Local adenoviral delivery of vascular endothelial growth factor C induces lymphangiogenesis in the conjunctiva in rabbits

IntroductionThe purpose of this study was to determine if conjunctival lymphangiogenesis can be induced using adenoviral delivery of VEGF-C. MethodsSeventeen White New Zealand rabbits received a subconjunctival injection containing 3.5x10<^>7 plaque forming units of an adenoviral vector containing the gene encoding VEGF-C (Ad-VEGF-C). The contralateral eye was used for control experiment (the same volume of either saline or an empty vector). After two weeks the animals were examined with trypan blue conjunctival lymphangiography and the eyes were harvested for histology and immunohistochemistry (podoplanin and CD31). ResultsTrypan blue conjunctival lymphangiography revealed significantly more extensive conjunctival vessel network in the Ad-VEGF-C group compared with control: 1.35 & PLUSMN;0.67 versus 0.28 & PLUSMN;0.17 vessel length / analysed area (P=<0.0001). This finding was confirmed with immunohistochemistry where a significant increase in the number of lymphatic vessels was found compared to control, 34 & PLUSMN;9 per mm2 versus 13 & PLUSMN;8 per mm2 (P=0.0019). There was furthermore a significant increase in lymphatic cross-sectional area; 32500 & PLUSMN;7900 & mu;m2 per mm2 versus 17600 & PLUSMN;9700 & mu;m2 per mm2 (P=0.0149). Quantification of blood vessels revealed no significant difference in blood vessel density between Ad-VEGF-C and control, 19 & PLUSMN;9 per mm2 versus 14 & PLUSMN;8 per mm2 (P=0.1971). There was no significant difference in total blood vessel area, 13200 & PLUSMN;7600 & mu;m2 per mm2 versus 7100 & PLUSMN;3000 & mu;m2 per mm2 (P=0.0715). Eyes treated with an adenoviral vector (VEGF-C or empty vector) responded with a reactive cellular response, predominantly lymphocytes, towards the vector. Discussion/ConclusionThe study demonstrates the feasibility of inducing conjunctival lymphangiogenesis with a single subconjunctival injection of Ad-VEGF-C. Future studies will explore how this can be used with a therapeutic purpose.