Microvascular hyperpermeability contributes to multiple organ dysfunctions associated with infection, sepsis, diabetes, and trauma and ischemic-reperfusion injury. Activation of polymorphonuclear neutrophils (PMNs) and releasing agents capable of disrupting endothelial (EC) barrier integrity play an essential role in microvascular leakage during inflammation. Development of effective therapies that can directly target endpoint cellular processes has been hampered, owing to our incomplete understanding of the precise molecular events underlying endothelial barrier injury. The goal of this project is o define the endothelial-specific mechanisms of PMN-elicited microvascular hyperpermeability. Our previous studies in this area have established a central role of non-muscle myosin light chain kinase (nmMLCK) in mediating endothelial paracellular leakage. Further investigation has led to the identification of several novel effectors in nmMLCK signaling, including the endothelial transmembrane protein CD44, focal adhesion protein kindlin-2 (KDL2), and cell-cell adherens junction protein ?-catenin (?CAT). Recently, in an effort to characterize the microvascular effects of a newly discovered bactericidal mechanism known as neutrophil extracellular traps (NETs), we obtained evidence that implicates NETs in endothelial barrier dysfunction. Built on these novel and significant findings, this application proposes a comprehensive analysis of microvascular permeability in response to PMN-EC interactions and NET production during inflammation. The central hypothesis to be tested is that PMNs/NETs activate the CD44-nmMLCK-KDL2-?CAT pathway in the endothelium resulting in paracellular hyperpermeability.
Three aims are proposed to examine 1) PMN-EC interactions and NET effects in microcirculation, 2) endothelial CD44 receptor mechanisms, and 3) intracellular signaling via nmMLCK-KDL2-?CAT. We design a complementary approach that integrates isolated microvessels and cultured endothelial cells into physiological analyses of microcirculation in genetically altered mice. Innovative experimental models and new molecular tools will be developed and tested. Data derived from this work will not only contribute to the advancement of leukocyte/endothelial biology, but also provide new mechanistic insights into the pathophysiology of microvascular inflammation. The study has the potential to identify novel therapeutic targets for future development of effective treatments against inflammatory diseases.

Public Health Relevance

Inflammation is a complex biological response of the body to harmful pathogens or diseases such as sepsis, diabetes, trauma, and ischemia-reperfusion injury. It is characterized by white blood cell (neutrophil) sticking to the wall of blood vessels nd causing fluid leakage into tissues, leading to organ dysfunction. Development of therapies to directly treat this problem has been hampered, owing to our incomplete understanding of the molecular reactions that occur at the endothelium, a cell barrier lining the inner surface of blood vessels that controls the transport of blood cells and fluid across the vessel wall. This research project is designed to provide a comprehensive analysis of the precise mechanisms by which neutrophils cause vascular endothelial barrier injury. Identification of key molecules responsible for blood vessel leakage will contribute to the development of novel therapeutic targets for effective treatments of inflammatory diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL126646-04
Application #
9686785
Study Section
Hypertension and Microcirculation Study Section (HM)
Program Officer
Reid, Diane M
Project Start
2016-05-01
Project End
2020-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
4
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of South Florida
Department
Physiology
Type
Schools of Medicine
DUNS #
069687242
City
Tampa
State
FL
Country
United States
Zip Code
33617
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Meegan, Jamie E; Yang, Xiaoyuan; Coleman, Danielle C et al. (2017) Neutrophil-mediated vascular barrier injury: Role of neutrophil extracellular traps. Microcirculation 24:
Beard Jr, Richard S; Yang, Xiaoyuan; Meegan, Jamie E et al. (2016) Palmitoyl acyltransferase DHHC21 mediates endothelial dysfunction in systemic inflammatory response syndrome. Nat Commun 7:12823