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

描述(由申请人提供)：血管灌流受损是许多疾病进展、组织/器官移植失败以及伤口修复相关并发症的主要因素。目前，涉及内皮前体的基于细胞的血管生成策略继续显示出希望，但存在促进治疗细胞的血管形成的定位、存活率和可预测的指导的次优传递策略。此外，缺乏对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可变性。这项拟议工作的长期目标是开发一种基于胶原蛋白的细胞输送基质，通过可调的生物物理和血管诱导功能，可预测地诱导三维(3D)血管形成。这项拟议的工作独特地将创新的胶原聚合物工程方法结合在一起，专注于自然产生的胶原分子间交联物和内皮细胞集落形成细胞(ECFC)，ECFC是一种特定的内皮前体，由其高增殖和血管形成能力定义。该提案的目标是定义交联链如何调节胶原组装，并定义特定的基质生物物理特征，这些特征可以被调节以指导体外和体内ECFC血管的形成。拟议的活动涉及三个目标：1)确定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，并有助于已知对血管形态发生重要的纤维和基质水平的生物物理特征的可调性；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、硬度和生物降解性)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-MMP4，以及它们作为ECFC感知和响应基质生物物理信号和调控血管形成的潜在途径的分子机制。纯化的胶原蛋白聚合物将从猪皮和肌腱中分离和制备，具体说明其分子间交联组成。胶原蛋白的浓度和交联剂的组成将被系统地改变，以确定这些聚合参数如何改变合成基质的组装动力学和生物物理性质。然后，这些胶原蛋白将用于悬浮ECFC，以确定特定的基质生物物理特征如何在体外和体内影响血管形成和持久性。最后，将进行涉及从外到内和从内到外的扰动策略的实验，以确定ECFC感知、区分优先顺序和响应基质生物物理线索的信号机制的关键节点。总而言之，所获得的知识和观点有望通过细化胶原蛋白的特征、标准化和应用于血管诱导基质的基本设计而显著影响组织工程学和再生医学。 与公共健康相关：我们的创新和多学科方法将提供第一个深入研究，记录分子间交联如何构成有效的胶原聚合物设计参数，以调整已知的对血管形态发生重要的基质生物物理特性。具有内皮细胞集落形成细胞(ECFC)高增殖潜能和血管形成能力的新型胶原构建块的界面有望对组织工程和再生医学领域产生积极影响，因为它将提供1)新型胶原基递送基质，旨在原位聚合以定位人ECFC，并为其提供定制的生物物理血管诱导线索；2)血管化组织模块，以支持临床规模工程化组织/器官替代物的开发和生产。

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. PUBLIC HEALTH RELEVANCE: Our innovative and multidisciplinary approach will provide the first in-depth study documenting how intermolecular cross-links constitute a valid collagen polymer design parameter for tuning matrix biophysical properties known to be important to vessel morphogenesis. The interface of a novel set of collagen building blocks with the high proliferative potential and vessel forming capacity of endothelial colony forming cells (ECFC) is expected to positively impact the fields of tissue engineering and regenerative medicine by providing 1) novel collagen-based, delivery matrices designed to polymerize in situ so to localize human ECFC and provide them with customizable biophysical vascular-inductive cues and 2) vascularized tissue modules to support development and production of clinical-scale engineered tissue/organ replacements.