Agonist Induced Regulation of Micro-Mechanical Properties of Pulmonary Endothelium: Role of Paxillin

The pulmonary endothelium functions as a semipermeable barrier separating vascular contents from the interstitium and airspaces of the lung. Increased endothelial permeability is a characteristic pathophysiologic feature of inflammatory conditions resulting in pulmonary edema and respiratory failure. Disruption of the pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event in highly morbid inflammatory conditions such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Actin cytoskeleton, an essential regulator of EC stiffness and permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. I studied the effects of 2 stimuli, Sphingosine-1- Phosphate, an endogenous bioactive lipid and LPS, a lipoglycan on the barrier function of human lung micro-vascular EC monolayer using the Atomic Force Microscope (AFM), Electronic Cell Substrate Impedance System (ECIS) and the FITC Dextran Permeability Assay. Measurements from the AFM indicated cell stiffness while the ECIS and FITC Dextran Assay gave a measure of permeability. Also, I investigated the role of paxillin, a focal adhesion-associated, phosphotyrosine-containing protein that plays a role in cell adhesion and migration pathways. Time scaled measurements on control and paxillin mutants (Y31/118) show that treatment with S1P increases the cell stiffness and trans-endothelial resistance reducing permeability. LPS conversely has antagonistic effects, increasing permeability of the monolayer. In conclusion, S1P has a barrier enhancing effect and LPS has a barrier disruptive effect, while paxillin mutation mitigates the role of each agonist by affecting the tyrosine phosphorylation pathway which plays a critical role in agonist induced barrier function.