Fig. 1

GW-788388 inhibits endothelial fibrosis both in vitro and in vivo at 24 and 72 h, respectively. Representative images (upper panels) and densitometric analyses (lower panels) from western blot experiments performed for the detection of VE-Cadherin (a), α-SMA (b), and fibronectin (c) in cultured endothelial cells treated with GW-788388 (5 μg/mL) in the presence or absence endotoxin (20 μg/mL) after 24 h of exposure. Protein levels were normalized against tubulin and expressed relative to vehicle-treated condition in the absence of endotoxin. Values are expressed as the mean ± SD. **p < 0.05, assessed by two-way ANOVA and the Bonferroni post-test (N = 4). d Cell viability assay in cultured endothelial cells treated with GW-788388 (0, 0.5, 5, 50, and 100 μg/mL) for 0, 12, 24, 48, 72, 96 h. Values are expressed as the mean ± SD. *p < 0.05; **p < 0.01. Every GW-788388 concentration was compared with the vehicle-treated condition at every time by two-way ANOVA and the Bonferroni post-test (N = 4). Representative images from fluorescence immunocytochemistry experiments performed for the detection of VE-Cadherin and α-SMA in endothelial cells cultured in the following conditions: vehicle-treated (e), endotoxin-treated (f), GW-788388-treated/endotoxin-treated (g), and GW-788388-treated (h). Scale bar represents 40 μm. (N = 4). i Fluorescence quantification of VE-Cadherin (left panel) and α-SMA (right panel) was performed from experiments (N = 4) as showed in e–h. Values are expressed as the mean ± SD. *p < 0.05; **p < 0.01, assessed by two-way ANOVA and the Bonferroni post-test. (N = 4). Representative images (upper panels) and densitometric analyses (lower panels) from western blot experiments performed for the detection of p-smad-2 and total smad-2 in cultured endothelial cells treated with GW-788388 (5 μg/mL) in the presence or absence endotoxin (20 μg/mL) after 6 h of exposure (j). p-smad-2 protein levels were normalized to those of total smad-2 and expressed relative to those in the vehicle-treated condition in the absence of endotoxin. Values are expressed as the mean ± SD. ***p < 0.001, assessed by two-way ANOVA and the Bonferroni post-test (N = 3). Representative images from fluorescence immunohistochemistry experiments performed for the detection of VE-Cadherin and Fibronectin in mesenteric artery (k–n), aorta artery (o–r) and renal vein (s–v) extracted from rats subjected to the following conditions: vehicle-treated/saline-treated (Vehicle/Saline) (k: mesenteric artery, o: aorta artery, s: renal vein), vehicle-treated/endotoxemic (Vehicle/Endo) (l: mesenteric artery, p: aorta artery, t: renal vein), GW-788388-treated/endotoxemic (GW/Endo) (m: mesenteric artery, q: aorta artery, u: renal vein), and GW-788388-treated/saline-treated (GW/Saline) (n: mesenteric artery, r: aorta artery, v: renal vein). Scale bar represents 50 μm. (N = 5). w Fluorescence quantification of VE-Cadherin (upper panels) and fibronectin (lower panels) was performed from several experiments as showed in mesenteric artery, aorta artery and renal vein. Values are expressed as the mean ± SD. *p < 0.05; **p < 0.01, assessed by two-way ANOVA and the Bonferroni post-test. (N = 5). Representative images (upper panels) and densitometric analyses (lower panels) from western blot experiments performed for the detection of VE-Cadherin (x), α-SMA (y), and fibronectin (z) in mesenteric primary endothelial cells (RMEC) extracted from rats subjected to the following conditions: vehicle-treated/saline-treated (Veh/Sal), vehicle-treated/endotoxemic (Veh/Endo), GW-788388-treated/endotoxemic (GW/Endo), and GW-788388-treated/saline-treated (GW/Sal). GW-788388 was administered by gavage (5 mg/kg a day) 24 h before and during endotoxemia. Endotoxemia was induced by the IP injection of LPS (20 mg/kg) for 72 h. Values are expressed as the mean ± SD. *p < 0.05; **p < 0.01, assessed by two-way ANOVA and the Bonferroni post-test. (N = 3)