Fig 1.
Acute increase of lymphatic endothelial cells via VEGFR3 (Flt4) after transient focal cerebral ischemia in Sprague Dawley (SD) rats: Male SD rats were subjected to 100 min of transient focal cerebral ischemia.
A, B. Flow cytometry analysis showed that VEGF-C appeared to be increased in CD31 + endothelial cells without significant changes appearing in GFAP+ astrocytes and CD68 + microglia/macrophage (n = 3). **P < 0.01. C. Western blot showed release of VEGF-C into CSF after focal ischemia in SD rats. D-F. Intracerebroventricular (i.c.v.) infusion of dextran-FITC (40 kDa) or 15 μm micro-beads-FITC together with intravenous (i.v.) injection of Evans blue in brains of normal rats confirmed CSF drainage pathway via perivascular space. G, H. Male SD rats were subjected to 100 min of transient focal cerebral ischemia, then meningeal area, followed by isolating peri-infarct cortex, was isolated. Flow cytometry analysis demonstrated that Flt4 positive lymphatic endothelium were significantly increased in meninges at 24 hr after stroke, but they were barely changed in peri-infarct cortex. (n = 4-7). **P < 0.01. All values are mean + /- SEM.
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.
Fig 3.
An inhibition of flt4 tyrosine kinase with MAZ51 decreased brain swelling after focal cerebral ischemia.
A. Flt4 tyrosine kinase inhibitor, MAZ51 (50 ng/5 µL) was intraventricularly injected immediately after 100 min of transient focal ischemia, then meninges in ipsilateral hemisphere were isolated at 24 hr after stroke onset for FACS analysis. B. Lymphatic endothelial markers, Flt4 and LYVE-1 positive cells were significantly decreased by MAZ51 treatment (n = 7-8). *P < 0.05, values are mean + /- SEM. C-E TTC staining showed no significant change in infarct size. Box-and-whisker plots in D-E show reduced brain swelling in the ipsilateral hemisphere with MAZ51 (n = 7–8, boxes = 25th–75th percentiles, line = median, whiskers = min–max, P < 0.05).