Cycloastragenol Inhibits Experimental Abdominal Aortic Aneurysm Progression

Figures

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  Figure 1. Induction of the surgical steps. (A) Isolated infrarenal aorta, (B) placing catheter in the infrarenal aorta, (C) intraluminal porcine pancreatic elastase infusion, (D) isolated AAA on day 28 post surgery.

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  Figure 2. The effect of CAG on AAA growth. Upper panel shows representative ultrasound recordings at baseline and post-surgery days 7, 14, 21, 28 for both vehicle and CAG treated rats. The abdominal aorta (Ab. Aorta) just distal from the left renal artery was used as a reference point for developmental aortic expansion in the experimental period (day 0 and post-surgery day 28). D1: shows aortic diameter. Below, the relative increase in maximal aortic aneurysm diameter adjusted for weight at entry from day 0–28 measured by ultrasound in CAG treated group and vehicle treated group (n = 10/10). Values are mean ± standard deviation. **** indicates p < 0.0001.

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  Figure 3. Elastin assessment in abdominal aortic aneurysms (AAA) (A) Representative micrographs of Miller’s elastin stain (black) from abdominal aorta, vehicle AAA and CAG AAA at day 28 (enlargements correspond to black square on the left image). Scale bar (black box) in micrographs to the left: 100 μm and micrographs to the right: 50 μm (B) Percentage of elastin in medial layer in vehicle and CAG group AAA at day 28 (n = 8/7). (C) Scoring of aneurysmal wall elastin disruption in vehicle and CAG group 1–4, 4 being severe wall disruption and 1 minimal wall disruption (n = 8/7; p = 0.0003). (D) Elastin related mRNA coding for LOX gene (n = 10/10) normalized to RPL41 mRNA levels. Values are median ± inter quartile range. *** Indicates p < 0.001.

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  Figure 4. Quantity and activity of matrix metalloproteinases (MMPs) in AAAs. Assessment of messenger RNA (mRNA) from aneurysmal wall samples from vehicle and CAG group coding for (A) MMP-2 (n = 9/9), (B) MMP-9 (n = 9/9), and (C) MMP-12 (n = 9/9). (D) Zymography and quantification of zymography showing significantly increased activity of MMP-2 in vehicle compared to CAG group (n = 10/10; p = 0.02). (E) Displays weak MMP-2 labeling in a subset of vascular smooth muscle cells of the aneurysm wall from both vehicle- and CAG-treated rats (n = 8/7), scalebar = 50 µm. Values in (A–C) are median ± interquartile range and normalized to RPL41 mRNA levels. Values in (D) are mean ± SD, * indicates p = 0.02.

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  Figure 5. Immune cells in abdominal aortic aneurysms. (A) Relative mRNA levels from aneurysmal wall samples from vehicle and CAG treated group of common lymphocyte marker CD45 (n = 9/9), and (B) macrophage/monocyte marker F4-80 (n = 10/10). All RNA data were normalized to RPL41 mRNA levels. (C) Semi-quantification of aneurysmal CD68 positive cells in AAA of each group per mm2 in the adventitial layer (n = 8/7). (D) Representative micrographs of CD68 positive cells in vehicle and CAG AAAs on day 28 (enlargement represents black square on the left image). Arrows mark positive cells (n = 8/7). Scale bar (black box) in micrographs to the left: 100 μm and in micrographs to the left: 50 μm. Values are median ± interquartile range.

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  Figure 6. Markers for M1 and M2 macrophages and cytokine expression in the aneurysm wall after 28 days on vehicle or CAG treatment. (A) Representative micrographs of CD206 positive staining from vehicle and CAG AAAs at day 28 (enlargement represent black square on the left image). Arrows mark positive cells (n = 8/7). Scale bar (black box) in micrographs to the left: 100 µm and in micrographs to the right: 50 μm. (B) Semi-quantification of CD206 positive cells in each group per mm2 in adventitia. (C) Relative mRNA levels of IL-10 (n = 10/10), (D) inducible NO synthase (iNOS) (n = 10/10), and (E) IL-6 (n = 9/9) in the aneurysm tissue. All RNA data is normalized to RPL41 mRNA levels. All values are median ± interquartile range.

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  Figure 7. Effect of CAG on aneurysmal oxidative stress. (A) Relative mRNA levels of nuclear factor erythroid 2–related factor (Nrf2) (n = 9/9) and (B) relative mRNA levels of Heme oxygenase (HO)-1 (n = 9/9) in the aneurysm tissue. All values are median ± interquartile range. All RNA data is normalized to RPL41 mRNA levels.

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  Figure 8. The effect of CAG treatment on aneurysm calcification. (A) Representative micrographs of calcium deposits (Black) in the aneurysm wall visualized by Von Kossa’s staining (enlargement represent black square on the left image). Scale bar (black box) in micrograph to the left: 100 μm and in micrograph to the right: 50 μm. (n = 7/7). (B) Donut plot of percentage of Von Kossa’s positive aneurysm sections in CAG and vehicle treated groups (n = 7/7; p = 0.018). All values are median ± interquartile range. All RNA data is normalized to RPL41 mRNA levels. * Indicates p < 0.05.

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  Figure 9. CAG does not affect α-actin positive area in the aneurysm wall. (A) Representative micrographs of α-actin staining from vehicle and CAG AAAs at day 28 (enlargement represent black square on the left image, (n = 8/7). Scale bar (black box) in micrographs to the left: 100 µm and in micrographs to the right: 50 μm. (B) Semi-quantification of α-actin positive area of total AAA area. Values are mean ± SD.