Decreased adipokine CTRP4 in CAD patients: CTRP4 attenuates atherosclerosis via inhibition of RAGE and TLR4

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  FIGURE 1. Reduction of C1q/TNF‐related protein 4 (CTRP4) level in the serum of patients with coronary artery disease (CAD), and in adipose tissue of mice with high‐fat feeding. (A) Serum CTRP4 levels in patients with CAD (n = 323) and non‐CAD controls (n = 315) were detected using enzyme‐linked immunosorbent assay (ELISA). (B) CTRP4 protein levels in the heart, kidney, liver, lung and adipose tissue of C57BL/6 mice (n = 3) fed a chow diet were detected by Western blot. (C) Quantification of the data in (B) (n = 3). (D) CTRP4 protein levels in the heart, kidney, liver, lung and adipose tissue of C57BL/6 mice (n = 3) fed a Western diet were detected by Western blot. (E) Quantification of the data in (D) (n = 3). (F) Western blot was used to detect the levels of CTRP4 protein in the para‐epididymal adipose tissue (pEAT), mesenteric adipose tissue (MAT) and subcutaneous adipose tissue (SAT) of C57BL/6 mice fed a chow diet and those fed a Western diet (n = 3 per group). (G) Quantification of the data in (F) (n = 3). (H) Immunohistochemical staining was used to observe the levels of CTRP4 MAT of C57BL/6 mice fed a chow diet and those a Western diet. Scale bar, 50 µm. (I) Quantification of CTRP4…

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  FIGURE 2. Decreased C1q/TNF‐related protein 4 (CTRP4) levels in epicardial adipose tissue of patients with coronary artery disease (CAD). (A) Transcriptional expression of C1QTNF4 was assessed in human epicardial adipose tissue surrounding atherosclerotic coronary arteries (n = 3) and non‐atherosclerotic coronary arteries (n = 3) by bulk RNA sequencing. (B) Gene Ontology (GO) enrichment analysis of differentially expressed genes. (C) Heatmap of differentially expressed genes. (D) Western blot was used to detect the expression of CTRP4 in epicardial adipose tissue surrounding atherosclerotic coronary arteries from patients with CAD undergoing coronary artery bypass grafting (CABG) surgery (n = 9) and non‐atherosclerotic coronary arteries from non‐CAD patients receiving other cardiac surgery (n = 9). (E) Quantification of the data in (D). (F) Immunohistochemical and immunofluorescence staining of CTRP4 in epicardial adipose tissue surrounding atherosclerotic coronary arteries from patients with CAD undergoing CABG surgery (n = 9) and non‐atherosclerotic coronary arteries from non‐CAD patients receiving other cardiac surgery (n = 9). Scale bar, 50 µm. (G and H) Quantification of CTRP4 protein…

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  FIGURE 3. Deficiency of C1q/TNF‐related protein 4 (CTRP4) promotes atherogenesis in ApoE−/− mice. (A) Experimental procedure: ApoE−/− and CTRP4−/−/ApoE−/− mice (male) were fed a chow diet for 8 weeks, followed by a Western diet for 12 weeks to establish atherosclerosis models (n = 10 per group). (B) En face analysis of aortic plaque area in ApoE−/− and CTRP4−/−/ApoE−/− mice. (C) Quantification of the data in (B). (D) Oil Red O, Masson's and macrophages/monocytes (MOMA‐2) staining were performed to evaluate plaque area, collagen content, necrotic core size (scale bar, 500 µm) and macrophage infiltration (scale bar, 100 µm) in the aortic roots of ApoE−/− and CTRP4−/−/ApoE−/− mice. (E‒H) Quantification of the data in (D) (n = 10 per group). Data are presented as mean ± SD. Data in (C), (E), (F), (G) and (H) were analysed using unpaired Student's t‐test. ApoE, apolipoprotein E.

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  FIGURE 4. Supplementation of C1q/TNF‐related protein 4 (CTRP4) through perivascular adipose tissue transplantation from wide‐type mice attenuates carotid atherosclerosis in CTRP4−/−/ApoE−/− mice. (A) Experimental procedure: CTRP4−/−/ApoE−/− mice (male) were fed a chow diet for 8 weeks. Then, mesenteric adipose tissue (MAT) of wild‐type and CTRP4−/− mice (6‒8 weeks old) was transplanted onto the carotid artery of these CTRP4−/−/ApoE−/− mice, and Western diet was continued for 14 days to establish the carotid atherosclerosis model (n = 10 per group). (B) Schematic diagram of adipose tissue transplantation. (C) MAT from wild‐type and CTRP4−/− mice was transplanted onto the carotid artery of CTRP4−/−/ApoE−/− mice, and were fed a Western diet for 14 days. Representative images of carotid artery plaques were shown. Scale bar, 2 mm. (D) Quantification of the data in (C) (n = 10 per group). (E) Haematoxylin and eosin (H&E), Masson's (scale bar, 100 µm) and macrophages/monocytes (MOMA‐2) (scale bar, 50 µm) staining of carotid artery plaques in CTRP4−/−/ApoE−/− mice after transplantation of MAT from wide‐type and CTRP4−/− mice. (F‒H) Quantification of data in (E) (n = 10 per group). Data are…

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  FIGURE 5. Injection of recombinant C1q/TNF‐related protein 4 (CTRP4) inhibits atherogenesis while its mutant has attenuated anti‐atherogenic effect in CTRP4−/−/ApoE−/− mice. (A) Experimental procedure: 8‐week‐old male CTRP4−/−/ApoE−/− mice were injected through the tail vein with recombinant protein CTRP4 and CTRP4Mut (10 µg/mouse, once every other day) or saline, and the atherosclerosis models were constructed after 12 weeks of Western diet (n = 10 per group). (B) En face analysis of aortic plaque area in CTRP4−/−/ApoE−/− mice injected with saline, recombinant CTRP4 or CTRP4Mut. (C) Quantification of the data in (B) (n = 10 per group). (D) Atherosclerotic plaque area, collagen content, necrotic core diameter (scale bar, 500 µm) and macrophage infiltration (scale bar, 100 µm) were evaluated by Oil Red O, Masson's and macrophages/monocytes (MOMA‐2) staining of aortic roots in CTRP4−/−/ApoE−/− mice injected with saline or recombinant proteins CTRP4 and CTRP4Mut (n = 10). (E‒H) Quantification of the data in (D) (n = 10 per group). Data are presented as mean ± SD. Data in (C), (E), (F), (G) and (H) were analysed by using one‐way ANOVA followed by Bonferroni post hoc tests. ApoE, apolipoprotein…

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  FIGURE 6. C1q/TNF‐related protein 4 (CTRP4) inhibits inflammatory pathways in macrophages. (A) Volcano plot of differentially expressed genes in bulk RNA sequencing of mesenteric adipose tissue (MAT) of wide‐type (n = 3) and CTRP4−/− mice (n = 3). Adjusted p‐value < .05, and |log2(fold change)| > 1. (B) Gene Ontology (GO) enrichment analysis of differentially expressed genes. (C) Heatmap of differentially expressed genes in inflammatory pathways. (D) Bone marrow‐derived macrophages (BMDMs) of CTRP4−/− mice were treated with phosphate‐buffered saline (PBS) or CTRP4 recombinant protein (1 µg/mL) for 3 h, and then stimulated with lipopolysaccharide (LPS) (50 ng/mL) for 0, 10, 20 and 40 min, respectively. The phosphorylation levels of ERK, p38, JNK and p65 were detected by Western blot. (E‒H) Quantification of data in (D) (n = 3). (I) BMDMs from CTRP4−/− and wild‐type (WT) mice were treated with LPS (50 ng/mL) for 0, 3, 6, 12 and 24 h. Western blot was used to detect the levels of inflammatory factors interleukin (IL)‐1β, tumour necrosis factor‐alpha (TNF‐α) and IL‐6. (J‒L) Quantitative analysis of data in (I) (n = 3). (M) BMDMs from CTRP4−/− and WT mice were treated with LPS of increasing…

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  FIGURE 7. C1q/TNF‐related protein 4 (CTRP4) binds and interacts with receptor for advanced glycation end products (RAGE) and Toll‐like receptor 4 (TLR4). (A) Experimental procedure: HEK293 cells were transfected with His‐CTRP4 overexpression plasmids. After 36 h, the cell lysate was incubated with the extract of lipopolysaccharide (LPS)‐stimulated THP‐1 cells, along with an anti‐His antibody and magnetic beads, to enrich CTRP4. The eluted proteins, including potential CTRP4‐associated proteins, were subsequently analysed using mass spectrometry. (B) Cellular localisation of proteins interacting with CTRP4. Blue indicates membrane proteins, red indicates nuclear proteins, and orange indicates cytoplasmic proteins. (C and D) Gene Ontology (GO) and KEGG enrichment analysis of proteins interacting with CTRP4. (E) Intersection of the genes in the positive regulation of NF‐κB transcription factor activity pathway (GO enrichment) and lipids and atherosclerosis pathway (KEGG enrichment). (F and G) Characteristic peptide spectra of RAGE and TLR4 pulled down by CTRP4. (H‒K) HEK293 cells were co‐transfected with His‐CTRP4 and Flag‐RAGE or Flag‐TLR4, and co‐immunoprecipitation experiments were…

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  FIGURE 8. Perivascular adipose tissue transplantation from mice with C1q/TNF‐related protein 4 (CTRP4) mutants fails to inhibit carotid atherosclerosis in CTRP4−/−/ApoE−/− mice. (A) Experimental procedure: adeno‐associated viruses overexpressing wild‐type CTRP4 (AAV‐CTRP4) and CTRP4Mut (AAV‐CTRP4Mut) and control virus (AAV‐Vector) were injected intraperitoneally into male CTRP4−/− mice (6‒8 weeks old) for 2 weeks. Then, mesenteric adipose tissue (MAT) of these mice was transplanted onto the carotid arteries of 8‐week‐old male CTRP4−/−/ApoE−/− mice. Western diet was given for 14 days to induce a carotid atherosclerosis model (n = 10 per group). (B) Representative images of carotid atherosclerotic plaques after transplantation of MAT from CTRP4−/− mice infected with AAV‐Vector, AAV‐CTRP4 or AAV‐CTRP4Mut onto the carotid artery of CTRP4−/−/ApoE−/− mice. The recipients were fed a Western diet for 14 days. Scale bar, 2 mm. (C) Quantification of the data in (B). (D) Haematoxylin and eosin (H&E), Masson's (scale bar, 100 µm) and macrophages/monocytes (MOMA‐2) (scale bar, 50 µm) staining of carotid atherosclerotic plaques in CTRP4−/−/ApoE−/− mice receiving adipose tissue transplantation from…

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  FIGURE 9. C1q/TNF‐related protein 4 (CTRP4) exerts anti‐inflammatory effects by engaging and inhibiting receptor for advanced glycation end‐products (RAGE) and toll‐like receptor 4 (TLR4) signalling pathways. Adipokine CTRP4, secreted by adipose tissue, inhibits the expression of inflammatory cytokines by binding and inhibition of RAGE and TLR4 in macrophages. This may lead to a reduction in inflammation and atherosclerosis.