Microenvironmental Control of Capillary Morphogenesis

描述（由申请人提供）： 血管生成，即从现有的脉管系统中长出新的毛细血管，是一个复杂的生物学过程，对于许多病理的治疗和组织工程的成功至关重要。促进治疗性血管形成的基于细胞的策略已经显示出临床前和临床成功，特别是对于严重肢体缺血的治疗，并且通常涉及将单一间充质细胞类型递送至缺血部位以刺激血管生成。相比之下，使工程化组织血管化的策略通常涉及两种细胞类型，其中内皮细胞（或其祖细胞）与某种支持基质细胞类型组合并通过基于水凝胶的细胞外基质（ECM）递送。然而，基质细胞和ECM的选择在研究中有很大差异。在之前的资助期间，我们使用了体外3D细胞培养模型和体内皮下植入物的组合，发现新生血管的形成受ECM的生物物理特性和支持基质细胞的特性的调节。我们的数据表明，不同来源的基质细胞差异控制ECM蛋白水解过程中血管生成发芽，并控制ECM分解的速度是至关重要的，以产生稳定的，功能性的血管。在这个竞争性的更新应用中，我们建议机械地研究本地微环境的这两个关键的指导元素（即，基质细胞和ECM）影响新脉管系统的数量、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蛋白水解和毛细血管形态发生速率的影响，使用主动微流变学监测局部ECM力学随时间的变化。目标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蛋白水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将研究基质细胞特性和ECM蛋白水解敏感性如何影响临床相关缺血模型中新生血管的数量和质量。这些研究的成功完成将提高微环境在毛细血管形态发生中的作用的当前理解，并最终指导明智地选择用于治疗缺血性疾病的细胞和ECM的理想组合。

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.