Intracellular Thiol Redox Status Regulates Lymphangiogenesis and Dictates Corneal Limbal Graft Survival

PURPOSE: Compounds regulating intracellular thiol redox status, such as N,N-diacetyl-L-cystine dimethylester (NM(2)), were shown to prolong corneal graft survival in a penetrating keratoplasty (PKP) model. However, the effect of NM(2) on hemangiogenesis and lymphangiogenesis has not been investigated. The effect of manipulating ambient thiol redox status on riskier (higher rejection rate) transplantation models, such as limbal graft survival and hemangiogenesis and lymphangiogenesis in a corneal suture model, were investigated. METHODS: C57BL/10 mice that received BALB/c corneas were treated by subconjunctival injection of NM(2), and limbal graft survival was assessed. Sutured C57BL/6 received daily intraperitoneal injections of NM(2), glutathione diethylester (GSH-OEt), or PBS. Lymphatic endothelial cell (LEC) and peritoneal mps were treated with NM(2) or GSHOEt, and then VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression were analyzed. Supernatants were collected for analysis of TNF-alpha and VEGF-A levels by ELISA. RESULTS: Significantly less cellular infiltration was detected in mice with corneal limbal transplant-treated NM(2)-treated mice. Hemangiogenesis and lymphangiogenesis were suppressed in the NM(2)-treated mice. NM(2) treatment of mps led to reduced levels of VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression compared with PBS- or GSHOEt- treated mps, lower levels of TNF-alpha, and increased secretion of VEGF. Moreover, NM(2)-treated LECs had reduced levels of LYVE-1 and Prox-1. CONCLUSIONS: Reduction of ambient redox status reduced inflammatory cell infiltrates. Consequently, reduced inflammatory response might have contributed to both the observed prolonged corneal limbal graft survival and the attenuated hemangiogenesis and lymphangiogenesis in cornea.