FIGURE 4.

Administration of VEGF‐C_156S improves cardiac lymphangiogenesis and edema after pressure overload. (A) Schematic of the methods for treatment of WT mice with saline or VEGF‐C_156S at doses of 33 (VEGF‐C_‐L) and 100 (VEGF‐C_‐H) ng/g and TAC for 6 weeks. (B) Heart sections stained with an anti‐LYVE‐1 (red) or anti‐VEGFR‐3 antibody (green) (left, scale bar: 50 μm) and quantification of LYVE‐1⁺ or VEGFR‐3⁺ lymphatics (right, n = 6). (C) Heart sections stained with an anti‐LYVE‐1 antibody (red), TRITC‐labeled WGA (green) and DAPI (blue) (left, scale bar: 50 μm) and the LYVE‐1⁺ vessel to CM ratio (right, n = 6). (D) Immunohistochemical staining of heart sections with an antibody against LYVE‐1, VEGFR‐3 or Podoplanin (left, scale bar: 50 μm) and quantification of LYVE‐1⁺, VEGFR‐3⁺, and Podoplanin⁺ lymphatic vessels (right, n = 6). (E) Gravimetric assessment of cardiac water content (%) as determined by the cardiac dry weight to wet weight (n = 6). (F) Representative color‐coded images of cardiac MRI T2 mapping in which the turquoise/blue areas are normal tissues and the red/yellow areas exhibit cardiac edema (left) and MRI‐based quantification of cardiac water content (T2 map signal intensity, msec) (right, n = 4). (G) Immunoblot analysis of the VEGFR‐3, p‐AKT, AKT, p‐ERK1/2, ERK1/2, CaNA, NFATc1, FOX2C, and CX43 proteins in the heart (left) and quantification of these proteins (right, n = 4). GAPDH was used as an internal control. The data are presented as the mean ± SD, and n represents the number of animals per group. Statistical analysis was performed with one‐way ANOVA; *p < 0.05, **p < 0.01, and ***p < 0.001 versus sham; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 versus TAC + saline; ^$ p < 0.05, ^$$ p < 0.01, and ^$$$ p < 0.001 versus TAC + VEGF‐C_‐L