(94f) Correlation between Fluid Shear Stress and Morphological Behaviour of Valvular Endothelial Cells

Valvular endothelial cells (VECs) line the outer layer of heart valves and withstand oscillatory and laminar shear forces along with other mechanical stimuli. Compared to vascular endothelial cells, VECs are understudied and as a consequence their exact physiological behaviour is unclear. Thus, endothelial dysfunction mechanisms leading to valve sclerosis or calcification are yet unknown. In vitro studies of VECs cultured under physiological mechanical stimulation can uncover mechanisms of VEC mechanobiology. Previous studies have shown that vascular endothelial cells align parallel to fluid flow while, in contrast, VECs align perpendicular to blood flow. Recent work from our group has demonstrated that this behaviour varies for different ranges of shear stress values. Here, we aim to show that VEC behaviour does not depend exclusively on fluid shear. There may be several other factors influencing such behaviours, such as confluence levels, time exposed to shear, as well as the stiffness of the supporting scaffold.

This work is based on the study of behaviour of VECs under different shear stress levels and different times of exposure. VECs are cultured on soft substrates on a custom-made parallel-plate chamber. This chamber is specially designed for cultures on soft scaffolds while applying fluid shear on cells in monolayer. Polydimethylsiloxane gels are prepared in the chamber and have their surfaces modified using sulfo-SANPAH as a linker for collagen type I. After collagen treatment, 10⁴ VECs/cm² are seeded on the surface and allowed to adhere for 48 h without shear exposure. Upon cell adhesion, 2.6, 6.5, 13 and 20 dyne/cm² fluid shear are applied on the monolayer culture for either 24 h or 48 h. Cultures then undergo immunofluorescence processing prior to imaging and image analyses of cell aspect ratios, circularity, area, alignment, etc.

Our results demonstrate that with increasing fluid shear, VECs tend to orient in more diverse way, losing their traditional cuboidal morphology, as quantified by cell aspect ratio and circularity parameters. In addition, higher shear levels decrease VEC area, regardless of confluence level. In addition, we observed that, the threshold for cell alignment with shear is much higher than previously observed. High shear also increases VEC differentiation and mesenchymal transition, as validated by F-actin and interstitial cell phenotypes. These results increase the current understanding of VEC physiology and this knowledge is key for the future engineering of replacement heart valves.