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

靶向消除中性粒细胞中的 FXII-uPAR-pAkt2 轴以治疗慢性伤口

• 批准号: 10542838
• 负责人: Evi X. Stavrou
• 金额: $ 39.03万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-15 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10542838
• 关键词: Acceleration; Adhesions; Autoimmune Diseases; Basic Science; Binding; Binding Sites; Biological; Biological Assay; Blood; Cells; Chemotaxis; Chronic; Development; Diameter; Event; Fab Immunoglobulins; Factor XII; Formulation; Foundations; Funding; Future; Goals; Growth Factor; Health Care Costs; Healthcare; Healthcare Systems; Impaired wound healing; In Vitro; Infection; Inflammatory; Interphase Cell; Kinetics; Leukocyte Elastase; Ligands; Lipids; Liquid substance; Maps; Mechanics; Mediating; Morbidity - disease rate; Nanotechnology; Neutrophil Activation; Neutrophil Infiltration; Nutritional Support; Pathology; Peptides; Pharmaceutical Preparations; Phase; Phosphorylation; Population; Proliferating; Proteins; Recombinants; Research; Sampling; Signal Transduction; Site; Site-Directed Mutagenesis; Surface; System; Testing; Therapeutic; Therapeutic Agents; Therapeutic Effect; Topical application; Treatment Efficacy; Urokinase Plasminogen Activator Receptor; Wound models; acute wound; alpha 1-Antitrypsin; autocrine; blocking factor; care burden; chronic wound; clinical efficacy; clinical translation; cost; design; diabetic patient; extracellular; healing; improved; in vivo; innovation; mortality; mouse model; mutant; nanoformulation; nanomedicine; nanovesicle; neutrophil; non-healing wounds; novel; novel strategies; novel therapeutics; peptide drug; preclinical study; protein aminoacid sequence; receptor binding; targeted delivery; technological innovation; therapeutically effective; translational approach; translational study; tumor progression; wound; wound healing

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.

项目概要 该提案的总体目标是表征因子 XII (FXII) 和尿激酶之间的相互作用 纤溶酶原激活剂受体 (uPAR) 设计扰乱中性粒细胞信号传导的策略进行治疗 慢性伤口。慢性、不愈合的伤口是一项重大的医疗负担，造成 250 亿美元的损失 每年都会增加美国的医疗保健费用，并且与高死亡率相关。目前受损伤口的治疗方法 治愈主要关注可控治愈因素的优化，例如机械保护、营养 支持和清除感染。迄今为止已经制定了有针对性的方法，包括主题 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），但不能阻止上游事件，例如 中性粒细胞过度激活、中性粒细胞胞外陷阱 (NET) 形成或中性粒细胞不平衡 蛋白水解活性，所有这些都是不愈合伤口中持续存在的标志性事件。在这个框架下，我们 建议通过有针对性地破坏 FXII-uPAR- 来下调中性粒细胞激活和 NET 形成 pAkt2 轴。我们发现 FXII-uPAR 上调中性粒细胞功能。具体来说，我们已经证明 中性粒细胞激活后，自分泌 FXII 通过 uPAR 发出信号，导致 Akt2 在 Ser474 上磷酸化 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 加快伤口愈合。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 轴对于治疗慢性伤口将具有治疗效果。 在此应用中，我们的目标是：1) 使用重组 FXII 删除在 FXII 上绘制 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）使用独特的纳米囊泡平台 专门结合活化的中性粒细胞并在伤口部位递送 FXII 抑制肽。我们首先会 在体外表征这些负载的纳米囊泡的生物效应，随后我们将确定它们的 在小鼠体内伤口愈合模型中的治疗效果。我们将 3) 将这些临床前研究关联起来 使用糖尿病患者的血液和伤口样本确定 FXII-uPAR-pAkt2 的组成活性。 最终目标是显示 FXII-uPAR-pAkt2 轴和下游效应器在非 治愈伤口病理学，这将为未来抑制其作用的转化研究奠定基础。 我们的科学创新是对中性粒细胞中 FXII-uPAR-pAkt2 信号轴的机制阐明 介导的病理学。我们的技术创新是开发肽纳米医学策略 阻断该轴对慢性伤口有治疗作用。如果成功，该策略将引入新颖且更安全的 治疗慢性伤口和美国人口中常见疾病的疗法。

项目成果

Thromboinflammatory effects of RBC microvesicles.

红细胞微泡的血栓炎症作用。

• DOI: 10.1182/blood.2020004985
• 发表时间: 2020
• 期刊: Blood
• 影响因子: 20.3
• 作者: Stavrou,EviX

Glycated albumin modulates the contact system with implications for the kallikrein-kinin and intrinsic coagulation systems.

• DOI: 10.1016/j.jtha.2022.12.015
• 发表时间: 2023-04
• 期刊: JOURNAL OF THROMBOSIS AND HAEMOSTASIS
• 影响因子: 10.4
• 作者: Hardy, Lewis J.;Bohinc, Dillon;Bane, Kara L.;Heal, Samantha L.;Hethershaw, Emma;Ali, Majid;Palmer-Dench, Thomas;Foster, Richard;Longstaff, Colin;Renne, Thomas;Stavrou, Evi X.;Philippou, Helen
• 通讯作者: Philippou, Helen

Beyond the thrombus: Platelet-inspired nanomedicine approaches in inflammation, immune response, and cancer.

• DOI: 10.1111/jth.15733
• 发表时间: 2022-07
• 期刊: Journal of thrombosis and haemostasis : JTH

Factor XII contributes to thrombotic complications and vaso-occlusion in sickle cell disease.

XII 因子导致镰状细胞病的血栓并发症和血管闭塞。

• DOI: 10.1182/blood.2022017074
• 发表时间: 2023
• 期刊: Blood
• 影响因子: 20.3
• 作者: Sparkenbaugh,EricaM;Henderson,MichaelW;Miller-Awe,Megan;Abrams,Christina;Ilich,Anton;Trebak,Fatima;Ramadas,Nirupama;Vital,Shantel;Bohinc,Dillon;Bane,KaraL;Chen,Chunsheng;Patel,Margi;Wallisch,Michael;Renné,Thomas;Gruber,Andr
• 通讯作者: Gruber,Andr

Monitoring DOACs with a Novel Dielectric Microsensor: A Clinical Study.

• DOI: 10.1055/s-0040-1715589
• 发表时间: 2021-01
• 期刊: Thrombosis and haemostasis
• 影响因子: 6.7
• 作者: Maji D;Opneja A;Suster MA;Bane KL;Wilson BM;Mohseni P;Stavrou EX
• 通讯作者: Stavrou EX