Lymphatic endothelial cells adapt their barrier function in response to changes in shear stress

Abstract

Background: Lymphatic endothelial cells form an important barrier necessary for normal lymph formation and propulsion. However, little is known about how physical forces within lymphatic vessels affect endothelial barrier function. The purpose of this study was to characterize how laminar flow affects lymphatic endothelial barrier function and to test whether endothelial cells respond to flow changes by activating the intracellular actin cytoskeleton to enhance barrier function.

Methods and results: Cultured adult human dermal microlymphatic endothelial cells (HMLEC-d) were grown on small gold electrodes arranged within a flow channel, and transendothelial electrical resistance (TER), an index of barrier function, was determined. Laminar flow was applied to the cells at a baseline shear stress of 0.5 dynes/cm(2), and was increased to 2.5, 5.0, or 9.0 dynes/cm(2), causing a magnitude-dependent increase in barrier function that was reversed 30 min later when the shear stress was returned to baseline. This response was abolished by blockade of actin dynamics with 10 microM phalloidin, and significantly inhibited by blockade of Rac1 activity with 50 microM NSC23766. Blockade of protein kinase A (10 microM H-89) did not inhibit the response. Mathematical modeling based on our impedance data showed that the flow-induced changes in TER were primarily due to altered current flow between cells and not beneath cells.

Conclusions: 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 Rac1-mediated actin dynamics.

FIG. 1.

Diagram of the ECIS flow system.

FIG. 2.

Step increases in fluid shear stress cause rapid enhancement of barrier function in lymphatic endothelial cell monolayers. Measurements of TER (y-axis) of HMLEC-d monolayers were initially taken under no-flow conditions, after which a step increase in laminar flow was applied, generating a shear stress of 10 dynes/cm². The flow was then turned off after 30 min. (A) A rapid increase in TER occurred after the step increase was applied. TER then gradually decreased but remained elevated until the flow was turned off, which caused a rapid drop. (B) This response was similar when the cells were bathed with EBM rather than EGM2-MV. (C) Application of a pulsatile flow (2 s on, 2 s off ) with a peak shear stress of 10 dynes/cm² also caused a similar initial increase in TER. The dotted horizontal line indicates the initial baseline TER level just prior to turning flow on. N = 8 electrodes for each group.

FIG. 3.

Live cell imaging of HMLEC-d during TER measurmement under baseline and elevated shear stress. (A) TER from a single electrode during baseline τ (0.5 dynes/cm²) and during elevated τ (9 dynes/cm²). The images in (B), (C), and (D) were taken at the time points indicated on the tracing. The circle (250 μm diameter) in the center is the cell-covered measuring electrode. The images are representative of 4 experiments.

FIG. 4.

The laminar flow-induced increase in TER is dependent upon the magnitude of the shear stress. (A) Time-course of changes in TER in response to increased laminar flow at varying magnitudes of shear stress. A baseline level of 0.5 in dynes/cm² was initially applied, followed by step increases to 2.5, 5.0, and 9.0 dynes/cm² for 30 min. The increases to 5.0 and 2.5 dynes/cm² were repeated. Each step increase caused a rapid elevation of TER, followed by a gradual decrease in TER, and finally a rapid decrease in TER when shear stress was returned to 0.5 dynes/cm². The tracing shown is the average of 14 electrode measurements. (B) displays the mean changes in resistance from baseline 6 min after the corresponding step increases shown in (A). (C) shows the ratio of the peak TER to baseline TER observed during various levels of elevated shear stress. (D) shows the time to reach the peak TER after various step increases in shear stress. *p < 0.01 vs. baseline (0.5 dynes/cm²). ^†p < 0.01 vs. 2.5 dynes/cm² group. ^§p < 0.01 vs. 2.5 dynes/cm²-repeat group. N = 14 for all groups.

FIG. 5.

Normal actin cytoskeleton dynamics are needed for shear stress-induced enhancement of lymphatic endothelial barrier function. Laminar flow was applied to HMLEC-d monolayers initially to produce a baseline shear stress of 0.5 dynes/cm². Application of a step increase to 9 dynes/cm² for 30 min caused an increase in TER until the shear stress was reduced back to 0.5 dynes/cm². Application of 1 μM phalloidin, an actin stabilizer, caused a slight, initial drop in TER. In addition, the shear stress-induced increase in TER in the presence of phalloidin was significantly inhibited (inset; *p < 0.05; N = 16).

FIG. 6.

Rac1 mediates shear stress-induced changes in HMLEC-d barrier function. Laminar flow was applied to HMLEC-d monolayers initially to produce a baseline shear stress of 0.5 dynes/cm². Application of a step increase to 9 dynes/cm² for 30 min caused an increase in TER until the shear stress was reduced back to 0.5 dynes/cm². Application of the Rac1 specific inhibitor NSC23766 (50 μM) caused a transient drop in TER by itself, which recovered within 20 min. NSC23766 also significantly inhibited the increase in HMLEC-d TER in response to a step increase in shear stress (inset; *p < 0.05; N = 16).

FIG. 7.

PKA does not mediate the shear stress induced rapid increase in lymphatic endothelial barrier function. Laminar flow was applied to HMLEC-d monolayers initially to produce a baseline shear stress of 0.5 dynes/cm². Application of a step increase to 9 dynes/cm² for 30 min caused an increase in TER until the shear stress was reduced back to 0.5 dynes/cm². Application of the specific PKA inhibitor H-89 (10 μM) caused a marked drop in TER, however, did not prevent the elevated shear-stress-induced increase in TER. A comparable increase in TER was observed both in the absence and presence of H-89 (inset; NS = not significant; N = 8).

FIG. 8.

Shear stress-induced enhancement of HMLEC-d barrier function is due mainly to the tightening of junctions between cells. The top tracing shows HMLEC-d TER, and arrows show when step changes in fluid shear stress were performed. The relative contributions of cell-matrix adhesion (α, middle tracing) and cell-cell adhesion (R_b, bottom tracing) to the overall TER (top tracing) were resolved using a mathematical model (see Materials and Methods; N = 8 electrodes).