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Thomas Clahsen, Christian Büttner, Birgit Regenfuss, Tim Gabriel, Felix Bock, André Reis, Claus Cursiefen; Tyrosinase is a novel endogenous inhibitor of lymphangiogenesis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):942.
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© ARVO (1962-2015); The Authors (2016-present)
Lymphangiogenesis is involved in tissue fluid balance, immune responses, corneal graft rejection, and dry eye disease. In previous work, Tyrosinase was identified as responsible factor for significant increase in lymph vessel area in albino C57BL/6 mice compared to C57BL/6 mice. However, the endogenous regulation is poorly understood. Here we analyze in vitro the influence of Tyrosinase on lymphangiogenesis.
To analyze the effect of low Tyrosinase concentrations on human dermal lymphatic endothelial cells (HDLECs) knock-down experiments were performed by using siRNA. Knock-down efficiency was determined by PCR or after each transfection. Proliferation of HDLECs was measured by using IncuCyte Zoom. To analyze if exogenous Tyrosinase induces apoptosis in HDLEC, the HDLECs were incubated with full medium supplemented with 3.5μg/ml Tyrosinase or only full medium and afterwards stained for AnnexinV and analyzed by flow cytometry. Fibromodulin and VEGF-D expression were quantified in primary human dermal lymphatic endothelial cells in vitro using qRT-PCR.
Comparison of Tyrosinase expression in human dermal lymphatic endothelial cells (HDLEC) and malignant melanoma cell line A-375 revealed that Tyrosinase is expressed in both cell types. LEC transfected with the Tyrosinase siRNA, showed a significant reduction in TyrosinaseRNA, compared to cells transfected with non-coding siRNA. Silencing Tyrosinase expression revealed a significant increase in proliferation of HDLECs compared to control HDLECs. On the other hand, treatment of HDLECs with recombinant Tyrosinase did not induce apoptosis but led to inhibition of proliferation rate of HDLECs in a dose-dependent manner. In addition, quantitative RT-PCR revealed that stimulation with Tyrosinase leads to significantly reduced mRNA expression of FMOD and VEGF-D.
Taken together, here we identify Tyrosinase as novel endogenous inhibitor of lymphangiogenesis. Tyrosinase reduces the expression of the downstream modulator FMOD and the lymphangiogenic factor VEGF-D. This opens up novel treatment options for diseases associated with pathological lymphangiogenesis, like corneal graft rejection.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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