FIG. 3.

Enhanced inflammation and vascular malformation in obese apelin KO mice. A: Increased CD11b⁺ macrophages (green) in the subcutaneous adipose layer of skin from apelin KO mice. B: Morphometric analysis showed that the number of CD11b⁺ macrophages was significantly increased in apelin KO mice. Double immunofluorescence staining for podoplanin (C, red) or meca-32 (D, red) and for APJ (green) revealed upregulated expression of APJ in lymphatics of skin of HFD-fed mice. E: Double immunofluorescence staining of skin for LYVE-1 (green) and meca-32 (red) revealed enlargement (arrowheads) and enhanced formation of LYVE-1⁺ lymphatic vessels and meca-32⁺ blood vessels in apelin KO mice. F: Computer-assisted morphometric analyses of lymphatic and blood vessels in skin. G: Immunofluorescence staining for claudin-5 in a whole-mount of ear skin. Lymphatic vessels in apelin KO mice were partly dilated compared with controls. Double immunofluorescence staining of claudin-5 (green) and podoplanin (red) confirmed that the nonuniform vessels were podoplanin-positive lymphatic vessels. H: Immunofluorescence analysis of mouse skin for podoplanin (red) revealed lymphatic hyperplasia in the adipose layer of apelin KO mice. I: Intradermal injection of Evans blue dye visualized enhanced leakiness of enlarged lymphatic vessels and lymphatic backflush (arrowheads) in apelin KO mice at 1 and 5 min after the injection. Scale bars indicate 200 μm except in (G), which indicates 100 μm. Data are expressed as mean values ± SD. **P < 0.01, ***P < 0.001.