Screening Of Small Molecular Compounds For Wet AMD Treatment And Researching Of Related Biological Mechanisms

Age-related Macular Degeneration(AMD)is a disease caused by multiple reasons and result in losing of central vision.Dry AMD and Wet AMD are the two main types of advanced stage AMD.There is no treatment for dry AMD available yet.VEGF antibodies turn to be the gold standard treatment of wet AMD.However,the existence of anti-VEGF treatment non-responder suggest that other signaling pathways are involved in the pathogenesis of wet AMD too.Therefore,non-VEGF related treatment is in urgent need for wet AMD.Vascular endothelium,which composed of single layer of endothelial cell,is a semipermeable barrier between inside and outside of blood vessel.Vascular endothelial cell monolayer plays an important role in maintaining the stability of decrease its barrier function,and lead to hyperpermeability,leaking,and tissue edema.The tight junction between endothelial cells is formed through cell surface protein VE-cadherin.Once VE-cadherin is internalized into cytoplasm by endocytosis will lose endothelial cells’ junction,decrease vascular stability.The goals of this thesisis are to set up high-throughput drug screening platform and identify novel treatments for wet AMD.We will setup a novel small molecule screening platform utilizing real-time cell analysis(RTCA)to discover the compound that can stabilize retinal endothelium vascular barrier function.The discovered compound might have potential usage for wet AMD therapy in the future.Our screening platform consists of two components: primary screening utilizes electric cell substrate impedance sensing(ECIS)to detect changes in endothelial barrier function;the secondary screening is a direct paracellular permeability assay using a transwell system.We try to find compounds that can inhibit inflammatory cytokine IL-1β-induced human retinal vascular endothelial cell(HRMVEC)monolayer hyperpermeability.Structure-activity relationship(SAR)of hits will be studied.In vitro cellular assay is utilized to study the mechanism of compound enhanced vessel stability.And compound’s in vivo therapeutic effect was confirmed in mouse model.Our experimental results demonstrate that the top two hits can enhance vascular stability by inhibiting IL-1β –induced Arf6 activation and VE-cadherinen docytosis.Animal model study confirmed that the compounds can enhance retinal blood vessels stability,and reduce Evans Blue leakage into retinal tissue.The above results indicate that our compounds are likely to be potential candidate for wet AMD treatment.