Mitigation of human iris angiogenesis through uPAR/LRP-1 interaction antagonism in an organotypic ex vivo model

Abstract

Rubeosis Iridis (RI) is characterized by an increase in neovascularization and inflammation factors in the iris. During angiogenesis, the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a pivotal role in extracellular matrix remodeling, where uPAR regulates endothelial cell migration and proliferation through assembly with transmembrane receptors. Here, in the context of hypoxia-induced angiogenesis, the uPA/uPAR system blockage was investigated by using UPARANT in a novel ex vivo human iris organotypic angiogenesis assay. The effects of uPA/uPAR system antagonism in the humanized model of ocular pathologic angiogenesis were analyzed by sprouting angiogenesis and protein assays (western, dot blots, and co-immunoprecipitation) and correlated to vascular endothelial growth factor (VEGF) inhibition. Phosphoprotein and co-immunoprecipitation assay illustrated an unidentified antagonism of UPARANT in the interaction of uPAR with the low-density lipoprotein receptor-related protein-1 (LRP-1), resulting in inhibition of β-catenin–mediated angiogenesis in this model. The effects of uPA/uPAR system inhibition were focal to endothelial cells ex vivo. Comparison between human iris endothelial cells and human retinal endothelial revealed an endothelial-specific mechanism of β-catenin–mediated angiogenesis inhibited by uPA/uPAR system blockage and not by VEGF inhibition. Collectively, these findings broaden the understanding of the effects of the uPA/uPAR system antagonism in the context of angiogenesis, revealing non-canonical β-catenin downstream effects mediated by LRP-1/uPAR interaction.