Meningeal lymphatic-associated brain swelling in acute stroke
Fig 2
Meningeal lymphatic endothelial angiogenesis through VEGF signaling in vitro.
A. LYVE-1 antibody-conjugated magnetic beads were used for lymphatic endothelial isolation. Scale: 50 μm. B. FACS analysis demonstrated that the isolated LYVE-1 positive cells highly expressed a lymphatic endothelium marker Flt4 (VEGF-C receptor), and VEGF-A receptor, Flk1. C. In vitro tube formation assay revealed that VEGF-C effectively induced lymphatic endothelial angiogenesis in vitro compared to VEGF-A (n = 3). Scale: 200 μm. D. Western blot showed that brain endothelium (RBE.4.) produced VEGF-C and VEGF-A into the cultured media following oxygen-glucose deprivation (OGD). E, F. Western blot analysis confirmed VEGF depletion from brain endothelial conditioned media following immunoprecipitation for each target (VEGF-C or VEGF-A). G. Schematic for media transfer experiment. Brain endothelium-derived conditioned media were transferred into meningeal lymphatic endothelial cells to assess lymphangiogenesis. H, I. After 24 hours, VEGF-C depletion significantly reduced the ability of endothelial conditioned media to promote angiogenic response in meningeal lymphatic endothelial cells, but VEGF-A depletion did not influence lymphangiogenesis compared to control IgG (n = 3). Scale: 200μm. *P < 0.05. All values are mean + /- SEM.