Flow‐Induced Changes in Lymphatic Endothelial Barrier Function

Abstract

The purpose of this study was to determine how shear stress affects barrier function of cultured lymphatic endothelial cells. We determined the transendothelial electrical resistance (TER) of cultured human dermal microlymphatic endothelial cells (HMLEC‐d) to evaluate quantitative, real‐time changes in barrier function, during no‐flow and various flow conditions. Cells were exposed to continuous or pulsing (2 s on, 2 s off) flow at various shear stresses (2.5, 5, 9, 19, or 38 dynes/cm2) for 30 min with periods of no‐ or low‐ (0.5 dynes/cm2) flow in between. We observed a shear‐dependent increase in TER that was reversed upon turning flow off or to low at 30 min. The increase in TER with 9 dynes/cm2 was abolished by blockade of actin dynamics with 10 μM phalloidin, and significantly inhibited by blockade of Rac with 50 μM NSC23766 (9.1 ± 1.1 % increase for control vs. 2.5 ± 2.0 % with NSC23766, P<0.05). Blockade of PKA (10 μM H‐89), or ROCK (5 μM Y‐27632) did not inhibit the response. A mathematical model based on our impedance data shows that the flow‐induced changes in TER were primarily due to altered current flow between cells and not beneath cells. These results suggest that lymphatic endothelial cells dynamically alter their morphology and barrier function in response to changes in shear stress by a mechanism dependent upon Rac‐mediated actin dynamics.