Targeted Abrogation of the FXII-uPAR-pAkt2 Axis in Neutrophils for Treatment of Chronic Wounds

项目总结 这项提议的总体目标是描述凝血因子XII(FXII)和尿激酶之间的相互作用 纤溶酶原激活剂受体(UPAR)设计干扰中性粒细胞信号传导的策略用于治疗 慢性伤口的症状。慢性、无法愈合的伤口是医疗保健的主要负担，耗资250亿美元 每年在美国的医疗保健费用，并与高死亡率有关。损伤创面的治疗现状 治愈的重点主要是优化可控的愈合因素，例如机械保护、营养 支持和清除感染。到目前为止，已经制定了有针对性的方法，包括专题 然而，生长因子的应用，临床疗效有限。此外，这些方法只会影响 伤口愈合终点(例如，增殖和重塑)，但不阻止上游事件，如 中性粒细胞过度激活，中性粒细胞胞外陷阱(Net)形成，或中性粒细胞失衡 蛋白分解活性，所有这些都是未愈合伤口的持续标志事件。在这个框架中，我们 建议通过靶向干扰FXII-uPAR-来下调中性粒细胞的激活和净形成 PAkt2轴。我们发现FXII-uPAR上调中性粒细胞功能。具体地说，我们已经证明了 中性粒细胞激活后，通过uPAR自分泌FXII信号导致Ser474上Akt2的磷酸化 中性粒细胞黏附、趋化和网状形成。中性粒细胞中FXII信号的中断 伤口愈合得更快。基于这些发现，我们的中心假设是选择性抑制FXII- 中性粒细胞中的uPAR-pAkt2轴在治疗慢性创面方面将是有效的。 在本申请中，我们的目标是：1)使用重组FXII缺失来定位FXII上的uPAR结合位点 突变体和定点突变。这些研究将为FXII的设计提供结构细节 干扰FXII-uPAR相互作用的抑制性多肽；2)使用独特的纳米胶囊平台，能够 特异性结合激活的中性粒细胞，并将FXII抑制肽输送到伤口部位。我们将首先 在体外表征这些负载的纳米微囊的生物学效应，随后我们将确定它们的 对小鼠体内创面愈合模型的治疗作用。我们将把这些临床前研究与 用糖尿病患者的血液和伤口标本测定FXII-uPAR-pAkt2的构成活性。 最终目标是显示FXII-uPAR-pAkt2轴和下游效应器在非 愈合伤口病理，这将为未来的翻译研究奠定基础，以抑制其作用。 我们的科学创新是对中性粒细胞FXII-uPAR-pAkt2信号轴的机制阐明。 中介病理学。我们的技术创新是发展多肽纳米医学战略，以 阻断这一轴，对慢性创面有治疗作用。如果成功，这一战略将引入新的和更安全的 治疗慢性伤口的疗法，这种疾病在美国人中很常见。

PROJECT SUMMARY The overall goal of this proposal is to characterize the interaction between Factor XII (FXII) and urokinase plasminogen activator receptor (uPAR) to design strategies that disrupt their signaling in neutrophils for treatment of chronic wounds. Chronic, non-healing wounds represent a major health care burden, costing 25 billion dollars annually in US health care costs, and are associated with high mortality. Current treatments for impaired wound healing focus mainly on optimization of controllable healing factors, e.g., mechanical protection, nutritional support and clearance of infections. Targeted approaches have been developed to date, including topical application of growth factors however, with limited clinical efficacy. Moreover, these approaches only influence wound healing end-points (e.g. proliferation and remodelling) but do not prevent upstream events such as excessive neutrophil activation, neutrophil extracellular trap (NET) formation, or unbalanced neutrophil proteolytic activity, all of which are persistent hallmark events in non-healing wounds. In this framework, we propose to downregulate neutrophil activation and NET formation through targeted disruption of the FXII-uPAR- pAkt2 axis. We identified that FXII-uPAR upregulate neutrophil functions. Specifically, we have shown that following neutrophil activation, autocrine FXII signals through uPAR leading to phosphorylation of Akt2 on Ser474 and to neutrophil adhesion, chemotaxis, and NET formation. Disruption of FXII signaling in neutrophils resulted in faster wound healing. Based on these findings, our central hypothesis is that selective inhibition of the FXII- uPAR-pAkt2 axis in neutrophils will be therapeutically effective in treating chronic wounds. In this application, our goals are to: 1) map the uPAR binding sites on FXII using recombinant FXII deletion mutants and site-directed mutagenesis. These studies will provide the structural details for the design of FXII inhibitory peptides that interfere with the FXII-uPAR interaction; 2) use a unique nanovesicle platform that is able to bind exclusively on activated neutrophils and deliver FXII inhibitory peptides at wound sites. We will first characterize the biologic effects of these loaded nanovesicles in vitro, and subsequently we will determine their therapeutic effect in murine models of wound healing in vivo. We will 3) correlate these preclinical studies and determine the constitutive activity of FXII-uPAR-pAkt2 using blood and wound samples from diabetic patients. The end goal is to show the relative abundance of the FXII-uPAR-pAkt2 axis and downstream effectors in non- healing wound pathology which will lay the foundation for future translational studies to inhibit its action. Our scientific innovation is the mechanistic elucidation of the FXII-uPAR-pAkt2 signaling axis in neutrophil- mediated pathology. Our technological innovation is the development of peptide nanomedicine strategies to block this axis for therapeutic benefit in chronic wounds. If successful, this strategy will introduce novel and safer therapies to treat chronic wounds, morbidities that are common among the U.S. population.