Vaccine targeting ANGPTL3 ameliorates dyslipidemia and associated diseases in mouse models of obese dyslipidemia and familial hypercholesterolemia

Figures

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  Figure 1. Development of an ANGPTL3 vaccine (A) Schematic diagram of ANGPTL3 protein. Epitope candidates (E1–E3) are denoted by black bars, and functional domains are indicated. (B) Schematic showing vaccine injection protocol (black arrowheads) and animal sacrifice (open arrowheads). (C–J) Analyses of ob/ob mice 6 weeks after the first immunization. (C) Antibody titer following injection of indicated peptides as assessed by ELISA (n = 7–12 per group). Values are reported as the serum dilution giving half-maximal binding (optical density: OD50%). (D–H) Western blots probed with sera from indicated mice (D–G) or with anti-BSA antibody (H). All 5 blots are identical and contain a molecular weight marker (lane 1), recombinant ANGPTL3 (lane 2), E1 peptide conjugated to BSA (E1-BSA) (lane 3), E2-BSA (lane 4), and E3-BSA (lane 5). IB, immunoblot; rANGPTL3, recombinant ANGPTL3. (I and J) Angptl3 transcript levels in liver of ob/ob mice (n = 6–11 per group) (I) and circulating ANGPTL3 concentrations in ob/ob mice (n = 7–12 per group) (J). Transcript levels in the KLH group were set to 1. (C and J) Results are expressed as median ± IQR. (I) Results are expressed as mean ± SEM n.s., not significant;…

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  Figure 2. Improved lipid profiles in ob/ob mice following ANGPTL3 vaccination (A–F) Levels of (A) fasting triglycerides (TG), (B) non-fasting TG, (C) fasting LDL-C, (D) fasting sd-LDL-C, (E) fasting HDL-C, and (F) fasting total cholesterol (TC) in circulation of mice 6 weeks after the first immunization (n = 7–12 per group). (A–D) Results are expressed as mean ± SEM (E and F) Results are expressed as median ± IQR. ∗p < 0.05 and †p < 0.01 versus KLH (control) group.

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  Figure 3. Decreased TG accumulation in liver of ob/ob mice following ANGPTL3 vaccination (A–K) Analyses of ob/ob mice 6 weeks after the first immunization. (A) Body weight (n = 7–12 per group). (B) Liver weight/body weight (%) (n = 7–12 per group). (C) TG content in liver (n = 7–12 per group). (D) Representative images of hematoxylin and eosin (H&E)-stained liver sections. Scale bars, 500 (left) and 100 μm (right). (E) Nonalcoholic fatty liver disease activity score (NAS), as estimated based on H&E-stained sections (n = 7–12 per group). (F and G) Quantitative real-time PCR in liver tissue of mRNAs encoding factors functioning in hepatic lipogenesis (KLH: n = 9, E3: n = 11). KLH group values were set to 1. (H) Circulating free fatty acid (FFA) levels (KLH: n = 10, E3: n = 12) . (I) Serum alanine aminotransferase (ALT) levels (KLH: n = 10, E3: n = 12). (J and K) Transcript levels of (J) IL-6 and (K) TNF-α in liver tissues (KLH: n = 9, E3: n = 11). KLH group values were set to 1. (A–C, F, and H–K) Results are expressed as mean ± SEM (E and G). Results are expressed as median ± IQR. n.s., not significant; ∗p < 0.05 and †p < 0.01 versus KLH (control) group.

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  Figure 4. Evaluation of cytotoxic autoimmune responses following ANGPTL3 vaccination in ob/ob mice (A–J) Analyses of T cells and liver from ob/ob mice 6 weeks after the first immunization. (A) T cell proliferation assay of immunized mouse spleen cells performed 48 h after supplementation of culture media with vehicle, E3, KLH, or PHA (n = 5 per group). (B–E) Concentrations of (B) IFN-γ, (C) IL-2, (D) IL-4, and (E) IL-10 in culture supernatants (n = 5 per group). (F and G) Representative images of Azan-Mallory-stained liver sections from (F) KLH and (G) E3 groups. Scale bars, 200 μm. (H) Percentages of aniline blue-positive interstitial areas indicate degree of liver fibrosis. (I and J) Transcript levels of (I) Col1a1 and (J) Col3a1 in liver. Transcript levels were normalized to Rps18 levels. KLH group values were set to 1. (A, B, D, E, I, and J) Results are expressed as mean ± SEM (C) Results are expressed as median ± IQR. Veh, vehicle; n.s., not significant; ∗p < 0.05, †p < 0.01 versus control.

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  Figure 5. Improved lipid profiles in severe familial hypercholesterolemia model mice following ANGPTL3 vaccination (A–I) Blood samples from Apoeshl mice fed a high-cholesterol diet were analyzed 6 weeks after the first immunization (n = 10 per group). (A) Titers of circulating antibodies. Values are reported as the serum dilution giving half-maximal binding (optical density: OD50%). (B) Circulating ANGPTL3 concentrations. (C–I) Levels of (C) fasting TG, (D) non-fasting TG, (E) fasting VLDL-C, (F) fasting LDL-C, (G) fasting sd-LDL-C, (H) fasting HDL-C, and (I) fasting TC in circulation. (J and K) Evaluation of atherosclerotic plaques in aorta from Apoeshl mice fed a high-cholesterol diet. Analysis was performed 22 weeks after the first immunization (n = 6 per group). (J) Representative photographs of Oil red O-stained aortas of indicated mice. (K) Quantitative comparison of disease severity based on atherosclerotic lesion area. (A, C, and H) Results are expressed as median ± IQR. (B, D–G, I, and K) Results are expressed as mean ± SEM. ∗p < 0.05 and †p < 0.01 versus KLH (control) group.

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  Figure 6. ANGPTL3 vaccine durability as assessed in C57BL/6J mice (A) Circulating antibody titers of mice at 6, 20, and 30 weeks after the first E3 immunization. Values are reported as the serum dilution giving half-maximal binding (optical density: OD50%). p < 0.01 for interaction between week and E3 treatment (n = 11 per group). (B) Non-fasting TG levels in circulation of mice before immunization and at 6, 20, and 30 weeks after the first E3 immunization. p < 0.01 for interaction between week and E3 treatment (n = 11 per group). (C) Body weight of mice before immunization and at 6, 20, and 30 weeks after the first E3 immunization (n = 11 per group). p = 0.88 for interaction between week and E3 treatment. Results are expressed as mean ± SEM n.s., not significant. ∗p < 0.05 versus KLH (control) group. †p < 0.01 versus KLH (control) group.

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  Figure 7. Effects of ANGPTL3 vaccine on circulating ANGPTL8 levels (A and B) Circulating ANGPTL8 protein levels in (A) ob/ob (KLH [control]: n = 10, E3: n = 8) and (B) Apoeshl (n = 10 per group) mice at 6 weeks after the first immunization. Results are expressed as median ± IQR., n.s., not significant., ∗p < 0.05 versus KLH group.