A New Assay Platform to Screen for Chemicals Adversely Affecting VEGFR Signaling in Endothelial Cells

Human development depends on Vascular Endothelial Growth Factor ReceptorVEGFRsignalingwhich regulates survivalmigrationand proliferation of endothelial cellsthe primary component of the vasculatureImpairment of VEGFR signaling is a main cause of infant deaths due to congenital malformations and decreased birth weightVEGFR signaling inhibition leads to nephropathypreeclampsiaand hypertensionwhile pathological activation is a hallmark of cancerarterial venous malformationsand macular degenerationSince some drugs and industrial chemicals can affect VEGFR signalingthe ability to identify such substances is of primary importance for the protection of human healthHowevercurrentlythere is no efficient toxicity screening system employing physiologically relevant cellular models for this purposeWe propose to commercialize a novel high throughputHTplatform enabling evaluation of inhibition of endothelial VEGFRdependent functions using human endothelial colony forming cellsECFCsOur proprietary cell growth media VecstemTM allows efficient isolationrobust expansionand long term cryopreservation of ECFCsThuswe have establisheddonor specific ECFCswhich we will commercialize as a unique model of endothelial functionIn the proposed productwe have integrated endothelial VEGFR dependent cytotoxicitytube formationandD migration assays into a single platformallowing assessment of a variety of functions important to vascular healthOur cytotoxicity assay is more specific than commercial analoguesOur tube formation assay hastimes more throughput andtimes shorter than similar commercial productsOurD migration assay has no commercial analoguesIn Phase Iwe will test the feasibility of our assay and benchmark it against other commercial assays by comparing the responses of our ECFC lines to commercial endothelial cellse gHUVECsand human fibroblastsnegative control cellsusinganti angiogenic chemicals including known inhibitors of VEGFR signalingIn Aimwe will demonstrate the effect of antiangiogenic chemicals on VEGFR dependent viability and proliferation of ECFCsIn Aimwe will demonstrate the effect of anti angiogenic chemicals on VEGFR dependent tube formation andD migration of ECFCsMetrics evaluated will include concentration response profilesinhibitory concentrationICvaluesand the coefficient of variationCVThe success of this study will justify progression to Phase IIwhere we will develop and validate awell HT high content imaging assay protocol usingchemicalsidentified in silico as potentially anti angiogenicin several ECFC linescorroborating the results in a zebrafish antiangiogenesis assayWe plan toaprovide a screening service to regulators and other businessesblicense our platform to drug discovery companiesandcmake available stand alone commercial product sOur assay platform has an excellent commercial perspective as U Sand European regulatory agencies are insisting on the use of alternative methods that replace reduce the use of animals in pre clinical toxicology and drug development Inhibition of Vascular Endothelial Growth Factor ReceptorVEGFRsignaling may cause embryonal deathdevelopmental abnormalitiesnephropathypreeclampsiahypertensionetcIt is currently unknownwhich of andgtfederally registered industrial chemicals can inhibit VEGFR signalingMoreoveranti VEGFR drugs are increasingly used to treat various retinopathies and cancersWe propose to commercialize a new highly relevant and efficient endothelial cell based assay platform to identify chemicals inhibiting VEGFR signaling