Regulation and Enhancement of Angiogenesis in Dense Fibrin Matrices

描述(申请人提供)：血管生成，即从现有的血管系统中长出新的毛细血管，是一个复杂的生物学过程，对多种病理的治疗和组织工程的成功至关重要。在许多有前景的促进组织工程血管生成的策略中，包括通过精确的空间和时间控制来控制促血管生成分子的输送。另一种方法是提供一种合适的细胞类型，这种细胞类型可以提供更具生理性的促血管生成信号，从而加速宿主血管的招募。与两位资深合作研究人员合作，PI已经开始在一种健壮的、基于纤维蛋白的毛细血管形态发生的三维体外模型中探索后一种策略。使用这个模型系统，它支持形成稳定的(&gt；3周)具有明确管腔的毛细血管样结构，我们已经表明，增加纤维蛋白基质密度显著抑制了毛细血管的形态发生。此外，我们已经证明，这种纤维蛋白密度的阻断部分是由于内皮细胞无法上调局部重塑基质和维持毛细血管侵袭所需的一组基质金属蛋白酶(MMPs)。然而，将骨髓间充质干细胞(MSCs)添加到三维组织结构中显著增强了毛细血管的形态发生，通过上调这一亚群MMPs的表达和/或活性，部分克服了基质密度增加造成的抑制。在这些初步发现的基础上，这项新的研究人员生物工程研究拨款提案试图测试这一假设，即骨髓间充质干细胞可以在体外和体内刺激致密的3-D纤维蛋白基质中的血管生成。这些目标将通过以下三个具体目标来实现：1.量化植入致密纤维蛋白基质中的MSCs在体外促进毛细血管样网络形成的能力。2.)探讨MMPs亚群(MMPs-2、MMPs-9和膜型MMP1-MMPs)在MSCs促血管生成中的作用。3.)在SCID-小鼠模型中，确定MSCs刺激体内皮下部位血管形成的能力。这些研究的成功完成将有助于我们从根本上了解ECM和MMPs在血管生成中的作用，并最终影响合成ECM类似物的设计和干细胞在组织工程中的应用。

DESCRIPTION (provided by applicant): Angiogenesis, the sprouting of new capillary blood vessels from existing vasculature, is a complex biological process of critical importance to the treatment of numerous pathologies and the success of tissue engineering. Amongst the many promising strategies to promote angiogenesis in tissue engineering includes the controlled delivery of pro-angiogenic molecules with precise spatial and temporal control. An alternative approach is to deliver an appropriate cell type that can provide a more physiologic mixture of pro- angiogenic cues that will accelerate the recruitment of host vessels. In conjunction with two senior co- investigators, the PI has begun to explore this latter strategy in a robust, fibrin-based 3-D in vitro model of capillary morphogenesis. Using this model system, which supports the formation of stable (> 3 weeks) capillary-like structures with well-defined lumens, we have shown that capillary morphogenesis is significantly inhibited by increasing fibrin matrix density. Furthermore, we have shown that this fibrin density block is partially due to the inability of endothelial cells to upregulate a subset of matrix metalloproteinases (MMPs) required to locally remodel the matrix and sustain capillary invasion. However, the addition of bone marrow-derived mesenchymal stem cells (MSCs) to the 3-D tissue construct significantly enhances capillary morphogenesis, partially overcoming the inhibition caused by increased matrix density by upregulating the expression and/or activity of this subset of MMPs. Building on these preliminary findings, this new investigator Bioengineering Research Grant proposal seeks to test the hypothesis that MSCs can stimulate angiogenesis within dense 3-D fibrin matrices both in vitro and in vivo. These objectives will be achieved via three specific aims as follows: 1.) Quantify the ability of MSCs embedded throughout dense fibrin matrices to enhance capillary-like network formation in vitro. 2.) Probe the roles played by a subset of MMPs (MMPs-2 and -9 and the membrane-type MT1-MMP) in the angiogenic enhancement by MSCs. 3.) Determine the ability of MSCs to stimulate vascularization in vivo in a subcutaneous site in a SCID-mouse model. Successful completion of these studies will contribute to our fundamental understanding of the role of the ECM and MMPs in angiogenesis, and will ultimately impact the design of synthetic ECM analogs and the use of stem cells in tissue engineering applications.