This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our objective is to study the role of matrix metalloproteinases (MMPs) in vasculogenesis and angiogenesis using a model system in which dermal fibroblasts are cocultured in 3-dimensional matrices along with hemangioblasts (CD34+ stem cells - vasculogenesis model) or with differentiated vascular endothelial cells (HUVEC -angiogenesis model). Different 3-dimensional matrices (collagen, Matrigel, fibrin) will be used that are similar to the different extracellular matrices that endothelial cells encounter in vivo. In addition, a composite matrix composed of collagen, chondroitin sulfate, and chitosan will be used that has already been successfully used in the tissue engineering of blood vessels. This proposal is based on observations suggesting that a variety of MMPs may play sometimes opposing roles in endothelial cell differentiation, the formation of vessels, and the maintenance of vessels. We hypothesize that different MMPs will be involved in vessel formation depending on whether endothelial cells or stems cells are used in experiments and depending on the choice of 3-dimensional matrix. Specifically we will: 1) Demonstrate that when CD 34+ stem cells enriched from peripheral blood mononuclear cells or HUVEC are co-cultured with dermal fibroblasts, capillary-like tubular structures form in 3-dimensional matrices. 2a) Use general MMP inhibitors to determine whether MMPs are involved in the formation (and maintenance) of these structures. 2b) Identify the specific MMPs that are present during the development of capillaries from HUVEC and CD34+ stem cells. 2c) Use stable mRNA silencing delivered by lentiviral vectors to determine whether the same MMPs are functionally involved in capillary formation by HUVEC and by CD34+ stem cells. 3) Use DNA microarray analysis to identify novel genes that are involved in capillary formation by HUVEC and/or CD34+ stem cells and whose expression is altered when total MMP activity is inhibited or when the expression of specific MMPs is blocked. The better understanding of the redundant cell types and molecular mechanisms involved in angiogenesis/vasculogenesis that will be revealed by our experiments will help sort out the roles that different cell types, different MMPs, and different ECM proteins play in vessel formation. In this manner, our studies may lead to clinical applications including: blocking tumor angiogenesis, promoting vascularization of myocardial infarcts, and forming tissue engineered vascular grafts.
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