Fibroblasts dynamically regulate lymphatic barrier functions through localization of junctional proteins

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, we first investigated the effect of secreted factors (secretomes) from normal human dermal fibroblasts (NHDFs) on human LECs (hLECs) and found enhanced junctional integrity. Co-culture with growth factor-activated, NHDF(G) and -inactivated, NHDF(B) fibroblasts increased transendothelial electrical resistance (TEER) by 140% and 110%, respectively. Confocal imaging revealed ZO-1 expression increased by 2.1- and 2.0-fold, while VE-cadherin rose by 1.6- and 1.3-fold in the NHDF(G) and NHDF(B) groups, compared to controls. Dextran transport dropped by 24% with NHDF(G) and 10% with NHDF(B), confirming reduced permeability. Junction analyzer program (JAnaP) showed more continuous ZO-1 junctions in NHDF co-cultures with a decrease in punctate and perpendicular junctions. Thrombin treatment disrupted hLEC junctions, with stronger effects in the presence of NHDFs. These data suggest that in homeostasis, fibroblasts maintain collecting vessel phenotypes critical for preventing lymphatic leakage in skin. Surprisingly, lung fibroblast’s secretomes showed opposite effect in lymphatic junction permeability revealing tissue-specific stromal regulation that may explain differential lymphatic responses in pulmonary versus dermal pathologies. The RNA-seq data showed upregulation of lymphatic markers in fibroblast-induced hLECs, junctional and actin-related genes, suggesting potential modulation of junctional integrity and cytoskeletal organization. In contrast, genes associated with endothelial adhesion to surrounding cells and extracellular matrix, were downregulated, indicating reduced cell-cell and cell-matrix interactions. Overall, our study highlights the role of fibroblasts as tissue determinants in regulating lymphatic barrier function by modulating endothelial cell-cell junctions. NEW AND NOTEWORTHY This exciting work demonstrates that fibroblasts play a role in actively regulating lymphatic barrier functions, and that this happens in a tissue dependent manner. The authors demonstrate that fibroblasts induce changes in lymphatic endothelial cells relating to extracellular matrix modification, cell-cell junctions, cell adhesion, and transport. Competing Interest Statement The authors have declared no competing interest. Footnotes We have edited the manuscript and added additional data to the supplement