Transglutaminase-Based Imaging of Wound Healing

描述（由申请人提供）：伤口愈合并发症是一个重要的健康问题，可能与糖尿病、褥疮和感染有关。不能在伤口部位上形成稳定的临时基质是伤口愈合不良的常见标志。没有稳定的基质，炎症反应细胞如内皮细胞、中性粒细胞和巨噬细胞的迁移就不可能产生新的血管1和抵抗感染。该项目的目标是开发一种成像方法来量化的稳定性， 在早期伤口愈合期间的临时基质。这种生物学过程在很大程度上是不可见的，预计这项工作将为延迟伤口愈合的分子事件提供重要的见解。 伤口愈合涉及一组复杂的精确定时过程，其开始为纤维蛋白凝块的形成，随后为炎症反应，并最终以广泛的组织重塑和伤口闭合结束。在炎症反应过程中，临时基质部分通过转氨酶（TG）酶交联血浆蛋白（如纤维蛋白原和玻连蛋白）的作用产生。我们实验室最近的工作表明，给予TG的红外荧光标记底物（包括蛋白质和肽），可产生体内成像的强烈对比度。2在本R21项目中，我们将测试我们的方法是否可以通过执行以下特定目标来识别与动物模型中不稳定基质形成相关的问题：（1）开发成像策略以支持愈合级联中的多个时间点成像;（2）在小鼠模型中评估多个时间点光学成像方法的体内性能;和（3）通过在糖尿病小鼠的延迟伤口愈合模型中成像基质形成来比较临时基质稳定性。多时间点成像可以监测基质形成和降解过程的问题。与对照相比，缓慢的基质形成将与标记底物向伤口中的不良结合相关。与对照相比，过度基质降解将与伤口中标记底物的快速损失相关。 一个成功的项目将导致一个新的成像方法研究伤口愈合和评估伤口愈合反应。在短期内，这种方法将用于动物模型研究，以评估伤口愈合过程，并支持开发新的方法来促进伤口愈合。从长远来看，这种方法将扩展到临床研究，以确定受损愈合的机制，并帮助伤口管理。此外，由于动脉粥样硬化3和癌症4中涉及类似的交联过程，因此这种成像方法也将在伤口愈合之外找到更广泛的应用。

DESCRIPTION (provided by applicant): Wound-healing complications are an important health concern that can be associated with diabetes, bed sores, and infection. The inability to form a stable provisional matrix over the wound site is a common hallmark of poor wound healing. Without a stable matrix, the migration of inflammation responsive cells such as endothelial cells, neutrophils and macrophages, needed to produce new blood vessels1 and fight infections is not possible. The goal of this project is to develop an imaging method to quantify the stability of the provisional matrix during early-stage wound healing. This biological process has been largely invisible and the proposed work is expected to provide significant insight into molecular events that delay wound healing. Wound healing involves an intricate set of precisely timed processes that begin with the formation of a fibrin clot, followed by an inflammatory response, and eventually ending with extensive tissue remodeling and wound closure. During the inflammatory response, the provisional matrix is produced in part through the action of transglutaminase (TG) enzymes cross-linking plasma proteins such as fibrinogen and vitronectin. Recent work in our laboratory has shown that administration of infrared fluorescent-labeled substrates for the TGs, including both proteins and peptides, produces strong contrast for in vivo imaging.2 In this R21 project we will test whether our method can identify problems associated with unstable matrix formation in animal models by executing the following Specific Aims: (1) develop imaging strategies to support multiple time-point imaging in the healing cascade; (2) evaluate the performance of the multiple time point optical imaging method in vivo in a mouse model; and (3) compare provisional matrix stability through imaging matrix formation in a delayed wound healing model of the diabetic mouse. Multiple time-point imaging can monitor problems with both matrix formation and degradation processes. Slow matrix formation will be associated with poor incorporation of labeled substrate into the wound compared with controls. Excessive matrix degradation will be associated with rapid loss of labeled substrates in the wounds compared with controls. A successful project will lead to a new imaging method for studying wound healing and evaluating wound- healing response. In the near term, this method would be used in animal model studies to evaluate the wound- healing process and support the development of new approaches to promote wound healing. In the longer term, this method would be extended to clinical studies to identify the mechanisms of compromised healing and help in wound management. Furthermore, because similar cross-linking processes are involved in atherosclerosis3 and cancer,4 this imaging method will also find broader applications beyond wound healing.