A peptide vaccine targeting angiotensin II attenuates the cardiac dysfunction induced by myocardial infarction

Figures

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  Figure 1. The induction of antibody production by the Ang II vaccine. (a) The protocols to investigate the antibody production induced by the Ang II vaccine and the effect of the treatment with the Ang II vaccine. (b) The serum anti-Ang II antibody titer. Sham + vehicle, n = 9; Sham + KLH, n = 5; Sham + Ang II vaccine, n = 5; MI + KLH, n = 11; MI + Ang II vaccine, n = 14. *p < 0.05 vs. Sham + KLH; #p < 0.05 vs. MI + KLH. Tukey–Kramer post hoc test. ND, not detected. (c) The serum anti-Ang II antibody titer in the MI + Ang II vaccine (post-MI) group. MI + KLH, n = 11; MI + Ang II vaccine (post-MI), n = 10. *p < 0.05 by the Student’s t-test. ND, not detected. (d) Comparison of the reactivity of commercial anti-Ang II monoclonal antibody and immunized serum derived from Ang II vaccine-injected rats.

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  Figure 2. The safety of the Ang II vaccine. (a) A representative photomicrograph of hematoxylin-eosin staining in various organs (magnification 200×; scale bars: 100 μm). (b) The plasma cystatin C level on day 56. Sham + vehicle, n = 7; Sham + Ang II vaccine, n = 5. NS, not significant. (c) The plasma BNP-45 level on day 56. Sham + vehicle, n = 8; Sham + Ang II vaccine, n = 5. (d) The serum albumin level on day 56. Sham + vehicle, n = 8; Sham + Ang II vaccine, n = 5. (e) The mean blood pressure. Sham + vehicle, n = 5; Sham + Ang II vaccine, n = 5.

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  Figure 3. The effects of each of the treatment approaches on cardiac dysfunction after MI. (a) Representative M-mode echocardiograms on day 56. A quantitative analysis of (b) the left ventricular ejection fraction and (c) the end-systolic left ventricular diameter. Sham + KLH, n = 5; Sham + Ang II vaccine, n = 5; MI + KLH, n = 11; MI + Ang II vaccine, n = 13; MI + Ang II vaccine (post-MI), n = 10; MI + losartan, n = 7. *p < 0.05 by the Tukey–Kramer post hoc test. (d) The correlation between the left ventricular ejection fraction and the serum anti-Ang II antibody titer in the MI + Ang II vaccine and MI + Ang II vaccine (post-MI) groups.

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  Figure 4. The effects of each of treatment approaches on post-MI remodeling. (a) Representative photomicrographs of Mallory-stained cross-sections from each group (magnification, 10×; scale bars: 1 mm) on day 56. (b) The quantitative data of the infarct thickness. Sham + KLH, n = 5; Sham + Ang II vaccine, n = 5; MI + KLH, n = 11; MI + Ang II vaccine, n = 14; MI + Ang II vaccine (post-MI), n = 10; MI + losartan, n = 7. (c) A representative photomicrograph of the FITC-conjugated WGA staining of the non-infarct area (magnification 400×; scale bars: 50 μm) and (d) a quantitative comparison of the cross-sectional size of the cardiomyocytes in each group on day 56. Sham + KLH, n = 4; Sham + Ang II vaccine, n = 4; MI + KLH, n = 6; MI + Ang II vaccine, n = 7; MI + Ang II vaccine (post-MI), n = 6; MI + losartan, n = 7. *p < 0.05 by the Tukey–Kramer post hoc test. (e) A representative photomicrograph of CD68 immunostaining (magnification 200×; scale bars: 100 μm) and (f) the number of infiltrating macrophages (CD68-positive cells) in the peri-infarct zone on day 56. Sham + KLH, n = 5; Sham + Ang II vaccine, n = 5; MI + KLH, n = 6; MI + Ang II vaccine, n = 6; MI + Ang II vaccine (post-MI), n = 6; MI…

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  Figure 5. The duration of antibody production induced by the Ang II vaccine. (a) The protocol to investigate the duration of antibody production after the administration of the Ang II vaccine. (b) The long-term changes in the serum anti-Ang II antibody titer after vaccination. n = 4. (c) The exponential decay of the serum anti-Ang II antibody titer.

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  Figure 6. The potency of the antibody produced by the Ang II vaccine on cardiac dysfunction after MI. (a) The protocol to evaluate the potency of immunized serum administration on cardiac dysfunction induced by MI. (b) The serum anti-Ang II antibody titer. MI + control serum, n = 7; MI + immunized serum, n = 7; MI + immunized serum + Ang II, n = 7. *p < 0.05 by the Tukey–Kramer post hoc test. (c) Representative M-mode echocardiograms on day 35. A quantitative analysis of (d) the left ventricular ejection fraction and (e) the end-systolic left ventricular diameter. MI + control serum, n = 7; MI + immunized serum, n = 7; MI + immunized serum + Ang II, n = 7. *p < 0.05 by the Tukey–Kramer post hoc test. (f) The correlation between the left ventricular ejection fraction and the serum anti-Ang II antibody titer in the MI + immunized serum and MI + immunized serum + Ang II groups.

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  Figure 7. The potency of the antibody produced by the Ang II vaccine on post-MI remodeling-associated responses in cardiac fibroblasts. (a) The protocol to evaluate the potency of immunized serum addition on Ang II signaling. (b) Representative Western blots of NADPH oxidase 4 (NOX4), phospho-c-Jun, phospho-nuclear factor-κB p65 subunit (NF-κB p65), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in neonatal rat cardiac fibroblasts. The relative protein expression of (c) NOX4, n = 4 each; (d) phospho-c-Jun, n-4 each; and (e) phospho-NF-κB p65, n = 5-6 each. *p < 0.05 by the Tukey–Kramer post hoc test. The relative mRNA expression of (f) angiotensin type 1a receptor (AT1aR), n = 5-6 each; and (g) angiotensinogen, n = 5–6 each. *p < 0.05 by the Tukey–Kramer post hoc test. (h) A representative image of gelatin zymography for matrix metalloproteinase (MMP)-2 and MMP-9 in the conditioned media of cultured cardiac fibroblasts. The relative activity of (i) MMP-2, n = 6 each; and (j) MMP-9, n = 6 each. *p < 0.05 by the Tukey–Kramer post hoc test. (k) The relative collagen level in cardiac fibroblasts, n = 12 each; and (l) the proliferation ratio of cardiac fibroblasts, n = 12 each. *p < 0.05…