Formation and Regulation of Lymphatic Valves during Inflammation

Abstract

Purpose: Lymphatic dysfunction is associated with a wide array of disorders from transplant rejection to cancer metastasis. To date, there is little effective treatment for lymphatic diseases. The cornea provides an ideal tissue for lymphatic research due to its unique features. We have recently provided the first evidence showing that lymphatic valves are formed during corneal inflammatory lymphangiogenesis (LG) via the up-regulation of integrin α9 (Truong et al. 2011). These structures play a critical role in directing proper lymph flow within the vessels. The purpose of this study is to further characterize the time course of valve formation in relation to vessel formation during corneal LG. In addition, integrin α9 antibody therapy was evaluated to determine whether integrin α9 blockade would interfere with the process of lymphatic valve formation during inflammatory corneal LG.

Methods: Our standardized suture-induced corneal inflammatory LG murine model was used. Whole-mount corneas were harvested every 2 weeks for up to 8 weeks post-suture for immunofluorescent microscopic analysis. Additionally, mice were randomized to receive subconjunctival injections of either integrin α9 antibody or isotype control twice a week for 2 weeks. Whole-mount corneas were harvested for immunofluorescent microscopic analysis. Digital images were analyzed by NIH Image J software to quantify lymphatic vessels as well as valve distribution.

Results: Corneal lymphatic vessel invasion area at 4, 6, and 8 weeks were significantly less than the peak at 2 weeks post-suture. In contrast, the ratio of corneal lymphatic valves to vessel invasion area was at its lowest at 2 weeks with a peak around 6 weeks post-suture. Moreover, therapeutic intervention with integrin α9 antibody significantly reduced the number of lymphatic valves after suture-induced corneal LG (p<0.05).

Conclusions: This study presents new insight into the time course of corneal lymphatic valve morphogenesis during inflammatory processes. Additionally, we have shown that it is possible to inhibit corneal valve formation via integrin α9 interference. Since lymphatic valves are closely associated with vessel function, they may serve as an important indicator of vessel maturation for future research and provide a novel therapeutic approach for lymphatic related diseases, such as inflammation and transplant rejection.

Keywords: 480 cornea: basic science • 557 inflammation • 609 neovascularization