Abstract
Purpose :
Lymphangiogenesis is critically involved in tissue fluid balance, immune responses, corneal graft rejection, and dry eye disease. The healthy cornea is an avascular and alymphatic tissue with a distinct limbal lymphatic vascular arcade. The genetic background significantly influences the architecture of this limbal lymphatic arcade and corneal lymphangiogenesis. By using the BALB/c x C57BL/6 intercross for QTL analysis we identified and functionally characterized a novel potential candidate gene responsible for the differences in the limbal lymphatic vessel architecture on chromosome 17, the cystathionine b-synthase (CBS) gene.
Methods :
To analyze the effect of CBS on human dermal lymphatic endothelial cells (HDLECs) aminooxyacetic acid (AOAA), a pharmacological inhibitor for CBS, was used in vitro. Proliferation and migration of HDLEC after treatment with AOAA was measured by using IncuCyte Zoom and the expression of lymphangiogenic factors was quantified by using qRT-PCR.
In vivo, three 11-0 nylon sutures were placed into the cornea stroma of C57BL/6 mice for 14 days. The treatment group received AOAA as eye drops three times per day for 14 days. Control mice received an equal amount of PBS. Vessel area was analyzed by using cellF software.
Results :
In vitro experiments showed that the inhibition of CBS with the inhibitor AOAA in HDLECs resulted in a significant dose-dependent decrease in proliferation compared with control HDLECs. HDELCs treated with the inhibitor show also significantly delayed wound closure compared to the control. In addition, qRT-PCR revealed that the treatment with AOAA influences the expression of lymphangiogenic factors and their receptors. Fourteen days after the inflammatory insult in vivo, the total surface area of the vessel ingrowth into the cornea of AOAA treated and control treated C57BL/6 mice was assessed and the AOAA treated C57BL/6 mice showed a significantly lower lymphatic surface area compared to control treated C57BL/6 mice, indicating that CBS is also involved in inflammation-induced lymphangiogenesis in vivo.
Conclusions :
In this study we identified cystathionine b-synthase as a novel endogenous regulator of lymphangiogenesis. CBS reduces not only proliferation and migration, but it also influences the expression of lymphangiogenic factors and their receptors. This opens new treatment avenues in diseases associated with pathologic lymphangiogenesis, such as corneal graft rejection.
This is a 2021 ARVO Annual Meeting abstract.