Papel da trombospondina-1 no contexto endotelial dos cavernomas cerebrais: estudos fenotípicos e funcionais

Abstract

Many studies have used in vitro models to investigate cellular mechanisms of diseases, such as cerebral cavernous malformation (CCM) or cerebral cavernomas. Cavernomas are associated with overactivation of the RhoA/ROCK signaling pathway in endothelial cells, leading to excess stress fibers that promote the opening of interendothelial adherent junctions, changes in junctional proteins such as zona occludin-1 (ZO-1) and cadherin vascular endothelium (VE-cadherin), with consequent extravasation of blood fluids and formation of bullous structures that are called “caves”. The disease results from loss-of-function mutations in one of the genes CCM1(KRIT-1), CCM2 and CCM3/PDCD10, which negatively regulate the RhoA/ROCK pathway, in addition to being associated with a reduced expression of thrombospondin-1 (TSP-1). TSP-1 is a matricellular protein that can both inhibit and stimulate angiogenesis through interactions with its multiple functional domains, being involved in numerous biological activities. To understand the complex role of CCM proteins and TSP-1 in this pathological context, experimental models of mimetic cells of the cavernomatous phenotype have been developed, through the silencing of CCM proteins expression. In the present work, we chose to performthe silencing the expression of the CCM3 isoform. Immortalized human microvascular endothelial cells (HMEC-1) and human umbilical vein primary endothelial cells (HUVECs) were used in the assays. CCM3 expression in these endothelial cells was confirmed by immunofluorescent staining. The two endothelial cell types showed different susceptibilities to the electroporation procedure, which could be optimized for each cell type. HUVECs and HMEC-1 showed silencing peaks at 48 hours post-transfection and with siRNA concentration (specific and “non-targeting”) of 300 nM. Cells were analyzed for the acquisition of the cavernomatous phenotype, through functional assays carried out in both wild-type and silenced cells: analysis of the organization of the actin cytoskeleton; assessment of endothelial cell proliferation; analysis of myosin light chain (MLC) phosphorylation status and analysis of tubulogenic differentiation. Our results showed that CCM3 silencing resulted in cells that reproduce some important features of cavernomatous endothelial cells, such as increased longitudinal stress fibers and increased MLC phosphorylation. Furthermore, a reduction in VE-cadherin was observed at the silencing peak (48 hours), probably related to the increased recycling of this adherent junction receptor (AJs). We observed a reduction in TSP-1 expression, with a concomitant increase in N-terminal fragments, of a pro-angiogenic nature. Our results suggest that the generation of angiogenic proteolytic fragments from TSP-1, as opposed to the intact form of this protein, may be part of the mechanism that leads to the destabilization of AJs in cavernomatous endothelial cells.