TITLE: NOVEL PARACRINE MECHANISMS FOR CELL-BASED THERAPY OF INJURED LUNGS SUMMARY. We have recently described a potentially paradigm-shifting signal pathway for repair of injured alveolar epithelium by mesenchymal stem (stromal) cells (MSCs). In injured distal airspaces, increased PAI-1 levels depress local fibrinolytic activity. We demonstrated that MSC-released keratinocyte growth factor (KGF) up-regulates fibrinolysins, which cleave epithelial sodium channels (ENaC). Based on our Preliminary Data, we hypothesize that KGF-activated fibrinolysins in MSCs constitute a novel signal cascade for normalizing alveolar fluid homeostasis and re-epithelialization in injured lungs. We propose that the KGF/uPA/plasmin/ENaC pathway is critical in KGF-mediated edema fluid resolution. This hypothesis is supported by a body of very recent observations: 1) MSC culture medium restores amiloride-inhibitable fluid re- absorption in injured lungs; 2) KGF stimulates expression of uPA, a predominate fibrinolysin in MSCs, and extracellular fibrinolytic activity during repair; 3) depolarization of epithelial cells improves wound healing by facilitating migration, and this depolarization is mainly determined by ENaC activity; 4) knockout of mouse uPA inhibits transepithelial fluid re-absorption; 5) both uPA and plasmin activate ENaC function by cleaving substrate-like extracellular motifs of ENaC; and finally, 6) plasmin augments alveolar fluid clearance in human lungs, and intratracheal delivery of uPA improves edema fluid resolution in acid aspiration injured mice.
Aim 1 will test the beneficial role of this novel KGF/fibrinolysins/ENaC pathway in MSC-based repair.
This aim will utilize an in vitro 3D model of cytokine/hypoxia-challenged primary human alveolar epithelial type 2 cells (AT2), acid injured mice, and ex vivo perfused human lungs infected with live bacteria;
Aim 2 will determine the molecular mechanisms by which the KGF/uPA/plasmin/ENaC pathway enhances reabsorption of alveolar edema fluid.
This aim will utilize a clinically relevant in vivo mouse lungs injured with acid, 3D primary AT2 cultures exposed to cytomix plus hypoxia, and expression systems. We will test the hypotheses 1) that MSC-derived KGF rebuilds the fibrinolytic niche for injured lung epithelium, 2) that elevated fibrinolysins cleave ENaC proteolytically following physical intermolecular regulation, and 3) that extracellular cleavage sites in ENaC proteins are identical to favorite catalytic substrates of fibrinolysins in sequence.
This aim will also test if the KGF/uPA/plasmin/ENaC pathway regulates proliferation, migration, and differentiation of AT2 cells. The robust and unbiased results of these studies will identify novel mechanisms and causal relationships for MSC and KGF-mediated restoration of normal alveolar fluid clearance in lung injury models, and determine how fibrinolytic proteases activate ENaC function to improve edema fluid resolution. Our studies are both basic and translational, as the results will enhance our understanding of the paracrine factors involved in MSC-based clinical therapy, and should provide novel insights into the systematic mechanisms of MSC-based cell therapy for acute respiratory distress syndrome.

Public Health Relevance

. We have discovered a novel mechanism, namely, the KGF/uPA/plasmin/ENaC signaling pathway for stem cell-based therapeutic interventions of lung injury associated with bacterial infection, endotoxin, and acid aspiration. Stem cell-released KGF stimulates the uPA/plasmin system, which then facilitates repair of injured epithelial layer and edema fluid re-absorption by activating ENaC.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL134828-01A1
Application #
9379277
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Aggarwal, Neil Raj
Project Start
2017-09-01
Project End
2021-08-31
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Texas Health Center at Tyler
Department
Type
Overall Medical
DUNS #
800772337
City
Tyler
State
TX
Country
United States
Zip Code
75708
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