Evaluation of Junctional Protein Transition in Postsurgical Lymphedema

Abstract

Background: Lymphedema occurs in up to 30% of patients undergoing mastectomy with axillary lymph node dissection, involving upper extremity swelling due to compromised lymphatic drainage and fibroadipose deposition. Lymphedema has no cure. Lymphatic endothelial cells (LECs) have specialized cell-cell junctions: buttons exist in lymphatic capillaries and are discontinuous to allow for fluid absorption from the interstitial space; zippers are continuous to prevent fluid leaking out of a lymphatic collecting vessel. Inflammation alters lymphatics structurally and functionally. This study aims to assess the composition of button and zipper junctions of LECs in a murine tail lymphedema model to better determine the mechanism of disease and provide novel treatment targets. Methods: Lymphedema was induced in mice with a 3mm full-thickness skin excision and surgical disruption of collecting lymphatic vessels of the tail. Structures of LEC junctions was accessed by immunostaining with tight/adherens junction associated proteins colocalizing with LYVE1 (lymphatic endothelial marker) on post-operative day 14. Occludin expression levels were quantified using immunofluorescent signal intensity from random (n=5) locations from experimental mice tails proximal and distal to the surgical site. Quantification of junction protein transcription was determined through qRT-PCR using primers with SYBR green fluorescence quantification. Results: mRNA transcripts of adherens (VE-Cadherin, β-catenin, and p120-catenin) and tight junction proteins (Claudin-5) were significantly upregulated in lymphedema mice tails (n=4) when compared to normal (n=4, p<0.05). Increased expression of occludin was observed in lymphedema induced mice tail (post-op day 14) when compared to normal (p<0.01). LYVE-1, a lymphatic marker, remained unchanged between the two groups, indicating that changes in junctional protein composition was not secondary to increased total number of LECs. Conclusions: This study shows that lymphatic cell junctions transition from buttons to zippers in the murine tail model of secondary lymphedema. Determining the mechanism of this pathway may provide novel therapeutic targets for lymphedema.