The lymphatic system plays a dual role: 1) draining interstitial fluid from tissues and returning it to the blood and, 2) participating in the immune response. All vascularized tissues, except the central nervous system (CNS), are invested with lymphatic vessels. Although significant progress has recently been made in understanding the molecules that regulate lymphangiogenesis, very little is known about factors or conditions that deter lymphatic growth. We, thus, propose to investigate this question and to test the hypothesis that the absence of lymphatic vessels in the CNS is the result of an active inhibitory process. We propose that suppression of lymphatic vessels in the CNS is mediated by Sema3F binding to NRP2, which acts to block the pro-lymphatic effects of VEGF-C and VEGF-D. We propose to test this hypothesis with the following aims three aims. (1) To examine the expression of Sema3F, NRP2 and VEGF-C in the retina and to further characterize the lymphatic-like structures in the retina. Sema3F and 3B will be examined by in situ hybridization in postnatal mouse retinas and in the adult. NRP2 expression will be assessed using NRP2-LacZ mice and by in situ hybridization. Lymphatic-like structures will be examined over time, beginning at P0, and will be assessed for expression of other lymphatic markers, including NRP2, VEGFR3, podoplanin and Prox-1. (2) To determine if blocking Sema3F leads to the formation of lymphatic vessels in the retina. The action of Sema3F will be neutralized by administration of soluble NRP2 or neutralizing Sema3F. In addition, the effect of VEGF- C overexpression will be assessed. (3) To determine if the Sema3F administration can suppress lymphangiogenesis in a model of corneal inflammation/wound healing. The ability of Sema3F to block the formation of lymphatic vessels outside of the retina will be examined by injection of Sema3F protein in a well-characterized corneal suture model.
