MATRIX METALLOPROTEINASE IN VASCULOGENESIS & ANGIOGENESIS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们的目的是利用真皮成纤维细胞与血管母细胞(CD34+干细胞-血管生成模型)或分化的血管内皮细胞(HUVEC-血管生成模型)在三维基质中共培养的模型系统，研究基质金属蛋白酶(MMPs)在血管生成和血管生成中的作用。将使用类似于内皮细胞在体内遇到的不同细胞外基质的不同三维基质(胶原、基质、纤维蛋白)。此外，还将使用由胶原、硫酸软骨素和壳聚糖组成的复合基质，该基质已成功用于血管组织工程。这一建议是基于观察表明，各种MMPs在内皮细胞分化、血管形成和血管维持中有时可能扮演相反的角色。我们假设，不同的MMPs将参与血管形成，这取决于实验中使用的是内皮细胞还是干细胞，也取决于三维基质的选择。具体地说，我们将：1)证明从外周血单个核细胞或人脐静脉内皮细胞中提取的CD34+干细胞与真皮成纤维细胞共培养时，可以在三维基质中形成毛细血管样的管状结构。使用普通的基质金属蛋白酶抑制剂来确定基质金属蛋白酶是否参与这些结构的形成(和维持)。2b)确定在HUVEC和CD34+干细胞的毛细血管发育过程中存在的特定MMPs。2C)使用慢病毒载体提供的稳定的mRNA沉默来确定相同的MMPs是否在功能上参与了HUVEC和CD34+干细胞的毛细血管形成。3)利用DNA微阵列分析，寻找与HUVEC和/或CD34+干细胞的毛细血管形成有关的新基因，当总的MMPs活性被抑制或特定MMPs的表达被阻断时，其表达发生变化。我们的实验将有助于更好地了解参与血管生成/血管生成的冗余细胞类型和分子机制，有助于理清不同细胞类型、不同MMPs和不同ECM蛋白在血管形成中所起的作用。通过这种方式，我们的研究可能会导致临床应用，包括：阻断肿瘤血管生成，促进心肌梗死的血管形成，以及形成组织工程血管移植物。

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.