(M-508) Synthetic Hyaluronic Acid Coating Preserves the Phenotypes of Lymphatic Endothelial Cells

Introduction:: Lymphatic endothelial cells (LECs) play a critical role in the formation and maintenance of the lymphatic vasculature, which is essential for the immune system, fluid balance, and tissue repair. However, LECs are often difficult to study in vivo; and in vitro models that accurately mimic their behaviors and phenotypes are limited. In a variety of diseases, such as cancer and chronic inflammation, the integrity of LECs is disrupted, leading to lymphatic dysfunction and immune suppression.^{1, 2} To understand many of these mechanisms and pathophysiology, it is imperative to have a reliable in vitro culture system that can preserve the phenotype and characteristics of LECs. Another important aspect of having a robust in vitro culture system for LECs is tissue engineering. Nowadays there've been tremendous efforts to generate lymphatic vessels using functional biomaterials like hydrogels.^{3, 4} Often it is not realized that just having a more complex and physiologically relevant 3D system will not result in better tissue engineering unless the rudimentary 2D culture is optimized as well. Therefore, it is important to culture LECs on a coating that supports their proliferation, survival, and functional activity over extended periods of time.

Currently LECs are cultured mostly on treated plastics or on conventional coatings like fibronectin or collagen. Compared to other ECM proteins, the use of Hyaluronan (HA) has been shown to hold promise in the culture of vascular endothelial cells. Here, we describe the development of a novel synthetic coating based on HA to preserve the phenotypes and characteristics of LECs.


Materials and Methods:: Dopamine-modified HA (HA-DP) is generated using sodium hyaluronate solution reacting with dopamine in presence of DMTMM and MES buffer. Conjugation of dopamine group to HA was confirmed using 1H-NMR. HA-DP was coated on tissue culture plates through polymerization of dopamine under basic conditions. HA-DP was dissolved in deionized water at 5 mg/ml. Prior to adding HA-DP to wells for coating, 20 μL of 10 M NaOH was added per 1 mL of HA-DP. After incubating at 37 °C overnight, the wells were vigorously washed with deionized water before cell culture. HA-DP coating was characterized and quantified using toluidine blue assay, as previously described⁵. Briefly, HA-DP coated plate were incubated with 1 ml Toluidine blue O (TBO, Sigma 198161-5G), supernatant was aspirated, and samples were washed properly with 10mM NaOH. At this stage a picture was taken to show TBO staining of coated wells compared to non-coated ones.


Human adult and juvenile lymphatic endothelial cells (PromoCell, Heidelberg, Germany) were expanded and used for experiments between passages 4 and 8, as previously described^{6, 7}. Briefly, LECs were maintained at 37ºC with 5% CO₂ in Endothelial Cell Growth Medium (EGM-MV2, C-22022, PromoCell). For phenotypic expression flow cytometry, PCR and immunofluorescence assay were utilized. Prox1, Podoplanin, LYVE-1, VEGFR3 were selected as LEC characteristic markers throughout the experiments. To visualize focal adhesion kinase and F Actin distribution FAK antibody (Abcam, 1:20) and phalloidin (Abcam, 1:50) were used. Conjugated YAP and TAZ antibodies were used to visualize and quantify spatial distribution of YAP/TAZ.


Results, Conclusions, and Discussions:: While it is a common practice to culture LEC on tissue culture plastic according to established protocols, we noticed difference in cell morphology and proliferation over different passages. Our FACS data shows that both adult and juvenile LECs lose their characteristic expression and surprisingly fast. This led us to consider alternatives to traditional coatings. Though HA is one of the most abundant components of the extracellular matrix (ECM), a naturally occurring HA based matrix is not commercially available, unlike many other constituents like fibronectin (FBN) and collagen. One top of that as HA itself is negatively charged, just solution of HA is incapable of forming any type of coating on tissue culture plastic. To resolve the issue, we synthesized a dopamine conjugated functional HA polymer (HA-DP). After coating the TC plate with the described method, we utilized toluidine blue, a reagent widely used for negative polymer staining, for quantification (Fig.2).

Many of the regulatory proteins LYVE-1, Podoplanin, VEGFR3 and transcription factor Prox1 collectively control LEC identity, characteristics, proliferation, and migration. Among the known receptors, LYVE-1 interacts with HA and that suggests that we can modulate this aspect and control LEC phenotype. We started culturing LEC on HA coated surface over different passages and found that interaction of LYVE-1- HA not only facilitates better expression of LYVE-1 but other characteristics of LECs like Podoplanin and Prox1 as well (Fig.3).

Difference in ECM component often induces different mechanical force on the cells independent of stiffness. Though there is no difference in stiffness between fibronectin and HA-DP coating, we found that late passage LECs showed significantly higher mechanotransduction on fibronectin compared to HA-DP (Fig.4). It is evident that stress-mediated mechanotransducers YAP/TAZ are critically involved in maintaining lymphatic integrity during adulthood⁸. Strikingly, lymphatic YAP/TAZ directly but negatively regulates Prox1 transcription and modulate Prox1 activity and lymphatic plasticity ⁸. This suggests that comparatively reduced mechanotransduction on HA-DP coating causes suppressed YAP/TAZ activity in LEC (Fig.5) which inversely translates to better Prox1 expression. Collectively, HA-DP is a promising biomaterial to culture LECs in vitro toward their applications in basic lymphatic biology and lymphatic regeneration.




Acknowledgements (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-35630012 to D.H.-P.), National Science Foundation (2047903 to D.H-P.), and National Institute of Health (R35-GM-143055 to D.H.-P.).


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