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.

Public Health Relevance

Chronic wounds are taking a staggering toll in all healthcare systems. Current treatments have shown modest efficacy and funding for wound research is exiguous. We are taking a new approach based on basic research and nanotechnology to inhibit the functions of a protein in inflammatory cells so we design the next generation of therapeutic agents to treat chronic, non-healing wounds.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL137695-01A1
Application #
9657141
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Klauzinska, Malgorzata
Project Start
2019-01-15
Project End
2023-12-31
Budget Start
2019-01-15
Budget End
2019-12-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106