Suppression of uPA and uPAR Attenuates Angiogenin Mediated Angiogenesis in Endothelial and Glioblastoma Cell Lines

Abstract

BackgroundIn our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood.Methodology/Principal FindingsIn the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells.Conclusions/SignificanceTaken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.

Adipose-Derived Mesenchymal Stromal Cells From Aged Patients With Coronary Artery Disease Keep Mesenchymal Stromal Cell Properties but Exhibit Characteristics of Aging and Have Impaired Angiogenic Potential

“…Therefore, we can speculate that activation of the uPA system in ADSCs from older patients might be a compensatory response to the reduction of proangiogenic factor secretion. It was shown that uPA and uPAR downregulation suppresses angiogenesis in endothelial cells partially by downregulation of angiogenin and inhibition of the angiopoietin-1/ AKT/FKHR pathway, and treatment of endothelial cells with recombinant uPA increased ANG secretion [55].…”

VEGF is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting

“…ADM signals angiogenesis by binding calcitonin-receptor-like receptor, which is widely expressed on normal and hypoxic endothelial cells (49,52). Angiogenin was another molecule previously demonstrated to be angiogenic, yet without consequence in TetON-HIF-1:VEGF Δ mice (28,29). In addition to these direct proangiogenic HIF targets, molecules contributing accessory functions such as matrix degradation and its modulation, MMP9, MMP3, and PAI-1, and vascular dilatation, iNOS, were also up-regulated in TetON-HIF-1:VEGF Δ mice.…”

“…Previous work has highlighted the angiogenic potential of several direct and indirect HIF-1 transcriptional targets (23)(24)(25)(26)(27)(28)(29). To determine whether these targets were induced by HIF-1 in the absence of VEGF, whole skin extracts were analyzed by using RT-PCR, ELISA, or antibody microarrays (Fig.…”

Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments

“…This is in accordance with Bronckaers et al [23] who found that FGF-2 was abundantly present in DPSC lysates, but was not secreted into the medium, as well as with Aranha et al [43] who found that hypoxia elevates secretion of VEGF, but not FGF-2, in DPSC cultures. Finally, uPA, which belongs to the serine proteases family, catalyzes plasminogen to plasmin, which is involved in proteolysis and activation of MMPs and growth factors, promoting EC invasion [52]. Antiangiogenic factors, on the other hand, comprise a heterogeneous group of molecules, acting primarily through their ability to bind and sequester proangiogenic factors, thus blocking their interaction with ECs [50].…”