Poster L5 - Effects of Modulating YAP Signaling on Lymphatic Endothelial Cell Morphology and Cell-Cell Junction Formation

Thursday, October 24, 2024

2:45 PM – 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Zhyan N. Noble

Undergraduate
Worcester Polytechnic Institute
Worcester, Massachusetts, United States

Co-Author(s)

Brian Ruliffson (he/him/his)

PhD Candidate
Worcester Polytechnic Institute
Worcester, Massachusetts, United States

Primary Investigator(s)

Catherine F. Whittington

Assistant Professor
Worcester Polytechnic Institute, United States

Introduction: Lymphatic vessels are a significant part of the immune system and includes lymphatic capillaries that are directly attached to the underlying extracellular matrix (ECM) and respond to ECM changes. Yes associated protein 1 (YAP) is a mechanosensitive transcription factor, and ECM stiffness and YAP activity can affect growth of lymphatic endothelial cells (LEC), the cells that make up lymphatic capillaries. ECM stiffness has been implicated in YAP activation and lymphatic vessel formation, and we have observed that increased ECM stiffness results in increased cell area and junction formation of VE-Cadherin, an adhesion molecule between LECs that helps control fluid flow across capillary walls (i.e., vessel function). Stressors can lead to changes in LEC actin cytoskeleton dynamic, which then affects YAP activity, cell morphology, and possibly VE-Cadherin. Yet, less is known about how YAP activity affects LEC morphology and capillary function. We aim to investigate whether cell morphology and VE-Cadherin expression change in response to mevalonic acid and simvastatin treatments that promote and inhibit activity downstream of YAP. This approach simulates stiffness-mediated changes in YAP activity. We hypothesize that mevalonic acid will increase cell area, perimeter, and VE-Cadherin, while simvastatin will decrease cell area, perimeter, and VE-Cadherin.

Materials and

Methods: YAP modulation treatments were applied to primary human dermal lymphatic endothelial cells (LEC; PromoCell) to evaluate effects on LEC cell morphology and function. Treatments include promotion using mevalonic acid and inhibition using simvastatin. LECs were cultured in MV2 endothelial cell supplemented media (PromoCell) and seeded in a 96-well plate at 20,000 cells/cm2 to analyze morphology and VE-cadherin. Cells were divided into 3 categories: Control, YAP promotion, and YAP inhibition, with each treatment category made up of a low (n=3; below recommended dosage), medium (n=3; recommended dosage), and high (n=3; above recommended) dosage. Mevalonic acid treatment groups were: 0.25 mM, 0.5 mM, and 1 mM within MV2 media. The simvastatin treatment groups were: 0.5 µM, 1 µM, and 2 µM in MV2 media. Treatments were applied 24 hours after initial cell seeding and treated for an additional 24 hours. Cells were fixed and stained with Phalloidin (F-Actin), DAPI (nucleus), and anti-VE-Cadherin antibody and imaged using the Keyence BZ800 Fluorescence Microscope. Fluorescence images were analyzed in CellProfilerTM on cell shape metrics (area, perimeter, form factor). Using GraphPad 10, Shapiro-Wilks tests were used to determine normality and a one-way Anova with a post-hoc Tukey’s test was used to determine differences between groups.

Results, Conclusions, and Discussions: Qualitative evaluation of treated LECs did not show significant morphological differences (Figure 1). However, after analyzing samples, the mevalonic acid treatment (YAP promoter) showed a slight decrease in cell area and no significant change in cell perimeter compared to the no-treatment control (Figure 2). In the simvastatin treatment (YAP inhibitor), there was an increase in cell perimeter at a high dosage and decreased form factor, while cell area remained unchanged when compared to the no-treatment control. These shape trends combine to suggest that higher YAP inhibition made cells more irregularly shaped toward a more characteristic LEC oak leaf shape. These data contradict what previous studies have observed when YAP nuclear accumulation (i.e., activation) was modulated with substrate stiffness. The discrepancy of results highlight the need for further research into elucidating the mechanisms by which stiffness and YAP modulate cellular morphology. Our VE-Cadherin results were inconclusive due to a short culture time that did not allow for junction formation (Figure 1). In future, the pre-treatment culture time will be extended to allow junctions to form prior to YAP modulation. In those experiments, it is expected that promoter groups will result in VE-Cadherin junctions with tighter, zipper-like cell junctions. In the inhibition group, the expectation is to see discontinuous and button-like VE-Cadherin junctions, implying looser cell junctions. Additional studies will be designed to link YAP activity, ECM stiffness, and lymphatic capillary function. Those studies will help solidify knowledge on how YAP alters junction formation and subsequent capillary function, which will guide researchers in new lymphatic endothelial research toward understanding factors that affect function, as well as growth.

Acknowledgements (Optional): I would like to thank Professor Whttington, Brian Ruliffson, Athenia Jones, and Stephen Larson for their assistance in this project. This work was supported by NSF Engineering Research Initiation Award (2138841), NIH/NCI R03 (5R03CA267449-02), Genentech Research Award, and NIH URISE@WPI (1T34GM153552-01).