Angiogenesis is a dynamic process which encompasses the formation of new blood vessels from preexisting ones. Recent studies suggest that the Notch signaling pathway may regulate the process of neovascularization. Direct contact between macrophages and endothelial cells may activate the Notch signaling pathway resulting in the spreading of endothelial cells. However, the mechanisms that regulate endothelial cell spreading and what other cell types may be involved in this process remain unknown. Several studies have functionally related mast cells to the process of angiogenesis. It is also known that mast cells express Notch. Our studies with SVEC4-10 endothelial cells and P815 mast cells have shown that the tryptases mMCP-6 and 7 have the capacity to accelerate the formation of endothelial tubes as well as to induce the release of pro- and anti-angiogenic factors. In the presence of rmMCP-7, Dll-4 and ADAMTS1 (a desintegrin and metalloproteinase with thrombospondin motif) were identified in the culture supernant of endothelial cells during in vitro angiogenesis. Dll-4 is the receptor for Notch-1 and for activation via the Notch pathway, Notch-1 must be cleaved by ADAMTS1. With a basis in these results, it is of interest to investigate if the Notch/Dll-4 signaling pathway is also involved in mast cell - endothelial cell interaction during angiogenesis. Initially SVEC4-10 cells will be co-cultured with P815 mast cells on Geltrex® and the express of the Notch/Dll-4 complex will be evaluated by immunofluorescence during in vitro angiogenesis. In order to verify that the Notch signaling pathway is necessary for mast cell induced endothelial cell spreading and tube formation, SVEC4-10 enothelial cells and P815 mast cells with be co-cultured with and with and without silencing the Notch and/or Dll-4 genes. Our preliminary findings also demonstrate that rmMCP-7 may degrade integrin subunits on the surface of endothelial cells and facilitate their movement over the substrate. Whether integrin degradation is a direct or indirect action of rmMCP-7 will be investigated. Assays for direct degradation will be performed using recombinant integrins and rmMCP-7 in vitro. The indirect degradation of integrins via endosomes will be investigated using SVEC4-10 cells incubated with or without an inhibitor of the ubiquitin/proteosome system in the presence of rmMCP-7. The results of the effect of rmMCP-7 on angiogenesis in vitro will be confirmed in vivo using Balb/c mice and the DIVAA" (Directed in vivo Angiogenesis Assay) system. This study will contribute to the understanding of the process of neovascularization and the molecules involved that may serve as future therapeutic targets. (AU)
Articles published in Agência FAPESP Newsletter about the research grant:
FREITAS FILHO, EDISMAURO GARCIA;
MARIN JACA, LUIZ AUGUSTO;
BAEZA, LILIAN CRISTIANE;
DE ALMEIDA SOARES, CELIA MARIA;
BORGES, CLAYTON LUIZ;
JAMUR, MARIA CELIA.
Proteomic Analysis of Lipid Rafts from RBL-2H3 Mast Cells.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES,
AUG 2 2019.
Web of Science Citations: 0.