Figure 3.

The levels of targeted proteins in tissues of both experimental groups. A, At d 4 after surgery, equal amounts of total protein extraction were immunoblotted using p‐AMPKα (Thr172), AMPKα, PPAR‐γ, PGC‐1α, VEGF, and β‐actin antibodies. Representative immunoblots and quantitative data show reduced levels of p‐AMPKα, VEGF, PPAR‐γ, and PGC‐1α proteins in ischemic muscles of stressed mice. Data are mean±SE (n=3). *P<0.01, NS, not significant by ANOVA and Tukey's post hoc tests. B, Reversed aorta rings (1–2 mm) (upper left panel) were cultured with EBM‐2 containing 50 ng/mL of VEGF on growth factor‐reduced Matrigel for 7 d, and then microvascular sprouting was characterized by fluorescent staining with FITC‐labeled mouse anti‐CD31 (lower left panel). The aortas (including thoracic aorta and aortic arch) were subjected to vascular cellular senescence by β‐galactosidase (β‐gal) staining. C and D, Quantitative data for endothelial cell sprouting areas and β‐Gal⁺ areas (×200). The endothelial sprouting areas and β‐Gal⁺ areas are expressed as the percentage of pixels per image occupied by vessels in the quantitative area. E, Representative Western blots and quantitative data showing the levels of PPAR‐γ and PGC1‐α in the aortas of stressed and control mice. Data are mean±SE (n=4–6). *P<0.01 by Student unpaired t test or ANOVA and Tukey's post hoc tests. Scale bar: 50 μm. ANOVA indicates analysis of variance; EBM, endothelial basal medium; FITC, fluorescein isothiocyanate; p‐AMPKα, phospho‐AMP‐activated protein kinase α; PGC‐1α, PPAR‐γ co‐activator 1α; PPAR‐γ, peroxisome proliferator‐activated receptor‐γ; VEGF, vascular endothelial growth factor.