Pregnancy-Associated Plasma Protein-A Accelerates Atherosclerosis by Regulating Reverse Cholesterol Transport and Inflammation

Abstract

Background: Recent studies have suggested that pregnancy-associated plasma protein-A (PAPP-A) is involved in the pathogenesis of atherosclerosis. This study aim is to investigate the role and mechanisms of PAPP-A in reverse cholesterol transport (RCT) and inflammation during the development of atherosclerosis.

Methods and Results: PAPP-A was silenced in apolipoprotein E (apoE^−/−) mice with administration of PAPP-A shRNA. Oil Red O staining of the whole aorta root revealed that PAPP-A knockdown reduced lipid accumulation in aortas. Oil Red O, hematoxylin and eosin (HE) and Masson staining of aortic sinus further showed that PAPP-A knockdown alleviated the formation of atherosclerotic lesions. It was found that PAPP-A knockdown reduced the insulin-like growth factor 1 (IGF-1) levels and repressed the PI3K/Akt pathway in both aorta and peritoneal macrophages. The expression levels of LXRα, ABCA1, ABCG1, and SR-B1 were increased in the aorta and peritoneal macrophages from apoE^−/− mice administered with PAPP-A shRNA. Furthermore, PAPP-A knockdown promoted RCT from macrophages to plasma, the liver, and feces in apoE^−/− mice. In addition, PAPP-A knockdown elevated the expression and secretion of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), tumor necrosis factor-α, and interleukin-1β through the nuclear factor kappa-B (NF-κB) pathway.

Conclusions: The present study results suggest that PAPP-A promotes the development of atherosclerosis in apoE^−/− mice through reducing RCT capacity and activating an inflammatory response.

Atherosclerosis is one of the leading causes of mortality worldwide. Dysregulation of lipid metabolism and inflammation are the pivotal etiologies for the progression of atherosclerosis.¹ When vascular endothelial cells are damaged, macrophages are recruited to the lumen and then transmigrate to the subintimal space, where they take up oxidized low-density lipoprotein (ox-LDL) and undergo foam cell formation, leading to the development of atherosclerosis.² Notably, macrophages accumulated in the sub-endothelium also secrete inflammatory factors to accelerate atherosclerosis.³ Reverse cholesterol transport (RCT) is the process whereby the excess cholesterol is removed from peripheral tissues, such as macrophages by high-density lipoprotein (HDL), and eventually delivered to the liver for metabolism and excretion through the bile and ultimately feces.^4,5 Previous studies from our laboratory and others have suggested that the increased expression in cholesterol transporters including ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor class B type I (SR-B1) in macrophages and peripheral tissues promotes the RCT and attenuates atherosclerosis.^6–9 Therefore, enhancing RCT capacity and mitigation of macrophage-related inflammation are important strategies for reducing the risk for atherosclerosis.

Pregnancy-associated plasma protein-A (PAPP-A) was first reported as a biomarker for pregnancy with genetic abnormality.¹⁰ Recently, PPAP-A was recognized as a new type of metalloproteinase in the insulin-like growth factor (IGF) system and has been implicated in the development of atherosclerosis.^11,12 Previous studies suggest that PAPP-A exists in the eroded and ruptured human atheromatous plaque that are enriched with activated macrophages and smooth muscle cells.^13,14 Our laboratory has also found that PAPP-A downregulates the expression of liver X receptor α (LXRα) through the IGF-I-mediated signaling pathway, decreasing the expression of cholesterol transporters and cholesterol efflux in THP-1 macrophage-derived foam cells.¹⁵ However, the role of PAPP-A in the development of atherosclerosis, particularly RCT and inflammation in vivo, remains unclear. Therefore, we used shRNA to knock down the expression of PAPP-A in apoE^−/− mice fed a Western-type diet and then examined the effects of PAPP-A knockdown on RCT, inflammation, and atherosclerosis in vivo and the underlying mechanisms.

Methods

Animal Studies

Male apoE^−/− mice (8 weeks old) were purchased from Changzhou Cavens Laboratory Animal Co. LTD (Jiang Su, China). ApoE^−/− mice were injected via the tail vein with 1×10¹¹ genome copies (GCs) of AAV-shRNA (TCTCGCTTGGGCGAGAGTA) or AAV-PPAP-A shRNA (GCAACAGATCCA CGCTACT) (Genechem, China) once at the beginning of the experiment. Animals were fed a Western-type diet (21% [wt/wt] fat, 0.3% cholesterol; Research Diets) for 8 weeks with free access to water. Peritoneal macrophages were obtained from the randomly selected mice (n=5) after the third day intraperitoneal injection with 4% Starch broth. All procedures were conducted in accordance with the Institutional Animal Ethics Committee and the University of South China Animal Care Guidelines for the Use of Experimental Animals.

Plasma Lipid Profiles

Blood samples from the apoE^−/− mice were collected into Ethylene Diamine Tetraacetic Acid (EDTA)-coated tubes, and plasma was isolated by centrifugation (5,000 rpm, 4˚C, 10 min). The levels of total cholesterol (TC), triglycerides (TG), and HDL-C in the plasma samples were detected in the clinical laboratory of the Affiliated Nanhua Hospital, University of South China (Hengyang, China). The levels of low-density lipoprotein cholesterol (LDL-C) were calculated by subtracting the levels of HDL-C and 0.2×TG from that of TC.

Reverse Cholesterol Transport Assay

ApoE^−/− mice were treated with control or PAPP-A shRNA and fed a Western-type diet for 8 weeks. J774 macrophages were treated with 50 μg/mL acetylated low-density lipoprotein (ac-LDL) (Yiyuan Biotechnologies, China) and loaded with 5 μCi/mL [³H] cholesterol for 24 h. J774 cells labeled with [³H] cholesterol (~4.5×10⁸ cells/mouse, n=5 mice/group) were intraperitoneally injected into an individual mouse, which was randomly selected from each group. After injection, mice were housed individually and plasmas samples were collected from mice at 6, 24, and 48 h after injection, followed by the measurement with a scintillation counter. Feces were collected continuously until 48 h, and dissolved in ethanol for counting. Mice were euthanized 48 h after the injection, and lipids were extracted from the liver for measurement. The data were shown as the percentage of counts.

Atherosclerosis Analysis

The aortas and hearts were collected immediately from euthanized mice and fixed using 4% paraformaldehyde. The whole aortas were stained with Oil Red O and the en face staining of the aortas was photographed with a digital camera. The aortic roots were frozen with optimal cutting temperature compound (O.C.T. compound; Sakura Finetek, USA, Inc., Torrance, CA, USA) and serial sections (8-μm thick) were cut throughout the three aortic valves. The sections were stained with Oil Red O, HE, and Masson’s trichrome staining for the quantification of plaque cross-sectional area. Lesion areas and percentages were quantified with Image Pro Plus software.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

To evaluate the gene expression in tissues or cells from mice, total RNAs were extracted using TRIzol reagent (Beyotime, China), following the manufacturer’s instructions. The complementary DNA was synthesized with a high-capacity cDNA reverse transcription kit (Takara, China). The primers were obtained from Biology Engineering Corporation in Shanghai, China. qRT-PCR analyses were performed on the iCycler IQ Real-Time Detection System (Bio-Rad, U.S.A) using the following primer pairs: ABCA1 Primers, Forward 5'-GGGTGGTGTTCTTCCTCATTAC-3' and Reverse 5'-GAATGACGAGGATGAGGATGTG-3'; ABCG1 Primers, Forward 5'-CCTGACACATCTGCGAATCA-3' and Reverse 5'-GAGGAACAGCATGGAGAAGAA-3'; SR-BI Primers, Forward 5'-TTTGGAGTGGTAGTAAAAAGGGC-3' and Reverse 5'-TGACATCAGGGACTCAGAGTAG-3'; LXRα Primers, Forward 5'-CTCAATGCCTGATGTTTCTCCT-3' and Reverse 5'-TCCAACCCTATCCCTAAAGCAA-3'; GAPDH Primers, Forward 5'-TGGATTTGGACGCATTGGTC-3' and Reverse 5'-TTTGCACTGGTACGTGTTGAT-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control.

Western Blotting Assay

Total proteins were extracted from cells or tissues using RIPA buffer (Beyotime) containing phenylmethylsulfonyl fluoride (Beyotime) to inhibit proteases. Protein concentrations were measured using a BCA Protein Assay Kit (Beyotime). Thereafter, the same amounts of total proteins were subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes (PVDF, Millipore Corporation, USA). After blocking in 5% dry milk, the PVDF membranes were incubated with antibodies against PAPP-A (ABCAM, 1:1,000), ABCA1 (ABCAM, 1:200), ABCG1 (ABCAM, 1:1,000), SR-B1 (SIGMA, 1:500), LXRα (ABCAM, 1:1,500), PI3K (CST, 1:1,000), Akt (CST, 1:1,000), or p65 (ABCAM, 1:30,000), followed by incubation with a secondary antibody: HRP-labeled Goat Anti-Rabbit IgG (H+L) (Beyotime, 1:1,000). Antibody binding was visualized with a chemiluminescence method using BeyoECL Plus (Beyotime) and quantified by Gel-Pro Analyzer software. β-actin was used as an internal control.

Enzyme-Linked Immunosorbent Assay (ELISA)

The ELISA kits were used to determine the levels of inflammation factors (MCP-1, IL-6, TNF-α and IL-1β) (ThermoFisher Scientific, USA) and IGF-1 (R&D Systems, UK) according to the standard protocol. Briefly, serum samples from apoE^−/− mice or cell culture medium were added into each well and incubated at room temperature for 120 min. Then, the biotin-conjugated antibody was added, followed by incubation for 90 min. Thereafter, the substrate solution was added into the well and incubated for 30 min. After washing five times with 0.01 mol/L tris buffered saline (TBS), 3,3',5,5'-tetramethylbenzidine (TMB) was added and incubated for 30 min in the dark. The absorbance at 450 nm was determined using a Bio-Rad iMark microplate reader.

Immunofluorescence (IF)

The frozen sections of aortic roots were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 20 min. No permeabilization was performed for ABCA1 and ABCG1 staining. The samples were blocked in goat serum for 30 min at room temperature, followed by an incubation with primary antibodies for CD68 (ab955, mouse monoclonal antibody, 1:200; ABCAM, UK), HSP70 (AF0189, rabbit polyclonal antibody, 1:200, Beyotime, China), ABCA1 (ab18180, mouse monoclonal antibody, 1:200; ABCAM), or ABCG1 (ab52617, rabbit polyclonal antibody, 1:100; ABCAM) for 3.5 h at room temperature or at 4℃ overnight. After washing, the sections were incubated with fluorescein isothiocyanate (FITC)-labeled Goat Anti-Mouse IgG (H+L) (A0568, 1:500; Beyotime, China), Cy3-labeled Goat Anti-Rabbit IgG (H+L) (A0516, 1:500; Beyotime, China), or Cy3-labeled Goat Anti-mouse IgG (H+L) (A0521, 1:200; Beyotime) for 1 h at room temperature. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, Solarbio, China). Fluorescence microscopy of HSP70, CD68, ABCA1, ABCG1, pJNK and pElk-1 in frozen atherosclerotic sections was performed using an OLYMPUS microscope (IX73P2F; Tokyo, Japan). The mean fluorescence intensity (MFI) for ABCA1 and ABCG1 on stained sections of aortic roots was measured using Image Pro Plus software.

Statistical Analysis

All data were expressed as means±SD and evaluated using Student’s t-test of two groups or one-way ANOVA with Tukey’s post hoc test or Dunnett’s post hoc. Statistical analyses were performed using GraphPad software (version 7.0). The difference was significant when P values were less than 0.05.

Results

PAPP-A Knockdown Attenuates Atherosclerotic Plaque Formation in ApoE^−/− Mice

To explore the role of PAPP-A in atherosclerosis in vivo, we knocked down the PAPP-A expression in apoE^−/− mice using adeno-associated virus-short hairpin RNA (AAV-shRNA). The mice were then fed a Western-type diet for 8 weeks. Injection of AAV-shRNA did not cause any changes in mortality (data not shown). The plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were also comparable among different groups (Supplementary Figure 1), indicating that AAV-shRNA did not cause obvious liver damage. The levels of PAPP-A in the plasma (Supplementary Figure 2), the aorta, and peritoneal macrophages (Supplementary Figure 3) were significantly increased in apoE^−/− mice on the Western-type diet compared to the wild-type mice on the Western-type diet and apoE^−/− mice on a regular chow diet. The expression of PAPP-A in the aorta and peritoneal macrophages was measured to confirm knockdown efficiency. We found that the levels of PAPP-A were dramatically reduced in mice injected with AAV-PAPP-A shRNA (Figure 1A,B). AAV-shRNA also significantly reduced plasma levels of PAPP-A in apoE^−/− mice on the Western-type diet (Supplementary Figure 2). Addition, we also measured the blood pressure, blood glucose and insulin of AAV-PAPP-A shRNA treated mice and the results showed that their levels didn’t change (Supplementary Table). We then examined the effects of PAPP-A knockdown on the development of atherosclerosis. Oil Red O staining revealed that the lipid-laden plaque areas in the aortic arch regions (Figure 1C) and the entire en face aorta (Figure 1D,E) were significantly decreased in apoE^−/− mice injected with PAPP-A shRNA. Consistently, HE, Oil Red O, and Masson staining of cross-sections of the aortic root showed that PAPP-A knockdown significantly reduced plaque formation in apoE^−/− mice (Figure 1F,G). CD-68-positive macrophages were also significantly reduced in the plaques of apoE^−/− mice injected with PAPP-A shRNA (Supplementary Figure 4). These findings suggest that PAPP-A plays an important role in atherosclerotic plaque formation.

Figure 1.

The pregnancy-associated plasma protein-A (PAPP-A) knockdown reduces atherosclerotic plaques in apolipoprotein E (apoE^−/−) mice. Male apoE^−/− mice were injected via the tail vein with saline only (Control), adeno-associated virus-shRNA (shRNA), or AAV-PAPP-A shRNA (PAPP-A shRNA) and fed on a Western diet for 8 weeks (20 mice per group). Western blot analysis was performed to determine PAPP-A levels in the aorta (A) and peritoneal macrophages (B) of apoE^−/− mice (n=3 mice/group). Plaques in aortic arches (C) of representative apoE^−/− mice were observed under a stereoscopic microscope. En face Oil Red O stained the whole aortas (D) and frozen sections of mouse aortic roots stained by HE, Oil Red O and Masson staining (F) from the apoE^−/− mice are shown. Mean atherosclerotic lesion areas of the aorta (E) and aortic root (G) were determined using Image J software. Representative images from each group were shown in panels (C), (D) and (E). Data in panels (F) and (G) are presented as the mean±SD of data collected from 10 mice from each group. **P<0.01 indicates a significant difference compared with Control groups.

PAPP-A Knockdown Increases the Efficiency of RCT in ApoE^−/− Mice

The dysregulation of lipid metabolism plays a critical pro-atherogenic role and the efficiency of RCT is associated with the progress of atherosclerosis. So, we examined the effect of PAPP-A on RCT in apoE^−/− mice. [³H]-cholesterol-loaded J774 cells were injected into Western diet-fed mice transduced with PAPP-A shRNA. The plasma levels of macrophage-derived [³H]-tracer in mice with PAPP-A shRNA were significantly increased 24 h and 48 h after injection (Figure 2A). The level of tracer was also increased in the liver and feces of PAPP-A knockdown apoE^−/− mice 48 h after injection of [³H]-cholesterol-loaded J774 cells (Figure 2B,C). Taken together, these observations indicate that PAPP-A reduces the efficiency of RCT to accelerate atherosclerosis in apoE^−/− mice.

Figure 2.

The reverse cholesterol transport (RCT) is increased in the apoE^−/− mice with PAPP-A knockdown. [³H]-cholesterol and acetylated LDL-loaded J774 macrophages were injected into apoE^−/− mice (n=5 mice/group). The amounts of [³H]-tracer in plasma (A), the liver (B), and feces (C) were determined by scintillation counting. Data are presented as the mean±SD. *P<0.05 (**P<0.01) indicates a significant difference compared with Control groups at the same time. Abbreviations as in Figure 1.

PPAP-A Knockdown Increases the Expression of ABCA1, ABCG1, and SR-B1 in ApoE^−/− Mice

Considering that the HDL is a major player of RCT and that cholesterol efflux is the first rate-limiting step in RCT, we detected plasma levels of lipids in mice and found that PAPP-A knockdown had no detectable effect on plasma levels of TC, LDL-C, and TG, but significantly increased the levels of HDL-C (Table). Given that ABCA1, ABCG1, and SR-B1 play important roles in HDL metabolism, we measured their expression in apoE^−/− mice and found that knockdown of PAPP-A expression significantly increased their expression in the aorta (Figure 3A–C), peritoneal macrophages (Figure 4A–C), and plaques (Figure 5). Similarly, the expression of ABCA1 was increased in the liver of apoE^−/− mice injected with PAPP-A shRNA (Supplementary Figure 5). SR-BI expression, however, was not altered in the liver by PAPP-A knockdown (Supplementary Figure 6). In contrast, the expression of LXRα and its target genes such as CYP7a1, SREBP1c, and IDOL was increased in the liver of PAPP-A knockdown apoE^−/− mice (Supplementary Figure 5). We then detected the levels of cholesterol efflux in the primary peritoneal macrophages and found that knockdown of PAPP-A promoted cholesterol efflux (Figure 4H,I). Our data suggest that PAPP-A reduces the levels of HDL-C and the expression of ABCA1, ABCG1, and SR-B1 to suppress the cholesterol efflux and RCT, leading to the promotion of atherosclerosis.

Table. Effects of the PAPP-A Knockdown on the Plasma Lipids of Western Diet-Fed Apolipoprotein E (apoE^−/−) Mice

	BW	TC	HDL-C	LDL-C	TG
Control	33.19±0.78	30.22±3.59	1.54±0.39	28.39±3.69	1.44±0.36
shRNA	32.11±0.91	29.56±3.16	1.50±0.42	27.76±3.22	1.50±0.41
PAPP-A shRNA	33.75±1.01	31.38±4.04	2.08±0.33*	28.97±4.17	1.67±0.34

Enzymatic methods were used to measure the levels of plasma lipids (mmol/L) including TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride. BW, body weight (g); PAPP-A, pregnancy-associated plasma protein A. All values are shown as mean±SD (n=10), *P<0.05 vs. Control .

Figure 3.

Expression of ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1) and scavenger receptor class B type 1 (SR-B1) is enhanced through the insulin-like growth factor (IGF)-1-PI3K-Akt pathway in the aorta of the apoE^−/− mice with PAPP-A knockdown. Total RNA was isolated from the aorta and then subjected to quantitative real-time polymerase chain reaction (qRT-PCR) to quantify mRNA levels of ABCA1 (A), ABCG1 (B), SR-B1 (C), and LXRα (G). The levels of each target were normalized to that of GAPDH at the same condition. The same amount of protein lysate isolated from aorta homogenate was subjected to western blot analysis to detect ABCA1 (A), ABCG1 (B), SR-B1 (C), p-PI3K (E), p-Akt (F) and LXRα (G). The levels of IGF-1 in serum (D) measured by enzyme-linked immuno sorbent assay are shown. Data are presented as the mean±SD (n=5 mice/group). *P<0.05 (**P<0.01) indicate significant difference compared with Control groups. Abbreviations as in Figure 1.

Figure 4.

Expression of ABCA1, ABCG1 and SR-B1 is increased through the IGF-1-PI3K-Akt pathway in peritoneal macrophages isolated from the PAPP-A knockdown apoE^−/− mice. Peritoneal macrophages were isolated from apoE^−/− mice with different treatments (n=5 mice/group). Total RNA was isolated and subjected to qRT-PCR to detect the mRNA levels of ABCA1 (A), ABCG1 (B), SR-B1 (C) and LXRα (G), which were normalized to that of GAPDH. Whole cell lysate was prepared from the isolated peritoneal macrophages. The same amount of total proteins was subjected to Western blot analysis to detect the protein levels of ABCA1 (A), ABCG1 (B), SR-B1 (C), p-PI3K (E), p-Akt (F) and LXRα (G). The levels of IGF-1 in medium (D) were measured by an enzyme-linked immunosorbent assay (ELISA). Peritoneal macrophages isolated from mice from each group were incubated with acLDL and [³H]-cholesterol. Culture medium and cells were collected separately and subjected to liquid scintillation counting. Cholesterol efflux to apolipoprotein A-I (apoA-I) (H) or high-density lipoprotein (HDL) (I) was calculated as the percentage of [³H] cholesterol in medium to the total [³H] cholesterol in medium and cells. Quantification data are presented as the mean±SD (n=3/group). *P<0.05 (**P<0.01) indicates a significant difference compared with Control groups. Abbreviations as in Figures 1–3.

Figure 5.

Expression of ABCA1, ABCG1 and SR-B1 is inhibited in atherosclerotic plaques of the apoE^−/− mice with PAPP-A knockdown. Representative immunofluorescence staining was shown for ABCA1 (A), ABCG1 (C) and SR-B1 (E) in atherosclerotic plaques of apoE^−/− mice. Quantification fluorescence intensity of ABCA1 (B), ABCG1 (D) and SR-B1 (F) in plaques was shown. Data are presented as the mean±SD (n=5 mice/group). *P<0.05 (**P<0.01) indicates a significant difference compared with Control groups. Abbreviations as in Figures 1,3.

PAPP-A Knockdown Enhances the Expression of ABCA1, ABCG1 and SR-BI Through the IGF-1/ PI3K/Akt Pathway in ApoE^−/− Mice

We previously reported that PAPP-A decreases the expression of ABCA1, ABCG1 and SR-B1 through the IGF-1/PI3K/Akt/LXRα pathway in THP-1 macrophage-derived foam cells.¹⁵ To define the regulatory mechanism in vivo, we detected the levels of IGF-1 in serum from PAPP-A shRNA-treated apoE^−/− mice and the culture medium of peritoneal macrophages isolated from apoE^−/− mice with PAPP-A knockdown. Our results showed that the IGF-1 was significantly increased in both conditions (Figures 3D,4D). The levels of p-PI3K and p-Akt were both decreased in the aorta (Figure 3E–G) and peritoneal macrophages (Figure 4E–G) of the PAPP-A shRNA-treated apoE^−/− mice. In contrast, the expression of LXRα was increased in the aorta and peritoneal macrophages. It indicates that PAPP-A knockdown inhibits the IGF-1/PI3K/Akt signal pathway and upregulates the LXRα expression to increase the expression of ABCA1, ABCG1 in vivo.

PAPP-A Knockdown Suppresses the Production of Inflammatory Cytokines by Inhibiting the NF-κB Pathway

Given that inflammation plays an important role in the development of atherosclerosis, we tested whether PAPP-A affected inflammation. First, we measured the levels of MCP1, TNF-α, IL-6, and IL-1β in serum (Figure 6A) and found that these pro-inflammatory cytokines were all decreased in the apoE^−/− mice injected with PAPP-A shRNA. To confirm the role of PAPP-A in macrophages, we isolated peritoneal macrophages and examined the expression and secretion of pro-inflammation cytokines and the nuclear translocation of p65. Our results revealed that both expression and secretion of MCP-1 (Figure 6B), TNF-α (Figure 6C), IL-6 (Figure 6D) and IL-1β (Figure 6E) were reduced in mouse meritoneal macrophages (MPMs) isolated from apoE^−/− mice with PAPP-A knockdown in response to lipopolysaccharide (LPS). In addition, we found that the nuclear translocation of p65 was blocked when the PAPP-A was downregulated (Figure 6F), suggesting the involvement of the NF-κB pathway in PAPP-A-induced inflammation.

Figure 6.

PAPP-A knockdown decreases inflammation through the NF-κB pathway. 1×10⁴ MPMs were incubated with 100 ng/mL LPS and the supernatants were collected to detect the levels of inflammatory factors. The levels of pro-inflammatory factors (A) in the serum were determined by ELISA kits (n=5 mice/group). The mRNA and secretion of MCP-1 (B), TNF-α (C), IL-6 (D) and IL-1β (E) in MPMs isolated from apoE^−/− mice treated with PAPP-A shRNA (n=5 mice/group). The nuclear translocation of p65 (F) was shown. Quantification data are presented as the mean±SD (n=5 mice/group). *P<0.05 (**P<0.01) indicates a significant difference compared with Control groups. Abbreviations as in Figures 1,4.

Discussion

Lipid accumulation and local inflammation in the vascular wall play a pivotal role in the progression of atherosclerosis. Therefore, inhibiting inflammation and elevating RCT efficiency could limit the plaque lesion formation and maintain plaque stability which are potential strategies to treat atherosclerosis.¹⁶ It was reported that the high PAPP-A levels are associated with atherosclerosis, and PAPP-A has been proposed as a new marker of acute coronary syndromes.^17,18 It was also reported that PAPP-A is highly expressed in atherosclerotic plaques and can accelerate lipid accumulation in cells.^13,19 The current study revealed that PAPP-A aggravates the development of atherosclerosis in apoE^−/− mice through inhibiting the expression of cholesterol transports including ABCA1, ABCG1 and SR-B1, reducing RCT efficiency, and enhancing the expression and secretion of pro-inflammation factors. Together with our previous findings that recombinant PPAP-A can activate the IGF-1/PI3K/Akt signal pathway to inhibit the expression of ABCA1, ABCG1 and SR-B1 in THP-1-derived macrophages,¹⁵ our results strongly support the conclusion that PAPP-A contributes to plaque formation in atherosclerosis. Thus, novel strategies aimed at PAPP-A, which is a relatively accessible extracellular enzyme, might prevent atherosclerosis.^14,20

It is well known that enhanced macrophage RCT is one of the crucial mechanisms by which HDL exerts its atheroprotective effect. There are three processes in RCT. Cholesterol efflux is the first step and a key component in RCT, which plays an important role in reducing the accumulation of lipids in the arterial wall and preventing the development of atherosclerosis. Cholesterol efflux depends on the expression of specific transport proteins including ABCA1, ABCG1, and SR-BI to mediate the efflux of excess cellular cholesterol from macrophages to apoA-I and nascent HDL.^21,22 However, ABCG1 possesses the function to redistribute intracellular cholesterol to plasma membrane domains, which are accessible for removal by HDL, but not lipid-poor apoA-I.²³ SR-BI mediates cholesterol efflux to HDL and cholesterol influx from HDL as well, but it does not alter cellular cholesterol mass. In addition, SR-BI mediates cholesterol efflux to large but not smaller HDL particles.^24,25 The second step of RCT is that free cholesterol in HDL is esterified by lecithin cholesterol acyl transferase (LCAT) to form cholesteryl ester (CE), which can be directly transferred to hepatocytes through SR-B1 or indirectly transferred to apoB-containing lipoproteins via CE transfer protein (CETP) and then delivered to the liver via the low-density lipoprotein Receptor (LDLR). Finally, hepatic cholesterol can be converted to bile salts or directly excreted to the bile. When ABCA1, ABCG1 and SR-B1 expression was increased, the levels of HDL-C were increased. In the current study, we indeed found that the expression of ABCA1, ABCG1, and SR-B1 was increased in peritoneal macrophages, atherosclerotic plaque, and aorta from the apoE^−/− mice treated with PAPP-A shRNA. The expression of ABCA1, ABCG1 and SR-B1 in other peripheral cells such as smooth muscle cells and endothelial cells were also upregulated when the PAPP-A was knocked down in the apoE^−/− mice. Further, the expression of ABCA1 but not SR-BI was increased in the liver of PAPP-A knockdown apoE^−/− mice (Supplementary Figures 5,6). Hepatic ABCA1 is critical for HDL biogenesis. Together, these findings may at least partially explain the mechanisms by which HDL-C levels were increased in PPAP-A shRNA-treated groups. However, the metabolism of HDL-C is complex. For example, lipoprotein lipase including endothelial lipase (EL) plays an important role in HDL metabolism, and CETP mediates the exchange of cholesterol ester from HDL to apoB-containing lipoproteins such as LDL.^26,27 Given that the apoE^−/− mice treated with PAPP-A shRNA exhibited a significant improvement in HDL-C levels and RCT efficiency, it is also possible that these pathways contribute to the effect of PAPP-A on HDL-C metabolism. Further studies are needed to explore these possibilities.

PAPP-A has been proven to regulate the IGF signaling transduction through degradation of IGF-binding protein (IGFBP), which increases local IGF bioavailability and receptor activation.^28,29 Notably, the role of IGF-1 in cardiovascular disease has attracted much attention of many researchers. The levels of IGF-1 are associated with cardiovascular disease risk.^30,31 It is known that IGF-1 activates the PI3K/Akt pathway and promotes the synthesis of nitric oxide to prevent cardiovascular disease.^32,33 Several studies have also demonstrated that the PI3K/Akt pathway participates in the regulation of metabolism balance through LXRα that can upregulate the expression of ABCA1, ABCG1 and SR-B1.^34–36 Our previous research indicated that PAPP-A might first downregulate the expression of LXRα through the IGF-1/PI3-K/Akt signaling pathway and decrease the expression of ABCA1, ABCG1, SR-B1 and cholesterol efflux in THP-1 macrophage-derived foam cells.¹⁵ In addition, we observed that the efficiency of RCT was improved in the apoE^−/− mice treated with PAPP-A shRNA. Thus, it is possible that PAPP-A inhibits the expression of the cholesterol transporters via the IGF-1/PI3K/Akt/LXRα pathway, reducing the efficiency of cholesterol efflux and RCT, and consequently leading to the development of atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the arterial wall, in which inflammation contributes to plaque formation. However, the effect of PAPP-A on inflammation and the underlying mechanism are unclear. Several groups indicate that the activation of NF-κB could be triggered by the PI3K/Akt signaling.^37,38 It was also reported that NF-κB is activated by several stimuli during the atherosclerotic process. This might be responsible for the expression of inflammatory proteins and cytokines that actively participate in the process, leading to plaque disruption and acute coronary events. Several previous studies have shown that IGF-1 regulates the immunity and immune responses via the NF-κB pathway in response to activation of the PI3K/Akt signaling.^39–41 Our findings also suggest that PAPP-A may increase inflammation through the IGF-1/PI3K-Akt/NF-κB signaling pathway to reduce plaque stability; the exact contribution of IGF-1/PI3K-Akt/NF-κB signaling needs to be further confirmed.

In summary, we have addressed the missing link between PAPP-A and atherosclerosis by providing evidence that the downregulation of PAPP-A is positively associated with RCT and negatively related with the inflammation, which is possibly mediated by inhibiting the expression of cholesterol transporters. Meanwhile, PAPP-A releases IGF-1 from IGF binding proteins and activates the PI3K/Akt pathway by IGF-1R. The activated signaling may inhibit the expression of ABCA1, ABCG1, and SR-B1 by suppressing LXRα, thereby reducing the efficiency of RCT and leading to the atherosclerotic plaque formation. In contrast, the PI3K/Akt pathway can trigger NF-κB activation to facilitate the secretion of pro-inflammation cytokines, accelerating atherosclerosis. Thus, the selective modulation of PAPP-A may provide the foundation to develop a novel therapeutic approach.

Acknowledgments

The authors gratefully acknowledge the financial support from the Natural Science Foundation of Hunan Province (2017JJ4076), the National Natural Sciences Foundation of China (81770461) and Hunan Provincial Innovation Foundation for Postgraduate (CX2018B61).

Supplementary Files

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-18-0700

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