Carla Motta1, Gabriella Lupo2, Dario Rusciano3, Melania Olivieri3, Liliana Lista4, Mario De Rosa5, Vincenzo Pavone4, Carmelina Daniela Anfuso2. 1. Bioos Italia Srl, Catania, Italy. 2. Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy. 3. Sooft Italia Spa, Roma, Italy. 4. Department of Chemical Science, University of Naples "Federico II" via Cintia, Italy. 5. Department of Experimental Medicine, Second University of Naples, Napoli, Italy.
Abstract
PURPOSE: To investigate the molecular mechanisms of the antiangiogenic activity of UPARANT, an antagonist of the urokinase-type plasminogen activator receptor (uPAR), on primary human retinal endothelial cells (HREC) as a model of in vitro angiogenesis. METHODS: The antiangiogenic activity of UPARANT was evaluated on endothelial cell migration, invasion, and tube formation. Human REC were further analyzed for viability, transendothelial electrical resistance (TEER), and tight junction (TJ) expression at the protein and mRNA levels. Vascular endothelial growth factor-related signaling molecules were also analyzed by Western and northern blots. RESULTS: UPARANT inhibited in a dose-dependent fashion HREC motility, invasion, and tube formation stimulated by VEGF-A, in a range of doses (1-100 nM) that had no effect on cell viability and proliferation. UPARANT also prevented the loss of permeability induced by VEGF-A, restoring normal TEER values and TJ protein expression. At the molecular level, UPARANT inhibited VEGFR-2 and STAT3 phosphorylation, thus decreasing VEGF and hypoxia-inducible factor 1-alpha expression, finally resulting in decreased activation of MEK/ERK, JNK, p38, and AKT signaling proteins. CONCLUSIONS: These findings indicate that UPARANT exerts its antiangiogenic effects through the inhibition of the downstream signaling activated by angiogenic factors such as VEGF-A.
PURPOSE: To investigate the molecular mechanisms of the antiangiogenic activity of UPARANT, an antagonist of the urokinase-type plasminogen activator receptor (uPAR), on primary human retinal endothelial cells (HREC) as a model of in vitro angiogenesis. METHODS: The antiangiogenic activity of UPARANT was evaluated on endothelial cell migration, invasion, and tube formation. Human REC were further analyzed for viability, transendothelial electrical resistance (TEER), and tight junction (TJ) expression at the protein and mRNA levels. Vascular endothelial growth factor-related signaling molecules were also analyzed by Western and northern blots. RESULTS: UPARANT inhibited in a dose-dependent fashion HREC motility, invasion, and tube formation stimulated by VEGF-A, in a range of doses (1-100 nM) that had no effect on cell viability and proliferation. UPARANT also prevented the loss of permeability induced by VEGF-A, restoring normal TEER values and TJ protein expression. At the molecular level, UPARANT inhibited VEGFR-2 and STAT3 phosphorylation, thus decreasing VEGF and hypoxia-inducible factor 1-alpha expression, finally resulting in decreased activation of MEK/ERK, JNK, p38, and AKT signaling proteins. CONCLUSIONS: These findings indicate that UPARANT exerts its antiangiogenic effects through the inhibition of the downstream signaling activated by angiogenic factors such as VEGF-A.
Authors: Filippo Locri; Noemi A Pesce; Monica Aronsson; Maurizio Cammalleri; Mario De Rosa; Vincenzo Pavone; Paola Bagnoli; Anders Kvanta; Massimo Dal Monte; Helder André Journal: J Mol Med (Berl) Date: 2020-09-17 Impact factor: 4.599