BDA-410 administration suppressed ascending aortic medial break in calpain-1 +/+ and −/− mice.

<p>Representative anterior ascending aortic tissue sections from AngII+vehicle (<b>A–D</b>) and AngII+BDA-410 (<b>E–H)</b> administered calpain-1 +/+ and calpain-1 −/− mice stained with Movat’s pentachrome. Arrow indicates medial break. Scale bars correspond to 50 µm. A,C,E and G = 40×; B,D,F and H = 200×.</p

BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A in aortas of LDL receptor −/− mice.

<p>Filamin A protein was detected by Western blotting in aortic tissue extracts from saline and AngII infused mice administered either vehicle or BDA-410 (n = 4). β-actin was shown as the loading control. Results are represented as means ± SEMs; Statistical analyses were performed using two-way ANOVA with a Holm-Sidak multiple comparison post-hoc test. * represent significance of <i>P</i><0.05 when vehicle compared to BDA-410.</p

BDA-410 administration reduced macrophage accumulation in ascending aortas of calpain-1 +/+ and −/− mice.

<p>Representative anterior ascending aortic tissue sections from AngII+vehicle (<b>A–D</b>) and AngII+BDA-410 (<b>E–H)</b> administered calpain-1 +/+ and calpain-1 −/− mice immunostained for CD68 (<b>E–H)</b>. CD68+ cells stain red. Arrow indicates positive staining with CD68. Scale bars correspond to 50 µm. A,C,E and G = 40×; B,D,F and H = 200×.</p

BDA-410 administration attenuated AngII-induced ascending aorta dilation and aortic medial thickness in calpain-1 deficient mice.

<p><b>A.</b> Measurement of intimal areas of ascending aortas on day 28 (n = 10–12). <b>B.</b> Measurement of medial thickness measured from internal to external lamina (n = 10–12). Open circles (calpain-1 +/+) and gray circles (calpain-1 −/−) represent individual mice, diamonds represent means, and bars are SEMs. Statistical analyses were performed using Two way ANOVA with a Holm-Sidak multiple comparison post-hoc test. * denotes <i>P</i><0.05 when comparing vehicle versus BDA-410.</p

BDA-410 administration suppressed abdominal aortic medial disruption in calpain-1 +/+ and −/− mice.

<p>Representative suprarenal aortic tissue-sections from AngII+vehicle (<b>A–D</b>) and AngII+BDA-410 (<b>E–H)</b> administered calpain-1 +/+ and calpain-1 −/− mice stained with Movat’s pentachrome. Arrow indicates medial break. Scale bars correspond to 50 µm. A,C,E and G = 40×; B,D,F and H = 200×.</p

Effects of BDA-410 administration on calpain-1 deficiency in male LDL receptor −/− mice infused with AngII.

<p>Values are represented as means ± SEMs. Body weights and plasma cholesterol concentrations were determined at termination. There were no significant differences between the calpain-1 genotypes for body weight and plasma cholesterol concentrations.</p

Elevated calpain activity in AngII infused calpain-1 +/+ and calpain-1 −/− aortas.

<p>Spectrin breakdown products (<b>A</b>) were detected by Western blotting in tissue extracts from aortas in calpain-1 +/+ x LDL receptor −/− and calpain-1 −/− x −/− LDL receptor −/− mice infused with either saline or AngII for 7 days as a measure of calpain activity. β-actin was shown as loading control. Calpain activity (<b>B</b>) was also measured by a fluorimetric assay in aortic tissue extracts from saline and AngII infused calpain-1 +/+ and −/− mice (n = 4). Calpastatin (<b>C</b>) was detected by Western blotting in aortic tissue extracts. Spectrin (<b>A</b>) and calpastatin (<b>C</b>) protein abundance was quantified by image analysis. Images are representative out of 4 independent experiments. Results are represented as means ± SEMs; Statistical analyses were performed using two-way ANOVA with a Holm-Sidak multiple comparison post-hoc test (<b>A</b>–<b>C</b>). * and horizontal bars represent significance of <i>P</i><0.05.</p

BDA-410 administration attenuated AngII-induced abdominal aortic luminal dilation and expansion in calpain-1 deficient mice.

<p><b>A</b>. Ultrasonic measurements of abdominal aortic diameters of calpain-1 +/+ and −/− mice administered with either vehicle or BDA-410 were measured on day 0 and after 28 days of AngII-infusion (n = 10–12). <b>B</b>. Measurements of maximal external width of abdominal aortas on day 28 (n = 10–12). Open circles (calpain-1 +/+) and gray circles (calpain-1 −/−) represent individual mice, diamonds represent means, and bars are SEMs. Statistical analyses were performed using repeated measures ANOVA (<b>A</b>), or Two way ANOVA with a Holm-Sidak multiple comparison post-hoc test (<b>B</b>). Horizontal bars represent significance of <i>P</i><0.05 when comparing day 0 and day 28. * denotes <i>P</i><0.05 when comparing vehicle versus BDA-410.</p

BDA-410 administration reduced macrophage accumulation in abdominal aortas of calpain-1 +/+ and −/− mice.

<p>Representative suprarenal aortic tissue-sections from AngII+vehicle (<b>A–D</b>) and AngII+BDA-410 (<b>E–H)</b> administered calpain-1 +/+ and calpain-1 −/− mice immunostained for CD68 (<b>E–H)</b>. CD68+ cells stain red. Arrow indicates positive staining with CD68. Scale bars correspond to 50 µm. A,C,E and G = 40×; B,D,F and H = 200×.</p

Calpain-1 deficiency had no effect on AngII-induced abdominal AA formation.

<p><b>A</b>. Ultrasonic measurements of abdominal aortic diameters were measured on day 0 and after 28 days of AngII-infusion (n = 15–20). <b>B</b>. Measurements of maximal external width of abdominal aortas on day 28 (n = 20–21). Open circles (calpain-1 +/+) and gray circles (calpain-1 −/−) represent individual mice, diamonds represent means, and bars are SEMs. Statistical analyses were performed using repeated measures ANOVA with a Holm-Sidak multiple comparison post-hoc test (<b>A</b>), or nonparametric Mann-Whitney Rank sum test (<b>B</b>). * denotes <i>P</i><0.05 when comparing saline versus AngII-infused mice.</p