Abstract
Purpose :
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 a context of hypoxia-induced angiogenesis, the antagonist effects of UPARANT onto the uPA/uPAR system were investigated using a novel ex vivo human iris organotypic angiogenesis assay. Additionally, to further elucidate whether UPARANT acts on epithelial or endothelial cells, in vitro assays were performed on human iris epithelial cells (hIEC) versus human retinal endothelial cells (hREC).
Methods :
hIEC, hREC and human iris organotypic cultures were kept in normoxia (20% O2) or exposed to hypoxia-stimulated angiogenesis (1% O2), and treated with UPARANT and control-vehicle. UPARANT anti-angiogenic effects were analyzed by in vitro wound healing and spheroid sprouting assays, ex vivo human iris sprouting assay, immunofluorescence, quantitative PCR and protein assays (western and dot blots, and co-immunoprecipitation). The effects of UPARANT in human ex vivo iris angiogenesis were focal to endothelial cells.
Results :
UPARANT does not affect iris epithelial wound recovery yet reduces sprouting angiogenesis in hREC spheroids and ex vivo human iris organotypic cultures. Western blot analysis demonstrates that UPARANT leads to a decrease of hypoxia-inducible factor (HIF)-1α protein levels, concomitantly with a decrease in phosphorylation levels of protein kinase B (AKT) and cyclic AMP response element-binding protein (CREB) when compared to vehicle treated.
Phospho-protein array illustrated an unidentified antagonism of UPARANT in the interaction of uPAR with the low-density lipoprotein receptor-related protein-1, resulting in inhibition of b-catenin–mediated angiogenesis in this model.
Conclusions :
These findings corroborate the role of UPARANT as a multifactor inhibitor of ocular vascular endothelial cells, which could benefit the treatment of RI in patients afflicted with multiple other ocular neovascular diseases.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.