Subconjunctival Lymphatics Respond to VEGFC and Anti-Metabolites in Rabbit and Mouse Eyes

Abstract

Purpose To characterize and pharmacologically influence subconjunctival lymphatics in rabbit and mouse eyes. Methods Rabbits received subconjunctival injections of trypan blue or fixable fluorescent dextrans. Bleb-related outflow pathways were quantified. Immunofluorescence for vessel-specific markers (lymphatics [podoplanin and LYVE-1] and blood vessels [CD31]) were performed in native rabbit conjunctiva and after fixable fluorescent dextran injection. Vascular endothelial cell growth factor-C (VEGFC) was injected subconjunctivally in rabbits. mRNA and protein were assessed for the above markers using RT-PCR and Western blot. Alternatively, mouse studies used Prox1-tdTomato transgenic reporter mice. Subconjunctival injection conditions included: no injection, balanced salt solution (BSS), VEGFC, 5-fluorouracil (5FU) and two concentrations of mitomycin-C (MMC). Two mouse injection protocols (short and long) with different follow-up times and number of injections were performed. Mouse eyes were enucleated, flat mounts created, and subconjunctival branching and length assessed. Results Rabbit eyes demonstrated clear bleb-related subconjunctival outflow pathways that were distinct from blood vessels and were without nasal/temporal predilection. Immunofluorescence against vessel-specific markers showed lymphatics and blood vessels in rabbit conjunctiva, and these lymphatics overlapped with bleb-related subconjunctival outflow pathways. Subconjunctival VEGFC increased lymphatic ( P = 0.004–0.04) but not blood vessel ( P = 0.77–0.84) mRNA or protein in rabbits. Prox1-tdTomato transgenic reporter mice demonstrated natively fluorescent lymphatics. Subconjunctival VEGFC increased murine lymphatic branching and length ( P ≤ 0.001–0.004) while antimetabolites ( P ≤ 0.001–0.043) did the opposite for the long protocol. Discussion Subconjunctival lymphatics are pharmacologically responsive to both VEGFC and antimetabolites in two animal models studied using different methodologies. These results may be important for bleb-forming glaucoma surgeries or ocular drug delivery.

VEGF-C and 5-Fluorouracil Improve Bleb Survival in a Rabbit Glaucoma Surgery Trabeculectomy Model

“… 27 – 29 Gene expression analysis in this study showed that VEGF-C successfully promoted lymphatics without a change in blood vessels. This replicated prior findings by our group 26 and others. 33 Then, the VEGF-C combined with 5-FU condition resulted in larger blebs and significantly prolonged bleb survival associated with IOP behavior that decreased over time.…”

“…Our previous research demonstrated that subconjunctival injection of VEGF-C promoted the proliferation of conjunctival lymphatic vessels without affecting blood vessels. 26 Therefore, the results of this study should replicate prior work, and we performed RT-qPCR mRNA analysis (podoplanin and LYVE-1 for lymphatics and CD31 for blood vessels) using filtration bleb tissue collected on postoperative day 30 ( Table 4 ).…”

“… 10 , 12 – 14 The most commonly used antifibrotic agents are 5-FU and mitomycin C. 10 , 12 , 14 Simultaneously, Guo et al 17 hypothesized that subconjunctival lymphatics play an important role in the drainage of GFS blebs. We followed this work by showing that subconjunctival lymphatic presence can be increased by agents such as VEGF-C. 26 Therefore, in this study, we tested the impact of VEGF-C added to 5-FU on glaucoma surgery in a rabbit GFS trabeculectomy model. We chose 5-FU as the antimetabolite as multiple prior reports had studied 5-FU in conjunction with biologics with established protocols.…”

“… 25 Given VEGF-C's purpose, our previous research demonstrated that subconjunctival delivery of VEGF-C in rabbit and mouse eyes significantly increased the number of subconjunctival lymphatic vessels without altering subconjunctival blood vessels. 26 However, it is unknown whether this VEGF-C–induced proliferation of subconjunctival lymphatics can lead to improved aqueous outflow from GFSs and consequently greater GFS success.…”

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

“…The same approach was tested in 3 rabbits, but instead of imaging and quantifying lymphatic vessel, Western blot was used to demonstrate an increased protein expression of a lymphatic endothelial marker at the time of sacrifice (10 days). In contrast to the study by Lee et al [14], we provide structural and functional evidence that our approach with gene therapy results in lymphangiogenesis for at least 4 weeks. Treatment with the pure VEGF-C protein is limited by rapid turnover of the protein.…”