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.

项目摘要 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）设计破坏中性粒细胞信号的策略以进行治疗 慢性伤口。慢性，非治疗伤口代表着重大的医疗负担，耗资250亿美元 每年的美国医疗保健费用，并与高死亡率有关。当前对伤口受损的治疗 康复主要关注可控愈合因子的优化，例如机械保护，营养 支持和清除感染。迄今已开发了有针对性的方法，包括局部 但是，生长因子的应用，临床功效有限。而且，这些方法只会影响 伤口愈合的终点（例如增殖和重塑），但不要阻止上游事件，例如 过度嗜中性粒细胞活化，中性粒细胞外陷阱（净）形成或不平衡的中性粒细胞 蛋白水解活性，所有这些都是非愈合伤口中持续的标志性事件。在这个框架中，我们 提议通过针对FXII-UPAR的靶向破坏来下调中性粒细胞激活和净形成 PAKT2轴。我们确定FXII-UPAR上调中性粒细胞功能。具体来说，我们已经表明 嗜中性粒细胞激活后，通过UPAR的自分泌FXII信号导致Ser474上的Akt2磷酸化 以及中性粒细胞粘附，趋化性和净形成。中性粒细胞中FXII信号的破坏导致 在更快的伤口愈合中。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轴将在治疗治疗慢性伤口方面有效。 在此应用程序中，我们的目标是：1）使用重组FXII删除在FXII上绘制UPAR绑定位点 突变体和定向诱变。这些研究将为FXII设计提供结构细节 干扰FXII-UPAR相互作用的抑制性肽； 2）使用能够 仅在激活的中性粒细胞上结合并在伤口部位递送FXII抑制性肽。我们将首先 表征这些负载的纳米接受体的生物学作用，随后我们将确定它们 体内伤口愈合模型中的治疗作用。我们将3）将这些临床前研究和 使用糖尿病患者的血液和伤口样本来确定FXII-UPAR-PAKT2的本构活性。 最终目标是显示非fxii-upar-pakt2轴和下游效应子的相对丰度 治愈伤口病理学将为将来的翻译研究奠定基础，以抑制其作用。 我们的科学创新是中性粒细胞中FXII-UPAR-PAKT2信号轴的机械阐明 介导的病理。我们的技术创新是肽纳米医学策略的发展 阻止此轴以在慢性伤口中获得治疗益处。如果成功，此策略将引入小说和更安全 治疗慢性伤口的疗法，在美国人群中常见的病态。

项目成果

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

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

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