Quantifying Lymphatic Endothelial Cell Morphological Changes in Response to Fluid Shear Stress, Cyclic Strain, or Combined Stress and Strain In Vitro

The lymphatic system performs vital transport of fluid, lipids, immune cells, and macromolecules. Lymphatic endothelial cells (LECs), which are highly mechanosensitive, are involved in modulating lymphatic contraction, permeability, etc. LECs are sensitive to the fluid shear stress (FSS) caused by lymph flow and the cyclic strain caused by wall contraction and relaxation. The effects of these mechanical forces on LECs are less understood compared to blood vascular endothelial cells, (BVEC), yet the relative changes in these forces on LECs are considerably greater in situ. To the best of our knowledge, no previous in vitro studies have applied simultaneous FSS and cyclic stretching to LECs. To address this information gap, we tested the hypothesis that both FSS and cyclic stretching will alter LEC structure and function. Toward this goal, we developed a novel bioreactor to apply simultaneous, independently-controlled FSS and cyclic uniaxial stretching to LECs on silicone membranes, and a second bioreactor for applying only uniaxial cyclic stretch. After subjecting LECs to FSS alone, stretch alone, or combined FSS and stretch in these systems, we imaged the cells using conventional or confocal fluorescence techniques and measured alterations in orientation and actin fiber alignment of the cultured LECs. Our results show statistically significant changes in LEC orientation compared to controls, aligning in the direction of low steady FSS (0.2 dynes/cm^2) after 12-24 hours. LECs subjected to the low steady flow combined with a 7.5% cyclic stretch, exhibited a more dramatic alignment that was parallel to flow and perpendicular to stretch after only 6 hours. These data indicate that, like BVEC, LEC morphological alignment is sensitive to both FSS and stretching, with a synergistic response when both are applied simultaneously. Similar levels of alignment were seen in LECs subjected to only cyclic stretch after 6 or 12 hours, but rather than aligning perpendicular to stretch, the distribution of cell angles was bimodal, with most cells aligning about +/- 15 degrees from perpendicular. This slightly off-axis alignment could be a function of applying stretch without fluid flow, or may be a response to the higher magnitude of stretch in those studies (40% compared to 7.5%). There was some visual indication of alignment of actin fibers in LEC subjected to combined FSS and stretching, but it was more difficult to determine clear trends due to spatial variation when imaging different regions of the membrane, and further study is therefore required. Taken together, our results show that application of FSS and strain differentially alter LEC alignment and morphology and provides an important platform to further investigate the mechanisms underlying the LEC response to complex physiologically relevant mechanical environments.