Collagen Cross-links Direct Matrix-Induced Vasculogenesis Via Tunable Pathways

描述（由申请人提供）：血管灌注受损是与许多疾病状态进展、组织/器官移植失败和伤口修复相关并发症相关的主要因素。目前，涉及内皮前体的基于细胞的血管发生策略继续显示出希望，但存在促进定位、存活和治疗细胞对血管形成的可预测指导的次优递送策略。此外，缺乏基本治疗细胞属性的定义，这可能导致临床前和临床结果的观察差异。这项工作的长期目标是开发一种基于胶原蛋白的细胞传递基质，通过可调节的生物物理、血管诱导特征，可预测地诱导三维（3D）血管形成。提出的工作独特地结合了创新的胶原聚合物工程方法，该方法专注于自然发生的胶原分子间交联和内皮集落形成细胞（ECFC），这是一种由其高增殖和血管形成能力定义的内皮前体的特定群体。该提案的目标是定义交联如何调节胶原蛋白组装，并定义特定的基质生物物理特征，这些特征可以用于指导体外和体内ECFC血管形成。提出的活动涉及三个目标：1)定义胶原聚合物的分子间交联组成如何调节胶原-纤维基质的分子组装，并有助于纤维和基质水平生物物理特征的可调性，这些特征对血管形态发生很重要；2)定义纤维和基质水平的设计特征，包括纤维密度、纤维间分支、刚度和生物降解性如何独立和相互依赖地调节体外和体内ECFC血管形成的早期和晚期过程；3)定义基质-整合素-细胞骨架信号轴中的关键节点，包括21-整合素、FAK、Cdc42和MT1-MMP，以及它们作为ECFC感知和响应基质生物物理线索和调节血管形成的潜在途径的分子机制的作用。纯化的胶原蛋白聚合物，根据其分子间交联组成来指定，将从猪皮和肌腱中分离和制备。胶原浓度和交联组成都将系统地改变，以确定这些聚合参数如何改变组装动力学和合成基质的生物物理性质。然后，这些胶原将用于悬浮ECFC，以确定特定基质生物物理特征如何影响体外和体内血管形成和持久性。最后，将进行由外向内和由内向外扰动策略的实验，以确定ECFC感知、优先考虑和响应基质生物物理线索的信号机制的关键节点。总的来说，所获得的知识和观点有望通过改进胶原蛋白的特征、标准化和应用于血管诱导基质的基本原理设计，对组织工程和再生医学产生重大影响。

DESCRIPTION (provided by applicant): Compromised vascular perfusion is a major factor associated with progression of many disease states, failure of tissue/organ transplants, and complications related to wound repair. At present, cell-based vasculogenesis strategies involving endothelial precursors continue to show promise but suffer from suboptimal delivery strategies that promote localization, survival, and predictable guidance of vessel formation by therapeutic cells. Furthermore, definition of essential therapeutic cell attributes is lacking and likely contributes to observed variability in preclinical and clinical outcomes. The long-term goal of the proposed work is to develop a collagen- based, cell-delivery matrix that predictably induces three-dimensional (3D) vessel formation through tunable biophysical, vascular-inductive features. The proposed work uniquely interfaces an innovative collagen polymer engineering approach that focuses on naturally-occurring collagen intermolecular cross-links and endothelial colony forming cells (ECFC), a specific population of endothelial precursors defined by their high proliferative and vessel forming capacities. The proposal objective is to define how cross-links modulate collagen assembly and define specific matrix biophysical features that can be tuned for guiding ECFC vessel formation in vitro and in vivo. The proposed activities involve three aims 1) Define how the intermolecular cross-link composition of collagen polymers modulates the molecular assembly of collagen-fibril matrices and contributes to tunability of fibril- and matrix-level biophysical features known to be important to vessel morphogenesis; 2) Define how fibril- and matrix-level design features including, fibril density, interfibril branching, stiffness, and biodegradability work independently and interdependently to modulate early- and late-stage processes of ECFC vessel formation in vitro and in vivo; and 3) Define critical nodes within the matrix-integrin-cytoskeleton signaling axis, including 21-integrin, FAK, Cdc42, and MT1-MMP and their roles as molecular mechanisms by which ECFC sense and respond to matrix biophysical cues and potential pathways to modulate vessel formation. Purified collagen polymers, specified in terms of their intermolecular cross-link composition, will be isolated and prepared from pig skin and tendon. Both collagen concentration and cross-link composition will be systematically varied to define how these polymerization parameters alter assembly kinetics and biophysical properties of resultant matrices. These collagens will then be used to suspend ECFC to define how specific matrix biophysical features affect vessel formation and persistence in vitro and in vivo. Finally, experiments involving outside-in and inside-out perturbation strategies will be conducted to identify critical nodes of signaling mechanisms by which ECFC sense, prioritize, and respond to matrix biophysical cues. Collectively, the knowledge and perspective gained is expected to significantly impact tissue engineering and regenerative medicine by refining how collagens are characterized, standardized, and applied to the rationale design of vascular-inductive matrices.