Abstract
Purpose :
Lymphangiogenesis is critically involved in immune responses and corneal graft rejection. Recently tyrosinase has been identified as a novel regulator of developmental and inflammation-induced lymphangiogenesis. However, the underlying mechanisms are still unknown. Here we analyze the influence of tyrosinase on proliferation and expression of vascular growth factors in vitro and in vivo and test whether tyrosinase influences graft survival.
Methods :
Proliferation of human dermal lymphatic endothelial cells (HDLEC) with/without tyrosinase was measured by using IncuCyte Zoom. Expression of vascular endothelial growth factors was quantified in HDLECs in vitro and in central cornea in vivo by using qRT-PCR. Balb/c mice were subjected to corneal transplantations and received a 1.5 mm graft from C57B/6 mice or albino C57BL/6 (B6N-TyrcBrd) mice. Graft survival was determined over 8 weeks and immune cell frequencies in draining lymph nodes were analyzed 3 days and 8 weeks post-transplantation.
Results :
Treatment of HDLECs with recombinant tyrosinase inhibited proliferation in a dose-dependent manner. The treatment of HDLECs with recombinant Tyrosinase resulted in a significantly reduced mRNA expression of VEGF-D, VEGF-R2, and -R3 in vitro.
In order to further analyze the influence of tyrosinase on lymphangiogenesis in vivo, the limbal lymph vessel architecture of naive C57BL/6 and B6N-TyrcBrd mice was compared. B6N-TyrcBrd mice showed a significant increase in lymph vessel area and a significantly higher number of endpoints and branch points. The quantitative RT-PCR of isolated central corneal RNA showed a significantly higher expression of VEGF-A, -C, VEGF-R2, and -R3 in B6N-TyrcBrd mice compared to C57BL/6 animals. BALB/c mice receiving grafts from B6N-TyrcBrd mice showed significantly reduced graft survival compared to animals grafted from C57BL/6 mice. No significant differences in the infiltration of immune cells into draining lymph nodes could be observed between the two groups.
Conclusions :
In this study, we identify tyrosinase as a novel regulator for the expression of vascular endothelial growth factors in vitro and in vivo. Furthermore, tyrosinase seems to be a key player in regulating limbal lymphatic vessel architecture and corneal graft survival. A potential mechanism of how tyrosinase is involved in corneal graft rejection is this novel lymphangiogenesis modulating activity.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.