glucose,and fatty acids.As research progressed,irisin was found to be expressed in different systems and organs.It can play a protective role in the liver,lung,heart,intestine,and other organs during ischemiareperfusion injury,mainly through reducing oxidative stress,inhibiting cell apoptosis,reducing inflammation,and maintaining the intercellular barrier.ROP and PDR are also considered a class of eye diseases in which ischemia triggers a series of responses within the retina that eventually lead to neovascularization.In recent clinical studies,irisin levels in the vitreous fluid and serum of PDR patients were found to be lower than those of nonproliferative diabetic retinopathy(NPDR)and the control group,suggesting that irisin may play a role in retinal pathological angiogenesis.However,whether irisin exists during normal vascularization,whether its expression changes when abnormal angiogenesis occurs in the retina and what role it plays in pathological angiogenesis remains unknown.Further exploration and clarification are needed.Based on the literature research and previous work,this subject uses mice of different ages,adopts the oxygen induced retinopathy(OIR)and the neonatal oxygen induced retinopathy(NOIR)mice model,employs western blot,q RT-PCR,retinal wholemount staining,frozen section,immunofluorescence,and other methods,conducts animal experiments in vivo and cell experiments in vitro,to investigate the development of retinal vessels and the expression of irisin during development,and thence the change,role,and mechanism of irisin in retinal pathological angiogenesis.The subject may provide new ideas and possible intervention strategies for basic research and clinical treatment of retinal neovascular diseases.Object:To observe the process of retinal vascularization and the expression of irisin during this process,to investigate the expression and role of irisin in OIR,and to explore the mechanism of irisin in pathological angiogenesis.Methods:Animal experiments1.C57BL/6 mice at postnatal(P)1,4,7,12,14,and 17 days were sacrificed,and retinal wholemount staining was conducted to observe the changes in centrifugal migration of astrocytes and the blood vessels;in mice of the same age,the growth of blood vessels in different layers of the retina was observed by IB4 staining in the frozen sections of the eyeballs.2.Extract the proteins of C57BL/6 mice retina at P1,P4,P7,P12,P14,and P17.Western blot was conducted to detect the expression of irisin,and immunofluorescence staining of the frozen sections was conducted to observe its localization.3.OIR model was established in C57BL/6 mice.The expression of irisin and VEGFA in the OIR mice retina was detected by western blot at P14 and P17.Immunofluorescence staining observed the localization of irisin and VEGFA at the corresponding times.4.The OIR mice were administered with exogenous irisin.At P17,retinal wholemount staining with IB4 and CD31 was used to observe retinal neovascularization.Retinal proteins were also extracted at P17,and PCNA(a marker of cell proliferation)and VEGFA expressions were detected by western blot.Retinal cryosections were stained with TUNEL to observe cell apoptosis.Retinal RNA was extracted at the corresponding time,and gene expressions of the angiogenesis and inflammation-related index(TNF-α,IL-1β,MCP-1)were detected by q RT-PCR.5.NOIR model was established in C57BL/6 mice,and exogenous irisin was supplied.At P7,retinal wholemount staining with IB4 and PDGFRα was used to observe retinal vasculature and astrocyte migration.Immunofluorescence staining was conducted to observe the expression of PH3,a marker of cell proliferation,and western blot was employed to detect PDGFRα and VEGFA expressions.Cell experiments1.Chemical hypoxia injury was induced in cultured retinal astrocytes.The concentration of VEGFA in the medium was detected by ELISA,and the expression of VEGFA in the cells was detected by q RT-PCR and western blot.2.Irisin was added to the astrocytes insulted by hypoxia for 24 hours.ELISA was performed to detect VEGFA concentration in the medium,and q RT-PCR and western blot were performed to detect VEGFA expression in the cells.3.The astrocyte conditioned medium of each group was collected,1:1 mixed with the fresh medium,and used to treat human microvascular endothelial cells 1(HMECs-1).Transwell and tube formation experiments were conducted to observe the effect of the astrocyte-conditioned medium on HMECs-1 migration and angiogenic ability.4.PBS(solvent),irisin,irisin+integrin inhibitor,and integrin inhibitor were added to the hypoxic astrocytes.The concentration of VEGFA in the medium was detected by ELISA,and protein expressions of VEGFA,HIF-1α,and HIF-2α in the cells were detected by western blot.Results:Animal experiments1.During the development of the mouse retina,blood vessels occurred after astrocytes entering into the retina,which migrated centrifugally and covered the retina at P7 and was distributed in the nerve fiber layer;the expression of irisin gradually increased after birth and reached a peak at P14,and its localization was only found in the nerve fiber layer before P7.2.At P14 and P17,the levels of irisin m RNA and protein in the retina of the OIR mice significantly decreased compared with the normoxic group,while the levels of VEGFA significantly increased.3.Exogenous irisin significantly reduces the percentages of the neovascular and avascular areas in the OIR mice retina.In addition,irisin can inhibit the apoptosis of retinal neurons in the OIR mice and downregulate TNF-α,IL-1β,and MCP-1 gene expressions.The protein expressions of PCNA and VEGFA were significantly lower in the retina of the irisin treated OIR mice than the control group.4.Exogenous irisin supplementation significantly reduced the vascularization area and endothelial cell proliferation in the NOIR mice retina,as did the vascular density and length.Cell experiments1.In hypoxic retinal astrocytes,the concentration of VEGFA in the medium was significantly higher than in the control group,as did VEGFA m RNA and protein expression in the cells.2.Irisin can inhibit the upregulation of VEGFA in astrocytes induced by hypoxia.Meanwhile,irisin-treated ACM significantly inhibited the migration and tube formation of HMECs-1 compared with the control ACM,and the inhibitory effect can be rescued by VEGFA supplementation.3.Integrin inhibitors reversed the irisin-induced decrease in VEGFA,HIF-1α,and HIF-2α protein levels in astrocytes.Conclusions:This study was the first to observe the expression of irisin in normal retinal vascularization and its changes in OIR mice and to confirm that irisin can alleviate pathological neovascularization by inhibiting the expression of VEGFA in astrocytes.In vivo experiments have determined the expression of irisin in retinal vascular development and OIR.The negative regulatory effect of irisin on pathological angiogenesis has been verified via OIR and NOIR mice models,and this effect may be associated with astrocytes.Subsequently,through the culture of retinal astrocytes in vitro,we observed the impact of irisin on the expression of VEGFA in hypoxic retinal astrocytes.We proposed that irisin downregulates HIF-1α and HIF-2α expressions via integrin receptors,thus inhibiting astrocytic VEGFA expression and vascular endothelial cell migration and tube formation.Together,this study explored the change of irisin in retinal physiological and pathological vascularization and investigated the role and mechanism of irisin in OIR for the first time.The subject helps deepen the understanding of irisin’s function and retinal neovascularization,which may provide new ideas and experimental evidence for widening the clinical treatment of such diseases.