Project Summary Lymphatic research denotes an explosive field of new discovery and lymphatic dysfunction has been found in myriad of diseases. To date, there is still a significant lack of effective treatments for lymphatic diseases in general. The cornea offers an ideal site for lymphatic research due to its accessible location, transparent nature, and lymphatic-free and -inducible features. It is also found that corneal lymphatic vessels develop luminal valves as lymphangiogenesis (LG) proceeds. Studies on corneal LG bears direct implications to transplant rejection in both lamellar and penetrating transplantation settings. The rejection rate of high-risk transplants can be as high as 90%, irrespective of current treatment modalities. However, many patients who are blind from corneal diseases fall into this category after an inflammatory, infectious, traumatic or chemical insult. Our long-term goal is to elucidate the underlying mechanisms of LG using various in vivo corneal models and in vitro cell culture systems, a necessary prerequisite to the development of new therapeutic protocols. Our main hypothesis is that corneal LG is mediated by specific lymphatic factors and the interactions between lymphatic endothelial cells (LECs) and extracellular matrix (ECM), which can be modulated to interfere with LG and graft rejection. This proposal is based on the most recent advances in technology and lymphatic research, and a large amount of preliminary data we have generated during the past few years. Our specific aims are: 1) investigate LG and valve formation in lamellar corneal transplantation using lymphatic fluorescent transgenic mice, live imaging technology, and neutralizing antibodies; 2) define the interaction between LECs and ECM using LEC culture system and mutant mice; and 3) assess the role of Wnt5a in high-risk corneal transplantation using conditional knockout mice and siRNAs. Research on corneal LG has broader clinical implications beyond the treatment of ocular diseases alone since lymphatic dysfunction is associated with a wide array of disorders, which include but are not limited to cancer metastasis, diabetes, inflammation, infection, obesity, hypertension and lymphedema.