The Effect of SEMA3F on Lymphangiogenesis in the Cornea

Abstract

Purpose: : We have previously shown that macrophages (Mps) contribute to the formation of lymphatic vessels in the inflamed cornea. Semaphorin 3F (SEMA3F) has recently been shown to suppress tumor lymphangiogenesis, but the effects on non-tumor lymphatics have not been investigated. We therefore investigated the effect of SEMA3F on lymphangiogenesis in murine corneal suture model and on macrophage function in vitro.

Methods: : Using male C57BL/6 (8-10 wk) (n=5) and stromal incisions that encompassed more than 120 degrees of the corneal circumference, three 11-0 nylon sutures were placed intrastromally. SEMA3F (1µg/20μl) or PBS as a control were injected on days 0 and 3 after suture placement. Seven days later, corneas were collected, and stained for lymphatic vessels with anti-LYVE-1 and for blood vessels with CD31. The areas covered by vessels were determined using NIH image software. For studies of macrophages in vitro, macrophages collected from peritoneal cavity 4 days following thioglycollate injection were treated with SEMA3F (100ng/ml) for 24 hr and levels of VEGFR3 mRNA, podoplanin and LYVE-1 determined by RT-PCR and western blotting. For studies of lymphatic endothelium in vitro, lymphatic endothelial cells were cultured under SEMA3F stimulation for 48 hrs and cell proliferation was assessed using a Coulter Counter.

Results: : SEMA3F significantly suppressed lymphangiogenesis in the cornea compared to control mice (P=0.0209) whereas blood vessel growth was unaffected. SEMA3F treatment of peritoneal macrophages down-regulated VEGFR3, podoplanin, and LYVE-1 expression. Also, SEMA3F significantly suppressed lymphatic endothelial cell proliferation in vitro.

Conclusions: : SEMA3F can block lymphatic growth associated with corneal wound healing. We speculate that the effect of SEMA3F on lymphatic growth is due not only to a direct effect on lymphatic endothelial but also to an inhibition of macrophage contribution by blocking the induction of lymphatic markers.

Keywords: cornea: basic science • neovascularization • inflammation