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Beyond LDL-C lowering: Distinct molecular sphingolipids are good indicators of proprotein convertase subtilisin/kexin type 9 (PCSK9) deficiency

Open AccessPublished:April 26, 2013DOI:https://doi.org/10.1016/j.atherosclerosis.2013.03.029

      Highlights

      • Characteristic molecular lipid changes for PCSK9 inhibition observed with sophisticated molecular lipidomics technology
      • Distinct molecular lipid species rather than LDL-C may serve as specific efficacy readout for PCSK9 inhibition.
      • New insight to PCSK9 inhibition and its consequences.
      • This paper challenges the current view of LDL-C as the one and only gauge of lipid lowering.

      Abstract

      Objectives

      Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has been proposed to be a potential new therapeutic target for treatment of hypercholesterolaemia. However, little is known about the effects of PCSK9 inhibition on the lipidome.

      Methods

      We performed molecular lipidomic analyses of plasma samples obtained from PCSK9-deficient mice, and serum of human carriers of a loss-of-function variant in the PCSK9 gene (R46L).

      Results

      In both mouse and man, PCSK9 deficiency caused a decrease in several cholesteryl esters (CE) and short fatty acid chain containing sphingolipid species such as CE 16:0, glucosyl/galactosylceramide (Glc/GalCer) d18:1/16:0, and lactosylceramide (LacCer) d18:1/16:0. In mice, the changes in lipid concentrations were most prominent when animals were given regular chow diet. In man, a number of molecular lipid species was shown to decrease significantly even when LDL-cholesterol was non-significantly reduced by 10% only. Western diet attenuated the lipid lowering potency of PCSK9 deficiency in mice.

      Conclusions

      Plasma molecular lipid species may be utilized for characterizing novel compounds inhibiting PCSK9 and as sensitive efficacy markers of the PCSK9 inhibition.

      Keywords

      1. Introduction

      Plasma low-density lipoprotein cholesterol (LDL-C) is an established risk factor for coronary artery disease (CAD). Generally, high blood cholesterol levels are treated with statins. However, in some patients, LDL-C treatment goals are not achieved with statins alone and in other patients, aggressive treatment may cause intolerable muscular side effects [
      • Sirtori C.R.
      • Mombelli G.
      • Triolo M.
      • Laaksonen R.
      Clinical response to statins: mechanism(s) of variable activity and adverse effects.
      ,
      • Fernandez G.
      • Spatz E.S.
      • Jablecki C.
      • Phillips P.S.
      Statin myopathy: a common dilemma not reflected in clinical trials.
      ]. Thus, new LDL-C lowering methods are needed for efficient treatment of hypercholesterolaemia.
      It has been shown that proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a central role in the regulation of plasma LDL-C levels [
      • Abifadel M.
      • Varret M.
      • Rabes J.P.
      • et al.
      Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.
      ]. PCSK9, which belongs to a family of serine proteases, the proprotein convertases [
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • et al.
      The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
      ], decreases the availability of LDL receptors (LDLR) expressed on the hepatocyte surface by facilitating their lysosomal degradation [
      • Horton J.D.
      • Cohen J.C.
      • Hobbs H.H.
      PCSK9: a convertase that coordinates LDL catabolism.
      ]. Intriguingly, individuals with loss-of-function mutations in the PCSK9 gene have reduced plasma LDL-C levels and also have lower risk for CAD [
      • Cohen J.
      • Pertsemlidis A.
      • Kotowski I.K.
      • Graham R.
      • Garcia C.K.
      • Hobbs H.H.
      Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.
      ,
      • Cohen J.C.
      • Boerwinkle E.
      • Mosley Jr., T.H.
      • Hobbs H.H.
      Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.
      ,
      • Benn M.
      • Nordestgaard B.G.
      • Grande P.
      • Schnohr P.
      • Tybjaerg-Hansen A.
      PCSK9 R46L, low-density lipoprotein cholesterol levels, and risk of ischemic heart disease: 3 independent studies and meta-analyses.
      ]. Interestingly, even a modest LDL-C reduction seems to confer significant cardiovascular protection on the loss-of-function subject. In contrast, gain-of-function mutations in the PCSK9 gene have been shown to be associated with elevated plasma LDL-C levels and premature CAD [
      • Abifadel M.
      • Varret M.
      • Rabes J.P.
      • et al.
      Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.
      ]. These observations have positioned PCSK9 as a potential target in the treatment of hypercholesterolaemia [
      • Seidah N.G.
      • Prat A.
      The proprotein convertases are potential targets in the treatment of dyslipidemia.
      ].
      Some unexpected effects of PCSK9 in lipid and lipoprotein metabolism including increased apoB synthesis and triglyceride secretion have been recorded in the transgenic mouse model [
      • Soutar A.K.
      Unexpected roles for PCSK9 in lipid metabolism.
      ]. Thus, we assumed that the effect of PCSK9 inhibition may not be limited only to LDL-C and applied a sophisticated molecular lipidomics workflow to investigate the effect of PCSK9 deficiency on plasma lipidomes both in mice and humans in order to identify characteristic molecular lipid changes of PCSK9 inhibition. Based on the results of this study we propose that distinct molecular lipid species rather than LDL-C may serve as specific efficacy readout for PCSK9 inhibitors and potentially serve also as indicators of off-target effects.

      2. Materials

      2.1 Animals and treatments

      All the procedures were approved by the bioethics committee for animal care of Clinical Research Institute of Montreal. C57BL/6 wild-type (Wt) mice were obtained from the Jackson Laboratory. The hepatocyte-specific PCSK9 homozygote knock-out (Pcsk9−/−) and PCSK9 heterozygote knock-out (Pcsk9+/−) animals mice were generated as described earlier [
      • Zaid A.
      • Roubtsova A.
      • Essalmani R.
      • et al.
      Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low-density lipoprotein receptor degradation and critical role in mouse liver regeneration.
      ]. Each group had 18 male mice aged 3 months. Mice were first on regular chow (2018 Teklad Global, Harlan Laboratories) diet for two weeks after which three mice from each group were sacrificed for another study on tissue lipidomics (day 15). The remaining mice were switched to standard Western (TD.88137 Harlan Teklad) diet for a period of two weeks (day 30). The Western diet contained 34%, 21%, and 0.2% of sugar, fat, and cholesterol, respectively, whereas the regular chow diet contained 5%, 6%, and 0% of these ingredients.

      2.2 Mouse sample preparation

      Mice were kept fasted for 4 h before bleeding. Cheek bleeds of about 250 μl were drawn using the 500 μl microcontainers (BD) containing EDTA. The blood samples were centrifuged at 3000 rpm for 15 min at 4 °C. The supernatants (50–100 μl) were transferred to clean Eppendorf tubes. The samples were frozen immediately upon sampling and stored at −80 °C prior to lipidomic analyses.

      2.3 Clinical samples

      To investigate the translation from mouse to man, human serum samples obtained from the participants of the Ludwigshafen Risk and Cardiovascular Health (LURIC) Study (n = 988) were analysed for molecular lipids. LURIC is an ongoing prospective study of environmental, biochemical, and genetic risk factors for CAD in a hospital-based cohort of Caucasians [
      • Winkelmann B.R.
      • Marz W.
      • Boehm B.O.
      • et al.
      Rationale and design of the LURIC study–a resource for functional genomics, pharmacogenomics and long-term prognosis of cardiovascular disease.
      ]. The study was approved by the institutional review board at the "Ärztekammer Rheinland-Pfalz". Informed written consent was obtained from each of the participants. LURIC study subjects have earlier been genotyped on the Affymetrics 6.0 array. We pulled out PSCK9 genotypic data for the known loss-of-function mutation (rs 11591147, R46L) [
      • Abifadel M.
      • Varret M.
      • Rabes J.P.
      • et al.
      Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.
      ] from the LURIC database (n = 22).

      3. Methods

      3.1 Lipid extraction

      For Shotgun lipidomic analyses 10 μl of mouse plasma (n = 1 per sample) was used for lipid extraction. For quantification of ceramides and cerebrosides 50 μl of mouse plasma (n = 1) was used lipid extraction. Briefly, lipids were extracted using a modified Folch lipid extraction [
      • Ekroos K.
      Unraveling glycerophospholipidomes by lipidomics.
      ] performed on a Hamilton Microlab Star robot (Hamilton Robotics, Switzerland) [
      • Jung H.R.
      • Sylvanne T.
      • Koistinen K.M.
      • Tarasov K.
      • Kauhanen D.
      • Ekroos K.
      High throughput quantitative molecular lipidomics.
      ]. Samples were spiked with known amounts of non-endogeneous synthetic internal standards purchased from Larodan Fine Chemicals, Avanti Polar Lipids, Matreya, and CDN Isotopes/QMX Laboratories. After lipid extraction, samples were reconstituted in chloroform:methanol (1:2, v/v) and synthetic external standards were post-extract spiked to the extracts. For quantification of free cholesterol, an aliquot of each lipid extract was treated with acetyl chloride to derivatize FC to modified cholesteryl ester species according to Liebisch et al. [
      • Liebisch G.
      • Binder M.
      • Schifferer R.
      • Langmann T.
      • Schulz B.
      • Schmitz G.
      High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).
      ]. The extracts were stored at −20 °C prior to MS analysis.

      3.2 Mass spectrometric analyses

      In Shotgun lipidomics, lipid extracts were analysed on a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500) equipped with a robotic nanoflow ion source (NanoMate HD) according to Ståhlman and colleagues [
      • Stahlman M.
      • Ejsing C.S.
      • Tarasov K.
      • Perman J.
      • Boren J.
      • Ekroos K.
      High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry.
      ]. Molecular lipids were analysed in both positive and negative ion modes using multiple precursor ion scanning (MPIS) and neutral loss scanning (NLS) based methods [
      • Liebisch G.
      • Binder M.
      • Schifferer R.
      • Langmann T.
      • Schulz B.
      • Schmitz G.
      High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).
      ,
      • Ekroos K.
      • Chernushevich I.V.
      • Simons K.
      • Shevchenko A.
      Quantitative profiling of phospholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer.
      ,
      • Ekroos K.
      • Ejsing C.S.
      • Bahr U.
      • Karas M.
      • Simons K.
      • Shevchenko A.
      Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation.
      ].
      Sphingolipids were analysed by reverse phase ultra-high pressure liquid chromatography (UHPLC) using an Acquity BEH C18, 2.1 × 50 mm column with a particle size of 1.7 μm (Waters, Milford, Massachusetts, USA). A 25 min gradient using 10 mM ammonium acetate in water with 0.1% formic acid (mobile phase A) and 10 mM ammonium acetate in acetonitrile:2-propanol (4:3, v/v) containing 0.1% formic acid (mobile phase B) was used. Sphingolipids were monitored on a 4000 Q TRAP® mass spectrometer (Applied Biosystems/MDS Analytical Technologies, Concord, Ontario, Canada) equipped with an UHPLC system: CTC PAL autosampler (Leap Technologies, Carrboro, NC, USA) and Rheos Allegro UHPLC (Flux Instruments AG, Basel, Switzerland) using multiple reaction monitoring (MRM) [
      • Merrill A.H.J.
      • Sullards M.C.
      • Allegood J.C.
      • Kelly S.
      • Wang E.
      Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry.
      ].

      3.3 Data processing and quality controlling

      The MS data files were processed using Lipid Profiler™ and MultiQuant™ softwares for producing a list of lipid names and peak areas. For each platform, a stringent cutoff was applied for separating background noise from actual lipid peaks. Each sample was controlled and only accepted when fulfilling the acceptance criteria. Masses and counts of detected peaks were converted into a list of corresponding lipid names. Lipids were normalized to their respective internal standard and sample volume to retrieve their concentrations. The concentrations of molecular lipids are presented as μM for plasma.
      Quality control (QC) samples were utilized to monitor the overall quality of the lipid extraction and mass spectrometry analyses [
      • Jung H.R.
      • Sylvanne T.
      • Koistinen K.M.
      • Tarasov K.
      • Kauhanen D.
      • Ekroos K.
      High throughput quantitative molecular lipidomics.
      ]. The QC samples were mainly used to remove technical outliers and lipid species that were detected below the lipid class based lower limit of quantification (LLOQ).

      3.4 Statistical analyses

      Wilcoxon rank-sum tests were conducted for comparing the plasma lipid profiles of the three mouse genotype groups on chow and Western diet. Lipid class concentrations were calculated by summing up the concentrations of corresponding molecular lipids averaged across the technical replicates. Statistical analyses of the clinical samples were performed using the unpaired Student t-test for LURIC dataset and paired t-test for analysis of statin trial samples. A p < 0.05 was considered significant. All analyses are performed using SAS 9.2 (SAS Institute).

      4. Experimental results

      4.1 PCSK9 deficiency resulted in reduction of the majority of lipid classes in plasma and liver in mice on chow diet

      In Pcsk9−/− and Pcsk9+/− mice on regular chow diet a decrease in most lipid classes was observed (Fig. 1A). In contrast, triacylglycerols (TAG) were shown to be significantly elevated by more than 50% in the plasma of both knockout (KO) strains when compared to the wild-type (Wt) mice. While most lipid changes in Pcsk9+/− mice were similar to the Pcsk9−/− the phosphatidylcholine (PC), lysophosphatidylcholine (LPC), sphingosine-1-phosphate (S1P), and sphinganine-1-phosphate (SA1P) levels did not decrease in the heterozygote KO mice. The absolute concentration values for each lipid class quantified in mouse plasma samples are presented in Supplement Table 1.
      Figure thumbnail gr1
      Fig. 1Differences in lipid class median concentrations in plasma. Samples between the PCSK9−/− mice and wildtype and the PCSK9+/− and wildtype mice on regular chow diet (A) and western diet (B). The significance of a change is marked as *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 for plasma samples (n ≥ 10). CE, cholesteryl ester; FC, free cholesterol; TAG, triacylglycerol; DAG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC O, alkyl-linked phosphatidylcholine; PC P, alkenyl-linked phosphatidylcholine; PE O, alkyl-linked phosphatidylethanolamine; PE P, alkenyl-linked phosphatidylethanolamine; SM, sphingomyelin; Cer, ceramide; Glc/GalCer, glucosyl/galactosylceramide; LacCer, lactosylceramide; SPH, sphingosine; S1P, sphingosine-1-phosphate; SPA, sphinganine; SA1P, sphinganine-1-phosphate.

      4.2 Western diet attenuated lipid lowering potency of PCSK9 deficiency

      On Western diet, Pcsk9−/− mice had only a small effect on plasma lipids when summed up at the lipid class level. Major changes in this setting were observed as significant elevations in sphingosine (SPH) and sphinganine (SPA) concentrations (Fig. 1B). However, Pcsk9+/− mice demonstrated greater and more significant plasma lipid responses to Western diet than Pcsk9−/− mice. While the SPH, SPA, S1P, and SA1P increased significantly up to 67%, Cer, glucosyl/galactosylceramides (Glc/GalCer), and LacCer decreased from 25% to 44% in Pcsk9+/− mouse plasma as compared to Wt (Fig. 1B). Furthermore, elevated plasma cholesteryl ester (CE, +40%) and FC (+21%) concentrations were recorded in Pcsk9+/− mouse plasma (Fig. 1B).

      4.3 Sphingolipids with short fatty acyl chains act as readout for PCSK9 inhibition

      Next, the effect of PCSK9 deficiency on molecular lipid species was studied in order to evaluate whether lipid class level observations are applicable to all molecular lipid species, or whether species specificity can be observed within each lipid class. In this analysis, especially short chain fatty acid species such as palmitic acid (C 16:0) containing sphingomyelin [SM(d18:1/16:0)], ceramide [Cer(d18:1/16:0)], glucosyl/galactosylceramide [Glc/GalCer(d18:1/16:0)], and lactosylceramide [LacCer(d18:1/16:0)] species appeared as the most affected molecular lipid species in both Pcsk9−/− and Pcsk9+/− mice when the animals were on regular chow diet (Fig. 2, respectively).
      Figure thumbnail gr2
      Fig. 2Percentage differences in molecular lipid median concentrations in plasma of PCSK9−/− mice on regular chow (A) and western (B) diet as compared to wildtype (WT).
      On Western diet a typical change in the plasma of PCSK9-deficient mice on Western diet appeared to be decreased Cer and Glc/GalCer species with long fatty acyl chains such as the Cer(d18:1/26:1), the Cer(d18:1/24:0), the Glc/GalCer(d18:1/26:1) and the Glc/GalCer(d18:1/24:0) (Fig. 2).

      4.4 Human PCSK9 loss-of-function carriers have lipid profiles comparable to Pcsk9−/− mice

      Next, we investigated the translation of the mouse findings to man. We analysed serum samples from human carriers of the known loss-of-function variant in the PCSK9 gene (R46L, n = 22). This genetic variation resulted in a 10.1% non-significant reduction of plasma LDL-C in non-statin treated subjects as compared to major allele carriers (Fig. 3). Intriguingly, the magnitude of reduction was much higher for many plasma molecular lipids species. In these subjects, the most significantly reduced lipid species included the palmitic and stearic acid containing CE, Glc/GalCer, LacCer, and SM species as observed also in the Pcsk9−/− and Pcsk9+/− mouse models (Fig. 2). In contrast to PCSK9 KO mice, some cholesteryl ester species including the CE 20:3 and the CE 20:4 were significantly reduced in fasting human serum due to the PCSK9-deficiency (−29% and −26%, respectively).
      Figure thumbnail gr3
      Fig. 3The percentage differences in molecular lipid median concentrations in serum of males (n = 22) who carry the R46L variant of the PCSK9 gene in comparison to the major allele carriers (n = 966). Each point corresponds to a lipid molecule.

      5. Discussion

      In the present experimental setting, PCSK9-deficiency resulted in decreased concentrations of a large variety of molecular lipid species in mouse plasma, especially when mice were on regular chow diet. Molecular lipidomic analyses of human serum from subjects carrying the R46L loss-of-function mutation of the PCSK9 gene also showed substantial lipid species reductions. In particular, distinct sphingolipid species were reduced by the PCSK9-deficiency in both mouse and man. Based on these observations one could suggest that there is an interaction between LDL receptor up-regulation and the levels of circulating ceramides. This could be due to subsequent inhibition of the Sterol Regulatory Element Binding Protein −1 and −2 mediated gene transcription and reduction in liver fatty acid synthesis due to increased hepatic uptake of lipids via LDL receptors [
      • Brito G.C.
      • Andrews D.W.
      Removing bias against membrane proteins in interaction networks.
      ,
      • Raghow R.
      • Yellaturu C.
      • Deng X.
      • Park E.A.
      • Elam M.B.
      SREBPs: the crossroads of physiological and pathological lipid homeostasis.
      ].
      The short chain fatty acid containing glucosyl/galactosylceramides, lactosylceramides, and sphingomyelins were good indicators of PCSK9 deficiency. This suggests that the short chain fatty acid containing sphingolipids, with the observed good translational potency, could be used as sensitive readout of PCSK9 inhibition. However, under high fat dietary conditions PCSK9 inhibition seemed less efficient and thus, it may be possible that dietary factors and metabolic states affecting lipid metabolism may also significantly modify lipid lowering effects in patients treated with PCSK9 inhibitors. Furthermore, we recorded significant increases in plasma TAG concentration due to PCSK9 deficiency in mouse models. However, this was not reproduced in human carriers of the loss-of-function mutation and, therefore, we believe that increase in plasma TAG was specific to used animal model and cannot be translated to human response to PCSK9 inhibition.
      It should be remembered that the studied loss-of-function mutation causes only modest reduction in plasma LDL-C levels and therefore it is of importance to validate the present results by analysing plasma of patients under treatment with PCSK9 inhibitors. Furthermore, the lipidomic profiles were evaluated in both mice and humans with a genetic defect or mutation, which may have induced some compensatory variation of genes involved in lipid metabolism during the embryogenesis. Thus, the effect observed in this sample does not necessarily reflect the effect that might be expected to see in patients treated with PCSK9 inhibitors.
      It is important to note that PCSK9 loss-of-function mutations may confer cardiovascular protection on the carriers without massive LDL-C reductions [
      • Cohen J.
      • Pertsemlidis A.
      • Kotowski I.K.
      • Graham R.
      • Garcia C.K.
      • Hobbs H.H.
      Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.
      ,
      • Cohen J.C.
      • Boerwinkle E.
      • Mosley Jr., T.H.
      • Hobbs H.H.
      Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.
      ,
      • Benn M.
      • Nordestgaard B.G.
      • Grande P.
      • Schnohr P.
      • Tybjaerg-Hansen A.
      PCSK9 R46L, low-density lipoprotein cholesterol levels, and risk of ischemic heart disease: 3 independent studies and meta-analyses.
      ]. Thus, extreme LDL-C reductions may in fact not be needed as it may be equally important to decrease plasma levels of low abundant, biologically active, molecular lipids such as distinct sphingolipids [
      • Cohen J.C.
      • Boerwinkle E.
      • Mosley Jr., T.H.
      • Hobbs H.H.
      Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.
      ,
      • Benn M.
      • Nordestgaard B.G.
      • Grande P.
      • Schnohr P.
      • Tybjaerg-Hansen A.
      PCSK9 R46L, low-density lipoprotein cholesterol levels, and risk of ischemic heart disease: 3 independent studies and meta-analyses.
      ]. Similar findings have earlier been reported by Meikle et al., who studied healthy subjects, stable CAD patients and acute coronary syndrome patients [
      • Meikle P.J.
      • Wong G.
      • Tsorotes D.
      • et al.
      Plasma lipidomic analysis of stable and unstable coronary artery disease.
      ]. They concluded that lipidomic markers may contribute to a new approach to risk stratification for unstable CAD. However, another explanation to cardiovascular protection could be the lifelong exposure to genetically pre-disposed low LDL-C levels in PCSK9 loss-of-function mutation carriers [
      • Lambert G.
      Unravelling the functional significance of PCSK9.
      ].
      In conclusion, PCSK9 inhibition results in diet dependant molecular lipid changes. Distinct sphingolipid species may be utilized for characterizing novel compounds inhibiting PCSK9 and as sensitive efficacy markers of the PCSK9 inhibition.

      Disclosure

      M.J., K.T., H.X.T., K.E., R.H., and R.L. are employees of Zora Biosciences Oy.

      Acknowledgements

      The research leading to these results has received funding from Finnish Funding Agency for Technology and Innovation , TEKES and the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 201668 ; AtheroRemo. Part of this work was supported by CIHR grants to NGS and AP (# MOP 102741 and # CTP 82946 ) and a Canada chair to NGS (#: 216684 ). The study was also supported with grants from the Competitive Research Funding of the Tampere University Hospital (Grant 9M048 and 9N035 for T.L.), the Emil Aaltonen Foundation (T.L.), and the Finnish Foundation for Cardiovascular Research (T.L).

      Appendix

      Supplement Table 1Plasma concentrations total lipid classes in wildtype, PCSK9 /− and PCSK9+/− mouse.
      Lipid classPlasma
      Regular chowWestern
      Concentration (μM)PCSK9−/− vs. WTPCSK9+/− vs. WTConcentration (μM)PCSK9−/− vs. WTPCSK9+/− vs. WT
      WTPCSK9−/−PCSK9+/−p-valuep-valueWTPCSK9−/−PCSK9+/−p-valuep-value
      CE2841.3861589.1932448.5654.37E-061.52E-013545.1753886.8865087.3973.79E-015.89E-02
      FC969.580496.730793.5774.37E-064.42E-031315.2761328.2161594.6824.73E-015.78E-01
      TAG252.737409.153397.9703.23E-02219.778277.4652.30E-01
      PC2231.9561567.5262243.2198.64E-064.58E-012681.4663083.0423440.2203.79E-011.47E-01
      PE8.5377.4908.6332.41E-019.70E-016.4597.1326.652
      PI24.22314.66621.2353.88E-042.62E-0124.91830.69426.5352.41E-015.39E-01
      PC P5.5073.6664.6705.19E-0112.2559.08610.1452.36E-014.63E-01
      LPC491.050401.294507.4091.23E-027.43E-01459.511508.983547.0663.79E-011.63E-01
      LPE4.6753.2785.4091.53E-018.04E-014.4552.6564.2218.94E-01
      SM69.54038.68656.2021.42E-051.81E-0260.25253.03272.5522.73E-014.64E-01
      Cer5.0343.1573.9362.64E-057.21E-027.8946.2634.4871.30E-011.36E-03
      Glc/GalCer9.7314.7187.3871.22E-057.89E-0317.89915.9729.9493.42E-013.40E-03
      LacCer0.1900.1100.1558.42E-073.06E-020.3190.2980.2391.93E-019.26E-04
      SPH0.1430.1710.1512.15E-019.08E-010.1390.1970.2291.51E-022.81E-05
      S1P0.0760.0680.0822.01E-011.00E+000.0640.0770.1061.49E-019.07E-05
      SPA0.0230.0210.0189.18E-015.20E-010.0210.0310.0342.21E-021.42E-04
      SA1P0.0030.0020.0037.57E-019.34E-010.0030.0030.0042.45E-011.42E-04

      References

        • Sirtori C.R.
        • Mombelli G.
        • Triolo M.
        • Laaksonen R.
        Clinical response to statins: mechanism(s) of variable activity and adverse effects.
        Annu Mediaev. 2011; 44: 419-432
        • Fernandez G.
        • Spatz E.S.
        • Jablecki C.
        • Phillips P.S.
        Statin myopathy: a common dilemma not reflected in clinical trials.
        Cleve Clin J Med. 2011; 78: 393-403
        • Abifadel M.
        • Varret M.
        • Rabes J.P.
        • et al.
        Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.
        Nat Genet. 2003; 34: 154-156
        • Seidah N.G.
        • Benjannet S.
        • Wickham L.
        • et al.
        The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
        Proc Natl Acad Sci U S A. 2003; 100: 928-933
        • Horton J.D.
        • Cohen J.C.
        • Hobbs H.H.
        PCSK9: a convertase that coordinates LDL catabolism.
        J Lipid Res. 2009; 50: S172-S177
        • Cohen J.
        • Pertsemlidis A.
        • Kotowski I.K.
        • Graham R.
        • Garcia C.K.
        • Hobbs H.H.
        Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.
        Nat Genet. 2005; 37: 161-165
        • Cohen J.C.
        • Boerwinkle E.
        • Mosley Jr., T.H.
        • Hobbs H.H.
        Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.
        N Engl J Med. 2006; 354: 1264-1272
        • Benn M.
        • Nordestgaard B.G.
        • Grande P.
        • Schnohr P.
        • Tybjaerg-Hansen A.
        PCSK9 R46L, low-density lipoprotein cholesterol levels, and risk of ischemic heart disease: 3 independent studies and meta-analyses.
        J Am Coll Cardiol. 2010; 55: 2833-2842
        • Seidah N.G.
        • Prat A.
        The proprotein convertases are potential targets in the treatment of dyslipidemia.
        J Mol Med. 2007; 85: 685-696
        • Soutar A.K.
        Unexpected roles for PCSK9 in lipid metabolism.
        Curr Opin Lipidol. 2011; 22: 192-196
        • Zaid A.
        • Roubtsova A.
        • Essalmani R.
        • et al.
        Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low-density lipoprotein receptor degradation and critical role in mouse liver regeneration.
        Hepatology. 2008; 48: 646-654
        • Winkelmann B.R.
        • Marz W.
        • Boehm B.O.
        • et al.
        Rationale and design of the LURIC study–a resource for functional genomics, pharmacogenomics and long-term prognosis of cardiovascular disease.
        Pharmacogenomics. 2001; 2: S1-S73
        • Ekroos K.
        Unraveling glycerophospholipidomes by lipidomics.
        in: Wang F. Biomarker methods in drug discovery and development. Humana Press, 2008: 369-384
        • Jung H.R.
        • Sylvanne T.
        • Koistinen K.M.
        • Tarasov K.
        • Kauhanen D.
        • Ekroos K.
        High throughput quantitative molecular lipidomics.
        Biochim Biophys Acta. 2011; 1811: 925-934
        • Liebisch G.
        • Binder M.
        • Schifferer R.
        • Langmann T.
        • Schulz B.
        • Schmitz G.
        High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).
        Biochim Biophys Acta. 2006; 1761: 121-128
        • Stahlman M.
        • Ejsing C.S.
        • Tarasov K.
        • Perman J.
        • Boren J.
        • Ekroos K.
        High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry.
        J Chromatogr B Analyt Technol Biomed Life Sci. 2009; 877: 2664-2672
        • Ekroos K.
        • Chernushevich I.V.
        • Simons K.
        • Shevchenko A.
        Quantitative profiling of phospholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer.
        Anal Chem. 2002; 74: 941-949
        • Ekroos K.
        • Ejsing C.S.
        • Bahr U.
        • Karas M.
        • Simons K.
        • Shevchenko A.
        Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation.
        J Lipid Res. 2003; 44: 2181-2192
        • Merrill A.H.J.
        • Sullards M.C.
        • Allegood J.C.
        • Kelly S.
        • Wang E.
        Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry.
        Methods. 2005; : 207-224
        • Brito G.C.
        • Andrews D.W.
        Removing bias against membrane proteins in interaction networks.
        BMC Syst Biol. 2011; 5: 169
        • Raghow R.
        • Yellaturu C.
        • Deng X.
        • Park E.A.
        • Elam M.B.
        SREBPs: the crossroads of physiological and pathological lipid homeostasis.
        Trends Endocrinol Metab TEM. 2008; 19: 65-73
        • Meikle P.J.
        • Wong G.
        • Tsorotes D.
        • et al.
        Plasma lipidomic analysis of stable and unstable coronary artery disease.
        Arteriosclerosis, Thromb Vasc Biol. 2011; 31: 2723-2732
        • Lambert G.
        Unravelling the functional significance of PCSK9.
        Curr Opin Lipidol. 2007; 18: 304-309