Efeito do domínio N-terminal da trombospondina-1 na modulação das propriedades funcionais das células endoteliais progenitoras humanas in vitro e in vivo: proposta de um produto de terapia celular

Abstract

Endothelial progenitor cells (EPCs) are recruited to sites of active neovascularization in adults, contributing to endothelial repair and vascular biology, a process called post-natal vasculogenesis. Thus, EPCs represent an important source of neovascularization therapy for peripheral arterial disease, myocardial ischemia and stroke. Whereas the EPCs potential functionality is limited by its scarse numbers in peripheral circulation, several pro-angiogenic molecules have been studied as pharmacological agents in order to improve the recruitment of bone marrow-progenitor cells into circulation, the capacity to adhesion to endothelium and thus the efficiency of transplantation. Thrombospondin-1 (TSP-1) is a glycoprotein found in ischemic situations, being able to both inhibit and stimulate angiogenesis. This dual biological activity is attributed to interactions of their domains with different cell surface receptors as well as with soluble molecules or proteins present in the extracellular matrix. The proangiogenic effect of TSP-1 is assigned to its N-terminal domain, which displays high affinities to heparin and proteoglycans. Previous data from our laboratory indicated two peptide sequences located within the N-terminal domain of TSP-1 (TSP-HepI, amino acids 17-35 e TSP-HepII, amino acids 78-94), were able to induce in vitro differentiation of mature endothelial cells (HUVECs) into capillary-like structures, by interacting with syndecan-4, a heparin sulfate proteoglycan, involved in focal adhesion. Our goal in this study was to investigate the potential of TSP-1 as a strategic molecule for promoting neovascularization in ischemic tissues induced by EPC using in vitro and in vivo assays. At first, we demonstrated that EPCs isolated from peripheral blood and expanded with a culture medium modified in our laboratory (EGM-2/ENR), exhibit phenotypic and functional characteristics of colonyforming endothelial cells (ECFCs) and high proliferative potential. Moreover, these progenitor cells (PB-ECFCs) secrete three-fold more fibronectin than HUVECs and exhibit a rearrangement of actin filaments, compatible with a migratory phenotype. We then showed that the peptide TSP-HepI and TSP-HepI stimulates key stapes of angiogenesis in vitro (chemotaxis, cell adhesion and differentiation into capillary-like structures), of PB-ECFCs and also of ECFCs isolated from umbilical cord blood. TSP-HepI peptide was able to enhance the in vivo neovascularization induced by FGF-2 a mice model of Matrigel plug. The preconditioning of ECFCs ex vivo isolated from umbilical cord blood with TSP-HepI stimulated their migration, increased expression of α6-integrin and increased its adherence to activated endothelium under physiological flow. This latter effect appeared to be mediated by syndecan-4, since that ECFC adhesion was significantly reduced by a syndecan-4-neutralizing antibody. ECFC migration and tubulogenesis were reduced by using a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI), or when HSPG was enzymatically disrupted, respectively. These findings together suggest that the N-terminal domain of TSP-1 could be provide a basis for a pharmaceutical molecule to increase the regenerative potential of ECFCs in new cell therapy protocols for the promotion of neovascularization.