Microenvironmental Control of Capillary Morphogenesis

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。以细胞为基础的促进治疗性血管形成的策略已经显示出临床前和临床上的成功，特别是在治疗严重的肢体缺血方面，通常包括将单一类型的间充质细胞输送到缺血部位以刺激血管生成。相比之下，工程化组织的血管化策略通常涉及两种细胞类型，内皮细胞(或其前体细胞)与某种支持基质细胞类型结合，并通过基于水凝胶的细胞外基质(ECM)传递。然而，基质细胞和细胞外基质的选择在不同的研究中差别很大。在之前的资助阶段，我们使用了体外3D细胞培养模型和体内皮下植入的组合，发现新生血管的形成既受细胞外基质的生物物理性质的调节，也受支持基质细胞的特性调节。我们的数据表明，不同来源的基质细胞在血管生成萌发过程中对ECM蛋白降解有不同的控制作用，控制ECM分解的速度对于产生稳定的、功能正常的血管至关重要。在这种相互竞争的更新应用中，我们建议从机制上研究局部微环境的这两个关键的指导元素(即基质细胞和细胞外基质)如何影响新血管的数量、功能质量和稳定性。目的1将量化基质细胞特性对基于纤维蛋白的3D共培养模型中ECM蛋白水解率和毛细血管形态发生的影响，使用活性微观流变学监测局部ECM机制随时间的变化。AIM 2将使用具有可调降解特性的工程生物材料平台来确定ECM蛋白降解敏感性对毛细血管网络的数量和功能质量的影响。目的3将在一个临床相关的缺血模型中研究基质细胞特性和细胞外基质蛋白分解敏感性如何影响新生血管的数量和质量。这些研究的成功完成将加强目前对微环境在毛细血管形态发生中的作用的理解，并最终指导明智地选择理想的细胞和细胞外基质组合来治疗缺血条件。

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. Cell-based strategies to promote therapeutic vascularization have shown pre-clinical and clinical success, particularly for the treatment of critical limb ischemia, and typically involve the delivery of a single mesenchymal cell type to ischemic sites to stimulate angiogenesis. By contrast, strategies to vascularize engineered tissues typically involve two cell types, with endothelial cells (or their progenitors) combined with some sort of supporting stromal cell type and delivered via a hydrogel-based extracellular matrix (ECM). However, the choices of stromal cells and ECM have varied widely across studies. In the prior funding period, we used a combination of 3D cell culture models in vitro and subcutaneous implants in vivo to discover that the formation of nascent vasculature is regulated by both the biophysical properties of the ECM and the identity of the supporting stromal cells. Our data suggest that stromal cells of different origins differentially control ECM proteolysis during angiogenic sprouting, and that controlling the rate o ECM breakdown is critical to yield stable, functional vessels. In this competing renewal application, we propose to mechanistically investigate how these two critical instructive elements of the local microenvironment (i.e., the stromal cells and the ECM) influence the quantity, functional quality, and stability of new vasculature. Aim 1 will quantify the impact of stromal cell identity on the rates of ECM proteolysis and capillary morphogenesis in a fibrin-based 3D co-culture model, using active microrheology to monitor local ECM mechanics as a function of time. Aim 2 will use an engineered biomaterial platform with tunable degradative properties to determine the impact of ECM proteolytic susceptibility on the quantity and functional qualities of capillary networks. Aim 3 will investigate how stromal cell identity and ECM proteolytic susceptibility affect the quantity and quality of neovasculature in a clinically relevant ischemic model. Successful completion of these studies will enhance current understanding of the role of the microenvironment in capillary morphogenesis, and ultimately guide the judicious selection of the ideal combination of cells and ECM for the treatment of ischemic conditions.