Screening Drugs To Enhance The Blood-retinal Barrier And Structure-Activity Relationship Study

Retinal vascular leakage and hemorrhage are primary causes of blindness.Transition of the retinal vascular barrier(BRB)play an essential role during the whole retinal diseases course.And retinal vascular barrier(BRB)is directly related to Diabetic retinopathy and age-related macular degeneration which are the most common retinal diseases.Therefore,we established a platform for screening drug to enhance the stability of vascular endothelial cells,and hope to find some small molecular compounds that can effectively inhibit the decline of the stability of vascular endothelial cells.Then the structure-activity correlation analysis of the special active compounds was carried out to uncover their structure-activity centers.In this experiment,the xCELLigene RTCA detection system of Eisen Biology Co.,Ltd.was used to make real-time detection to the variations of cell matrix resistance,called electric cell-substrate impedance sensing(ECIS).After the high-throughput screening platform aim to explore drugs for enhancing the stability of vascular endothelial cells monolayer was established,the library contains 2100 known active small molecule compounds were screened.Two different methods to observe the leakage of vascular monolayer endothelial cells,XPerT and Transwell,were used for the second cycle screening.Then subsequently,we further seek the molecular mechanism and structural activity of Apigenin that special to enhance vascular endothelial stability.In addition,we inquired the effects of small molecular compounds(Ethaverine hydrochloride,Apigenin,Deguelin and Dihydrocelastrol)on the permeability of single-layer human retinal microvascular endothelial cells induced by different cytokines and growth factors.We also extended the platform to stabilize the functional integrity of vascular endothelial barrier in various tissues and organs.The ECIS assay detected that IL-1beta could induce monolayer human retinal microvascular endothelial permeability transition.Later then,four small molecule compounds screened from 2100 small molecule compounds can efficaciously inhibit the increase of vascular endothelial permeability induced by IL-1beta.These four small molecule compounds could also inhibit vascular leakage induced by various cytokines and growth factors(TNFalpha,VEGF and Thrombin).Along with HRMVEC cells,these four small molecule compounds can inhibit the vascular leakage of HMVEC-d,HUVEC and HSMEC cells as well.And that,XPeT experiments showed that these four small molecule compounds could effectively inhibit the increase of vascular endothelial cell permeability induced by IL-1beta,TNFalpha and Thrombin in human retinal microvascular endothelial cells.Transwell leakage assay indicated that these four compounds could inhibit the increase of vascular endothelial cell permeability induced by IL-6 in HSMEC cells.Furthermore,when exploring the relationship of these four small molecule compounds with the proliferation of human retinal microvascular endothelial cells,it was manifested that drug concentration was negatively correlated with the proliferation of human retinal microvascular endothelial cells.Through a series of experiments,we certified that the xCELLigene RTCA real-time detection system of Eisen Biology Co.,Ltd.can screen small molecular compounds that contribute to the stability of vascular monolayer endothelial cells.we established a drug screening platform that was in virtue of detecting transition phenotypic traits in cell matrix resistance,and manifested some drugs could enhance the functional integrity of BRB.Eventually,four small molecule compounds were demonstrated have similar effects on other cytokines and growth factors that induce vascular endothelial permeability transition,and their role is conserved among various organs and tissues.