Local Adenoviral Delivery of Vascular Endothelial Growth Factor C Induces Lymphangiogenesis in the Conjunctiva in Rabbits

Abstract

Introduction: The purpose of this study was to determine if conjunctival lymphangiogenesis can be induced using adenoviral delivery of vascular endothelial growth factor C (VEGF-C). Methods: Seventeen New Zealand white rabbits received a subconjunctival injection containing 3.5 × 107 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 2 weeks, the animals were examined with trypan blue conjunctival lymphangiography, and the eyes were harvested for histology and immunohistochemistry (podoplanin and CD31). Results: Trypan blue conjunctival lymphangiography revealed significantly more extensive conjunctival vessel network in the Ad-VEGF-C group compared with control: 1.35 ± 0.67 versus 0.28 ± 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 ± 9 per mm2 versus 13 ± 8 per mm2 (p = 0.0019). Furthermore, there was a significant increase in lymphatic cross-sectional area; 32,500 ± 7,900 µm2 per mm2 versus 17,600 ± 9,700 µm2 per mm2 (p = 0.0149). Quantification of blood vessels revealed no significant difference in blood vessel density between Ad-VEGF-C and control; 19 ± 9 per mm2 versus 14 ± 8 per mm2 (p = 0.1971). There was no significant difference in total blood vessel area; 13,200 ± 7,600 µm2 per mm2 versus 7,100 ± 3,000 µm2 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. Conclusion: The 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.