Micromechanical Properties Of Fixed And Living Vascular Pulmonary Endothelial Cells Following Exposure To Barrier Enhancing And Barrier Disrupting Agents

Abstract

Disruption of pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event that occurs in multiple inflammatory disease processes. The actin cytoskeleton, an essential regulator of endothelial permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. We used atomic force microscopy (AFM) to characterize structural and mechanical changes in the F-actin cytoskeleton of cultured human pulmonary artery EC in response to both barrier-enhancing and barrier-disrupting conditions. The mechanical properties of both fixed and live cells were evaluated. Elastic modulus values in the range of 50-1000 kPa were typically measured for fixed cells, while much lower values of 1-40 kPa were characteristic of live cells. In fixed cells, a differential distribution of elasticity was observed after exposure to the barrier-enhancing compound S1P (sphingosine 1-phosphate) compared to that produced by the barrier-disrupting agonist, thrombin. After S1P, the elastic modulus was elevated primarily at the periphery, while thrombin treatment increased elasticity in the central region of the cell. These observations correspond with the distribution of F-actin in parallel-treated EC as detected by immunofluorescence. In living cells, thrombin generally increased the average elastic modulus over 60 minutes; however, the S1P response was more varied and subtle. Experiments are under way to confirm these preliminary observations. These results provide novel insights into the structural and mechanical properties that dynamically regulate pulmonary EC barrier function.