Figure 1.
EPA reduces aortic aneurysm formation.
Gross morphological and histological analyses of aortas were performed at 6 weeks after perivascular application of CaCl2 to the infra-renal aorta. A. Representative images of in situ infra-renal aortas (demarcated by the broken lines) from mice in the sham-operated, control diet or EPA diet groups. B. Quantitative analysis of the maximal external aortic diameters of aortas. n = 4 for sham, n = 12 for control diet and EPA diet groups. C. Histological analysis by EVG staining, showing preserved aortic wall structure of the aorta from EPA diet group compared to the aorta from control diet group. Elastin breaks were also quantified. Scale bars, 200 µm (upper panels) and 50 µm (lower panels). n = 5 for sham, n = 11 for control diet, and n = 12 for EPA diet groups. Representative images of at least three independent experiments are shown in A and C. *P<0.05.
Figure 2.
EPA suppressed aortic calcification after AAA-induction.
Aortic calcification was assessed by micro-CT imaging of in situ aortas 6 weeks after perivascular CaCl2 application. Both sagittal and transverse slices (A) show reduced overall calcification in the infra-renal aortas from EPA diet group compared to the control diet group, and this was consistent with the results of quantitative analysis of the total calcification volume in each aorta (B). n = 4 for control diet and EPA diet groups. Red arrowheads indicate the posterior wall of the infra-renal aorta. Representative images of two independent experiments are shown in A. *P<0.05 compared to control diet group.
Figure 3.
EPA attenuates Mmp9 and Tnfsf11 upregulation in CaCl2-induced AAA. A
. mRNA levels of the matrix metalloproteases Mmp2 and Mmp9, as well as their tissue inhibitors Timp1 and Timp2, in aortas at 1 and 3 weeks after perivascular CaCl2 application were analyzed using real-time RT-PCR. B. The mRNA levels of the factors known to be involved in the development of vascular calcification, Tnfsf11 and Tnfrsf11b, were also similarly analyzed using real-time RT-PCR. All expression levels were first normalized to 18s rRNA levels and then presented as fold change over the sham group. *P<0.05.
Figure 4.
MMP2, MMP9, and RANKL expression in AAAs.
A. Immunohistochemical staining for indicated proteins of serial sections of aortas one week after CaCl2 treatment. Elastic van Gieson staining is also shown. SM α-actin and F4/80 were stained to locate SMCs and macrophages, respectively. Shown are representative images of 4 or more samples in each group. Scale bars, 50 µm. B. Relative positive staining area of MMP2, MMP9, and RANKL in sections from control diet and EPA diet groups. n = 4–5. *P<0.05.
Figure 5.
EPA reduces Mmp9 expression in macrophages.
A. Gelatin zymography of aortic tissues one week after CaCl2 treatment together with quantitative analysis, showing reduced MMP9 activity in samples from the EPA diet group. Equal amounts of protein (20 µg) were loaded per aortic sample. For quantitation, n = 6–7 in each group. B. Gating strategy for the flow cytometric analysis of AAA macrophages. Macrophages were identified as Ly-6ClowCD11b+F4/80+Ly-6G− cells (full gating strategy shown in Figure S2 in File S1). C. The number of aortic macrophages per aortic sample. No statistically significant difference in the number of aortic macrophages between control diet and EPA diet groups was detected. D. The mRNA levels of Mmp9 in sorted aortic macrophages. Expression levels were first normalized to 18s rRNA levels and then further normalized to the level of control diet group. n = 5 in each group. E. RAW264.7 macrophages were cultured with either vehicle (10% BSA) or EPA (50 µmol/L) for 48 hours. The cells were then stimulated with recombinant mouse TNF-α (20 ng/mL) for a further 6 hours and harvested for analysis by RT-PCR. Expression levels were first normalized to 18s rRNA levels and then presented as relative expression compared to baseline vehicle sample. n = 3 per condition. *P<0.05 compared to control diet group in A and D or respective vehicle controls in E.