Regulação da diferenciação angiogênica endotelial por matrizes de gliomas humanos

Abstract

The extracellular matrix (ECM) is an essential component of angiogenesis, responsible for triggering crucial signaling pathways leading to endothelial cell migration, invasion, proliferation and survival. Tenascin-C (TN-C) is an iconic matricellular protein involved in the regulation of tumor cell migration and angiogenesis. ECM secreted by high grade (III and IV) astrocytomas contains elevated amounts TN-C, which interferes of the adhesive activity of fibronectin, by destabilizing focal adhesions, thus stimulating glioma proliferation. We have previously shown that TN-C rich matrices, produced by human astrocytoma cells, selected a tubulogenesis-defective endothelial cell subpopulation, while inducing anoikis – a particular type of cell death triggered by cell detachment - of another endothelial subpopulation. In Part I, we confirmed that the increase of TN-C by human astrocytes is a crucial part of tumoral transformation, since normal astrocytes, in contrast with astrocytoma cells, synthesize a FN-rich ECM, which does not elicit tubulogenic defects in endothelial cells. We also observed that cells able to adhere on U-373 MG astrocytoma matrix express more α9β1integrin, besides exhibiting altered kinetics for FAK and ERK activation, when compared to endothelial cells incubated with their autologous immobilized ECM. We also found that the incubation of endothelial cells with astrocytoma ECM led to a decrease of both FGFR1 and syndecan-4 expression, which work as partners in the activation of PKCα signaling. On the other hand, the activation of PKCδ in tubulogenesis-defective endothelial cells was increased in the same condition, suggesting an antagonic role for both PKC isoforms in endothelial tubulogenesis. These data were confirmed with the use of selective and specific inhibitors and activators of PKC isoforms. Additionally, we found that the astrocytoma matrix increases VEGFR2 expression and endothelial migration induced by VEGF-A. Suprisingly, the tubulogenic defect was potentiated by VEGF-A. The last result led us to hypothesize that the matrix secreted by astrocytoma cells promoted the enrichment of a tip cell-like endothelial phenotype. Tip cells are highly migratory endothelial cells, expressing high levels of VEGFR2 and specialized in guiding neo-vessels towards angiogenic cues. However, an excess of tip cells over stalk cells in a nascent structure precludes branch formation and stabilization. We performed a whole functional genome analysis (transcriptome sequencing) and found that endothelial cells primed by their incubation with TN-C-rich ECM expressed increased the expression of toolkit genes for tip cell phenotype, such as AQP1, DLL4, EFNB2, MMP14 e PLXD1. In Part II, we devoted attention to the endothelial cell subpopulation that undergoes anoikis when incubated with the matrix secreted by U-373 MG cells. Initially, we observed that the conditioned medium (CM) containing apoptotic cells stimulated tubulogenesis of TDECs (for tubulogenesis-defective endothelial cells), in a Matrigel assay. Further characterization of conditioned media showed a near three-fold increase in extracellular vesicles (EVs) in the CM of cells incubated on astrocytoma ECM, as compared with autologous ECM. EVs significantly contributed to tubulogenesis and migration of TDECs. Overall, our work strongly suggests that TN-C rich matrices play a fundamental role in the establishment of disfunctional vascular networks prevailing in high grade astrocytomas.