Microvascular leakage has been implicated in the pathogenesis of multiple organ dysfunctions in inflammation, sepsis, and ischemic reperfusion injury. The abnormality is largely attributable to neutrophil (PMN) activation and releasing edemagenic agents. The overall goal of this research project is to define the precise molecular mechanisms of PMN adhesion-dependent and adhesion-independent microvascular barrier injury. Our studies during the prior funding cycle have revealed a series of coordinated signaling and structural reactions at the microvascular endothelium during PMN stimulation, characterized by focal adhesion-supported cytoskeleton contraction and junction disorganization. Comparative analysis of the complex pathways points to a predominant role for myosin light chain (MLC)-mediated contractile force development as a triggering event to open the paracellular pathway for protein leakage across the endothelium. As a continuing effort, this study is designed to further characterize the endothelial contractile mechanism by testing the hypothesis that PMNs induce actin polymerization and MLC phosphorylation via p60Src-activation of endothelial-specific myosin light chain kinase (eMLCK) and RhoA-inhibition of the myosin targeting subunit of myosin light chain phosphatase (MLCP).
Three specific aims are proposed: 1) to study the eMLCK mechanism of PMN-elicited microvascular leakage, 2) to study the MLCP regulation of microvascular barrier function during PMN activation, and 3) to characterize the mechanical and molecular basis of endothelial contractile response in PMN-induced hyperpermeability. The research plan incorporates genetic, molecular, and pharmacological approaches into an integrative analysis of molecular reactions and barrier function at both the microvascular and subendothelial levels. Intravital microscopic studies will be complemented by in situ and in vitro experiments for a comprehensive evaluation of hydraulic conductivity and solute permeability. The study will provide novel mechanistic insights into the regulation of microvascular endothelial barrier function by PMNs, with broad implications in various types of vascular diseases or injuries.
|Beard Jr, Richard S; Haines, Ricci J; Wu, Kevin Y et al. (2014) Non-muscle Mlck is required for ?-catenin- and FoxO1-dependent downregulation of Cldn5 in IL-1?-mediated barrier dysfunction in brain endothelial cells. J Cell Sci 127:1840-53|
|Sun, Chongxiu; Beard Jr, Richard S; McLean, Danielle L et al. (2013) ADAM15 deficiency attenuates pulmonary hyperpermeability and acute lung injury in lipopolysaccharide-treated mice. Am J Physiol Lung Cell Mol Physiol 304:L135-42|
|Rigor, Robert R; Shen, Qiang; Pivetti, Christopher D et al. (2013) Myosin light chain kinase signaling in endothelial barrier dysfunction. Med Res Rev 33:911-33|
|Yuan, Sarah Y; Shen, Qiang; Rigor, Robert R et al. (2012) Neutrophil transmigration, focal adhesion kinase and endothelial barrier function. Microvasc Res 83:82-8|
|Lee, Eugene S; Van Spyk, Elyse N; Chun, Kevin C et al. (2012) Monocytic adhesion molecule expression and monocyte-endothelial cell dysfunction are increased in patients with peripheral vascular disease versus patients with abdominal aortic aneurysms. J Surg Res 177:373-81|
|Guo, Mingzhang; Yuan, Sarah Y; Frederich, Bert J et al. (2012) Role of non-muscle myosin light chain kinase in neutrophil-mediated intestinal barrier dysfunction during thermal injury. Shock 38:436-43|
|Sun, Chongxiu; Wu, Mack H; Lee, Eugene S et al. (2012) A disintegrin and metalloproteinase 15 contributes to atherosclerosis by mediating endothelial barrier dysfunction via Src family kinase activity. Arterioscler Thromb Vasc Biol 32:2444-51|
|Rigor, Robert R; Beard Jr, Richard S; Litovka, Olesya P et al. (2012) Interleukin-1?-induced barrier dysfunction is signaled through PKC-? in human brain microvascular endothelium. Am J Physiol Cell Physiol 302:C1513-22|
|Lee, Eugene S; Shen, Qiang; Pitts, Robert L et al. (2011) Serum metalloproteinases MMP-2, MMP-9, and metalloproteinase tissue inhibitors in patients are associated with arteriovenous fistula maturation. J Vasc Surg 54:454-9; discussion 459-60|
|Sun, Chongxiu; Wu, Mack H; Yuan, Sarah Y (2011) Nonmuscle myosin light-chain kinase deficiency attenuates atherosclerosis in apolipoprotein E-deficient mice via reduced endothelial barrier dysfunction and monocyte migration. Circulation 124:48-57|
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