Decreased APOE-containing HDL subfractions and cholesterol efflux capacity of serum in mice lacking Pcsk9
© Choi et al.; licensee BioMed Central Ltd. 2013
Received: 29 May 2013
Accepted: 22 July 2013
Published: 24 July 2013
Studies in animals showed that PCSK9 is involved in HDL metabolism. We investigated the molecular mechanism by which PCSK9 regulates HDL cholesterol concentration and also whether Pcsk9 inactivation might affect cholesterol efflux capacity of serum and atherosclerotic fatty streak volume.
Mass spectrometry and western blot were used to analyze the level of apolipoprotein E (APOE) and A1 (APOA1). A mouse model overexpressing human LDLR was used to test the effect of high levels of liver LDLR on the concentration of HDL cholesterol and APOE-containing HDL subfractions. Pcsk9 knockout males lacking LDLR and APOE were used to test whether LDLR and APOE are necessary for PCSK9-mediated HDL cholesterol regulation. We also investigated the effects of Pcsk9 inactivation on cholesterol efflux capacity of serum using THP-1 and J774.A1 macrophage foam cells and atherosclerotic fatty streak volume in the aortic sinus of Pcsk9 knockout males fed an atherogenic diet.
APOE and APOA1 were reduced in the same HDL subfractions of Pcsk9 knockout and human LDLR transgenic male mice. In Pcsk9/Ldlr double-knockout mice, HDL cholesterol concentration was lower than in Ldlr knockout mice and higher than in wild-type controls. In Pcsk9/Apoe double-knockout mice, HDL cholesterol concentration was similar to that of Apoe knockout males. In Pcsk9 knockout males, THP-1 macrophage cholesterol efflux capacity of serum was reduced and the fatty streak lesion volume was similar to wild-type controls.
In mice, LDLR and APOE are important factors for PCSK9-mediated HDL regulation. Our data suggest that, although LDLR plays a major role in PCSK9-mediated regulation of HDL cholesterol concentration, it is not the only mechanism and that, regardless of mechanism, APOE is essential. Pcsk9 inactivation decreases the HDL cholesterol concentration and cholesterol efflux capacity in serum, but does not increase atherosclerotic fatty streak volume.
KeywordsApolipoprotein E Atherosclerotic fatty streak Low-density lipoprotein receptor Macrophage foam cell Proprotein convertase subtilisin/kexin type 9
PCSK9 is a member of the proprotein convertase subtilisin/kexin family. Mutations in PCSK9 have been identified in familial autosomal dominant hypercholesterolemia patients, and gain-of-function mutations increase LDL cholesterol concentration [1, 2]. The major molecular function of PCSK9 in LDL cholesterol and lipid homeostasis is degradation of the LDL receptor (LDLR), VLDL receptor (VLDLR) and LDLR-related protein 8 (LRP8) [3–5]. In addition, several studies in mice and non-human primates have shown that PCSK9 is involved in HDL metabolism. Pcsk9 KO male mice on a B6 background fed a chow diet exhibited a 30% reduction in HDL cholesterol concentration . B6 male mice fed a high fat diet and then treated with a Pcsk9 antisense oligonucleotide inhibitor for 6 weeks showed a 54% reduction in HDL cholesterol concentration . In male cynomolgus macaques, treatment with neutralizing antibodies against PCSK9 reduced HDL cholesterol concentrations for the first seven days of treatment . Despite the accumulating evidence, the molecular mechanism by which PCSK9 regulates HDL cholesterol concentration has not been investigated. Previous studies reported decreased levels of circulating APOE and higher levels of LDLR, VLDLR, and LRP8 by PCSK9 inhibition [4–6]. APOE in lipoproteins acts as a ligand of LDLR family proteins and promotes lipoprotein particle clearance [9, 10]. APOE is an efficient cholesterol acceptor in HDL, and the binding of APOE in newly secreted HDL (also called nascent HDL) increases the particle size and cholesterol concentration [11, 12]. Thus, PCSK9-mediated regulation of APOE levels in HDL may be a key mechanism that determines HDL cholesterol concentration. In this study, we show that increased LDLR decreases APOE-containing HDL subfractions and HDL cholesterol concentrations in mice. We further demonstrate that, although LDLR plays an important role in PCSK9-mediated regulation of HDL cholesterol concentration, PCSK9 does not entirely rely on LDLR and that PCSK9-mediated regulation of HDL cholesterol concentration relies entirely on the presence of APOE. Finally, we show that, although Pcsk9 KO reduces HDL cholesterol concentration and cholesterol efflux capacity in serum, there is no significant impact on early atherogenesis.
PCSK9-mediated HDL cholesterol regulation is partially sex- and diet-dependent
APOE in non-HDL depleted serum (NHDS) is significantly reduced in Pcsk9 KO mice
To investigate how PCSK9 regulates HDL cholesterol concentration, we examined whether APOE composition and distribution in HDL were affected in Pcsk9 KO mice. We performed mass spectrometry using the NHDS of 8-week-old Pcsk9 KO and control mice. The NHDS was separated on a non-denaturing 4-30% polyacrylamide gel that exhibited 11 bands after staining with Coomassie Brilliant Blue R-250. Each band was excised as a 1-mm-wide slice for mass spectrometry to identify apolipoproteins; results were compared between Pcsk9 KO and control mice. As expected, APOA1, but not APOB, was present in all bands (data not shown). The presence of APOA1 indicated that the bands contained HDL, while the absence of APOB indicated that APOB-containing lipoproteins were effectively removed by the precipitation method. In Pcsk9 KO mice, APOE was not found in any bands, while in control mice, APOE was found in the five bands with a molecular weights above 272 kDa (See Additional file 1: Table S1). We next tested whether the absence of APOE results from decreased APOE production. Compared to wild-type controls, Pcsk9 KO mice showed no decrease in Apoe expression and APOE protein level in the liver (See Additional file 2: Figure S1) and in the peritoneal macrophages (data not shown) where APOE is mainly produced. Combined, these results suggest that the reduced APOE level in Pcsk9 KO NHDS is not due to decreased APOE production.
APOE-containing HDL subfractions are decreased in Pcsk9 KO mice
Increased LDLR leads to decreased levels of HDL cholesterol concentration and APOE-containing HDL subfractions
PCSK9 regulates HDL cholesterol concentration through LDLR and APOE
We also tested whether APOE was required for PCSK9-mediated HDL regulation. HDL cholesterol concentrations were measured in Apoe KO and Pcsk9/Apoe double-KO males and were found similar (101.6 ± 7.0 mg/dl vs 99.9 ± 4.5 mg/dl). These results indicate that PCSK9-mediated HDL regulation — either LDLR-dependent or LDLR-independent — completely relies on APOE.
Effects of Pcsk9 inactivation on the cholesterol efflux capacity of serum and atherosclerotic fatty streak volume in aortic sinus
In this study, we revealed the molecular mechanism by which PCSK9 controls HDL cholesterol concentration by regulating the APOE-containing HDL. We show that APOE and APOA1 in the same size of HDL subfractions were reduced in Pcsk9 KO mice. Combined with the result showing no impact on smaller 66-kDa sized HDL level, our data suggest that PCSK9 specifically controls APOE-containing HDL.
Because APOE-containing lipoproteins are mainly cleared via LDLR [10, 21] and Pcsk9 KO mice exhibiting 2- to 3-fold higher levels of LDLR in the liver [6, 22], we hypothesized that rapid clearance of APOE-containing HDL via LDLR might be the major cause of decreased HDL cholesterol concentration in Pcsk9 KO mice. This hypothesis was reinforced by the analysis of Tg(hLDLR) mice that overexpress LDLR. They exhibit very low levels of APOE-containing HDL, and their HDL cholesterol concentrations are reduced by 82% (versus about 47% in Pcsk9 KO mice). Because APOE is known to promote cholesterol loading on HDL , this dramatic 82% reduction likely reflects a lower HDL cholesterol concentration due to the faster clearance of the APOE-containing HDL and the poor cholesterol concentration in the remaining smaller lipid-free or lipid-poor HDL subfraction. Our data clearly points at the key role of LDLR in the clearance of HDL.
We also show that, although LDLR plays an important role in PCSK9-mediated HDL cholesterol regulation, it is not a unique factor implicated in this process. HDL cholesterol concentrations in Pcsk9/Ldlr double-KO mice were 21% lower than in Ldlr KO mice, revealing an LDLR-independent effect of PCSK9. No change of ABCA1 and SR-B1 levels in Pcsk9 KO mouse livers support that reduced HDL cholesterol concentration in Pcsk9 KO mouse serum is largely through the clearance of APOE-containing HDL subfractions. A possible LDLR-independent mechanism may be based on APOE binding to the VLDLR, as APOE is an efficient ligand of VLDLR. VLDLR is targeted for degradation by PCSK9 in HEK293 and NIH 3 T3 cell lines , and cell surface VLDLR levels were increased in the adipose tissue of Pcsk9 KO mice . We finally show that HDL cholesterol concentrations are similar between Apoe KO and Pcsk9/Apoe double-KO males, suggesting that the PCSK9-mediated HDL cholesterol regulation is dependent on the presence of APOE and that the role of APOE entirely depends on its ability to mediate the binding of HDL to LDLR or VLDLR.
Therapeutic inhibition of PCSK9 is a promising pro-atherogenic LDL cholesterol-lowering treatment. Co-treatment of PCSK9 inhibitors with drugs that suppress cholesterol synthesis is even more effective in reducing LDL cholesterol in hypercholesterolemia patients [23–25]. Inconsistent with recent clinical trials, studies in laboratory animals show that PCSK9 inhibition reduces HDL cholesterol concentration. The inconsistency might be due to different dosage of PCSK9 inhibition in different studies. Or, a reduction in HDL cholesterol concentration by PCSK9 inhibition might be restricted to species such as the mouse in which APOE-containing HDL level is elevated, compared to human [26–28].
Independently of the level of HDL cholesterol concentration, cholesterol efflux capacity is associated with atherosclerotic plaque formation in the coronary arteries . We found that Pcsk9 KO leads to a reduction in cholesterol efflux capacity of serum from THP-1 macrophage foam cells, but there was no significant impact on atherogenic-diet induced fatty streak volume in aortic sinus. Similar observation was made in a recent study showing no increase of atherosclerotic lesion size in the aortas of Pcsk9/Ldlr double-KO and Pcsk9/Apoe double-KO mice. We speculate that this might be because Pcsk9 inactivation reduces pro-atherogenic LDL level in the circulation and also reduces the accumulation of esterified cholesterol in the aortas .
In mice, PCSK9 controls circulating cholesterol concentrations by regulating both LDL and HDL levels through LDLR. Our data suggest that the regulation of HDL by PCSK9 is mainly through LDLR-mediated APOE-containing HDL clearance and that other targets of PCSK9 might be involved in the process [5, 30]. Our data validate that reduced HDL cholesterol concentration and cholesterol efflux capacity in serum by Pcsk9 inactivation do not have significant impact on the early stage of atherosclerosis development.
Mice, husbandry and diet
Descriptions of mice used in this study
Full name, description
B6;129P2-Apoa1 tm1Unc /J (JAX® 002055)
B6.129P2-Apoe tm1Unc /J (JAX® 002052) backcrossed to B6
Hemizygous male offspring of hemizygous B6;SJL-Tg(Mt1-LDLR)93-4Reh/AgnJ (JAX® #008850) males mated with B6SJLF1/J (JAX® #100012) females
Wild-type male littermates of Tg(hLDLR)
B6.129S7-Ldlr tm1Her /J (JAX® 002207) backcrossed to B6
B6;129S6-Pcsk9 tm1Jdh /J (JAX® 005993) backcrossed to B6
Pcsk9 KO IRCM
B6 mice that lack the Pcsk9 proximal promoter and exon 1 region
Apoe KO mice crossed to Pcsk9 KO IRCM mice
Ldlr KO mice crossed to Pcsk9 KO IRCM mice
HDL cholesterol measurement
At 8 and 16 weeks of age, mice were fasted from 07:00 am to 11:00 am and then retro-orbitally bled; 100–150 μl of blood was collected in a 1.5 ml tube for serum and in a 1.5 ml tube containing 5 μl of 200 μM ethylenediaminetetraacetic acid (EDTA) for plasma. Serum or plasma was isolated by centrifugation at 15,000 rpm for five minutes at room temperature within two hours of the bleed. Collected supernatant was stored at −20°C until the HDL cholesterol concentration was measured by the HDLD assay, using an enzymatic reagent kit (Beckman Coulter Inc., Palo Alto, CA) on a Beckman Synchron DXC (Beckman Coulter Inc., Palo Alto, CA). The HDL method used for HDLD assay was validated in mice and was used in our previous publications [32, 33]. At three months of age, plasma samples were obtained from Pcsk9/Ldlr double-KO, Pcsk9/Apoe double-KO, Pcsk9 KO IRCM, Ldlr KO, Apoe KO, and B6 males as previously described . At 34 weeks of age, non-fasting serum HDL cholesterol concentration was measured in the group of Pcsk9 KO and control mice that were fed an atherogenic diet for 10 weeks .
Preparation of non-HDL depleted serum (NHDS) and identification of APOE and APOA1
At eight weeks of age, mice were singly housed for four days. Blood was collected and serum was isolated as described above. The NHDS was collected using one-tenth volume of the chemical precipitation reagent containing dextran sulfate (10 g/L), magnesium ions (500 mM), and non-reactive ingredients with sodium azide (0.1%), where the binding of the reagent to serum precipitates the LDL and VLDL . After precipitation, the NHDS was collected and then total protein concentration (μg/μl) was determined using a Bradford assay (Sigma Life Sciences, St. Louis, MO). Fifteen μg of proteins in NHDS were run on a 4-30% non-denaturing polyacrylamide gradient gel , and proteins bands were visualized by Coomassie Brilliant Blue R-250. The molecular weight of the different bands was determined using a molecular weight kit (14–500 kDa) (Sigma Life Sciences, St. Louis, MO) according to the manufacturer’s instructions. To identify proteins, Coomassie Brilliant Blue R-250-stained bands were cut into 1 mm3 cubes, proteins in each band were digested in trypsin solution, and the tryptic peptides were subjected to LC-MS/MS. Mass spectrometry data were collected and MS spectra were searched against an IPI mouse protein sequence database (version 3.75) using SEQUEST (Bioworks software, v3.3.1; Thermo Electron) .
Proteins in whole liver were prepared in protein extraction buffer (T-PER reagent, Roche, Indianapolis, IN) and a protease inhibitors cocktail tablet (Roche, Indianapolis, IN). Fifteen μg of proteins in NHDS were electrophoresed by SDS-PAGE or native-PAGE, and western blotting was performed using antibodies for APOE (ab20874, polyclonal rabbit primary 1/1,000, Abcam, Cambridge, MA), APOA1 (ab20453, polyclonal rabbit primary 1/1,000, Abcam, Cambridge, MA), ABCA1 (MAB10005, monoclonal mouse primary 1/750, EMD Millipore, Temecula, CA), β actin (ab8227, polyclonal rabbit primary 1/25,000, Abcam, Cambridge, MA) and secondary antibodies for anti-rabbit IgG (7074S, HRP-linked secondary 1/5,000, Cell Signaling Technology Inc., Danvers, MA) and for anti-mouse IgG (AP308P, HRP-linked secondary 1/5,000, EMD Millipore, Temecula, CA). Protein levels were calculated by the protein band intensity that was obtained using ImageJ 1.44o (National Institute of Health, Bethesda, MD).
Quantitative PCR of Apoe
Total RNA in liver was extracted using a Trizol Plus RNA Purification kit (Invitrogen Life Technologies, Grand Island, NY). Complementary DNA (cDNA) was synthesized using an Omniscript RT kit (Qiagen, Valencia, CA) and used for qPCR with SYBR green (Applied Biosystems, Inc., Foster City, CA) and Apoe primers (Forward: 5′ AACCGCTTCTGGGATTACCTG 3′ and Reverse: 5′ TCAGTTCTTGTGTGACTTGGGA 3′ from Primerdesign Ltd., Southampton, UK). The Apoe expression level was normalized by Gapdh (Forward: 5′ TGGTGAAGGTCGGTGTGAAC 3′ and Reverse: 5′ CAATGAAGGGGTCGTTGATGG 3′ from Primerdesign Ltd., Southampton, UK). Relative expression differences were obtained using LinRegPCR (v11.0)  and the Relative Expression Software Tool (REST©) .
In vitro assessment of cholesterol efflux capacity of serum from macrophage foam cells
THP-1 and J774A.1 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in ATCC-formulated DMEM based media and RPMI-1640, respectively, at 5% CO2 atmosphere at 37°C. Low passage cells (p0–2) were used throughout the experiment. Both cell types were treated with 10 nM of phorbol myristate acetate (PMA) to differentiate into macrophages. As described previously , the differentiated macrophages were treated with 10 μM of fluorescent cholesterol mimic (F-cholesterol)  and 50 μg/ml oxidized LDL (Biomedical Technologies, Inc., Stoughton, MA) to induce macrophage foam cell formation. The macrophage foam cells were used to perform a cell-based, high-throughput screening assay for cholesterol efflux. Five μl of serum was added in 100 μl of cell culture medium (n = 5 per strain) in triplicate and left for 48 hours for J774A.1 macrophage foam cells and 1 hour for THP-1 macrophage foam cells. Fluorescent cholesterol mimic (F-cholesterol) in cells and media was measured in separate wells at 485/535 excitation/emission wavelengths. Cholesterol efflux capacity (%) was calculated as F-cholesterol efflux (%) = F-cholesterol in medium ∕ (F-cholesterol in medium + F-cholesterol in cells) × 100.
Histological assessment of atherosclerotic fatty streak volume
Fatty streak volume was assessed as previously described  with minor modifications. In brief, 24-week-old Pcsk9 KO and control males were fed an atherogenic diet for 10 weeks. Supplementation of 0.5% cholic acid in the diet increases intestinal cholesterol absorption, which then accelerates atherosclerosis development. Hearts were collected, embedded in optimal cutting temperature (OCT) compound, sliced in 10-μm thick sections, placed on glass slides and fixed in 10% formalin. Lipids and esterified cholesterol were stained with oil red O and counter-stained with Mayer’s hematoxylin . Images of slides were digitalized using a Nanozoomer (Hamamatsu, Bridgewater, NJ). To identify the identical histological region for all animals, the area in the aortic sinus where the coronary artery and ascending aorta join was used as a landmark. For each animal, individual digitalized images of 12 sections above and 12 sections below the landmark were loaded into FIJI (NIH, Bethesda, WD) and saved as Z-stacks. These Z-stacks were loaded into AutoAligner (Bitplane AG, Zürich, Switzerland) to align sections and then opened in Imaris (Bitplane AG, Zürich, Switzerland) to reconstruct 3-dimensional images and calculate the average volume of atherosclerotic fatty streak (mm3).
All data represent the mean ± SEM from the number of animals of each group. For comparisons of two groups, levels of significance were calculated with the two-sample t-test using JMP9 (SAS Institute, Inc., Cary, NC).
We thank Joanne Currer, Kyle Beauchemin and Dr. Kevin Mills for manuscript preparation; Beverly Macy for animal care and serum collection; Sue Grindle for HDL cholesterol measurement; Nick Gott and Mark Lessard for histological assessment of fatty streak lesion volume assay; Will Schott and Ted Duffy for flow-cytometry; Susan Sheehan, Kenneth Walsh, Christina Caputo, Ann Chamberland and Anna Roubtsova for general technical support. This work was supported by HL081162, HL077796 and HL095668 from the National Heart, Lung and Blood Institute and by the Canadian Institutes of Health Research grants 82946 and 102741. The Proteomics Core Facility is supported by the Vermont Genetics Network through NIH grant 8P20GM103449 from the INBRE program of the National Institute of General Medical Sciences (NIGMS) and the National Center for Research Resources (NCRR). BRP thanks the NIH (CA83831) for financial support. AP was supported by the Canadian Institutes of Health Research grants 82946 and 102741.
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