L10 - Hyaluronic Acid Coating and Oscillatory Shear Stress Synergistically Enhances Lymphatic Valve Phenotypes

Introduction: : The engineering of a functional lymphatic collecting vessel has become crucial due to the lack of cell-based therapies for diseases related to lymphatic dysfunction. Recent studies have demonstrated that the in vitro onset of lymphatic endothelial cells (LECs) to a lymphatic collecting vessel phenotype arises from the presence of physiological oscillatory shear stress (OSS) (Sabine et al., Dev Cell. 2012). This is specifically characterized by mechanotransduction responses in LECs which include gene expression variation and cellular morphological changes. However, to engineer a 3D functional lymphatic vessel, further optimization of the cells is required. While previous studies have used fibronectin (FN) coatings for lymphatic vessel maturation, the use of hyaluronic acid (HA) coatings has not been investigated though it has been previously demonstrated to preserve LEC phenotype by enhancing key lymphatic markers via LYVE-1 surface receptors. In this study, a dopamine-conjugated HA (HADP) coating will be used to conduct in-vitro flow experiments on LECs to synergistically influence and maintain the lymphatic collecting vessel phenotype that arises.

Materials and

Methods: : Human donor juvenile LECs were cultured onto ibidi μ-Slide I 0.6 Luer channel slides. Slides were coated with either 40 μg/mL FN or 5 mg/mL HADP. Synthesis and characterization of HADP was done as previously described (Saha et al., . Biomater. Sci. 2023). OSS (at 0.5 dyne/cm^2 and 10 dyne/cm^2) were applied to all LECs with the ibidi Pump System for 48 hours (see Figure 1). For oscillatory flow, the pump changed flow directions every 0.5 seconds. Immunostaining was conducted and imaging was done on a revolve fluorescence microscope and confocal microscope. Image analysis was conducted on ImageJ utilizing the analyze particle functions and the OrientationJ plugin (Rezakhaniha et al., Biomech Model Mechanbiol, 2011). Real-time PCR was conducted with human angiogenesis array panels. Statistical analyses were unpaired t-tests, two-way ANOVAs, and Kruskal-Wallis tests according to data type with P = 0.05 conducted on GraphPad Prism V10.

Results, Conclusions, and Discussions:: Quantification of the cellular morphology using VE-cadherin (VE-CAD) membrane staining and DAPI nuclear staining demonstrated that HA-DP influences the morphological response of LECs to varying OSS rates to a more corresponding localized shape compared to the FN coating (see Figure 2). The orientation distribution of the cytoskeleton of the HA-DP coated LECs were more distributed, especially in response to OSS, and more accumulated in the perinuclear region compared to the FN coated (see Figure 2). Furthermore, HA-DP enhances mechanotransduction pathways of key lymphatic valve formation and maintenance transcription factors in LECs that occurs in response to OSS flow rates compared to the FN coating (see Figure 3). Proliferative activity in HA-DP coated LECs were found to be less variable between static and OSS conditions compared to the FN coated LECs (see Figure 3). The treatment of cells in all conditions with a mechanotransduction inhibitor (10nM rapamycin) were found to similarly downregulate mTOR signaling and PROX1 intensity, thus confirming the synergistic behavior of HA and OSS on the LECs. The morphological properties and marker expressions obtained using a HA-derived coating when exposing LECs to low and high OSS flow rates demonstrates its efficiency in maturing into lymphatic-valve forming cells. The HA-coated LECs were able to respond to OSS, remodel its shape efficiently, and further locally distribute cytoskeleton alignment compared to FN-coated LECs. Moreover, the HA-coated LECs demonstrated a favorable change in the expression of genes primarily responsible for lymphatic valve development in response to different physiological OSS flow rates. The proliferative activity of HA-coated LECs confirmed its optimal OSS response. Lastly, the inhibition of mechanotransduction via mTOR signaling demonstrated the reliance of HA-coated LECs on key lymphatic markers and pathways. Overall, it was demonstrated that HA-coated LECs were able to enhance the mechanotransduction responses that are crucial during lymphatic valve development to better mature and grow lymphatic valves in lymphatic collecting vessels. This study provides insight that HA-DP is the optimal coating for in-vitro models and that HA-based 3D biomaterials could successfully be used to engineer a lymphatic collecting vessel for eventual in-vivo use.

Acknowledgements and/or References (Optional):: We acknowledge support from the University of Notre Dame through “Advancing Our Vision” Initiative in Stem Cell Research,
Harper Cancer Research Institute – American Cancer Society Institutional Research Grant (IRG-17-182-04), American Heart
Association through Career Development Award (19-CDA-34630012 to D.H.-P), National Science Foundation (2047903 to D.H-P.), National
Institutes of Health (1R35-GM-143055-01 to D.H.-P.), and GEM Fellowship to N.K.L.