Fig. 5: Endothelial PRL3 overexpression promotes HRMEC proliferation, migration and permeability by facilitating VEGF signaling.
From: PRL3-zumab as an anti-angiogenic therapy in neovascular eye diseases
a HRMEC-PRL3 exhibits a higher rate of cell proliferation compared to HRMEC-Vector (p = 0.018, n = 9 replicates from three independent batches of transduced HRMECs). b HRMEC-PRL3 have a greater cell migratory ability compared to HRMEC-Vector (p = 0.0499, n = 6 replicates from three independent batches of transduced HRMECs). c There is an increased permeability in HRMEC-PRL3 monolayer compared to HRMEC-Vector (p = 0.003, n = 16 replicates from three independent batches of transduced HRMECs). d HRMEC-PRL3 monolayer have diminished ZO-1 cell surface expression compared to HRMEC-Vector by immunofluorescence staining. Both HRMEC-Vector and HRMEC-PRL3 have reporter GFP gene for successful retroviral infection of the plasmids. In the PRL3 panel (upper, panel B), there is a mixture of PRL3 overexpressing and wildtype cells, and stronger ZO-1 expression is seen in the wildtype cells (white arrows point to intact ZO-1 expression). In the PRL3 panel (lower, panel D), the white arrows point to the PRL3 localisation in the cell membrane and cytoplasm. Scale, 20 µm. e Quantification of ZO-1 staining intensity (p = 0.032, n = 5 from three independent batches of transduced HRMECs). f Western blotting shows that HRMEC-PRL3 have increased levels in pERK1/2, pAKT, pSRC, pPaxillin and reduced expression of ZO-1, VE-Cadherin, and VEGFR2 compared to HRMEC-Vector. All data are representative images and western blots of three independent batches of transduced HRMECs for each experiment. g Quantification of Western blotting analysis (VE-Cadherin: p = 0.0028, ZO-1: p = 0.03, pERK1/2: p = 0.02, pAKT: p = 0.04, pSRC: p = 0.038, pPax: p = 0.039, VEGFR2: p = 0.0001). h Schematic model depicting the upregulation of endogenous PRL3 by VEGF via MEF2C, and PRL3’s involvement in the downstream VEGF signaling in a forced overexpression system that promotes the phosphorylation of SRC, AKT, ERK1/2 and Paxilin, downregulation of ZO-1 and VE-Cadherin and a negative feedback loop resulting in a reduced VEGFR2 expression (Created in BioRender73). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as loading control. All experiments were done using three independent batches of transduced HRMECs. The mean value was calculated by two-sided Student’s t-test (mean ± s.d., n = 3 replicates each). ***P < 0.001, **P < 0.01, *P < 0.05. Source data are provided as a Source Data file.
