Disassembly of adherens junctions (AJs) of lung endothelial cell (EC) monolayers by edemagenic agents causes microvascular hyper-permeability and protein-rich pulmonary edema formation, and if uncorrected, it leads to deterioration of lung gas exchange. These changes reflect the failure of the lung's intrinsic homeostatic mechanisms, and as such, they are the hallmarks of acute respiratory distress syndrome (ARDS). Despite our current understanding of the multi-faceted pathogenic mechanisms increasing lung vascular permeability, little is known about the molecular regulation of endothelial barrier restoration following lung injury. The focus of Project 4 will be on defining the role of what we postulate is a key regulatory enzyme phospholipase D2 (PLD2). We propose based on our Supporting Data that PLD2-activated signaling pathways are crucial in the formation of adhesive contacts between ECs via activation of homotypic interactions of VE-cadherin, and in turn restore lung endothelial barrier integrity. The underlying molecular mechanisms regulating VE-cadherin assembly and the closure of adherens junctions (AJs) however are not well understood. In Project 4 we will interrogate the central concept that that activation of PLD2 generates phosphatidic acid (PA), which in turn stimulates the activation of sphingosine kinase 1 (SphK1) in a phosphorylation-dependent manner that results in the localized generation of sphingosine-1-phosphate (S1P). Thereafter, efflux of S1P via the S1P transporter Spinster homologue-2 (Spns2) facilitates the ligation of S1PR1 in an autocrine/paracrine manner to induce cortactin-dependent formation of lamellipodia and re- annealing of AJs through VE-cadherin interactions. The signaling elements of this pathway, mechanisms of their activation, and how formation of membrane protrusions restores AJ integrity will be defined. Thus, in Project 4 we will pursue the following Specific Aims: (i) we will determine the obligatory role of PLD2 generation of PA in mediating trafficking of VE-cadherin, and thereby the formation of AJs and restoration of the lung endothelial barrier; and (ii) we will determine the role of the PLD2?SphK1 signaling axis in activating S1P efflux via the transporter Spinster homologue-2 (Spns2), a rate limiting step, and how the subsequent tyrosine phosphorylation of cortactin mediates the re-sealing of AJs. Together, these studies will delineate for the first time to our knowledge the required and sufficient roles of PLD2, Spns2, VE-cadherin and cortactin acting in concert in the formation of lamellipodia to facilitate resealing of endothelial gaps and restoration of endothelial barrier following lung injury. We expect that our studies will provide the framework for the deveolpment of novel therapeutic approaches to combat and treat ARDS patients with leaky lung vessels and intractable pulmonary edema.

Public Health Relevance

Disassembly of adherens junctions (AJs) of lung endothelial cell (EC) monolayers by edemagenic agents causes microvascular hyper-permeability and protein-rich pulmonary edema formation, and if uncorrected, it leads to deterioration of lung gas exchange, the hallmarks of acute respiratory distress syndrome (ARDS). The focus of Project 4 will be on defining the role of the phospholipase D2 (PLD2) activated signaling pathway in the formation of adhesive contacts between adjacent ECs in order to restore lung endothelial barrier integrity. We expect that our studies on the restoration of AJs will provide the framework for the deveolpment of novel therapeutic approaches to combat and treat patients with ARDS.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL060678-20
Application #
9970544
Study Section
Special Emphasis Panel (ZHL1)
Program Officer
Xiao, Lei
Project Start
2000-03-08
Project End
2021-06-30
Budget Start
2020-07-01
Budget End
2021-06-30
Support Year
20
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Type
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
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