Lymphatic Endothelial Cell Tubule Formation in 3D Culture is Inhibited by Increased Matrix Pressure
Wan Jiao, M.D., Ph.D., Gene K. Lee, M.D., Sun Young Park, M.S., Brian Shaw, B.S., Cynthia Sung, B.S., Bo Han, Ph.D., Young-Kwong Hong, Ph.D., Alex K. Wong, M.D..
Keck School of Medicine of USC, Los Angeles, CA, USA.
PURPOSE:Lymphedema affects over 140 million individuals worldwide and can be caused by surgical injury or resection of lymph nodes. Patients with lymphedema are at risk for infection, impaired extremity function, and in rare cases malignant transformation. Pathogenesis begins with dysfunctional lymphatic drainage which then leads to fluid stasis, inflammation, fibrosis, and progressive stiffness of the soft tissue. We hypothesize that a progressive increase in interstitial pressure has an adverse effect on lymphatic endothelial cell function, thus further contributing to disease progression. To test this hypothesis, we cultured lymphatic endothelial cells (LEC) in 3-D scaffolds of different stiffness conditions to determine the effect of interstitial pressure on LEC proliferation and tube formation.
METHODS: Human primary LECs were isolated from neonatal foreskin under an IRB approved protocol and cultured in Endothelial Cell Basal Medium (EBM, Lonza) with 15% FBS. LECs (1x104) were encapsulated in a range of percentages of biomimetic collagen hydrogels with physiologically relevant pressure including: 5.25 % (3.37 pKa), 6.0 % (5.57 pKa), 7.5 (12.8 pKa),and 9.0 % (15.31 pKa). Experiments were performed in quadruplicate. 3-D in vitro cultures were observed for their phenotypic behavior and analyzed at defined time points for three weeks. Two-way ANOVA (Tukey's multiple comparison test) was used for multi-group statistical analysis.
RESULTS: Lymphangiogenesis, lymphatic vessel-like branching, and formation of collateral lymphatic-like structures were observed at 3 days post cell-seeding in 3D hydrogel. Immunofluorescence demonstrated that these branch-like structures were positively for podoplanin an LECs specific marker. The optimal percentage of gel concentration which favored the formation of these structures was 6.0% gel. Using group comparisons, 6.0% and 7.5% groups were statistically different from 5.25% and 9.0 % groups (p = 0.0198). There was no statistical difference between 6.0% and 7.5 % groups, and 5.25% and 9.0 % groups.
CONCLUSION: The physiologic response to lymphatic injury and lymph fluid obstruction involves lymphangiogenesis and formation of collateral vessels to provide alternative drainage pathways. As lymphedema progresses towards a fibrotic stage, interstitial pressure increases. In this study, we used a 3D in vitro model to demonstrate that lymphangiogenesis occurs optimally at 6.0-7.5% gel (5.57 to 12.8 kPa), which is in the normal physiologic range. At lower (3.37 kPa) or higher (15.31kPa) pressures, lymphangiogenesis is less efficient. This knowledge may be used to guide medical/pro-lymphangiogenic and surgical approaches to the management of lymphedema.
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