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. 2023 May 17;72(2):242-252.
doi: 10.1538/expanim.22-0103. Epub 2022 Dec 5.

Loss of AKAP12 aggravates rheumatoid arthritis-like symptoms and cardiac damage in collagen-induced arthritis mice

Yanhui Ni  1 , Jingjing Cao  2 , Jing Yuan  1 , Xiaoran Ning  2
Affiliations

Loss of AKAP12 aggravates rheumatoid arthritis-like symptoms and cardiac damage in collagen-induced arthritis mice

Yanhui Ni et al. Exp Anim. .

Abstract

A-kinase anchoring protein 12 (AKAP12) has been identified as an anti-inflammatory and anti-fibrotic regulator in chronic inflammation and cardiovascular disease. However, the potential of AKAP12 in autoimmune disorders, rheumatoid arthritis (RA) and associated cardiac complications remains elusive. Here, a murine model of collagen-induced arthritis (CIA) was successfully induced, followed by adenovirus-mediated AKAP12 short hairpin RNA (shRNA) treatment. AKAP12 silenced mice displayed elevated clinical arthritis scores and significant ankle joint swelling. AKAP12 loss in CIA mice increased inflammatory cell infiltration and cartilage erosion, increased the levels of anti-IIC IgG and inflammatory cytokines IL-1β, IL-6, tumor necrosis factor (TNF)-α in serum, and upregulated the expression of cartilage-degrading enzymes MMP-1, MMP-3, MMP-13 in synovium, but reduced IL-10. The number of M1 macrophages and the expression of the markers (CCR7, IL-6, TNF-α and iNOS) was enhanced in synovial tissues, while M2 polarized macrophages and the makers (IL-10 and arginase-1) were reduced in response to AKAP12 loss. Moreover, low expression of AKAP12 was detected in the hearts of CIA mice. Loss of AKAP12 results in increased cardiac inflammation and fibrosis. This work suggests that AKAP12 loss aggravates joint inflammation likely through the promotion of M1 macrophage polarization and exacerbates inflammation-caused cardiac fibrosis.

Keywords: A-kinase anchoring protein 12 (AKAP12); heart; inflammation; macrophage polarization; rheumatoid arthritis.

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Conflict of interest statement

The authors claim no conflict of interest to disclose.

Figures

Fig. 1.
Fig. 1.
A-kinase anchoring protein 12 (AKAP12) level is decreased in a collagen-induced arthritis (CIA) mouse model. Analysis of RA progression was based on a CIA mouse model. (A) Experimental procedure was shown. (B) The clinical scores were recorded and paw thickness was measured every 3 days beginning 23 days after the initial immunization. (C) Photographs of hind paws 32 days after modeling. (D) AKAP12 mRNA expression in synovial tissues and fluid was determined by quantitative PCR. (E) Immunohistochemical staining of AKAP12 in synovial tissues. (F) Representative band images of AKAP12 via western blot. Data are shown as mean ± SD. **P<0.01, ***P<0.001.
Fig. 2.
Fig. 2.
Loss of A-kinase anchoring protein 12 (AKAP12) worsens collagen-induced arthritis (CIA) progression. One day after booster immunization, the mice received Ad-shAKAP12 or Ad-shNC (once per week) via tail vein injection. (A) Clinical assessment and paw thickness measurement on day 23, 26, 29 and 32. (B) Pictures of hind paws on day 32 were displayed. (C) Quantitative PCR and western blot analysis of AKAP12 expression in synovial tissues. (D) Detection of serum anti-IIC antibody level by ELISA. NT, not treated. Data are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001; +P<0.05, +++P<0.001.
Fig. 3.
Fig. 3.
Histopathological analysis of tissues. (A) Pathological observation of synovial tissues (hemotoxylin and eosin (H&E) staining) and subsequent scoring. * indicates inflammatory infiltration of synovium. (B) Evaluation of cartilage damage (box) in ankle joints by Safranin O-fast green staining, and the Osteoarthritis Research Society International (OARSI) scoring. NT, not treated. Data are indicated as mean ± SD. **P<0.01; ++P<0.01.
Fig. 4.
Fig. 4.
Loss of A-kinase anchoring protein 12 (AKAP12) affects the production of inflammatory cytokines and matrix metalloproteinases (MMPs) in collagen-induced arthritis (CIA) mice. (A) Serum levels of IL-1β, IL-6, tumor necrosis factor (TNF)-α and IL-10 were detected by ELISA. (B) Western blot analysis of MMP-1, MMP-3 and MMP-13 in synovial tissues. NT, not treated. Data are shown as mean ± SD. *P<0.05, ***P<0.001; +P<0.05, ++P<0.01, +++P<0.001.
Fig. 5.
Fig. 5.
Loss of A-kinase anchoring protein 12 (AKAP12) modulates M1 macrophage polarization in collagen-induced arthritis (CIA) mice. (A) Representative images of immunofluoresence double staining of CD68 (green) and CCR7 (red), and quantification of the number of CD68+ macrophages and CCR7+ M1 macrophages. (B) Quantitative PCR was utilized for mRNA expression levels of IL-6, TNF-α and iNOS, three markers of M1 macrophages. Data are represented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001; +P<0.05, ++P<0.01, +++P<0.001.
Fig. 6.
Fig. 6.
Loss of A-kinase anchoring protein 12 (AKAP12) inhibits M2 macrophage polarization. (A) Immunofluoresence double staining of CD68 (green) and CD206 (red), followed by quantification of CD68+/CCR7+ macrophages. (B) Detection of M2 macrophage markers (IL-10 and arginase-1) using quantitative PCR. Data are represented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001; +P<0.05, ++P<0.01, +++P<0.001.
Fig. 7.
Fig. 7.
Loss of A-kinase anchoring protein 12 (AKAP12) worsens cardiac damage of collagen-induced arthritis (CIA) mice. (A) Quantitative PCR and western blot assays were performed to measure AKAP12 expression level in hearts. (B, C) Representative photographs showed hemotoxylin and eosin (H&E) and Masson’s trichrome staining of heart tissues, and assessment of heart damage and fibrosis (scoring). NT, not treated. Data are represented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001; ++P<0.01, +++P<0.001.
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