Angiotensin‐converting enzyme 2 deficiency accelerates and angiotensin 1‐7 restores age‐related muscle weakness in mice

Abstract Background A pharmacologic strategy for age‐related muscle weakness is desired to improve mortality and disability in the elderly. Angiotensin‐converting enzyme 2 (ACE2) cleaves angiotensin II into angiotensin 1‐7, a peptide known to protect against acute and chronic skeletal muscle injury in rodents. Since physiological aging induces muscle weakness via mechanisms distinct from other muscle disorders, the role of ACE2‐angiotensin 1‐7 in age‐related muscle weakness remains undetermined. Here, we investigated whether deletion of ACE2 alters the development of muscle weakness by aging and whether angiotensin 1‐7 reverses muscle weakness in older mice. Methods After periodic measurement of grip strength and running distance in male ACE2KO and wild‐type mice until 24 months of age, we infused angiotensin 1‐7 or vehicle for 4 weeks, and measured grip strength, and excised tissues. Tissues were also excised from younger (3‐month‐old) and middle‐aged (15‐month‐old) mice. Microarray analysis of RNA was performed using tibialis anterior (TA) muscles from middle‐aged mice, and some genes were further tested using RT‐PCR. Results Grip strength of ACE2KO mice was reduced at 6 months and was persistently lower than that of wild‐type mice (p < 0.01 at 6, 12, 18, and 24‐month‐old). Running distance of ACE2KO mice was shorter than that of wild‐type mice only at 24 months of age [371 ± 26 vs. 479 ± 24 (m), p < 0.01]. Angiotensin 1‐7 improved grip strength in both types of older mice, with larger effects observed in ACE2KO mice (% increase, 3.8 ± 1.5 and 13.3 ± 3.1 in wild type and ACE2KO mice, respectively). Older, but not middle‐aged ACE2KO mice had higher oxygen consumption assessed by a metabolic cage than age‐matched wild‐type mice. Angiotensin 1‐7 infusion modestly increased oxygen consumption in older mice. There was no difference in a wheel‐running activity or glucose tolerance between ACE2KO and wild‐type mice and between mice with vehicle and angiotensin 1‐7 infusion. Analysis of TA muscles revealed that p16INK4a, a senescence‐associated gene, and central nuclei of myofibers increased in middle‐aged, but not younger ACE2KO mice. p16INK4a and central nuclei increased in TA muscles of older wild‐type mice, but the differences between ACE2KO and wild‐type mice remained significant (p < 0.01). Angiotensin 1‐7 did not alter the expression of p16INK4a or central nuclei in TA muscles of both types of mice. Muscle ACE2 expression of wild‐type mice was the lowest at middle age (2.6 times lower than younger age, p < 0.05). Conclusions Deletion of ACE2 induced the early manifestation of muscle weakness with signatures of muscle senescence. Angiotensin 1‐7 improved muscle function in older mice, supporting future application of the peptide or its analogues in the treatment of muscle weakness in the elderly population

Development of vaccine for dyslipidemia targeted to a proprotein convertase subtilisin/kexin type 9 (PCSK9) epitope in mice

<div><p>Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates expression of low-density lipoprotein (LDL) receptors via receptor internalization and subsequent lysosomal degradation. Thus, an anti-PCSK9 antibody is well known as an anti-hyperlipidemia drug. Here, we aimed to develop vaccine for a long-term treatment of dyslipidemia targeted to PCSK9. In This study, we designed a peptide vaccine for mouse PCSK-9, which consisted of short peptides conjugated to keyhole limpet hemocyanin (KLH) as a carrier protein. Vaccines were administered to male <i>apolipoprotein E (ApoE) deficient mice</i> with adjuvants and significantly elicited an antibody response against PCSK9. The PCSK9 vaccines were administered to mice three times in 2-week intervals, and antibody titers and lipoprotein levels were evaluated up to 24 weeks after the first immunization to determine the therapeutic effect. Anti-PCSK9 antibody titers reached peak levels 6 weeks after the first immunization, and theses titers were maintained for up to 24 weeks. Decreased plasma levels of total cholesterol, very low-density lipoprotein (VLDL), and chylomicron (CM) were maintained for up to 24 weeks. Immunized mice exhibited a significant increase in cell-surface LDL receptor expression. Stimulation with KLH, but not PCSK9, induced the production of INF-gamma and interleukin-4 (IL-4), as determined with ELISPOT assays, thus indicating that PCSK9 vaccine did not elicit T-cell activation in our vaccine system. The present anti-PCSK9 vaccine induced long-lasting anti-PCSK9 antibody production and improved lipoprotein profiles. Thus, anti-PCSK9 vaccine could become a new option for the treatment of dyslipidemia as a long-acting therapy in future.</p></div

Screening of PCSK9 peptide vaccines in male <i>ApoE-deficient mice</i>.

<p>Two candidate vaccines (V1 and V2 vaccines) or control (KLH) was injected (5 μg peptide per mouse) (N = 4 per group). (A) The antibody titers against candidate PCSK9 peptides conjugated with bovine serum albumin were evaluated pre-immunization (pre) and post-immunization (4 or 8 weeks), and the results are expressed as half-maximal binding (optical density: OD50%). Significance values were obtained with a 2-factor repeated-measure ANOVA with subsequent Tukey’s multiple comparisons tests. (B) Mouse plasma PCSK9 levels were measured at pre-immunization (pre) and post-immunization (4 weeks) time points. Significance values were obtained using two-way ANOVA with subsequent Tukey’s multiple comparisons test. (C and D) Mean values of TC and TG levels (mg/dL) were measured post-immunization (4 weeks). Significance values relative to KLH (*P<0.05) were obtained with one-way ANOVA with subsequent Tukey’s multiple comparisons tests. All data in this Figure are expressed as the means ± SEM. *P<0.05, **P<0.01, and ****P<0.0001.</p

Evaluation of the PCSK9 vaccine (V2 vaccine) in male <i>ApoE-deficient mice</i> (N = 5–7).

<p>PCSK9 vaccine (V2 vaccine) or control (Saline) was injected at different doses (Low; 5 μg and High; 50 μg peptides per mouse) three times in biweekly intervals (0, 2, and 4 weeks). (A) Anti-PCSK9 antibody titers (OD50%) were evaluated at pre-immunization (pre) and post-immunization time points (2, 4, 6, 8, 12, 16, 20, and 24 weeks). Data are presented as the average of each groups; error bars indicate the SEM. **P<0.01 and ****P<0.0001 show significant changes between low dose group and saline group. ††††P<0.0001 shows significant changes between high dose group and saline group. (B) PCSK9 levels in plasma samples from pre-immunized (pre) and post-immunized (6 weeks) mice. Bars represent mean levels of detected mouse PCSK9 levels in plasma samples, and error bars represent ± SEM. Significance values relative to saline group (*P<0.05, ***P<0.001) were obtained using two-way ANOVA with subsequent Tukey's multiple comparisons tests. (C) Cell-surface LDLR levels in liver hepatocytes were measured in immunized mice at 6 weeks post-immunization via ELISA. The results are presented as a fold-increase relative to saline-treated groups. Significance values relative to saline (****P<0.0001) were obtained with one-way ANOVA with subsequent Tukey’s tests for multiple comparisons. All data in this Figure are expressed as the means ± SEM.</p

Evaluation of long-term efficacy of PCSK9 vaccine (N = 5–7).

<p>PCSK9 vaccine (V2 vaccine) was injected at a low dose (5 μg) or high dose (50 μg) peptide per mouse three times in biweekly intervals (0, 2, and 4 weeks), and mice were followed up until 24 weeks. Plasma TC levels (A), detailed cholesterol lipoprotein profile (B), and plasma TG levels (C) were measured at the particular time points (pre-immunization, 6 and 24 weeks after first immunization). Significance values were obtained with a 2-factor repeated-measure ANOVA with subsequent Tukey’s multiple comparisons tests. All data in this Figure are expressed as the means ± SEM. **P<0.01 ***P<0.001, and ****P<0.0001.</p