Title: Matrix Stiffening Drives Lymphatic Endothelial Dysfunction via Upregulated IL-33/ST2 Signaling in Lymphedema

Neil Reddy, Wan Jiao, Ping Shu, Nihal Sriramaneni, Nitya Devisetti, Sun Young Park, Bo Han, Young-Kwong Hong, Alex K. Wong 

PURPOSE: Lymphedema is a progressive disorder characterized by impaired lymphatic transport, inflammation, and fibrosis. As clinical staging advances, extraluminal matrix stiffening alters lymphatic endothelial cell (LEC) function. We identified stiffness-dependent gene drivers of LEC dysfunction.

METHODS: Primary human LECs were encapsulated in collagen I hydrogels (5.25%–9.0%) modeling lymphedema stages and observed for 7–21 days. Plexus formation was assessed using viability, proliferation, and lymphatic markers. Structures were subjected to bulk RNA-seq to identify mechanosensitive pathways. Genes were validated by qPCR and Western blot in LECs, HUVECs, and fibroblasts. A murine tail lymphedema model (n=10) provided in vivo validation, with tissue harvested at postoperative days 3, 11, and 30.

RESULTS: LECs formed optimal plexuses at mildly supra-physiologic stiffness (6.0%–7.5%), while physiologic and markedly elevated stiffness impaired networks. RNA-seq identified IL-33 signaling as a top stiffness-regulated pathway. In vitro, LEC IL-33 transcripts increased with stiffness; HUVECs showed no trend, and fibroblasts exhibited inverse patterns. Western blotting confirmed increased IL-33 in LECs and HUVECs but decreased expression in fibroblasts. In vivo, IL-33 and ST2 were upregulated in affected tissue at all timepoints, with maximal differences by day 30.

CONCLUSION: Matrix stiffening activates IL-33/ST2 signaling in LECs, creating a feed-forward cycle of dysfunction. IL-33/ST2 represents a potential stage-specific therapeutic target or biomarker of secondary lymphedema.