Inhibitory Effects Of Decorin On Choroidal&Retinal Neovascularization

Purpose: Choroidal and retinal neovascularization are the main reason forvision loss in patients suffered retinal diseases. Hypoxia-induced vascularendothelial growth factor (VEGF) overexpression, which can increase theability of endothelial cells on cell migration and proliferation, is the commonpathological feature. However, there were few completely safe and effectivetreatments up to date. The key on treating choroidal and retinalneovascularization is how to inhibit VEGF overexpression under hypoxia.Decorin, a small leucine-rich proteoglycan, functions as a paracrinepan-inhibitor of receptor tyrosine kinases. Until now no obvious literature hasreported its effects on fundus neovascularization. Our project first focuses onthe effects and mechanisms of decorin on VEGF overexpression in humanretinal pigment epithelial cells (ARPE-19) under hypoxia; then a lentivirus vector carrying the human decorin open reading frame (ORF) is constructedand transduced to ARPE-19cells. After lentivirus transduction, the ARPE-19cells were cocultured with rhesus chororetinal vascular endothelial cells(RF/6A) to examine the influence of hypoxic ARPE-19cells on theproliferation, migration and tube formation ability of RF/6A cells; finally, weconstructed a self-complementary adeno-associated viral (scAAV) vectorcarrying the human decorin ORF to observe the inhibitory effects of decorinon retinal neovascluarization in mouse oxygen-induced retinopathy (OIR)model.Methods:(1) In vitro assays: first, find the appropriate decorin concentration(50nM) to inhibit the VEGF overexpression in ARPE-19cells under hypoxiawith CCK-8and ELISA assays; then western blot and qPCR assasy wereused to evaluate Met, Rac1, hypoxia-inducible factor-1alpha (HIF-1α) andVEGF expression in ARPE-19cells which were pretreated with decorin50nM at3,6,12and24h after hypoxia; then small interfering RNA (siRNA)was employed to silence the Met mRNA in ARPE-19cells to observe themechanism of decorin on VEGF overexpression in ARPE-19cells underhypoxia; finally, we constructed a lentivirus vector coding decorin ORF(LV-decorin) by recombinant DNA technique, and LV-decorin transducedARPE-19cells were cocultured with RF/6A cells to examine the influence on proliferation, migration and tube formation ability of RF/6A cells.(2) In vivoassays: we constructed a scAAV-CMV vector coding human decorin ORF,employed intravitreal injection, induced mouse OIR model, examinedHIF-1α, VEGF and decorin expression in retinas by western blot, qPCR andELISA, and retinal flatmount isolectin staining was applied to observe theneovascularization areas.Results:(1) In vitro part: Met, Rac1, HIF-1α and VEGF expression wereincreased in a time-dependent manner in ARPE-19cells under hypoxia,which could be inhibited by pretreatment of decorin50nM. The expressionof Met, Rac1, HIF-1α and VEGF were significantly decreased under hypoxiaafter Met siRNA transduction. There was no statistical difference between theMet siRNA-transduced group and the decorin-pretreated group. ARPE-19cells significantly expressed decorin of40KD molecular weight aftertransduction by LV-decorin. The LV-decorin transduced ARPE-19cells underhypoxia significantly inhibited the proliferation, migratin and tube formationability of cocultured RF/6A cells.(2) In vivo part: the plasmid ofscAAV-CMV-decorin was successfully constructed and packaged. Theneovacularization areas of retinas were significantly decreased inscAAV-CMV-decorin injected group comparing with the PBS injected groupat P17. The scAAV-CMV-decorin injected group overexpressed40KD decorin, and the expression of HIF-1α and VEGF were significanly dereasedcomparying with the PBS injected group.Conclusion: Decorin may inhibit VEGF overexpression in ARPE-19cellsunder hypoxia through antagonzing Met and downregulating Rac1andHIF-1α expression. Overexpression of decorin in hypoxic ARPE-19cells caninhibit angiogenic potential of choroid-retinal endothelial cells. Intravitrealinjection of the scAAV vector coding decorin ORF could significantlydecrease the retinal neovacularization areas in OIR mice.