PURPOSE: The initial stages of lymphedema are characterized by accumulation of interstitial fluid in the tissues. However, the functional effects of interstitial fluid accumulation on surrounding tissue lymphatics remain unknown. We therefore assessed the molecular effects of interstitial fluid collected from the lymphedematous tissues of patients with secondary lymphedema on cultured lymphatic endothelial cells (LECs).
METHODS: Extravasated lymph fluid from 4 patients with secondary lymphedema was aspirated and collected at the time of operative debulking of the affected limbs. Primary human dermal lymphatic endothelial cells (HDLECs) were treated in vitro with media supplemented with 15% volume of patient-derived lymph fluid for 12-72h (experimental); controls were treated with media alone. To assess tube forming capacity, HDLECs were seeded onto Matrigel basement membrane for 12-24h to assess tube forming capacity in response to treatment. Following 72h treatment with lymphatic fluid, HDLECs were harvested to assess changes in the expression of lymphatic growth regulators and modulators of vascular permeability via flow cytometry, western blot, and immunohistochemistry (IHC) analysis
RESULTS: Using western blot analysis, we found reduced expression of VEGFR3 in HDLECs treated with lymph from 3/4 patients and no changes in overall VEGFR2 quantities in 4/4 experimental groups. Interestingly, treated HDLECs simultaneously exhibited a significant upregulation of cell surface VEGFR3 localization, reduced membrane Neuropilin-2 expression, and no significant changes in amounts of membrane VEGFR2 (p=0.03, p=0.001, p=0.09 respectively). These findings suggest that post translational modifications of VEGFR3 such as alterations in receptor trafficking or internalization may be induced by exposure to lymphatic fluid. Consistent with increased cell surface expression of VEGFR3, we also noted increased AKT, ERK, and eNOS phosphorylation in cells stimulated with lymphatic fluid. However, cells cultured in lymphatic fluid did not exhibit significant differences in tubule formation or cellular proliferation. LECs exposed to lymphatic fluid demonstrated a marked increase in the expression of phospho-VE-Cadherin suggesting that exposure to lymphatic fluid increases permeability of HDLECs.
CONCLUSION: Exposure of HDLEC to lymphedema fluid in vitro promotes alterations in the expression and cellular localization of VEGFR3, thereby influencing the activation of downstream signaling pathways. Additionally, lymphedema fluid induces increased vascular permeability in HDLECs by disrupting the function of adherens and tight junction proteins. These findings provide a cellular mechanism by which lymphatic fluid accumulation in the skin can cause lymphatic dysfunction in the early stages of lymphedema.