Inhibition of the Notch-Delta Pathway at Early Steps of Endothelial Progenitor Differentiation Impairs Adhesion and Spreading to the ECM Via Integrin Modulation.
Authors: Caiado Francisco , Real Carla , Dias Sergio 
Published: April 2020

Abstract
Endothelial progenitor cells (EPC) have been proven essential in models of neoangiogenesis, where it was shown they differentiate at the angiogenesis site and incorporate the neo-vasculature. However, the mechanisms that mediate this differentiation process are not fully understood. Since members of the Notch-Delta (N-D) pathway are present on EPC (specifically N1, J1,J2 and Dll4), in this study, we exploited the possibility that N-D signaling might be involved in the early stages of EPC (bone marrow or humbilical chord blood derived) differentiation. First, using an optimized in vitro endothelial differentiation assay, the involvement of the N-D pathway in this process was evidenced by the increased expression of the downstream targets Hes1, Hey1 and Hey2 during EPC (Lin-, Sca-1+, Flk-1+/KDR+, CD133+ depending on the source) differentiation. We show that murine BM derived EPCs show a severe extracellular matrix (ECM) adhesion defect and a consequent impaired endothelial differentiation when exposed to Notch-Delta (N-D) pathway inhibitor (gamma-secretase inhibitor) during the first 6 days of endothelial differentiation. Early inhibition of the N-D pathway had no effect on BM derived EPC cells survival (apoptosis) or proliferation, although it reduced the number of adherent cells and inhibited their differentiation into mature endothelial cells, as determined by the reduced number of LDL, vWF, PE-CAM and Flk-1 positive cells. Similar results, evidencing a defect in EPC adhesion, were obtained from culturing BM derived EPC from Dll4+/− (heterozygous) mice. Transfection of BM derived EPC with a constitutively active form of Notch4 receptor promote their adhesion to the ECM and consequently increased the number of mature endothelial cells obtained at the end of the differentiation assay. These adhesion and spreading defects suggest an interplay between N-D pathway and integrin related pathways. Indeed, using human umbilical cord blood derived EPC, we show that early inhibition of N-D pathway leads to a decrease in a3 integrin surface expression, which strongly suggest a link with the EPC adhesion defect observed. In order to exploit the functional implications of this defect we sought to investigate whether N-D inhibition on BM-derived EPCs interfered with their ability to contribute towards endothelial recovery following wounding. Using a well established in vitro endothelial monolayer wounding assay, we observed that untreated EPCs adhere preferentially at the wounding site and to the endothelial cells at the wound leading edge, while EPCs treated with an N-D pathway inhibitor show a reduced ability to adhere at the wounding site, thus interfering with wound closure. Altogether these data suggest that N-D pathway is necessary for EPC endothelial differentiation and that its inhibition interferes with their ability to adhere/spread to the ECM, possibly via integrin a3 activation and cytoskeletal modulation, and consequently to differentiate into mature endothelial cells interfering with the reendothelization process on wounded endothelium.