Characterization of Endothelial Elastic Properties and Gap Closure under Barrier-Regulatory Agonists

Vascular integrity is primarily determined by endothelial cell (EC) cytoskeletal structure that is differentially regulated by natural barrier-promoting agents such as sphingosine 1-phosphate (S1P) and edemagenic agents such as thrombin. Direct quantification of cytoskeletal remodeling requires a reliable methodology for assessing cytoskeletal-driven force generation. In this study, we further explored mechanistically agonist-induced regulation of EC barrier function by measuring cellular elasticity with atomic force microscopy (AFM). Here atomic force microscopy is used to characterize structural and mechanical properties in the cytoskeleton of cultured EC in response to various stimuli. The measured Young’s modulus provides valuable insights about the cell elasticity variations under baseline, diseased and treatment conditions. I employed AFM to characterize Young’s modulus in cultured human pulmonary artery EC (HPAEC) and human lung microvascular EC (HLMVEC) in response to barrier protective agents S1P and HGF (hepatocyte growth factor) or the barrier disruptive antagonist thrombin. S1P structural analogue FTY720 since this compound is currently in clinical use as FDA-approved therapy. In studies presented here, HPAEC’s demonstrated a much higher Young’s modulus overall as compared to HLMVEC for each treatment condition. Results indicate a value of about 2.9 KPa for HPA control cells and 1.8 KPa for HLMV control cells with subsequent increases 10 and 30 minutes after S1P stimulation. S1P induced the highest Young’s modulus increase (6.1KPa) compared to the other barrier enhancing stimuli tested, HGF (5.8KPa) and FTY720 (4.1KPa), for the 30 minute treatment time. In contrast, the barrier disruptive agent thrombin decreased values from 2.5 KPa down to 0.7 KPa depending on the cell type and treatment time. AFM images also support the quantitative biophysical data regarding EC stiffness since there was cytoskeleton rearrangement observed toward the periphery by barrier enhancing agonists and away from the periphery by the barrier disruptive agent thrombin. We also developed a wound healing assay to examine the EC physiological response to physical injury. Injury by physical wounds, severe inflammation or mechanical ventilation, causes formation of paracellular gaps within the endothelium. EC migrate as a sheet into the injured area to reform a protective barrier. The wound healing assay, in which a scratch from a blade separates a cultured EC intact monolayer, enables quantification of cell migration rates that can be modulated by barrier enhancement agonists like S1P and HGF, or ectopic expression of the barrier regulatory cytoskeletal protein MLCK (myosin light chain kinase) adenovirus constructs (with and without stimulation). Cell migration begins with successive, broad protrusions of the cell membrane (called lamellipodia) that are powered by local polymerization of the underlying cytoskeleton. The same barrier agonists that increase cell stiffness in an intact monolayer may also promote lamellipodial extensions that lead to cell migration into paracellular gaps providing insights into biomechanical properties of EC that regulate vascular barrier function and have applicability to vascular leak syndromes and acute lung injury. We have compared gap closure rates in unstimulated and stimulated EC to evaluate the influence of barrier protective agonists on lamellipodial extension and cell migration. We also investigated the role myosin light chain kinase (MLCK) may have on cell migration through over-expression of MLCK by adenovirus constructs. Sealing of paracellular gaps can then restore cell stiffness and barrier integrity. The gap sizes ranged from 37 m to 357 m with average migration rates of 150 m2/min for control cells under absence and presence of S1P, 1930 m2//min and 4970 m2//min for MLCK infected cells under the absence and presence of a S1P respectively. These standardized assays can be used to model lamellipodia dynamics to enhance its tissue engineering application.