Estudo da adesão, sobrevivência e tubulogênese endotelial em modelo de células de glioma silenciadas para a expressão de Tenascina-C

Abstract

We have previously shown that extracellular matrices secreted by high-grade astrocitoma promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells, while also leading to the death, by anoikis, of another endothelial subpopulation. The use of defined adhesion substrata containing various levels of tenascin-C (TN-C) and fibronectin (FN) allowed us to confirm that the balance between TN-C and FN was a major cause of the selection of both endothelial phenotypes. However, this strategy did not allow us to address the potential role of other molecular components present in tumor matrices. In the present work, we studied the effect of a matrix produced by U-373 MG cells previously silenced for TN-C expression in endothelial cell adhesion, survival and tubulogenic differentiation, as compared to the matrix secreted by wild-type astrocitoma cells. U-373 MG cells silencing was confirmed by Western blotting, real time RT-PCR and ELISA, and cells remained silenced (≥ 90 %) throughout the 120 hours period necessary for generating immobilized native matrices required for endothelial cell function assays. Human umbilical vein endothelial cells (HUVECs) adhesion to the extracellular matrix secreted by astrocitoma cells silenced for TN-C expression was significantly increased by ≅18 %, as compared to wild-type tumor matrix. The number of HUVECs exhibiting picnotic nuclei a hallmark of advanced apoptosis has also been significantly decreased by ≅35 %, when endothelial cells were allowed to incubate with TN-C-depleted tumor matrix, as compared to the wild-type tumor matrix. Concerning angiogenic differentiation, endothelial cells incubated with the matrix produced by silenced U-373 cells were strongly attenuated for their tubulogenesis defect, as compared to HUVECs incubated with the TN-C-rich wild-type matrix. Thus, these data corroborated our previous observations that TN-C in astrocitoma matrices crucially interferes with endothelial cell differentiation. Besides adhesion, survival and tubulogenic differentiation, the responses of endothelial cells to astrocitomas matrices are also affected by cell cycle transitions. We have previously shown that endothelial cells undergoing anoikis had already transitioned through the S-phase of cell cycle at the moment of seeding. Thus, we decided to investigate the expression of p27 protein, an inhibitor of ciclin-dependent kinases (CKI) that has been already implicated in glioma angiogenesis. We found that HUVECs incubated with the matrix secreted by U-373 MG wild-type cells for 24 hours exbibited a 2 to 3-fold increase in p27 expression. In contrast to the other results discussed herein, these differences were not correlated with the expression of TN-C by U-373 MG cells, since the matrix produced by tumor cells silenced for TN-C did not alter the expression of p27 in endothelial cells. Overall, the present data suggest that, although TN-C in native tumor matrix does play a major role in endothelial cell angiogenic differentiation, other matrix components may act in concert with TN-C to modulate endothelial cell proliferation in tumor contexts.