Studies on the activation mechanisms of MMP-2 and ADAM17: identification of regulatory proteins, oxidant production pathways, epigenetics and proteolytic targets

Abstract

The role of Matrix Metalloproteinases (MMPs) in extracellular matrix degradation and remodeling is only one facet of this family of 27 members described since the 1960s. Once activated, MMPs cleave substrates such as matrix proteins, pro-inflammatory mediators and which activate several signaling pathways dependent on MAP kinases, important in processes such as maintenance of vascular tone, cardiac hypertrophy and response to injury. It is now known that activation of MMPs by agonists is rapid, and occurs via PKC, production of oxidants and other MMPs (such as membrane), but it is still necessary to know the activation / functions specifically of each MMP, mainly thanks to the large therapeutic potential of these metalloenzymes. In addition, it has been described that MMP-7 leads to an increase in oxidant concentrations, which is very important because it changes the paradigm from which the regulation of cardiovascular effects by MMPs and redox processes is commonly thought. In this project the aim is observe the mechanism of action and consequences in the production of intracellular oxidants, epigenetic regulation and to identify new substrates, specifically MMP-2 and ADAM17 in the cardiovascular system. In this study the objective is analyze the effects of increased rhMMP-2 on vascular function and production of oxidants in rabbit aortas. For this, the recombinant MMP-2 will be injected into a New Zealand rabbit aorta weighing between 2.2-3.5 kg from the University of São Paulo's Ribeirão Preto Animal Hospital. RhMMP-2 will be added to the interior of the arteries in bath with carbogen, and functional analyzes will be done in the presence and absence of inhibitors of the production of oxidants. The thoracic aorta will be divided into 3 segments for a posteriori studies: metaloproteinase activity by in situ zymography (using as DG-Gelatin substrate), immunohistochemistry for MMP-2 localization, structural histological analysis (eosin and hematoxylin, elastin and collagen staining), structural analysis by scanning microscopy, and the function of the arteries by isometric tension. Vascular function will be evaluated in the presence and absence of inhibitors of the production of oxidants such as Peg-Catalase and also inhibitor of MMP-2 (GM6001). (AU)