The IGF/IGFBP5 Signaling Pathway Mediates 9-cis Retinoic Acid-induced Lymphangiogenesis In Secondary Lymphedema

PURPOSE: Secondary lymphedema (SL) is a chronic, debilitating condition that most commonly arises after cancer associated with lymph node management. SL patients are managed via conservative approaches or surgical interventions; however, there are currently no FDA-approved pharmacologic therapies available. We have previously demonstrated that 9-cis retinoic acid (9-cisRA) promotes lymphangiogenesis in the setting of SL via lymphatic endothelial cell (LEC) dependent RXR-alpha signaling. However, the intermediate mechanistic pathways underlying this effect are not completely understood. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have previously been described in regulating vascular and lymphatic development. In this study, we propose a novel mechanistic pathway involving IGFBP5 and the IGF signaling axis as a contributor to the unique pro-lymphangiogenic effect 9-cisRA has on LECs. METHODS: Secondary lymphedema was induced in C57BL/6 mice using a circumferential tail excision model. Mice were treated with vehicle or 9-cisRA, and lymphedema was assessed through tail volume measurements and histologic evaluation. 10x Visium spatial transcriptomics was performed on control and treated sections of mouse tail tissue to identify genes differentially expressed in the distal treated region. The results were corroborated with in vitro studies: LECs, human umbilical vein endothelial cells (HUVECs), and fibroblasts were treated with increasing concentrations of 9-cisRA (0.5-2 M). Further characterization of the results was performed through immunofluorescence (IF), western blotting, and immunohistochemistry (IHC). RESULTS: In vivo, 9-cisRA treatment significantly reduced tail swelling and preserved dermal connective tissue architecture compared with untreated controls. Hematoxylin and Eosin staining confirmed mitigation of lymphatic engorgement and dermal thickening in treated animals. Spatial transcriptomics of distal tail sections identified IGFBP5 as significantly and differentially upregulated in LECs following 9-cisRA treatment. In vitro, RT-qPCR confirmed a ~10-fold upregulation of IGFBP5 in LECs (p=0.0149 for 2 M at 48 hours), with no significant change in HUVECs and fibroblasts. Western blotting corroborated increased IGFBP5 protein levels, peaking at intermediary doses of 9-cisRA. IGF2 expression was also significantly upregulated in LECs after 9-cisRA treatment (p=0.0075), whereas IGF1 levels remained unchanged. Conversely, HUVECs demonstrated decreased IGF2 expression under the same conditions (p=0.0101). IF and IHC staining confirmed the cell type specificity of IGFBP5 expression to LECs in vitro and in vivo. CONCLUSION: This study reveals that 9-cisRA promotes lymphangiogenesis through a mechanism that is specific to LECs, driven by selective induction of the IGFBP5/IGF signaling axis. Spatial transcriptomics and in vitro validation showed that 9-cisRA upregulates IGFBP5 and IGF2 exclusively in LECs, which is known to be independent of the classical VEGF-C/D-VEGFR3 signaling pathways. This study provides the first mechanistic link between 9-cisRA and IGFBP5 in the context of lymphangiogenesis, highlighting a promising therapeutic target for pharmacologic treatment of SL. *Source: https://ps-rc.org/meeting/Program/2026/117.cgi*