Fibroblasts dynamically regulate lymphatic barrier function by modulating cell-cell junctions

Abstract

Lymphatic dysfunction has been linked to several pathological conditions, including edema, inflammation, and cancer metastasis. The tight and adherens junction proteins between lymphatic endothelial cells (LECs) are important for preserving lymphatic vascular integrity. Despite the known role of fibroblasts in lymphangiogenesis, the direct impact of fibroblasts on LEC barrier function remains poorly understood. Here, normal human dermal fibroblast (NHDF) secretomes and non-contact co-culture models were used to examine how fibroblasts regulate human lymphatic endothelial cells (hLECs). Co-culture with dermal fibroblasts increased transendothelial electrical resistance (TEER) by > 110% and increased ZO-1 expression by >2-fold. VE-cadherin expression increased by at least 1.3-fold compared to controls. Furthermore, 4kDa dextran transport was markedly reduced, confirming an overall reduced hLEC permeability. Junction morphology analysis further showed a marked shift from discontinuous punctate and perpendicular junctions toward continuous ZO-1-rich borders, while actin anisotropy was reduced with redistribution away from aligned central stress fibers toward a more junction-supportive cytoskeletal architecture. Thrombin challenge revealed that fibroblasts also shape endothelial responses to inflammatory stress, indicating that stromal-endothelial crosstalk regulates both basal and stress-induced barrier behavior. Transcriptomic analysis identified two fibroblast-driven endothelial programs that converged on junctional remodeling but diverged in broader physiological features: activated NHDFs promoted lymphatic identity, junction stabilization, and a more quiescent endothelial state, whereas inactivated NHDFs favored extracellular matrix remodeling and partial EndoMT-like signaling while retaining some junction-supportive features. In contrast, lung fibroblast secretomes weakened barrier function, supporting the idea that stromal regulation of lymphatic permeability is tissue specific. Together, these findings identify fibroblasts as active regulators of lymphatic barrier physiology and reveal stromal heterogeneity as an important determinant of endothelial junction organization, transport, and tissue-specific lymphatic function. NEW AND NOTEWORTHYThis exciting work demonstrates that fibroblasts are active regulators of lymphatic endothelial barrier physiology during homeostasis. Dermal fibroblasts strengthen barrier function and reduce paracellular transport by promoting continuous junction organization, cytoskeletal remodeling, and extracellular matrix reprograming, while fibroblast state and tissue origin produce distinct endothelial programs.