Ischemia/reperfusion (I/R) injury triggers a series of inflammatory responses in the microcirculation characterized by plasma leakage and leukocyte diapedesis. The microvascular pathology largely stems from endothelial barrier dysfunction, a complex cellular process that has not been fully understood at the molecular level. Emerging evidence indicates that the barrier property of the microvascular wall is controlled by dynamic interactions of endothelial cell-cell junctions and cell-matrix focal adhesions;the latter are maily composed of transmembrane integrin receptors and associated proteins such as kindlins and focal adhesion kinase (FAK). These molecules not only provide structural support for endothelial barrier integrity but also transmit biochemical signals that regulate barrier function. The goal of this project is to elucidate the molecular mechanisms underlying FAK-mediated microvascular hyperpermeability during I/R injury. We hypothesize that I/R-elicited oxidative stress upregulates FAK signaling in the microvascular endothelium, inducing integrin (?5?1, ?v?3) internalization and kindlin- targeted ?-catenin nuclear translocation and junction dissociation, leading to weakened cell-matrix and cell-cell adhesions thereby impairing barrier integrity. This novel pathway will be tested in a series of mechanistic studies centered on intravital microscopic quantification of mesenteric microcirculation, leukocyte dynamics, and fluid/protein permeability in mice subjected to superior mesenteric artery occlusion followed by reperfusion. A newly developed mouse model of endothelial-specific conditional FAK knockout will be used in comparison with pharmacological inhibition of FAK. The in vivo studies will be complemented with imaging analyses and molecular assays in microvascular endothelial cells exposed to hypoxia/reoxygenation or oxidative stress. The studies will provide new mechanistic insights into I/R-induced microvascular injury contributing to the future development of targeted therapies to prevent tissue damage following resuscitation or reperfusion. Knowledge gained from this project may have broad implications in other diseases associated with microvascular barrier injury.

Public Health Relevance

Ischemia/reperfusion (I/R) injury occurs in a variety of clinical conditions including trauma, hemorrhage, stroke, myocardial infarction, thrombosis, mesenteric ischemia, bypass surgery and organ transplant. Enormous efforts have been devoted to the identification of inflammatory mediators and oxidative factors released from immune cells in response to systemic inflammation. However, therapies blocking individual upstream signals have demonstrated limited effectiveness in the prevention or treatment of reperfusion injury, prompting evaluations for alternative therapeutic modalities that target endpoint cellular processes, such as the microvascular barrier. Further understanding the molecular mechanisms of microvascular dysfunction at the cellular level will lead to the development of more efficient therapies against I/R injury. Successful completion of this project will not only bring our understanding of I/R pathophysiology to a new level, but also contribute to the future development of targeted and clinically applicable therapies to prevent tissue damage following resuscitation. In addition, insights gained from this study will have broad implications in other types of inflammatory disease or injury associated with endothelial dysfunction and microvascular hyperpermeability.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL120954-01A1
Application #
8767044
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Gao, Yunling
Project Start
2014-08-01
Project End
2018-06-30
Budget Start
2014-08-01
Budget End
2015-06-30
Support Year
1
Fiscal Year
2014
Total Cost
$373,750
Indirect Cost
$123,750
Name
University of South Florida
Department
Surgery
Type
Schools of Medicine
DUNS #
069687242
City
Tampa
State
FL
Country
United States
Zip Code
33612
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Haines, R J; Beard Jr, R S; Wu, M H (2015) Protein tyrosine kinase 6 mediates TNF?-induced endothelial barrier dysfunction. Biochem Biophys Res Commun 456:190-6