Role Of TGFβ1/pSMAD2/3 Pathway In Retinal Vascular Malformation And Neuronal Damage In Retinopathy Of Prematurity

Objectives:Retinopathy of prematurity(ROP)is defined as abnormal retinal neovascularization in preterm and low birth weight(BW)infants.A study in China showed that the incidence of ROP was 11.8%in preterm infants with a BW less than 2000 g and the incidences were 5.9%,23.6%,60.0%in infants with a BW of 1500-1999g,1000-1499g and less than 1000g,respectively.Common risk factors of ROP include mechanical ventilation,oxygen therapy and blood transfusion,etc.Previous studies mainly focused on superficial retinal vascular lesions,including growth arrest in ’phase 1’ and abnormal neovascularization in ’phase 2’,whereas deeper layers of blood vessels and other types of cells received less attention.The incidence of visual function damage in ROP patients is higher than the gestational age-matched ones and the malfunction persists years after the vascular lesions regress.Rod photoreceptor injury is the most prominent neuronal damage in ROP patients.Rod dominates the visual function in dark field and is one of the cell types that greatly rely on oxygen in the human body.As a result,rod is sensitive to hyperoxia,hypoxia and other stimulations,and is prone to oxidative stress injury.The Fulton’s team from Harvard University found that Rhodopsin(Rho),the biomarker of rod,dramatically increased after 25 weeks of gestational age,suggesting that rod developed rapidly after that and were vulnerable to insults of preterm birth.Electroretinogram(ERG),functional test of retina,revealed that the function of rod was damaged in ROP patients,indicated by abnormal a wave.While few studies refer to the mechanisms of rod damage.From the perspective of blood supply,it was found that the deep retinal plexus(DVP)and choroidal blood vessels collaboratively support the rod.Furthermore,DVP was reported to be damaged in ROP and Transforming growth factor β1(TGFβ3)was confirmed indispensable in DVP development,confirmed by loss of DVP in TGFβ1 knock-down animals.In addition,TGFβ1 was altered in ROP patients and animal models.Nonetheless,the role of TGFβ1 in ROP was not clear.We raised the hypothesis that the altered expression of TGFβ1 participated in the malformation of retinal DVP and resulted in rod photoreceptor malformation.The purpose was to explore the damage of DVP in oxygen-induced retinopathy(OIR)model in newborn rats and to investigate the developmental defects of rod,providing a new target for ROP intervention.Methods:1.Isolectin B4 was applied to investigate the retinal vascular development on D8(the eighth day after birth),D10,D12,D14,D16 and D18 in normal newborn rats.2.OIR model was established using newborn SD rats.Immunohistochemistry(IHC)was applied to compare the expression of TGFβ1 in the OIR and control group on D2,D4,D6,D8,D10,D12,D14,D16 and D18.Western-blotting was conducted to explore the expression of pSMAD2/3 on D14 and D18(critical time points of retinal vascular development).3.LY364947(inhibitor of the TGFβ1/SMAD2/3 pathway)was adopted and the four groups were established,namely the OIR,OIR+ LY364947,Normoxia,Normoxia +LY364947 groups.Isolectin B4 was applied in presentation of retinal vasculatures at D14 and D18;Western-blotting was implemented to explore the expression of CD31 and Rho(markers of vasculature and rod,respectively);Electron-microscopy was applied to investigate the development of DVP on D14,the phenotype of pericytes,the rod and synaptic contacts of rod with other cells.Results:1.The DVP developed during D10-D14 under normal condition.2.TGFβ1 in OIR rats was first upregulated and then downregulated.The downregulation occurred during D10-D18.In addition,TGFβ1 was absent in the outer plexus layer(OPL)where DVP located.Furthermore,pSMAD2/3 was downregulated on D14 and upregulated on D18.3.In the OIR and OIR+LY364947 groups,CD31 and Rho were downregulated on D14 and the inhibitor worsened it.Isolectin B4 staining demonstrated significant retardation of DVP in the OIR and OIR+LY364947 retinas and electron microscopy also showed absence of DVP in the peripheral retina at OPL.While the DVP were present in the Normoxia and Normoxia +LY364947 groups.In the OIR group,the number of photoreceptors decreased with increased cell volume and the perinuclear fibrosis was obvious,and the inhibitor further worsened the phenotypes.In the OIR and OIR+LY364947 groups,there was no synaptic triad.In the Normoxia group,typical triad structure was seen,while in the Normoxia +LY364947 group,the structure was abnormal.Conclusions:The study demonstrated the critical role of the TGFβ1/SMAD2/3 signaling pathway in DVP development and that its deficiency participated in the DVP and rod photoreceptor malformation.The data suggested a possible therapeutic target to prevent DVP and rod photoreceptor damage in ROP.