CTRP12 ameliorates atherosclerosis by promoting cholesterol efflux and inhibiting inflammatory response via the miR-155-5p/LXRα pathway

Abstract

C1q tumor necrosis factor-related protein 12 (CTRP12), a conserved paralog of adiponectin, is closely associated with cardiovascular disease. However, little is known about its role in atherogenesis. The aim of this study was to examine the influence of CTRP12 on atherosclerosis and explore the underlying mechanisms. Our results showed that lentivirus-mediated CTRP12 overexpression inhibited lipid accumulation and inflammatory response in lipid-laden macrophages. Mechanistically, CTRP12 decreased miR-155-5p levels and then increased its target gene liver X receptor α (LXRα) expression, which increased ATP binding cassette transporter A1 (ABCA1)- and ABCG1-dependent cholesterol efflux and promoted macrophage polarization to the M2 phenotype. Injection of lentiviral vector expressing CTRP12 decreased atherosclerotic lesion area, elevated plasma high-density lipoprotein cholesterol levels, promoted reverse cholesterol transport (RCT), and alleviated inflammatory response in apolipoprotein E-deficient (apoE^-/-) mice fed a Western diet. Similar to the findings of in vitro experiments, CTRP12 overexpression diminished miR-155-5p levels but increased LXRα, ABCA1, and ABCG1 expression in the aortas of apoE^-/- mice. Taken together, these results suggest that CTRP12 protects against atherosclerosis by enhancing RCT efficiency and mitigating vascular inflammation via the miR-155-5p/LXRα pathway. Stimulating CTRP12 production could be a novel approach for reducing atherosclerosis.

Fig. 1. Effects of CTRP12 on cholesterol efflux and lipid accumulation in macrophages.

A–G THP-1 macrophages were pretreated with or without 50 µg/mL ox-LDL for 48 h, and then transduced with PBS, LV-NC, or LV-CTRP12 for 72 h. A CTRP12 expression was determined by western blot. B, C Measurement of intracellular TC, FC, CE, and TG concentrations. D Representative images of Oil red O staining (×200). Scale bar = 20 μm. E, F Representative fluorescent images of NBD-cholesterol burden (×200). Cholesterol efflux mediated by apoA-I and HDL was quantified in these groups. Scale bar = 20 μm. G Representative fluorescent images of Dil-ox-LDL uptake (×200). Scale bar = 20 μm. Data are expressed as the mean ± SD from three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2. Effects of CTRP12 on the expression of cholesterol efflux and uptake markers.

A–D THP-1 macrophages were incubated with 50 µg/mL ox-LDL for 48 h, followed by treatment with PBS, LV-NC, or LV-CTRP12 for 72 h. A, B The expression of ABCA1 and ABCG1 was determined by qRT-PCR and western blot. C, D Detection of CD36 and SR-A expression using qRT-PCR and western blot. Data are expressed as the mean ± SD from three independent experiments. ****P < 0.0001; ns not significant.

Fig. 3. Involvement of LXRα in CTRP12-induced upregulation of ABCA1 and ABCG1 expression.

A THP-1 macrophages were treated with 50 µg/mL ox-LDL for 48 h, followed by transfection with PBS, LV-NC, or LV-CTRP12 for 72 h. LXRα expression was determined by qRT-PCR and western blot. B After transfection with scrambled siRNA or LXRα siRNA for 48 h, cell lysates were immunoblotted with indicated antibodies. C–E THP-1 macrophages were loaded with 50 µg/mL ox-LDL for 48 h, transfected with LXRα siRNA for another 48 h and then treated with LV-CTRP12 for 72 h. C The mRNA and protein levels of ABCA1 and ABCG1 were detected by qRT-PCR and western blot, respectively. D, E Representative fluorescent images of NBD-cholesterol burden (×200) and quantitative analyses of cholesterol efflux to apoA-I and HDL. Scale bar = 20 μm. Data are expressed as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4. Validation of LXRα as a direct target of miR-155-5p.

A Schematic of miR-155-5p binding site in the 3′ UTR of LXRα mRNA and corresponding mutation. B Calculation of free energy score by the RNAhybrid database. C The 293T cells were co-transfected with the luciferase reporter plasmids (LXRα-WT and LXRα-Mut) and miR-155-5p mimic or its negative control for 48 h. The luciferase activity was then determined. D, E After 48 h of treatment with ox-LDL at 50 µg/mL, THP-1 macrophages were transfected with miR-155-5p mimic/inhibitor or their negative controls for 48 h. D Analysis of miR-155-5p expression by qRT-PCR. E Detection of LXRα expression by both qRT-PCR and western blot. Data are represented as the mean ± SD from three independent experiments. ****P < 0.0001; ns not significant.

Fig. 5. CTRP12-induced upregulation of LXRα, ABCA1, and ABCG1 is mediated by miR-155-5p.

A THP-1 macrophages were treated with 50 µg/mL ox-LDL for 48 h and then transfected with PBS, LV-NC, or LV-CTRP12 for 72 h, followed by detection of miR-155-5p expression using qRT-PCR. B–D THP-1 macrophages were loaded with 50 µg/mL ox-LDL for 48 h, transfected with miR-155-5p mimic for another 48 h and then transduced with LV-CTRP12 for 72 h. The mRNA and protein levels of LXRα, ABCA1, and ABCG1 were detected by qRT-PCR and western blot, respectively. Data are represented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 6. Effects of CTRP12 on macrophage polarization and inflammatory cytokine expression.

A, B THP-1 macrophages were incubated with 50 µg/mL ox-LDL for 48 h, which was followed by transfection with PBS, LV-NC, or LV-CTRP12 for 72 h. The mRNA expression of iNOS, CD86, Mrc-1, Arg-1, MCP-1, TNF-α, and IL-10 was assayed using qRT-PCR. C–F THP-1 macrophages were loaded with 50 µg/mL ox-LDL for 48 h, transfected with LXRα siRNA or miR-155-5p mimic for another 48 h and then transduced with LV-CTRP12 for 72 h. The qRT-PCR was employed to measure the mRNA expression of iNOS, CD86, Mrc-1, Arg-1, MCP-1, TNF-α, and IL-10. Data are the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7. CTRP12 inhibits atherosclerosis in apoE^−/− mice.

A–C Western diet-fed apoE^−/− mice were injected via the tail vein with PBS, LV-NC, or LV-CTRP12 (n = 15 in each group). A Analysis of CTRP12 expression in the aortas by western blot (n = 10). B The plaques (green arrows) in the aortic arch under a stereoscopic microscope (n = 10); C Cryosections of the aortic root were stained with HE, Oil Red O, or Masson, followed by quantification of lesion area, lipid accumulation and collagen contents using Image-Pro Plus 7.0 software (n = 10). Scale bar = 100 μm. Data are represented as the mean ± SD. ****P < 0.0001; ns not significant.

Fig. 8. Effects of CTRP12 on plasma HDL-C levels and RCT in apoE^−/− mice.

A Plasma levels of TC, TG, HDL-C, and LDL-C were determined using the commercial kits (n = 10). B Mice were injected intraperitoneally with [³H]-cholesterol-labeled J774 macrophages. The radioactivity in the plasma, liver, and feces were assessed by a liquid scintillation counter (n = 5). C, D Detection of miR-155-5p, LXRα, ABCA1, and ABCG1 expression in the aortas by qRT-PCR and western blot (n = 10). Data are represented as the mean ± SD. *P < 0.05, ****P < 0.0001; ns not significant.