Introduction

Atherosclerosis is regarded as a pathological factor for various cardiovascular diseases such as coronary heart disease, stroke, and peripheral vascular disorder. The formation of foam cells is a hallmark of early atherosclerotic plaques (Yu et al. 2013). Multiple modulators are involved in the complicated process of atherosclerotic lesion formation. ATP-binding cassette (ABC) transporters, especially ABCA1 and ABCG1, have been shown to play important roles in the regulation of cholesterol homeostasis (Dean et al. 2001; Tarling and Edwards 2011). They mediate the efflux of cholesterol and phospholipids to lipid-poor apolipoproteins, a key event of reverse cholesterol transport (RCT) (Ouimet et al. 2019). Liver-X-receptor (LXR) is cholesterol-sensing nuclear receptor. The LXR pathway regulates lipid metabolism and transport (Wang and Tontonoz 2018). LXR consists of two subunits, LXR-α and LXR-β, which are encoded by two independent genes. Activation of LXR-α can upregulate the expression of ABCA1, ABCG1, and SR-BI, thus promoting macrophage cholesterol efflux (Bonamassa and Moschetta 2013). These transporters and receptors involved in RCT are potential therapeutic targets for atherosclerosis.

It has been reported that adipose tissue can release multiple bioactive adipokines, such as leptin, omentin, adiponectin, and C1q/tumor necrosis factor-related proteins (CTRPs) (Shibata et al. 2014; Fang and Judd 2018; Maruyama et al. 2012; Yu et al. 2018). These adipokines regulate glucose and lipid metabolism and elicit anti-inflammatory and anti-atherosclerotic effects. Recently, compelling studies have indicated that CTRP9, a newly identified paralog of adiponectin (Wong et al. 2009), plays a protective role in atherosclerosis. In patients with coronary atherosclerosis disease (CAD), serum CTRP9 level is significantly decreased (Sun et al. 2013). Similar results are observed in a mouse model of acute myocardial infarction (Wang et al. 2015). CTRP9 exerts its atheroprotective effects through inhibition of vascular inflammation (Jung et al. 2016), alteration of lipid metabolism (Yang et al. 2016), amelioration of endothelial dysfunction (Zheng et al. 2011; Sun et al. 2017), and regulation of vascular smooth muscle cell (VSMC) phenotype (Liu et al. 2017). However, the role of CTRP9 in RCT remains unclear.

Overexpression of mouse CTRP9 gene via a lentiviral expression system can prevent the development of atherosclerotic lesions in ApoE−/− mice fed with HFD (Huang et al. 2019). In the present study, we investigated the robust effect of subcutaneous injection of CTRP9 protein on atherosclerotic lesion formation and cholesterol efflux. Additionally, we investigated the mechanisms responsible for CTRP9-mediated atheroprotection.

Materials and Methods

Animal Models

Our study was conformed with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Shanxi Medical University (Taiyuan, China).

Eight-week-old male ApoE−/− mice were purchased from Laboratory Animal Center of Peking University (Beijing, China). All mice were maintained on a high-fat diet (21% [wt/wt] fat, 0.21% cholesterol) during the whole experimental period. Mice were randomized into control and CTRP9 groups. Ten weeks after feeding the high-fat diet, mice received vehicle or human CTRP9 (0.25 μg/g/day; Aviscera Bioscience, Santa Clara, CA, USA; catalog number: 00081-04-100) for additional 4 weeks via mini-osmotic minipumps (DURECT Corporation, Cupertino, CA, USA), which were inserted subcutaneously into the back of the mice.

In Vivo Macrophage RCT Assay

In vivo RCT assay was performed as previously described (Liang et al. 2017; Zhang et al. 2003). Ox-LDL (catalog number: 20605ES05; Yeasen Biotechnology, Shanghai, China) at a final concentration of 50 µg/ml was radiolabeled with [3H] cholesterol (5 µCi/ml). The mixture was bathed in water at 37 °C for 30 min, added into RAW 264.7 cell cultures and cultured for another 48 h. Cells were resuspended after centrifugation, and the count was adjusted to 10.0 × 109 cells/L. Radiolabeled cells (500 μl) with the radioactive activity of 6.2 × 106 cpm/ml were intraperitoneally injected into each mouse. Forty-eight hours later, the mice were anesthetized using 1% pentobarbital sodium at a dose of 50 mg/kg. Blood samples were obtained from retro-orbital plexus. After centrifuging for 5 min at 1200 r/min, 20 μL of plasma was extracted and used for liquid scintillation counting. Feces were homogenized in 50% ethyl alcohol overnight and subjected to liquid scintillation counting. The mice were sacrificed, and liver samples were collected for liquid scintillation counting.

Plasma Lipid Profile Analysis

Blood samples of mice were collected using tubes containing EDTA (2.0 mg/ml). After centrifuging at 2000×g for 15 min at 4 °C, plasma was separated. Triglyceride (TG; catalog number: BC0625), total cholesterol (TC; catalog number: BC1985), LDL-C (catalog number: BC5335), and HDL-C (catalog number: BC5325) were determined by enzymatic kits (Solarbio, Beijing, China) according to the manufacturer’s instructions.

Evaluation of Aortic Lesions

Aortic roots were dissected and fixed as previously described (Liang et al. 2017). Hearts were cut directly under and parallel to the leaflet, and the upper portions were embedded in OCT medium and kept at − 24 °C. The aortic sinus was cut into sections of 8 mm in thickness. Twenty sections were stained with Oil-red O (catalog number: O8010; Solarbio) for lipids. Lesion areas were determined by ImageJ software (National Institutes of Health, America). Data were recorded as lesion size ± SEM.

Cell Culture and Treatments

RAW 264.7 cells (Cell Bank of Chinese Academy of Sciences, Shanghai, China) were plated on 6-well plates in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 2% penicillin–streptomycin and cultured at 37 °C (5% CO2). Upon reaching 85% confluence, cells were transformed into foam cells by incubation with 50 μg/mL ox-LDL for 24 h. Foam cells were then treated with 0.3, 1, and 3 μg/ml CTRP9 for 3 h.

Small Interfering RNA (siRNA) Transfection

siRNA duplex oligonucleotides were designed for human cDNAs encoding AMP kinase-α1 (AMPK-α1) (GenePharma, Shanghai, China). The siRNA sequences were 5′-UGCCUACCAUCUCAUAAUAdTdT-3′ (sense) and 5′-UAUUAUGAGAUGGUAGGCAdT dT-3′ (antisense). After reaching 80% confluence, RAW 264.7-derived foam cells were transfected with AMPK-siRNA or negative control siRNAs at a final concentration of 50 nM using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA; catalog number: 13778150).

RNA Isolation and Real-Time Quantitative PCR

Animals were anesthetized with pentobarbital and livers were collected. Total RNA from tissues and cells was extracted using Trizol reagent according to the manufacturer’s protocol. The targeted genes and primer sequences are as follows: ABCA1 forward: 5′-AACAGTT TGTGGCCCTTTTG-3′, reverse: 5′-AGTTCCAGGCTGGGGTACTT-3′, ABCG1 forward: 5′-GTGACGCTGACTATAAGAGA-3′, reverse: 5′-AGGTGATTCGCAGATGTG-3′, GAPDH forward: 5′-CGCTCTCTGCTCCTCCTGTT-3′, reverse: 5′-CCATGGTGTCTGAGCGATGT-3′, LXR-α forward: 5′-CTACAACCACGAGACAGAA-3′, reverse: 5′-GGCGATAAGCAAGGCATA-3′, AMPK forward: 5′-UGCCUACCAUCUCAUAAUAdTdT-3′, reverse: 5′-UAUUAUGAGAUGGUAGGCAdTdT-3′. Real-time quantitative PCR using SYBR green was performed in an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Samples were subjected to 95 °C denaturation for 2 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Relative fold changes were determined using the formula 2−ΔΔCt.

Western Blotting Analysis

Protein was extracted from liver tissues and RAW 264.7-derived foam cells using lysis buffer containing protease inhibitors (catalog number: P8340; Sigma-Aldrich, St. Louis, MO, USA). Protein samples (30 μg) were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes. Nonspecific proteins were blocked by 5% skim milk in Tris buffered saline containing 0.1% Tween 20 for 1 h at room temperature. Membranes were incubated overnight with primary antibodies to ABCA1 (1:300; catalog number: ab307536), ABCG1 (1:500; catalog number: ab52617), AMPK (1:1000; catalog number: ab32047), LXRα (1:1000; catalog number: ab41902), or GAPDH (1:2000; catalog number: ab8245) (Abcam, Cambridge, MA, USA), and then with secondary antibodies (catalog number: ab270144 or catalog number: ab205719, Abcam) for 1 h. Images were captured and quantified using a Bio-Rad ChemiDoc XRS system. Immunoreactive protein bands were detected with ECL test. The relative protein levels were expressed as the ratio of protein to GAPDH densitometric values.

Cholesterol Efflux Assay

Cellular cholesterol efflux was measured as previously described (Liang et al. 2017). Macrophages were cultured and labeled with 0.2 μCi/mL [3H] cholesterol for 72 h. After washing, equilibrated [3H] cholesterol-labeled cells were incubated in efflux medium containing RPMI 1640 medium and 0.1% bovine serum albumin (BSA) with human plasma ApoA-I (20 μg/mL) for 6 h. Monolayers were lysed, and cellular lipids were extracted with isopropanol. Concentration of [3H] cholesterol in medium and cells was measured by liquid scintillation counting. The following formula was used to determine the cholesterol efflux: Cholesterol efflux (%) = [total media counts / (total cellular counts + total media counts)] × 100.

High-Performance Liquid Chromatography (HPLC)

HPLC analysis was conducted as previously described (Liang et al. 2017). Cells were collected and lysed fully by sonication. A 0.1-mL aliquot of cell solution (containing 5–20 μg protein) was used to measure the free cholesterol (FC), and another aliquot was used to detect total cholesterol (TC). A 15% KOH ethanol solution (50 μL) was prepared and heated for 2 h to allow for TC extraction. 300 μL ethanol solution was prepared for FC extraction. Then 500 μL N-hexane:isopropanol (4:1) was added and stirred for 1 min at room temperature. After centrifugation at 3600×g for 5 min, supernatant was collected and frozen in liquid nitrogen. 20 μL of dried samples was reconstituted into mobile phase (isopropanol:acetonitrile 50:50) and injected into the HPLC system (Waters Corporation, Milford, MA, USA). Analysis was performed using a C8 reversed-phase ODS2 HPLC column. Data were analyzed using Empower software (Waters Corporation).

Statistical Analysis

All data are presented as means ± standard deviation. Statistical comparisons were done by Student’s t test or one-way analysis of variance (ANOVA) with a post hoc multiple-comparison test. All statistical analyses were conducted using SPSS 22.0 software (SPSS, Chicago, IL, USA). P value less than 0.05 was considered as statistically significant.

Results

CTRP9 Decreases Lesion Area of Aortic Sinus Sections in ApoE−/− Mice Fed with HFD

We first investigated the effect of CTRP9 on the formation of atherosclerotic plaques in ApoE−/− mice fed with a high-fat diet. After 4-week administration of CTRP9, quantification of lipid accumulation in aortic sinus sections by Oil Red O staining revealed that CTRP9 significantly decreased lesion areas by 10.43% in ApoE−/− mice, compared with control group (Fig. 1). These data suggest that CTRP9 can attenuate the development of atherosclerosis.

Fig. 1
figure 1

CTRP9 decreases aortic sinus lesion area size in ApoE−/− mice fed with HFD. Representative staining of aortic sinus with Oil Red O in ApoE−/− mice fed with HFD for 10 weeks. Values were expressed as mean ± standard error of the mean (n = 10 per group). *P < 0.05 vs. control group

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CTRP9 Increases Cholesterol Efflux and Affects Plasma Lipid In Vivo

To further assess the effects of CTRP9 on RCT in vivo, [3H] cholesterol radiolabeled RAW 264.7 macrophages were intraperitoneally injected to ApoE−/− mice. Radioactivity of [3H] cholesterol was determined by liquid scintillation counting. The results showed that the radioactivities of plasma, liver, and feces samples were increased after the treatment with CTRP9 (Fig. 2).

Fig. 2
figure 2

CTRP9 increases reverse cholesterol transport in vivo. 3H-cholesterol radiolabeled ox-LDL-loaded macrophages were injected to each group. Blood, liver, and feces were collected after 48 h for the analysis of RCT. Data were expressed as the ratio of 3H-cholesterol tracer to total cpm tracer injected. A Percentage of plasma 3H-cholesterol tracer. B Percentage of hepatic 3H-cholesterol tracer. C Percentage of fecal 3H-cholesterol tracer. Values were expressed as mean ± standard deviation (n = 6 per group). *P < 0.05 vs. control group

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Next, we determined plasma lipid, including TG, TC, LDL-C, and HDL-C levels in each group. Our results indicated that CTRP9 upregulated plasma HDL-C levels and downregulated plasma TC and LDL-C levels, compared with control (Table 1). No difference in the TG levels between the CTRP9 and control groups was observed.

Table 1 CTRP9 regulates plasma lipid profile in ApoE −/− mice fed with HFD
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CTRP9 Upregulates ABCA1, ABCG1, and LXR-α Expression in ApoE−/− Mice Fed with HFD

In the progression of atherosclerosis, ABCA1 and ABCG1 play critical roles in mediating cholesterol efflux from macrophages to HDL. ABCA1 can assemble effluxed cellular cholesterol with apolipoprotein A-I (ApoA-I) to form nascent HDL. To further evaluate the impact of CTRP9 on the regulation of cholesterol efflux, we measured the mRNA expression of ABCA1 and ABCG1 in livers of ApoE−/− mice fed with HFD. As shown in Fig. 3A and B CTRP9 significantly increased the mRNA levels of ABCA1 and ABCG1 relative to the control group. Consistent results were observed when we analyzed the protein levels of ABCA1 and ABCG1 (Fig. 3D–F).

Fig. 3
figure 3

CTRP9 upregulates ABCA1, ABCG1, and LXR-α expression in livers of Apo E−/− mice fed with HFD. A, B, C Expression of ABCA1, ABCG1, and LXR-α mRNA was quantified by real-time quantitative PCR. D, E, F, G Protein levels were measured by Western blotting. Data were shown as the mean ± standard deviation from three independent experiments. *P < 0.05 vs. control group

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LXR-α is capable of stimulating the transcription of ABCA1 and ABCG1 (Costet et al. 2000). As demonstrated above, CTRP9 regulated the expression of ABCA1 and ABCG1 in RAW 264.7 macrophages. To further confirm whether CTRP9 has an impact on the expression of LXR-α, we performed real-time quantitative PCR and Western blot analyses to measure the mRNA (Fig. 3C) and protein level (Fig. 3D and G) of LXR-α. The results showed that administration of CTRP9 significantly induced the expression of LXR-α mRNA and protein in vivo.

CTRP9 Decreases Cholesterol Content and Increases Cholesterol Efflux to ApoA-I in RAW 264.7 Macrophage-Derived Foam Cells

To further elucidate the mechanism of CTRP9 on cholesterol regulation, we treated foam cells derived from RAW 264.7 macrophages with different concentration of CTRP9. The results showed that CTRP9 can reduce cellular TC (total cholesterol), FC (free cholesterol), and CE (cholesterol ester) in a dose-dependent way (Table 2). Next, we measured the cholesterol efflux to ApoA-I in each group by liquid scintillation counting. As shown in Fig. 4A, administration of CTRP9 increased cholesterol efflux to ApoA-I in a dose-dependent manner.

Table 2 CTRP9 regulates cholesterol content in RAW 264.7 macrophage-derived foam cells
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Fig. 4
figure 4

CTRP9 upregulates cholesterol efflux and expression of ABCA1, ABCG1, and LXR-α in RAW 264.7 macrophage-derived foam cells. A ApoA-I-mediated cholesterol efflux in RAW 264.7 macrophage-derived foam cells was analyzed by liquid scintillation counting assays. B, C, D Expression of ABCA1, ABCG1, and LXR-α mRNA was quantified by real-time quantitative PCR. E, F, G, H Protein levels were measured by Western blotting. Data were shown as mean ± standard deviation from three independent experiments. *P < 0.05 vs. control group

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CTRP9 Upregulates ABCA1, ABCG1, and LXR-α Expression in RAW 264.7 Macrophages-Derived Foam Cells

Given that cholesterol efflux was increased by CTRP9, we further investigated the expression of ABCA1 and ABCG1 by real-time quantitative PCR and Western blot analyses. Consistent with what we observed in vivo, CTPR9 treatment increased the expression of ABCA1, ABCG1, and LXR-α at the mRNA (Fig. 4B and C) and protein (Fig. 4E–G) levels in RAW 264.7macrophages-derived foam cells. Such increase was in a dose-dependent manner.

CTRP9 Upregulates AMPK Expression in RAW 264.7 Foam Cells

AMPK regulates cellular energy metabolism and acts downstream of CTRP9 signaling (Sun et al. 2017; Kambara et al. 2015). We next assessed the effect of CTRP9 on the expression and activation of AMPK. The results showed that CTRP9 stimulated the mRNA expression of AMPK in RAW 264.7 foam cells (Fig. 5A). Moreover, the phosphorylation of AMPK protein was enhanced upon CTRP9 treatment (Fig. 5B and C).

Fig. 5
figure 5

CTRP9 upregulates AMPK phosphorylation in RAW 264.7 macrophage-derived foam cells. A Phosphorylation of AMPK mRNA was quantified by real-time quantitative PCR. B, C Protein levels were measured by Western blot. Data were shown as the mean ± standard deviation from three independent experiments. *P < 0.05 vs. control group

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AMPK is Involved in the CTRP9-Mediated Regulation of ABCA1 and ABCG1 and Cholesterol Efflux

To test whether CTRP9 increases ABCA1 and ABCG1 expression by activating the APMK pathway, we knocked down AMPK using siRNAs (> 85%, Fig. 6A and B). When treated with AMPK-siRNAs, the upregulation of ABCA, ABCG1, and LXR-α by 3 μg/mL of CTRP9 was completely suppressed (Fig. 6C–F). Moreover, the cholesterol efflux to ApoA-I was suppressed when AMPK was depleted (Fig. 6G). Intracellular cholesterol content was increased in AMPK-depleted cells (Table 3). Taken together, these results suggested that the effect of CTRP9 on cholesterol efflux is dependent on the AMPK signaling pathway.

Fig. 6
figure 6

AMPK activation is involved in CTRP9 upregulation of ABCA1, ABCG1, LXR-α, and cholesterol efflux. A, B Knockdown of AMPK by siRNA was confirmed using Western blot analysis (> 80% knockdown). C, D, E, F AMPK knockdown blocked CTRP9-mediated upregulation of ABCA1, ABCG1, and LXR-α expression. G Cellular cholesterol efflux from RAW 264.7 macrophages was analyzed using liquid scintillation counting assays. Data were shown as mean ± standard deviation from three independent experiments. *P < 0.05 vs. control group, ***P < 0.001 vs. control group, #P < 0.05 vs. CTRP9 group

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Table 3 CTRP9 regulates cholesterol efflux in an AMPK independent manner
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Discussion

In this work, we suggest a novel delivery strategy and show that subcutaneous administration of CTRP9 yields atheroprotective effects in the ApoE−/− mouse model. CTRP9 attenuates atherosclerotic lesions and promotes cholesterol efflux in ApoE−/− mice. Previous studies have indicated the anti-inflammatory activity of CTRP9 in RAW 264.7 macrophages (Zhang et al. 2019). Our data provide direct evidence for the regulation of cholesterol efflux by CTRP9 in RAW 264.7 cells. Moreover, activation of AMPK signaling is responsible for CTRP9-dependent regulation of cholesterol efflux. Our results collectively point toward the potential of exogenous CTRP9 in preventing atherosclerosis.

As the closest paralog of adiponectin in CTRP family, CTRP9 is associated with atherosclerosis. In CAD patients, serum CTRP9 levels are negatively correlated with TNF-α and IL-6 levels, while positively correlated with high-density lipoprotein (HDL-C) levels (Wang et al. 2015). Decreased CTRP9 levels are an independent risk factor for thin fibrous cap atherosclerosis in patients with coronary heart disease (Liu et al. 2022). These studies suggest the contribution of CTRP9 to the occurrence of atherosclerosis. ApoE plays a crucial role in the removal of cholesterol-rich lipoproteins from the plasma due to its high affinity binding to low-density lipoprotein (LDL) receptor on the surface of hepatocytes. Its loss leads to hypercholesterolemia and atherosclerosis in mice fed with the HFD (Alagarsamy et al. 2022). The ApoE−/− mouse model has been commonly used to recapitulate the disrupted lipid metabolism and atherosclerotic development in humans. Hence, we utilized the ApoE−/− mouse model to evaluate the effect of exogenous CTRP9 on atherosclerosis. Lentiviral delivery of mouse CTRP9 can prevent the development of atherosclerotic lesions in ApoE−/− mice fed with HFD (Huang et al. 2019). Here, we delivered CTRP9 protein through a different route and demonstrated that subcutaneous administration of human CTRP9 attenuates HFD-induced atherosclerotic plaque formation in ApoE−/− mice.

Multiple studies have demonstrated that CTRP9 is antiatherogenic through various mechanisms. It has been reported that CTRP9 inhibits the uptake of cholesterol by VSMCs and protects VSMCs from cholesterol-induced damage (Liu et al. 2017). CTRP9 overexpression in ApoE knockout mice exhibit a significant reduction of lipid contents in carotid plaques (Huang et al. 2019). These studies indicate a protective role for CTRP9 in regulating lipid metabolism. Our in vivo study reveals that CTRP9 administration influences plasma lipid levels, especially cholesterol contents. The administration of CTRP9 significantly increases the serum level of HDL-C and decreases the serum levels of TC and LDL-C. Moreover, we show that CTRP9 significantly augments cholesterol efflux to serum, livers, and feces. CTRP9-mediated enhancement of cholesterol efflux is also observed in foam cells and VSMCs (Liu et al. 2017; Lei et al. 2021). These results suggest that exogenous CTRP9 improves lipid metabolism and thus ameliorates HFD-induced atherosclerotic plaque formation in ApoE−/− mice.

ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play crucial and complementary roles in mediating cholesterol efflux (Phillips 2014). While ABCA1 transfers cholesterol to lipid-free apolipoproteins, such as ApoA-I and ApoE, for the neogenesis of HDL in the early steps of cholesterol efflux from peripheral cells (Hong and Tontonoz 2014), ABCG1 transfers cholesterol from macrophage to large HDL particles (Westerterp et al. 2014). In atherosclerosis, the expression of ABCA1 and ABCG1 is decreased, which aggravates the accumulation of cholesterol and formation of foam cells. So, these two ABC transporters are key targets to regulate RCT in the progression of atherosclerosis. In our in vivo studies, we demonstrated that CTRP9 can upregulate ABCA1 and ABCG1 expression both in mRNA and in protein levels compared with control group in ApoE−/− mice fed with HFD. These findings suggested that CTRP9 may exert its atheroprotective properties by upregulating cholesterol efflux and decreasing foam cell formation, which is the key step in RCT. Consistent results were also observed in RAW 264.7-derived foam cells in our vitro study. LXRs, key sterol-sensitive transcription factors, are essential for regulating cholesterol homeostasis and lipogenesis. The activation of LXR inhibits the absorption of extracellular cholesterol, while it promotes the efflux of cholesterol from peripheral tissues (Phillips 2014). ABCA1 and ABCG1 expressions are transcriptionally regulated by multiple processes, including PKCα pathway (Liu et al. 2013), PKC-η/phospholipase D2 (PLD2) signaling pathway (Park et al. 2013), and activation of LXR in macrophages (Costet et al. 2000; Kennedy et al. 2005). We have shown in our study that CTRP9 upregulated LXR-α, ABCA1, and ABCG1expression both in vivo and in vitro. This suggests the regulation of CTRP9 on ABCA1 and ABCG1 in RCT is mediated via LXRα pathway.

AMPK is a central regulator of cellular energy homeostasis. Previous studies have shown that in AMI model or diet-induced obesity mice, overexpression of CTRP9 enhances AMPK activation, while the knockout of CTRP9 reduced AMPK activation (Kambara et al. 2015; Peterson et al. 2013; Wei et al. 2014). Administration of globular CTRP9 significantly reduces TNF-α and MCP-1 production via the AdipoR1/AMPK/NF-kB signaling pathway in RAW 264.7 macrophages (Kambara et al. 2015). The anti-contractile effect of CTRP9 in perivascular adipose tissues is mediated by activating the AMPK-eNOS pathway (Han et al. 2018). All these studies suggest that AMPK is a major modulator in CTRP9 signaling. Consistent with this, we demonstrate that CTRP9 upregulates AMPK mRNA and protein levels in RAW 264.7-derived foam cells. When AMPK is knocked down, the upregulation of ABCA1, ABCG1, and LXR-α by CTRP9 is blocked. Also, the promotion of cholesterol efflux by CTRP9 is also decreased. These results suggest an AMPK-dependent mechanism involved in the upregulation of cholesterol efflux by CTRP9. Taken together, our in vivo and in vitro studies indicate that CTRP9 may regulate reverse cholesterol transport via AMPK-LXR-α-ABCA1/ABCG1 pathway.

Although our results confirm the protective role of human CTRP9 in atherosclerosis development, some limitations of this study should be noted. First, the main target cells for exogenous CTRP9 are not characterized. Many cell types including macrophages, endothelial cells, and VSMCs can be modulated by CTRP9. (Zheng et al. 2011; Liu et al. 2017; Lei et al. 2021) Our in vitro study validates the modulation of macrophage cholesterol efflux by CTRP9. Further investigation should be done to corroborate the key cells that contribute to the effect of CTRP9 on atherosclerosis. Second, the receptor mediating the activity of CTRP9 during atherosclerosis is unclear. Finally, the pharmacokinetics and pharmacodynamics of human CTRP9 in ApoE−/− mice need to be further determined.

In conclusion, our study shows that CTRP9 prevents atherosclerotic plaque formation and enhances RCT in a mouse model of atherosclerosis. CTRP9 has the ability to stimulate the expression of ABCA1/ABCG1, and LXR-α through activation of AMPK signaling, consequently accelerating cholesterol efflux from foam cells. Therefore, delivery of CTRP9 may provide therapeutic benefits in the treatment of atherosclerosis.