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

项目摘要 该提案的总体目标是描述因子XII（FXII）和尿激酶之间的相互作用 纤溶酶原激活物受体（uPAR），以设计在中性粒细胞中破坏其信号传导的策略用于治疗 慢性创伤。慢性、不愈合的伤口是一个主要的卫生保健负担，耗资250亿美元 每年在美国的医疗保健费用，并与高死亡率。受损伤口的当前治疗方法 愈合主要集中在可控愈合因素的优化，例如，机械保护，营养的 支持和清除感染。到目前为止，已经制定了有针对性的方法，包括局部 然而，生长因子的应用具有有限的临床功效。此外，这些方法只影响 伤口愈合终点（例如增殖和重塑），但不能阻止上游事件， 中性粒细胞过度活化、中性粒细胞胞外陷阱（NET）形成或中性粒细胞失衡 蛋白水解活性，所有这些都是不愈合伤口中的持续标志事件。在这个框架内，我们 建议通过靶向破坏FXII-uPAR下调中性粒细胞活化和NET形成， pAkt 2轴。我们确定FXII-uPAR上调中性粒细胞功能。具体来说，我们已经证明， 在中性粒细胞活化后，自分泌FXII信号通过uPAR导致Ser 474上的Akt 2磷酸化， 以及中性粒细胞粘附、趋化性和NET形成。中性粒细胞中FXII信号传导的破坏导致 伤口愈合更快基于这些发现，我们的中心假设是选择性抑制FXII- 中性粒细胞中的uPAR-pAkt 2轴将在治疗慢性伤口中治疗有效。 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 突变体和定点诱变。这些研究将为FXII的设计提供结构细节 干扰FXII-uPAR相互作用的抑制肽; 2）使用能够 专门结合活化的嗜中性粒细胞并在伤口部位递送FXII抑制肽。我们将首先 表征这些负载的纳米囊泡在体外的生物学效应，随后我们将确定它们的 在体内伤口愈合的鼠模型中的治疗效果。我们将3）关联这些临床前研究， 使用来自糖尿病患者的血液和伤口样品测定FXII-uPAR-pAkt 2的组成活性。 最终目标是显示在非肿瘤细胞中FXII-uPAR-pAkt 2轴和下游效应子的相对丰度。 愈合伤口病理学，这将奠定基础，为未来的翻译研究，以抑制其作用。 我们的科学创新是对中性粒细胞中FXII-uPAR-pAkt 2信号传导轴的机制阐明， 介导的病理学我们的技术创新是肽纳米医学战略的发展， 在慢性伤口中阻断该轴以获得治疗益处。如果成功，这一战略将引入新颖和更安全的 治疗慢性伤口的疗法，美国人口中常见的疾病。

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.