Matrix metalloproteinases in Angiogenesis and Inflammation

The vasculature is in charge of optimally delivering nutrients and oxygen throughout the body and this is especially relevant in diseased tissues. Dynamic reshaping of the microvasculature is partially achieved by regression/pruning, the elimination of lower-blood flow capillaries by a not yet well-characterized process involving intraluminal endothelial cell migration. Our recent observations point to novel cellular and molecular players in capillary regression in vivo whose modulation may benefit revascularization and repair after tissue ischemia. The TFM will focus on the following objectives:

1. Identification of new molecular players in endothelial cell polarization and migration during vessel regression in vivo.  Retinas will be isolated from neonatal mice and stained for CD31/VE-cadherin, GM130/GLG1, F-actin and DAPI to analyze in the endothelial cells of the regression events (COL IV+/IB4-): i) polarization, by Golgi-nucleus vectors; and ii) migration, by VE-cadherin and F-actin junctional pattern.

2. Characterization of molecular actors coupling blood flow-induced polarization and migration in vitro and in vivo. Mouse aortic endothelial cells (MAEC) will be isolated from young mice. We will label MAEC with vital probes and analyze their migration upon laminar flow (20 dynes/cm2 for 12 h) in Ibidi chambers by time-lapse microscopy and Image J (https://imagej.nih.gov/ij/). At final time-point, we will fix and stain cells for VE-cadherin, GM130/GLG1 and DAPI and quantitate elongation and polarization. As a complementary in vivo approach, we will perform endothelial denudation in the abdominal aorta and quantitate migration against flow after 24 h (Mack et al, 2017). Molecular pathways candidates to be involved in endothelial cell migration under flow (nitric oxide, VEGFR2, etc) will be tested by treating MAEC with inhibitors.

We will use high- and super-resolution microscopy, 2D and 3D image analysis, proteomics, bioinformatics, protein modelling, lentiviral strategies and genetic mouse models to pursue these goals. We finally intend to apply this knowledge to develop novel angiotherapies aimed at improving capillary perfusion and tissue performance in several pathophysiological contexts.