Arteriosclerosis, Thrombosis, and Vascular Biology

CLINICAL AND POPULATION STUDIES

Overfeeding Saturated Fat Increases LDL (Low-

Density Lipoprotein) Aggregation Susceptibility
While Overfeeding Unsaturated Fat Decreases
Proteoglycan-Binding of Lipoproteins
Maija Ruuth ,* Mari Lahelma,* Panu K. Luukkonen, Martina B. Lorey , Sami Qadri, Sanja Sädevirta, Tuulia Hyötyläinen ,
Petri T. Kovanen, Leanne Hodson, Hannele Yki-Järvinen,† Katariina Öörni †

OBJECTIVE: We recently showed that measurement of the susceptibility of LDL (low-density lipoprotein) to aggregation is
an independent predictor of cardiovascular events. We now wished to compare effects of overfeeding different dietary
macronutrients on LDL aggregation, proteoglycan-binding of plasma lipoproteins, and on the concentration of oxidized LDL
in plasma, 3 in vitro parameters consistent with increased atherogenicity.

APPROACH AND RESULTS: The participants (36 subjects; age, 48±10 years; body mass index, 30.9±6.2 kg/m2) were randomized
to consume an extra 1000 kcal/day of either unsaturated fat, saturated fat, or simple sugars (CARB) for 3 weeks. We
measured plasma proatherogenic properties (susceptibility of LDL to aggregation, proteoglycan-binding, oxidized LDL)
and concentrations and composition of plasma lipoproteins using nuclear magnetic resonance spectroscopy, and in LDL
using liquid chromatography mass spectrometry, before and after the overfeeding diets. LDL aggregation increased in the
saturated fat but not the other groups. This change was associated with increased sphingolipid and saturated triacylglycerols
in LDL and in plasma and reduction of clusterin on LDL particles. Proteoglycan binding of plasma lipoproteins decreased
in the unsaturated fat group relative to the baseline diet. Lipoprotein properties remained unchanged in the CARB group.

CONCLUSIONS: The type of fat during 3 weeks of overfeeding is an important determinant of the characteristics and functional
properties of plasma lipoproteins in humans.

REGISTRATION: URL: [Link] Unique identifier NCT02133144.

GRAPHIC ABSTRACT: A graphic abstract is available for this article.

Key Words: atherosclerosis ◼ dietary fats ◼ low-density lipoprotein ◼ nutrition ◼ sphingomyelin

A
ccumulation of LDL (low-density lipoprotein)- enzymatic, proteolytic, and oxidative modifications, which
derived lipids in the arterial wall induces the devel- can induce aggregation of the modified LDL particles.2,3
opment of atherosclerotic lesions.1 LDL particles Both modified and aggregated lipoproteins are found in
enter the arterial intima from the circulation and, upon atherosclerotic lesions.4–6 Aggregation of modified LDL
encountering the tight network of extracellular matrix particles can contribute to atherogenesis by enhancing
in the arterial intima, are retained by intimal PGs (pro- LDL retention, by inducing foam cell formation, and by
teoglycans). The retained lipoproteins are exposed to promoting inflammation in the arterial intima.2,7–10

Correspondence to: Katariina Öörni, PhD, Wihuri Research Institute, Haartmaninkatu 8, 00290 Helsinki, Finland. Email [Link]@[Link]
*M. Ruuth and M. Lahelma share first authorship.
†H. Yki-Järvinen and K. Öörni share senior authorship.
The Data Supplement is available with this article at [Link]
For Sources of Funding and Disclosures, see page 2834.
© 2021 The Authors. Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health,
Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and
reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.
Arterioscler Thromb Vasc Biol is available at [Link]/journal/atvb

Arterioscler Thromb Vasc Biol. 2021;41:2823–2836. DOI: 10.1161/ATVBAHA.120.315766 November 2021   2823
 Ruuth et al Diet and Functional Properties of Lipoproteins
CLINICAL AND POPULATION

Nonstandard Abbreviations and Acronyms Highlights

STUDIES - AL

apo apolipoprotein • Three-week overfeeding of saturated fats but not

CARB simple carbohydrates unsaturated fats or simple sugars induces signifi-
DNL de novo lipogenesis cant metabolic changes in plasma lipoproteins.
• Overfeeding of saturated fats increases aggrega-
HDL high-density lipoprotein
tion susceptibility of LDL (low-density lipoprotein)
IDL intermediate-density lipoprotein particles.
LC-MS liquid-chromatography-mass • Overfeeding of unsaturated fats decreases binding
spectrometry of plasma lipoproteins to proteoglycans.
LDL low-density lipoprotein • Overfeeding of simple sugars does not change LDL
NMR nuclear magnetic spectroscopy aggregation susceptibility, proteoglycan binding of
plasma lipoproteins, or concentration of oxidized LDL.
oxLDL oxidized LDL
• The source of macronutrients during 3 weeks of
PG proteoglycan overfeeding is an important determinant of the prop-
SAT saturated fat erties of plasma lipoproteins in humans.
UNSAT unsaturated fat
VLDL very low-density lipoprotein
lipids (phosphatidylcholines, sphingomyelins, lysophospha-
tidylcholines, and ceramides). Aggregation-prone LDL par-
The importance of the interaction of LDL and PGs in ticles have a higher sphingomyelin and ceramide content.8
atherogenesis has been shown by mutating apoB (apo- Interestingly, plasma concentrations of sphingomyelins
lipoprotein B)-100 or by vaccination using antibodies are higher in patients with coronary artery disease than in
directed against the glycosaminoglycan chains of PGs.11,12 matched controls.28 Similarly, increased concentrations of
The affinity of LDL to PGs varies depending on the proper- plasma ceramides predict cardiovascular deaths.29,30 Since
ties of the LDL particles, particularly those that affect their a large proportion of plasma sphingolipids are carried by
charge, as recently reviewed.13 In addition, the presence of LDL particles, their increased plasma concentrations
apoE or apoC-III, 2 small exchangeable apolipoproteins, largely reflect their content in LDL particles.
can influence the PG-binding of plasma lipoproteins, apoE Sphingomyelins are synthesized de novo mostly
by interacting directly with glycosaminoglycans and apoC- from palmitoyl-CoA in a reaction catalyzed by serine-
III via an unknown mechanism.14–16 Patients with cardio- palmitoyl-transferase, with the last step in the synthesis
vascular disease or type 2 diabetes have LDL, which has of sphingomyelin being the addition of phosphocholine
higher affinity toward PGs than control subjects.17–20 This head group to ceramides. Therefore, consumption of diet
increased affinity can be lowered by treatment with cho- rich in palmitate and other saturated fats (SATs) could
lesterol-lowering medications and plant stanol esters.21–23 be expected to increase sphingolipids levels. Indeed, in
Oxidative modifications of LDL induce generation of mouse models, high-fat diet feeding has been found
oxidized phospholipids and other lipid mediators that are to increase both sphingomyelin and ceramide levels in
considered to be important in inflammation and athero- plasma.31,32 Interestingly, it was recently shown in humans
genesis.24 Oxidized LDL (oxLDL) particles are found in that overfeeding saturated but not unsaturated fat
atherosclerotic lesions,5,25 and the concentration of circu- (UNSAT) or carbohydrates, increased plasma concentra-
lating oxLDL particles is increased in patients with car- tions of ceramides and LDL cholesterol.33 The impact of
diovascular disease.24 overfeeding different macronutrients on proatherogenic
Oxidation and other modifications of LDL particles lead- properties of LDL have not, however, been studied.
ing to their chemical and structural changes can induce their Here, we examined effects of overfeeding 3 different
aggregation.2 We recently developed an assay to determine diets on the binding of LDL to PGs, levels of oxLDL in
the aggregation susceptibility of LDL particles and showed plasma, and the aggregation susceptibility of LDL par-
that this propensity varies significantly from donor to donor.8 ticles, 3 parameters associated with increased athero-
Although LDL aggregation does not correlate with LDL- genicity. In addition, we examined how the diets altered
cholesterol concentration, the aggregation susceptibility is plasma lipoproteins and LDL lipid and protein composi-
higher in children having familial hypercholesterolemia com- tion as determined nuclear magnetic resonance (NMR)
pared with normocholesterolemic children.26 Importantly, spectroscopy and liquid chromatography-mass spec-
the presence of aggregation-prone LDL in circulation was trometry (LC-MS).
shown in 2 independent studies to predict future cardio-
vascular events independently from classical risk factors for
atherosclerotic cardiovascular disease.8,27 MATERIALS AND METHODS
LDL particles consist of hydrophobic core lipids (cho- The data that support the findings of this study are available
lesteryl esters and triacylglycerols) and amphiphilic surface from the corresponding author upon reasonable request.

2824   November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2823–2836. DOI: 10.1161/ATVBAHA.120.315766
Ruuth et al Diet and Functional Properties of Lipoproteins

Subjects Binding of Plasma Lipoproteins to Proteoglycans

CLINICAL AND POPULATION

Subjects for this study were recruited by advertisements or by The binding of plasma lipoproteins to PGs was measured in
contacting subjects who had previously participated in meta- 96-well plates that had been coated with PG isolated from

STUDIES - AL
bolic studies. The recruitment and screening procedures and human aortas as previously described.22,37
exclusion criteria have been described previously33 and in the
Study flow chart (Figure I in the Data Supplement). Written
informed consent was obtained from all volunteers. The Ethics
Measurement of oxLDL
Committee of the Helsinki University Hospital had approved OxLDL concentrations were measured in human plasma
the study protocol and study was conducted in accordance with samples with the Mercodia, (Uppsala, Sweden) OxLDL sand-
the Declaration of Helsinki. wich ELISA (product No. 10-1143-01), which includes the
mouse monoclonal antibody 4E6 directed against a confor-
mational epitope in oxidized ApoB-100, following the manu-
Study Design facturer’s protocol.
Study participants were randomized to 3 groups to consume
extra 1000 kcal/day for 3 weeks in addition to their habit-
NMR Spectrometry
ual diet. The extra calories were predominately derived from
Plasma samples at baseline and after the interventions were
UNSAT group, SAT group, or simple sugars (CARB group) as
analyzed using a high-throughput NMR platform (AVANCE
shown in Table I in the Data Supplement.
500 MHz and AVANCE III 600 MHz, Bruker, Karlsruhe,
Compliance to the diet was monitored by food diaries and
Germany).38 The platform quantifies over 220 metabolites
by measuring the fatty acid composition of VLDL-TGs (very
including total lipids, TGs, phospholipids, total cholesterol, free
low-density lipoprotein triacylglycerols), which reflect hepatic
cholesterol, esterified cholesterol, apolipoproteins, 14 lipopro-
fatty acid composition in blood samples taken after an over-
tein subclasses and their composition, as well as other low-
night fast.34
molecular weight metabolites such as amino acids, glycolysis
De novo lipogenesis (DNL) was measured using gas chro-
related metabolites, and ketone bodies, as described in detail
matography–mass spectrometry based incorporation of deu-
in study by Würtz et al.38
terium from 2H2O in plasma water (Finnigan Gas-Bench-II;
Thermo Fisher Scientific, Loughborough, United Kingdom) into
VLDL-triacylglycerol palmitate as described in detail in study by Lipidomic Analyses by LC-MS
Luukkonen et al.33 Absolute DNL was calculated by multiplying
LDL samples were extracted using a modified version of
%DNL and the concentration of triacylglycerol in VLDL.35 the previously published Folch procedure.39 Briefly, 10 µL
The primary outcomes in the current study were LDL aggre- of 0.9% NaCl, 40 µL of CHCl3:MeOH (2:1, v/v), and 80 µL
gation, LDL oxidation, and PG binding of plasma lipoproteins. of the 2.25 µg/mL internal standard solution of chosen lipid
standards (for quality control and normalization purposes) were
LDL Isolation added to each 10 µL plasma sample. The internal standard
LDL (d=1.019–1.063 g/mL) was isolated from plasma sam- solution contained the following compounds: 1,2-diheptadec-
ples by D2O-based sequential ultracentrifugation.36 An aliquot anoyl-sn-glycero-3-phosphoethanolamine (PE [17:0/17:0]),
of 300 µL of plasma was used for the isolation, and 300 µL of N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine
LDL was collected after ultracentrifugation. The concentration (sphingomyelin [d18:1/17:0]), N-heptadecanoyl-D-
of LDL was measured using the Pierce BCA Protein Assay Kit erythro-sphingosine (Cer [d18:1/17:0]), 1,2-dihep-
(Thermo Scientific, Rockford). tadeca-noyl-sn-glycero-3-phosphocholine (PC [17:0/17:0]),
1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC
[17:0]), and 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phospho-
Susceptibility of LDL to Aggregation choline (PC [16:0/d31/18:1]). These were purchased from
The susceptibility of isolated LDL particles to aggregation was mea- Avanti Polar Lipids, Inc (Alabaster, AL). In addition, triheptadec-
sured essentially as described before.8,23 Briefly, isolated LDL sam- anoin (triacylglycerol [17:0/17:0/17:0]) was purchased from
ples were first diluted to a concentration of 200 µg of LDL protein/ (Larodan AB, Solna, Sweden). Samples were vortexed and incu-
mL. Aggregation was induced by addition of human recombinant bated on ice for 30 minutes after which they were, along with 60
acid SMase produced in-house23 to the LDL samples, and LDL µL from the lower layer of each sample, collected, and 60 µL of
aggregation was then determined by dynamic light scattering using CHCl3:MeOH (2:1, v/v) added to each sample.
Wyatt DynaPro Plate Reader II (Wyatt Technology) with paraffin The UHPLC-quadrupole time of flight mass spectrometer
coating. This allowed us to follow the increase in the apparent LDL analyses were performed as described earlier40 with some
size (ie, aggregate size) as a time-varying parameter. Aggregation modifications. The UHPLC-quadrupole time of flight mass
of LDL particles was followed by measuring particle size at various spectrometer system was from Agilent Technologies (Santa
time points for 6 hours and data were collected with Dynamics V7. Clara, CA) combining 1290 Infinity system and 6545 quadru-
LDL aggregate size data were analyzed with GraphPad Prism ver- pole time of flight mass spectrometer, interfaced with a dual jet
sion 8.0.1 (GraphPad Software, La Jolla, CA). The maximum aggre- stream electrospray (dual ESI) ion source. MassHunter B.06.01
gate size was limited to 3000 nm and the minimum size to 14 software (Agilent Technologies, Santa Clara, CA) was used for
nm. LDL aggregation curves were fitted with nonlinear regression all data acquisition and MZmine 2 was used for data process-
curve fit of the time versus aggregate size curves to determine their ing.41 Lipid identification was based on in-house spectral library
inflection points as a measure of LDL aggregation. with retention times. Lipids were normalized with lipid-class

Arterioscler Thromb Vasc Biol. 2021;41:2823–2836. DOI: 10.1161/ATVBAHA.120.315766 November 2021   2825
 Ruuth et al Diet and Functional Properties of Lipoproteins

specific internal standards and (semi) quantitation was per- ≥54% to the reference run were selected for further analy-
CLINICAL AND POPULATION

formed using lipid-class specific calibration curves. sis. The relative quantification was done with 1 peptide per
protein. Sample pools and individual samples were directly
STUDIES - AL

compared as before and after interventions, only quantitations

Proteomic Analyses by LC-MS/MS with P<0.05 were accepted, and known contaminants such
The proteins were digested in Amicon Ultra-0.5 centrifu- as keratins were excluded.
gal filters using a modified filter-assisted sample preparation
method.42,43 In brief, reduction and alkylation of samples were
achieved by the addition of tris (2-carboxyethyl) phosphine and Statistical Analyses
iodoacetamide to a final concentration of 2 and 50 mmol/L, The data are presented as mean and SD. Differences in out-
respectively, followed by incubation in the dark for 30 minutes. comes between the 3 diet groups were determined using
For digestion, trypsin in 50 mmol/L ammonium bicarbonate was 2-way ANOVA with repeated measures in which a signifi-
added in a ratio of 1:50 w/w and incubated overnight at room cant time×group interaction denotes a statistically significant
temperature. The peptides were cleaned using C18-reverse- change by the interventions. Tukey post hoc multiple compari-
phase ZipTipTM (Millipore), dried, re-suspended in 1% trifluo- sons test was used for between-group comparisons. Within-
roacetic acid, and sonicated in a water bath for 1 minute before group comparisons were performed with Šidak post hoc test.
injection into a nano-LC Q Exactive Plus LC-MS/MS system Before analysis, the data were log transformed. ANCOVA was
(Thermo Scientific). The peptides were separated on an Easy- used to analyze the potential effect of variation in baseline val-
nLC system (Thermo Scientific) equipped with a reverse-phase ues between the groups on changes in response to the inter-
trapping column Acclaim PepMapTM 100 (C18, 75 μm×20 vention, and 1-way ANOVA to compare absolute week 3 (after
mm, 3 μm particles, 100 Å; Thermo Scientific), followed by an intervention) values between the groups. The Spearman cor-
analytical Acclaim PepMapTM 100 RSLC reversed-phase col- relation coefficient was used to analyze the relationship LDL
umn (C18, 75 µm×250 mm, 2 µm particles, 100 Å; Thermo lipid composition and LDL aggregation susceptibility. False
Scientific). The injected sample analytes were trapped at a flow discovery rate correction was performed using the Benjamini
rate of 2 µL×min−1 in 100% of solution A (0.1 % formic acid). and Hochberg test. The principal component analysis was
After trapping, the peptides were separated with a linear gradi- performed for multivariable LDL lipidomic data to investigate
ent of 120 minutes comprising 96 minutes from 3% to 30% of the lipid species mainly responsible for the variation between
solution B (0.1% formic acid/80% acetonitrile), 7 minutes from samples. Before analysis, the lipidomic data were normalized
30% to 40% of solution B, and 4 minutes from 40% to 95% by median, log transformed, and Pareto scaled. The tests were
of solution B. Each sample run was followed by 2 empty runs performed using IBM SPSS Software (version 25.0, North
to reduce carryover. Castle, NY), GraphPad Prism version 8.0.1, or MetaboAnalyst
LC-MS acquisition data were acquired with the following software 4.0 (Xia Lab, McGill University, Montreal, QC, CA).44
settings: the resolution was set to 140 000 for MS scans, and P<0.05 was considered to be statistically significant.
17 500 for the MS/MS scans. A full MS was acquired from 350
to 1400 m/z, and the 10 most abundant precursor ions were
selected for fragmentation with 30 second dynamic exclusion RESULTS
time. Ions with 2+, 3+, and 4+ charges were selected for MS/
MS analysis. Secondary ions were isolated with a window of 1.2 Study Design and Compliance to the Diet
m/z. The MS AGC target was set to 3×106 counts, whereas the We overfed 36 overweight subjects for 3 weeks with
MS/MS AGC target was set to 1×105. Dynamic exclusion was 1000 extra kcal/day of either SAT, UNSAT, or simple sug-
set with a duration of 20 seconds. The NCE collision energy
ars (CARB).33 The baseline caloric intake of the partici-
was set to 28 kJ×mol–1.
Following LC-MS/MS acquisition, the raw files were quali-
pants in different groups was similar (ANOVA, P=0.613),
tatively analyzed by Proteome Discoverer (version 2.4, Thermo and the mean intakes were 2120±520 kcal (n=9) in
Scientific, United States). The identifications were performed the UNSAT group, 2040±880 kcal in the SAT group
against the Swissprot Human protein database (Human (n=13), and 2070±440 kcal in the CARB group (n=12).
Swissprot downloaded on January 21, 2021, with 20 406 At the end of the 3-week diet, the caloric intake based
entries) using the built-in SEQUEST HT engine. The following on the food diaries of the participants was 3030±700
parameters were used: 10 ppm and 0.02 Da were tolerance kcal in the UNSAT group, 3030±1010 kcal in the SAT
values set for MS and MS/MS, respectively. Trypsin was used group, and 3150±570 kcal in the CARB group (ANOVA,
as the digesting enzyme, and 2 missed cleavages were allowed. P=0.880). The changes in macronutrients in the 3
The carbamidomethylation of cysteines was set as a fixed mod- groups are shown in Figure II in the Data Supplement.
ification, while the oxidation of methionine and deamidation of
Compliance was assessed by measuring changes in the
asparagine and glutamine were set as variable modifications.
The false discovery rate was set to <0.01, and a peptide mini-
fatty acid composition of the VLDL-TGs. As expected,
mum length of 6 amino acids. the percentage of saturated fatty acids in VLDL-TGs
Relative quantification between samples using precursor increased in the SAT group (Figure III in the Data Supple-
ion intensities was performed with Progenesis QI (V 3.0) soft- ment). Consistent with carbohydrate-induced increase in
ware (Nonlinear Dynamics/Waters) and ProteinLynx Global de novo lipogenesis,33 which produces exclusively satu-
Server (PLGS V3.0). Chromatograms were aligned by the rated fatty acids,34,45 the percentage of saturated VLDL-
Progenesis QI software, and those with alignment scores TGs also increased in the CARB group (Figure III in the

2826   November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2823–2836. DOI: 10.1161/ATVBAHA.120.315766
Ruuth et al Diet and Functional Properties of Lipoproteins

Data Supplement). In the SAT group, the proportions of HDL-cholesterol increased (Figure 1). In addition, in the

CLINICAL AND POPULATION

both mono- and polyunsaturated VLDL-triacylglycerol SAT group but not in the other groups, concentrations of
fatty acids decreased (Figure III in the Data Supple- particles, total lipids, phospholipids, total and free choles-

STUDIES - AL
ment). DNL was similar between the groups at baseline terol, and cholesterol esters increased in XS-VLDL, IDL
(ANOVA, P=0.55). As expected, DNL increased during (intermediate-density lipoprotein), L-LDL, S-LDL, and all
CARB overfeeding (from 90±62 µmol/L [mean±SD] sizes of HDL particles (Figure IV in the Data Supplement).
before to 202±168 µmol/L after, P=0.05), but not dur-
ing UNSAT (53±45 µmol/L before versus 80±152
µmol/L after, P=0.59) or SAT (65±66.3 µmol/L before Overfeeding SAT Increases the Susceptibility of
versus 132.4±237.4 µmol/L after, P=0.24) overfeeding. LDL to Aggregate
Aggregation of LDL particles was induced with human
recombinant SMase and particle aggregation was followed
Diet-Induced Changes in Clinical and Metabolic by measuring the aggregate size at various time points.
Characteristics The aggregate size versus time curves at baseline and
The clinical characteristics of the study subjects are after the diet (Figure 2A) show that the UNSAT and CARB
shown in the Table. The groups were similar at baseline diets did not influence the susceptibility of LDL particles to
with respect to common cardiovascular disease risk fac- aggregate, while in the SAT group LDL aggregated much
tors, including BMI, blood pressure, age, and plasma lip- faster after the intervention than at baseline. The inflec-
ids. Weight gain was small and similar in all groups in tion point of the aggregate size versus time curves is used
response to overfeeding (Table) and averaged 1.2±0.2 as a measure of the aggregation susceptibility of the LDL
kg (1.3±0.2%). Concentrations of total cholesterol, LDL- particle and the faster the particles aggregate, the shorter
cholesterol, HDL (high-density lipoprotein)-cholesterol, is the time required to reach the inflection point (EC50)
and non-HDL-cholesterol increased during the SAT of the aggregation curves. At baseline, EC50 was similar
diet (Table). Plasma levels of triglycerides and glucose between the UNSAT (4.4±1.3 hour, mean±SD) and SAT
remained unchanged in all groups. (3.9±1.3 hour) groups and lower (2.6±1.2 hour, P<0.05)
Quantitative NMR profiling was used to analyze in in the CARB group. The change in the inflection point of
detail diet-induced changes in lipids and lipoproteins and the aggregation curve in each group is shown in Figure 2B.
metabolites. In the SAT group, the proportion of satu- There was a significant within-group difference in aggre-
rated fatty acids and concentrations of sphingomyelins, gation (P value for 2-way ANOVA Time×Group interac-
phosphoglycerides, cholines, apolipoprotein A1, and tion term=0.0077), which occurred in the SAT group. The

Table. Clinical Characteristics of the Study Subjects in UNSAT, SAT, and CARB Groups at Baseline and After the 3-wk Dietary
Intervention

UNSAT SAT CARB

After After After

Group Baseline intervention† Baseline intervention† Baseline intervention† P value*
Number (women/men) 11 (6/5) 13 (8/5) 12 (6/6) 0.842
Age, y 51±10 48±8 45±10 0.310
BMI, kg/m2 30.6±6.0 30.8±6.1 29.4±6.4 29.9±6.5‡ 32.8±6.0 33.2±6.0‡ 0.568
SBP, mm Hg 133±17 134±18 133±15 131±18 139±20 137±10 0.799
DBP, mm Hg 83±8 81±11 80±11 84±14 85±13 82±8 0.773
Cholesterol, mmol/L 5.3±0.8 5.2±0.5 5.1±1.3 5.6±1.1§ 5.4±0.9 5.2±1.1 0.001
HDL-C, mmol/L 1.6±0.5 1.7±0.5 1.6±0.4 1.9±0.5§ 1.5±0.4 1.4±0.3 0.001
LDL-C, mmol/L 3.4±0.8 3.3±0.7 3.1±1.1 3.5±1.0‡ 3.5±0.8 3.5±1.0 0.022
Non-HDL-C, mmol/L 3.7±0.9 3.5±0.7 3.4±1.4 3.7±1.2¶ 3.8±0.9 3.8±1.0 0.019
LDL-C/HDL-C 2.5±1.2 2.3±1.3 2.1±1.1 2.0±1.1 2.4±0.8 2.6±0.8 0.070
TG, mmol/L 1.1±0.4 1.2±0.5 1.1±1.0 1.1±0.8 1.4±0.6 1.4±0. 8 0.492
Glucose, mmol/L 5.7±0.6 5.6±0.7 5.5±0.6 5.6±0.5 5.9±0.7 6.0±0.6 0.629

Data are shown as mean±SD. Two-way ANOVA with repeated measures was used to test differences between groups during interventions. BMI indicates body
mass index; CARB, simple carbs; DBP, diastolic blood pressure; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; SAT, saturated fat; SBP, systolic blood pressure; TG,
triacylglycerol; and UNSAT, unsaturated fat.
*P values are reported for the effect of interaction term (time×group).
†Asterisks in the column represent P value for within-group changes calculated by Šidak post hoc test, except for sex (χ2 test) and age (1-way ANOVA).
‡P<0.01.
§P<0.001.
¶P<0.05.

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 Ruuth et al Diet and Functional Properties of Lipoproteins
CLINICAL AND POPULATION
STUDIES - AL

Figure 1. Change in the nuclear magnetic spectroscopy metabolomic profile during the 3-wk overfeeding of unsaturated fat
(UNSAT), saturated fat (SAT), and simple carbs (CARB).
Metabolomic analysis was performed from plasma samples collected at baseline and at the end of the intervention. The log-transformed
metabolite concentrations were compared using paired 2-sided Student t test with false discovery rate method described by Benjamini and
Hochberg. Differences were calculated by subtracting the mean before-value from the mean after-value. Thus, a positive difference indicates an
increase and a negative difference a decrease in the mean metabolite concentration or proportion. UNSAT, n=11; SAT, n=13; CARB, n=12.
ApoA1 indicates apolipoprotein A1; CARB, simple carbs; HDL, high-density lipoprotein; and SFA%, percentage of saturated fatty acids.

increase in LDL aggregation in the SAT group was also of oxLDL was 50.0±10.0 mU/L (mean±SD) in UNSAT
highly significant in ANCOVA (P=0.002) with baseline group, 40.3±15.1 mU/L in SAT group, and 44.9±10.6
aggregation as the covariate. At week 3 (after interven- mU/L in CARB group. After 3 weeks of overfeeding,
tion), absolute values of EC50 were statistically signifi- concentration of oxLDL was 42.7±7.6 mU/L in UNSAT,
cantly different between the UNSAT (4.4±1.2 hour), SAT 42.2±14.8 mU/L in SAT, and 44.8±12.6 mU/L in CARB
(2.6±0.8 hour), and CARB group (2.6±0.9 hour) (ANOVA, group. No statistically significant changes in oxLDL con-
P=0.004). Tukey post hoc test showed lower aggrega- centration were observed (Figure 2D), and there were no
tion in the UNSAT group compared with the SAT group significant differences in absolute week 3 (after inter-
(P=0.001) and CARB group (P=0.001), and no difference vention) values.
between the SAT and CARB group (P=0.981).

Effects of Overfeeding Diets on LDL Lipid and

Overfeeding UNSATs Decreases Proteoglycan- Protein Composition
Binding of Plasma Lipoproteins and Reduces Effects of the overfeeding diets on the lipid composition
oxLDL of LDL particles were analyzed in detail by LC-MS. Sig-
Effects of the different overfeeding diets on binding of nificant changes were seen only in SAT group, in which
plasma lipoproteins to human aortic PGs were measured in the core lipids of LDL the proportions of saturated TGs
using a solid-phase binding assay in which aliquots of increased while unsaturated TGs decreased (Figure V in
serum samples were incubated in PG-coated microtiter the Data Supplement).
wells. At baseline, PG-binding was 2.9±0.5 nmol/L per The lipid species of LDL detected by LC-MS were next
well (mean±SD) in UNSAT group, 2.2±0.5 nmol/L/well used as loadings in principal component analysis of LDL
in SAT group, and 2.5±0.4 nmol/L/well in CARB group. samples at baseline and after the diet period. The first and
After 3 weeks of overfeeding, PG-binding was 2.4±0.5 second principal component scores showed that at the
nmol/L/well in UNSAT, 2.3±0.5 nmol/L/well in SAT, and end of overfeeding, LDL lipid compositions of the groups
2.4±0.3 nmol/L/well in CARB group. The log2FC in PG- were diverged compared with baseline (Figure 3A and
binding in each group are shown in Figure 2C. There were 3B). Particularly, the UNSAT and the SAT groups were bet-
no significant differences in absolute week 3 (after inter- ter separated after the overfeeding period than at base-
vention) values in PG-binding between the groups, but line. The first and second principal components explained
the change from baseline in PG-binding was significantly 43.2% of the variance between samples (Figure 3B).
different between the groups (P value for 2-way ANOVA The first component had large positive associations with
interaction term=0.0077). Post hoc analysis showed the sphingomyelins, ceramides, lysophosphatidylcholines,
difference was because of a significant decrease in the cholesterol esters, and negative associations with unsat-
UNSAT group with no change in the other groups. urated TGs and phosphatidylcholines, while the second
We also measured the effect of the diets on the con- component had large negative associations with satu-
centrations of oxLDL in plasma. At baseline, concentration rated and monounsaturated TGs (Figure 3C).

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Figure 2. Changes in LDL (low-density lipoprotein) aggregation, lipoproteins proteoglycan-binding, and oxidized LDL (oxLDL)
during the 3-wk overfeeding of unsaturated fat (UNSAT), saturated fat (SAT), and simple carbs (CARB).
A, The aggregate sizes (median±interquartile range) at the various time points are shown in the UNSAT, SAT, and CARB groups. LDL was isolated
from plasma samples collected at the baseline and at the end of the diet. LDL aggregation was induced by addition of human recombinant
sphingomyelinase, and the increase in LDL aggregate size was followed by dynamic light scattering. Inflection point of the aggregate size vs time
curves was used as a measure of LDL aggregation susceptibility. B, Change in LDL aggregation susceptibility. C, Change in the proteoglycan-
binding of plasma lipoproteins. Aliquots of plasma samples collected at the baseline and at the end of the study were incubated in microtiter
wells coated with human aortic proteoglycans and the amount of cholesterol bound was measured after incubation for 1 h. D, Change in the
concentration of oxLDL. ELISA was used to measure oxLDL concentrations in plasma samples collected at the baseline and at the end of the
intervention. The changes in B–D are presented as mean±SE of mean of the log2 values of fold changes. A positive value indicates an increase
and a negative value a decrease in the value. Two-way ANOVA with repeated measures was used to test differences between groups during
interventions. P values (at the bottom) are reported for the effect of interaction term (time×group). Asterisks represent P value for within-group
changes calculated by Šidak post hoc test. The data were analyzed using GraphPad Prism version 8.0.1. **P<0.01; ***P<0.001.

The protein composition of the pooled groups was proteoglycan binding, we analyzed the individual LDL
analyzed by LC-MS/MS and normalized to ApoB-100 protein compositions of 5 participants of each group.
(Figure VI in the Data Supplement). Apolipoproteins were The participants were selected based on groupwise
mildly depleted in all groups after overfeeding, except similarity with respect to baseline characteristics such
ApoA-I, ApoA-IV, and ApoE, which showed a slight as sex, age, BMI, and LDL-cholesterol, and consid-
increase in the CARB group. ering the representative changes in the primary out-
comes, LDL-aggregation, and PG-binding, of the whole
respective group (Table II in the Data Supplement). To
Proteomics Comparison of Individuals From eliminate small individual changes, only proteins were
SAT and UNSAT Group included, which were identified in at least 4 out of 5
As the SAT group showed significant changes in participants and which had the log2FC in the same
LDL aggregation and the UNSAT group decreased direction within the 5 participants if the log2 fold

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Figure 3. Changes in LDL (low-density lipoprotein) lipidomics and proteomics during the 3-wk overfeeding of unsaturated fat
(UNSAT), saturated fat (SAT), and simple carbs (CARB).
A–C, Principal component analysis (PCA) demonstrating differences in LDL lipid composition before and after 3-wk overfeeding of UNSAT,
SAT, and CARB. Lipidomic analysis was performed using liquid chromatography–mass spectrometry, and percentage concentrations of
both core and surface lipids were introduced to analysis. Before analysis, samples were normalized by median, log transformed, and Pareto
scaled. Two outliers, one in the UNSAT group and one in the SAT group, were excluded. A, The PCA scores plot before intervention. The
first and second components explained 20.7%, and 17.8% of the variance between samples, respectively. The scores of the first and second
principal components with 95% CIs are presented for UNSAT (blue), SAT (red), and CARB (green) groups. B, The PCA scores plot after
intervention. The first and second components explained 26.5%, and 17.6% of the variance between samples, respectively. C, Loadings plot
showing influence of each lipid species to the first and second principal components after intervention. Each lipid species is colored with
distinctive color. The first component had large positive associations with sphingomyelins, ceramides, lysophosphatidylcholines, cholesterol
esters, and negative associations with unsaturated TGs and phospatidylcholines, while the second component had large negative associations
with saturated and monounsaturated TGs. D and E, Changes in LDL protein composition in (D) UNSAT and (E) SAT groups. The protein
composition was analyzed by liquid-chromatography-mass spectrometry/mass spectrometry after normalization to ApoB-100, only proteins
were included which were identified in 4 out of the 5 selected individuals and the change was in the same direction (increase/decrease) for
changes over log2 fold change >0.5. The log2 fold changes in protein concentrations are presented as mean±SD. The data were analyzed
using GraphPad Prism version 8.0.1 and MetaboAnalyst software 4.0. UNSAT, n=10; SAT, n=12; CARB, n=12. CE indicates cholesterol ester;
Cer, ceramide; FC, fold change; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; SM, sphingomyelin; TG, triacylglycerol.

change was larger than 0.5, indicating a fold change a diet-induced decrease in ApoA-I, ApoA-IV, ApoC-II,
of >1.42. As in the pooled samples, the pairs of both ApoF, and clusterin (Figure 3E).
groups exhibited a relative depletion in apolipoproteins,
especially for ApoA-IV and ApoC-II (Figure 3D and 3E). Plasma NMR Profiling, LDL Lipidomics, LDL
In the UNSAT group, pairwise comparison indicated
that the LDL particles were also relatively depleted of
Aggregation, Lipoprotein PG-Binding, and
ApoC-I, ApoD, ApoE, and acute phase protein serum oxLDL
amyloid A-4 after the diet period (Figure 3D). Pairwise We next analyzed the relationship between lipoprotein
comparison of the LDL particles from the SAT groups subclasses as determined using NMR spectroscopy
showed a small diet-induced increase in complement and LDL aggregability, lipoprotein PG-binding, and
component C4-A, serum paraoxonase/arylesterase 1, oxLDL. At baseline, both PG-binding and oxLDL were
antibody component immunoglobulin kappa constant, strongly associated with several apoB-100-containing
as well as platelet-activating factor acetylhydrolase and lipoprotein subclasses (Figure 4). Thus, PG-binding and

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Figure 4. Relationships between LDL (low-density lipoprotein) aggregation susceptibility, proteoglycan-binding of plasma
lipoproteins, and plasma oxidized LDL (oxLDL) concentrations, and plasma lipoprotein subclasses at baseline.
A, LDL aggregation, (B) the binding of plasma lipoproteins to proteoglycans, and (C) concentrations of oxLDL in plasma were determined as
described under Figure 3 and Methods. Quantitative nuclear magnetic spectroscopy metabolomics was used for the quantification of plasma
lipoprotein subclasses and their components. The associations for log-transformed metabolite concentrations were analyzed using Spearman
correlation coefficient analysis. In heatmaps, the positive correlations are represented with red and negative correlations with blue. Unsaturated
fat, n=11; saturated fat, n=13; simple carbs, n=12. *P<0.05, **P<0.01, ***P<0.001.

oxLDL correlated positively with concentrations and lipid Regarding LDL core and surface lipid composition, an
components of VLDL, IDL, and LDL subclasses apart increase in sphingomyelins and decrease in the phos-
from large VLDL. HDL correlated inversely with the phatidylcholine to lysophosphatidylcholine -ratio were
concentration of oxLDL. LDL aggregation did not cor- associated with an increase in LDL aggregation dur-
relate with any of the lipoprotein subclasses or their lipid ing overfeeding (Figure 6A). An increase in many TGs
components. In contrast, LDL aggregation at baseline containing unsaturated fatty acids was associated with a
showed a strong negative correlation with the proportion decrease in PG-binding (Figure 6B) and an increase in
of phosphatidylcholines and positive correlation with the oxLDL (Figure 6C).
proportion of sphingomyelins and lysophosphatidylcho-
lines on the surface of the particles (Figure VII in the
Data Supplement). DISCUSSION
Finally, we analyzed whether diet-induced changes in The impact of different diets on cardiovascular health is
plasma NMR profiles or the composition of the LDL lipi- still intensely debated.46 As LDL-cholesterol is a causal
dome were related to LDL aggregation, lipoprotein PG- factor in the development of atherosclerotic cardiovascu-
binding, or oxLDL. We observed a significant correlation lar disease,1 most studies have focused on effects of dif-
between the change in the proportion of saturated fatty ferent diets on concentrations of plasma LDL-cholesterol.
acids in plasma and LDL aggregation: an increase in However, for any given concentration of LDL-cholesterol,
the saturated fatty acids was associated with more pathogenic properties of LDL particles, such as the affin-
rapid LDL aggregation (Figure 5A). Changes in LDL ity of LDL to bind to arterial PGs, the presence of oxLDL,
aggregation were not associated with changes in lipo- and the susceptibility of LDL to aggregation have the
protein subclasses (not shown). An increased propor- potential to influence the atherogenicity of the LDL par-
tion of saturated fatty acids was also associated with ticles.47,48 In this study, we examined the effects of over-
an increase in PG-binding (Figure 5B). The association feeding 1000 excess calories as either SATs, UNSATs, or
between the change in the proportion of saturated fatty simple sugars for 3 weeks on these qualitative aspects
acids and the change in oxLDL was not significant and of LDL particles and show that excess consumption of
is shown in Figure 5C. SATs increases LDL aggregation, while consumption of

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Figure 5. Relationships between changes in LDL (low-density lipoprotein) aggregation susceptibility, proteoglycan-binding of
plasma lipoproteins, plasma oxidized LDL (oxLDL), and the proportions of plasma saturated fatty acids concentrations during
3-wk overfeeding of the unsaturated fat (UNSAT), saturated fat (SAT), and simple carbs (CARB) diets.
A, LDL aggregation, (B) the binding of plasma lipoproteins to proteoglycans, and (C) concentrations of oxLDL in plasma, were determined as
described under Figure 3 and Methods. Quantitative nuclear magnetic spectroscopy metabolomics was used for the quantification of changes
in the proportion of saturated fatty acids (SFA%) in plasma. The associations for log2 fold changes of metabolite concentrations or proportions
were analyzed using Spearman correlation coefficient analysis. UNSAT, n=11; SAT, n=13; CARB, n=12. 1Positive value indicates decrease in
LDL aggregation, negative value increase in LDL aggregation. R, Spearman correlation coefficient; FC, fold change.

UNSATs decreases PG-binding of plasma lipoproteins did not influence LDL lipid composition or LDL aggrega-
based on change from baseline diet. tion. These differences may explain why LDL aggrega-
LDL particles aggregate in the intima after proteo- tion remained unchanged in the CARB group. Of note,
lytic, enzymatic, or oxidative modifications and the higher baseline aggregation was higher in the CARB group.
the LDL concentration the faster is LDL aggregation.8 Although the change in LDL aggregation remained
Aggregation enhances binding of LDL to intimal PGs2 highly significant after adjusting for baseline values, and
and promotes foam cell formation.7,49–51 Uptake of aggre- absolute LDL aggregation was significantly higher at 3
gated LDL also induces secretion of matrix metallopro- weeks in the SAT than in the UNSAT group, the base-
tease 7, a protease associated with plaque rupture.8,52 In line difference nevertheless complicates interpretation of
line with the pathogenic properties of aggregated LDL, changes in this parameter in the CARB group.
we recently showed that the susceptibility of plasma The interaction of lipoproteins with PGs is one of the
LDL to aggregate associates with future cardiovascu- first steps in atherogenesis.56–59 Here, overfeeding of
lar events independently of conventional risk factors for the UNSAT diet decreased binding of plasma lipopro-
atherosclerotic cardiovascular disease.8,27 LDL particles teins to PGs based on change from baseline diet. Previ-
can be rendered more stable by addition of plant sta- ous studies have also shown that change in dietary fat
nol ester-enriched spread into the diet.53 Here, we show can influence PG-binding of plasma lipoproteins or iso-
that overfeeding of SATs renders LDL particles more lated LDL particles. Thus, intake of linolenic acid (18:3,
prone to aggregate and that this change correlates ω3)-rich Camelina sativa oil and consumption of plant
with increase in the proportion of saturated fatty acids stanol esters both decrease the PG-binding of plasma
in plasma. In addition, LDL-bound clusterin was signifi- lipoproteins.53,60 Similarly, Jones et al61 have shown that
cantly decreased in the SAT group after the diet period. both canola oil rich in oleic acid and corn/safflower oil
Clusterin has been shown to inhibit LDL aggregation and rich in linoleic acid decrease binding of isolated LDL
reduce atherogenesis in a mouse model.54,55 particles to PGs. In the present study, the PG-binding
The SAT diet, but not the other diets, significantly of plasma lipoproteins was strongly associated with the
increased lipids that increase aggregation of LDL, that concentration of small VLDL, IDL, and LDL particles
is, the content of sphingolipids in LDL and plasma sphin- and their lipid components, raising the possibility that
gomyelins.8,53 Principal component analysis of the LDL in addition to LDL, small VLDL and IDL particles may
samples after the diet period clearly separated particu- also have contributed to retention of lipoproteins. In the
larly the SAT and the UNSAT groups. In the SAT group, SAT group, the amounts of these apoB-100-containing
LDL was more enriched with sphingomyelins, cerami- lipoproteins increased; yet, the binding of the lipopro-
des, and saturated TGs, while LDL in the UNSAT group teins to PGs did not change indicating that there is a
was enriched with phosphatidylcholines and unsaturated reduction in the affinity of the lipoproteins to PGs. As
TGs. In the CARB group, carbohydrate-induced increase we did not observe any changes in apoE or apoC-III of
in DNL led to less pronounced changes in % saturated LDL, the 2 small exchangeable apolipoproteins known
fatty acids in plasma than did the SAT diet (Figure 5) and to modulate the interaction of lipoproteins to PGs,14–16 it

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Figure 6. Relationships between changes in LDL (low-density lipoprotein) lipid composition and changes in LDL aggregation
susceptibility, proteoglycan-binding of plasma lipoproteins, and plasma oxidized LDL (oxLDL) concentrations during 3-wk
overfeeding of the unsaturated fat (UNSAT), saturated fat (SAT), and simple carbs (CARB) diets.
Volcano plots showing associations between changes in LDL lipids and changes in (A) LDL aggregation susceptibility, (B) proteoglycan-
binding (PG) of plasma lipoproteins, and (C) oxLDL concentrations. The associations for log2 fold changes of metabolite concentrations were
analyzed using Spearman correlation coefficient analysis. Two outliers, 1 in the UNSAT group and 1 in the SAT group, were excluded from
analysis. Changes in the same direction indicate that both the measured LDL atherogenic property and the lipid concentration increased or
decreased, and changes in the opposite direction indicate that the other of these increased and the other decreased. UNSAT, n=10; SAT,
n=12; CARB, n=12. ρ, Spearman correlation coefficient; LPC indicates lysophosphatidylcholine; PC, phosphatidylcholine; SM, sphingomyelin;
and TG, triacylglycerol.

is likely that the changes in the PG-binding depend on In accordance, we show that increased concentration of
changes in the conformation of apoB-100, which has oxLDL was associated with increase in many unsatu-
been shown to be important in the PG-binding of LDL rated TGs, which were increased after the UNSAT diet.
particles.13 In contrast, in the UNSAT group, LDL-apoE Despite this, overfeeding of the UNSAT diet induced a
decreased after the diet, a finding that could at least nonsignificant decrease rather than an increase in the
partly explain the decrease in PG-binding in this group. concentration of oxLDL in plasma. As TGs are a minor
Consumption of unsaturated fatty acids has been component of LDL particles, it is possible that diet-
reported to increase the oxidizability of LDL particles.62 induced changes in other lipids outweigh the increase in

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 Ruuth et al Diet and Functional Properties of Lipoproteins

oxidation-prone unsaturated TGs. Importantly, increase in Affiliations

CLINICAL AND POPULATION

the concentration of oxLDL does not necessarily lead to Atherosclerosis Research Laboratory, Wihuri Research Institute, Haartmaninka-
tu, Helsinki, Finland (M.R., M.B.L., P.T.K., K.Ö.). Research Programs Unit, Faculty
increased atherosclerosis at least in an animal model.63
STUDIES - AL

of Medicine, University of Helsinki, Finland (M.R.). Minerva Foundation Institute

One reason for this could be related to reduced inflam- for Medical Research, Helsinki, Finland (M.L., P.K.L., S.Q., S.S., H.Y.-J.). Depart-
matory potential of lipoproteins after consumption of ment of Medicine, University of Helsinki and Helsinki University Hospital, Fin-
land (M.L., P.K.L., S.Q., S.S., H.Y.-J.). School of Science and Technology, Örebro
UNSATs.64,65 In line with these data, we show here that University, Örebro, Sweden (T.H.). Oxford Centre for Diabetes, Endocrinology
serum amyloid A in LDL was decreased after the UNSAT and Metabolism, University of Oxford, and National Institute for Health Re-
diet. Oxidation of circulating LDL particles takes place, search Oxford Biomedical Research Centre, Oxford University Hospital Trusts,
United Kingdom (L.H.).
at least in part, during oxidative stress reactions in endo-
thelial cells, which generate reactive oxygen and nitrogen Acknowledgments
species. We show here that at baseline, the concentra- We thank Maija Atuegwu, Anne Salo, Aila Karioja-Kallio, Päivi Ihamuotila, and
Daniel Duberg for excellent technical assistance and Dr Jenny Presto (Mercodia)
tion of oxLDL was associated positively with most VLDL for providing the oxLDL measurement kit.
and LDL subclasses and negatively with HDL particles
and lipids in HDL. This finding is in line with the ability of Sources of Funding
This research was funded by Jenny and Antti Wihuri Foundation that maintains
HDL to inhibit lipid oxidation in LDL.53
the Wihuri Research Institute. This study was also supported by grants from the
This study is the first in humans to compare effects Academy of Finland (grants No. 315568 and No. 332564), Novo Nordisk Fonden
of overfeeding 3 different diets on the properties of (NNF19OC0057411), and the Finnish Foundation for Cardiovascular Research
to K. Öörn and M.B. Lorey; British Heart Foundation Senior Research Fellow-
LDL and plasma lipoproteins linked with increased ath-
ship (FS/15/56/31645) to L. Hodson; Alfred Kordelin Foundation, Jalmari and
erogenicity. Limitations of our study as well as all of Rauha Ahokas Foundation, Paulo Foundation, and Finnish Medical Foundation
other dietary intervention studies hitherto performed66,67 to P.K. Luukkonen; the Academy of Finland (grant No. 309263) and the Sigrid
Juselius, EVO and Novo Nordisk foundations to H. Yki-Järvinen.
include small sample size and a relatively short dura-
tion of the study. However, longer periods of overfeed- Disclosures
ing might be considered unethical and would likely also M. Ruuth, K. Öörni, and P.T. Kovanen have applied for a patent on the LDL (low-
decrease compliance. It is also noteworthy that total fat density lipoprotein) aggregation assay. The funders had no role in the design of
the study, in the collection, analyses, or interpretation of data, in the writing of the
intake in the SAT and the UNSAT groups was almost article, or in the decision to publish the results.
60% of total energy intake with SATs comprising 33%
and 14% of total energy, respectively.33 Intake of SAT Supplemental Materials
Data Supplement Tables I–III
the SAT group thus clearly exceeded current dietary Data Supplement Figures I–VII
recommendations.68,69 As the study was performed in
overweight subjects with caloric excess, the results may
not be applicable to normal weight individuals consum- REFERENCES
ing eucaloric diets. LDL aggregation at baseline was 1. Ference BA, Ginsberg HN, Graham I, Ray KK, Packard CJ, Bruckert E,
faster in the CARB group than in the other 2 groups, Hegele RA, Krauss RM, Raal FJ, Schunkert H, et al. Low-density lipoproteins
cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epi-
which complicates interpretation of changes LDL demiologic, and clinical studies. A consensus statement from the European
aggregation in the CARB group. Regardless, changes Atherosclerosis Society Consensus Panel. Eur Heart J. 2017;38:2459–
in aggregation remained still highly significantly differ- 2472. doi: 10.1093/eurheartj/ehx144
2. Oörni K, Pentikäinen MO, Ala-Korpela M, Kovanen PT. Aggregation,
ent between the groups after adjusting for variation fusion, and vesicle formation of modified low density lipoprotein particles:
in baseline LDL aggregation, and the only significant molecular mechanisms and effects on matrix interactions. J Lipid Res.
within-group change was observed in the SAT group. 2000;41:1703–1714.
3. Öörni K, Rajamäki K, Nguyen SD, Lähdesmäki K, Plihtari R, Lee-Rueckert
In conclusion, the present data show that overfeed- M, Kovanen PT. Acidification of the intimal fluid: the perfect storm for ath-
ing SAT induces substantial metabolic changes that lead erogenesis. J Lipid Res. 2015;56:203–214. doi: 10.1194/jlr.R050252
to modification of LDL composition and enhance the 4. Tamminen M, Mottino G, Qiao JH, Breslow JL, Frank JS. Ultrastructure of
early lipid accumulation in ApoE-deficient mice. Arterioscler Thromb Vasc
susceptibility of LDL to aggregation, independent of the Biol. 1999;19:847–853. doi: 10.1161/[Link].19.4.847
concentration of LDL cholesterol. In addition, overfeed- 5. Lehti S, Nguyen SD, Belevich I, Vihinen H, Heikkilä HM, Soliymani R,
ing UNSAT decreases the binding of plasma lipoproteins Käkelä R, Saksi J, Jauhiainen M, Grabowski GA, et al. Extracellular lipids
accumulate in human carotid arteries as distinct three-dimensional struc-
to aortic PGs from baseline diet. This property is strongly tures and have proinflammatory properties. Am J Pathol. 2018;188:525–
associated with the concentrations of small VLDL, IDL, 538. doi: 10.1016/[Link].2017.09.019
and LDL subclasses in plasma. Thus, this study shows 6. Hoff HF, Morton RE. Lipoproteins containing apo B extracted from human
aortas. Structure and function. Ann N Y Acad Sci. 1985;454:183–194. doi:
that the type of fat during overfeeding affects the prop- 10.1111/j.1749-6632.1985.tb11857.x
erties of plasma lipoproteins associated with increased 7. Tabas I, Li Y, Brocia RW, Xu SW, Swenson TL, Williams KJ. Lipopro-
atherogenicity and extends the previous data on the tein lipase and sphingomyelinase synergistically enhance the asso-
ciation of atherogenic lipoproteins with smooth muscle cells and
harmfulness of excess intake of SATs. extracellular matrix. A possible mechanism for low density lipoprotein
and lipoprotein(a) retention and macrophage foam cell formation. J Biol
Chem. 1993;268:20419–20432.
ARTICLE INFORMATION 8. Ruuth M, Nguyen SD, Vihervaara T, Hilvo M, Laajala TD, Kondadi PK,
Gisterå A, Lähteenmäki H, Kittilä T, Huusko J, et al. Susceptibility of low-
Received December 8, 2020; accepted August 17, 2021. density lipoprotein particles to aggregate depends on particle lipidome, is

2834   November 2021 Arterioscler Thromb Vasc Biol. 2021;41:2823–2836. DOI: 10.1161/ATVBAHA.120.315766
Ruuth et al Diet and Functional Properties of Lipoproteins

modifiable, and associates with future cardiovascular deaths. Eur Heart J. hypercholesterolemia display changes in LDL and HDL function: a cross-

CLINICAL AND POPULATION

2018;39:2562–2573. doi: 10.1093/eurheartj/ehy319 sectional study. J Intern Med. In press. doi: 10.1111/joim.13383
9. Haka AS, Grosheva I, Chiang E, Buxbaum AR, Baird BA, Pierini LM, 27. Heffron SP, Ruuth MK, Xia Y, Hernandez G, Äikäs L, Rodriguez C, Öörni K,
Maxfield FR. Macrophages create an acidic extracellular hydrolytic com- Berger JS. Low-density lipoprotein aggregation predicts adverse cardiovas-

STUDIES - AL
partment to digest aggregated lipoproteins. Mol Biol Cell. 2009;20:4932– cular events in peripheral artery disease. Atherosclerosis. 2021;316:53–57.
4940. doi: 10.1091/mbc.e09-07-0559 doi: 10.1016/[Link].2020.11.016
10. Singh RK, Haka AS, Bhardwaj P, Zha X, Maxfield FR. Dynamic actin 28. Jiang XC, Paultre F, Pearson TA, Reed RG, Francis CK, Lin M, Berglund L,
reorganization and Vav/Cdc42-dependent actin polymerization promote Tall AR. Plasma sphingomyelin level as a risk factor for coronary artery
macrophage aggregated LDL (low-density lipoprotein) uptake and catab- disease. Arterioscler Thromb Vasc Biol. 2000;20:2614–2618. doi:
olism. Arterioscler Thromb Vasc Biol. 2019;39:137–149. doi: 10.1161/ 10.1161/[Link].20.12.2614
ATVBAHA.118.312087 29. Laaksonen R, Ekroos K, Sysi-Aho M, Hilvo M, Vihervaara T, Kauhanen D,
11. Skålén K, Gustafsson M, Rydberg EK, Hultén LM, Wiklund O, Innerarity TL, Suoniemi M, Hurme R, März W, Scharnagl H, et al. Plasma ceramides pre-
Borén J. Subendothelial retention of atherogenic lipoproteins in early ath- dict cardiovascular death in patients with stable coronary artery disease
erosclerosis. Nature. 2002;417:750–754. doi: 10.1038/nature00804 and acute coronary syndromes beyond LDL-cholesterol. Eur Heart J.
12. Soto Y, Acosta E, Delgado L, Pérez A, Falcón V, Bécquer MA, Fraga Á, 2016;37:1967–1976. doi: 10.1093/eurheartj/ehw148
Brito V, Álvarez I, Griñán T, et al. Antiatherosclerotic effect of an antibody 30. Öörni K, Jauhiainen M, Kovanen PT. Why and how increased plasma cerami-
that binds to extracellular matrix glycosaminoglycans. Arterioscler Thromb des predict future cardiovascular events? Atherosclerosis. 2020;314:71–
Vasc Biol. 2012;32:595–604. doi: 10.1161/ATVBAHA.111.238659 73. doi: 10.1016/[Link].2020.09.030
13. Hurt-Camejo E, Camejo G. ApoB-100 lipoprotein complex formation with 31. Eisinger K, Liebisch G, Schmitz G, Aslanidis C, Krautbauer S, Buechler C.
intima proteoglycans as a cause of atherosclerosis and its possible ex vivo Lipidomic analysis of serum from high fat diet induced obese mice. Int J Mol
evaluation as a disease biomarker. J Cardiovasc Dev Dis. 2018;5:E36. doi: Sci. 2014;15:2991–3002. doi: 10.3390/ijms15022991
10.3390/jcdd5030036 32. Boini KM, Zhang C, Xia M, Poklis JL, Li PL. Role of sphingolipid mediator
14. Olin-Lewis K, Krauss RM, La Belle M, Blanche PJ, Barrett PH, Wight TN, ceramide in obesity and renal injury in mice fed a high-fat diet. J Pharmacol
Chait A. ApoC-III content of apoB-containing lipoproteins is associated with Exp Ther. 2010;334:839–846. doi: 10.1124/jpet.110.168815
binding to the vascular proteoglycan biglycan. J Lipid Res. 2002;43:1969– 33. Luukkonen PK, Sädevirta S, Zhou Y, Kayser B, Ali A, Ahonen L, Lallukka S,
1977. doi: 10.1194/jlr.m200322-jlr200 Pelloux V, Gaggini M, Jian C, et al. Saturated fat is more metabolically harm-
15. Hiukka A, Ståhlman M, Pettersson C, Levin M, Adiels M, Teneberg S, ful for the human liver than unsaturated fat or simple sugars. Diabetes Care.
Leinonen ES, Hultén LM, Wiklund O, Oresic M, et al. ApoCIII-enriched LDL 2018;41:1732–1739. doi: 10.2337/dc18-0071
in type 2 diabetes displays altered lipid composition, increased suscepti- 34. Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ.
bility for sphingomyelinase, and increased binding to biglycan. Diabetes. Sources of fatty acids stored in liver and secreted via lipoproteins in patients
2009;58:2018–2026. doi: 10.2337/db09-0206 with nonalcoholic fatty liver disease. J Clin Invest. 2005;115:1343–1351.
16. Mahley RW, Weisgraber KH, Innerarity TL. Interaction of plasma lipopro- doi: 10.1172/JCI23621
teins containing apolipoproteins B and E with heparin and cell surface 35. Santoro N, Caprio S, Pierpont B, Van Name M, Savoye M, Parks EJ. Hepatic
receptors. Biochim Biophys Acta. 1979;575:81–91. doi: 10.1016/0005- de novo lipogenesis in obese youth is modulated by a common variant in
2760(79)90133-4 the GCKR gene. J Clin Endocrinol Metab. 2015;100:E1125–E1132. doi:
17. Camejo G, Acquatella H, Lalaguna F. The interaction of low density lipopro- 10.1210/jc.2015-1587
teins with arterial proteoglycans. An additional risk factor? Atherosclerosis. 36. Hallberg C, Hådén M, Bergström M, Hanson G, Pettersson K, Westerlund C,
1980;36:55–65. doi: 10.1016/0021-9150(80)90198-7 Bondjers G, Ostlund-Lindqvist AM, Camejo G. Lipoprotein fractionation in
18. Fagerberg B, Wiklund O, Agewall S, Camejo G, Wikstrand RJ. Multifactorial deuterium oxide gradients: a procedure for evaluation of antioxidant binding
treatment of hypertensive men at high cardiovascular risk and low-density and susceptibility to oxidation. J Lipid Res. 1994;35:1–9.
lipoprotein cholesterol affinity to human arterial proteoglycans. Eur J Clin 37. Sneck M, Kovanen PT, Oörni K. Decrease in pH strongly enhances binding
Invest. 1996;26:960–965. doi: 10.1046/j.1365-2362.1996.2030543.x of native, proteolyzed, lipolyzed, and oxidized low density lipoprotein parti-
19. Linden T, Bondjers G, Camejo G, Bergstrand R, Wilhelmsen L, Wiklund O. cles to human aortic proteoglycans. J Biol Chem. 2005;280:37449–37454.
Affinity of LDL to a human arterial proteoglycan among male survivors of doi: 10.1074/jbc.M508565200
myocardial infarction. Eur J Clin Invest. 1989;19:38–44. doi: 10.1111/j. 38. Würtz P, Kangas AJ, Soininen P, Lawlor DA, Davey Smith G, Ala-Korpela M.
1365-2362.1989.tb00193.x Quantitative serum nuclear magnetic resonance metabolomics in large-
20. Garces F, López F, Niño C, Fernandez A, Chacin L, Hurt-Camejo E, Camejo G, scale epidemiology: a primer on -omic technologies. Am J Epidemiol.
Apitz-Castro R. High plasma phospholipase A2 activity, inflammation mark- 2017;186:1084–1096. doi: 10.1093/aje/kwx016
ers, and LDL alterations in obesity with or without type 2 diabetes. Obesity 39. Folch J, Lees M, Sloane Stanley GH. A simple method for the isola-
(Silver Spring). 2010;18:2023–2029. doi: 10.1038/oby.2010.9 tion and purification of total lipides from animal tissues. J Biol Chem.
21. Wiklund O, Bondjers G, Wright I, Camejo G. Insoluble complex formation 1957;226:497–509.
between LDL and arterial proteoglycans in relation to serum lipid levels 40. O’Gorman A, Suvitaival T, Ahonen L, Cannon M, Zammit S, Lewis G,
and effects of lipid lowering drugs. Atherosclerosis. 1996;119:57–67. doi: Roche HM, Mattila I, Hyotylainen T, Oresic M, et al. Identification of a plasma
10.1016/0021-9150(95)05628-9 signature of psychotic disorder in children and adolescents from the Avon
22. Ahmed O, Littmann K, Gustafsson U, Pramfalk C, Öörni K, Larsson L, Longitudinal Study of Parents and Children (ALSPAC) cohort. Transl Psy-
Minniti ME, Sahlin S, Camejo G, Parini P, et al. Ezetimibe in combination chiatry. 2017;7:e1240. doi: 10.1038/tp.2017.211
with simvastatin reduces remnant cholesterol without affecting biliary lipid 41. Pluskal T, Castillo S, Villar-Briones A, Oresic M. MZmine 2: modular
concentrations in gallstone patients. J Am Heart Assoc. 2018;7:e009876. framework for processing, visualizing, and analyzing mass spectrometry-
doi: 10.1161/JAHA.118.009876 based molecular profile data. BMC Bioinformatics. 2010;11:395. doi:
23. Ruuth M, Janssen LGM, Äikäs L, Tigistu-Sahle F, Nahon KJ, Ritvos O, 10.1186/1471-2105-11-395
Ruhanen H, Käkelä R, Boon MR, Öörni K, et al. LDL aggregation suscepti- 42. Scifo E, Szwajda A, Soliymani R, Pezzini F, Bianchi M, Dapkunas A, Dębski J,
bility is higher in healthy South Asian compared with white Caucasian men. Uusi-Rauva K, Dadlez M, Gingras AC, et al. Proteomic analysis of the pal-
J Clin Lipidol. 2019;13:910–919.e2. doi: 10.1016/[Link].2019.09.011 mitoyl protein thioesterase 1 interactome in SH-SY5Y human neuroblas-
24. Trpkovic A, Resanovic I, Stanimirovic J, Radak D, Mousa SA, Cenic- toma cells. J Proteomics. 2015;123:42–53. doi: 10.1016/[Link].2015.
Milosevic D, Jevremovic D, Isenovic ER. Oxidized low-density lipoprotein 03.038
as a biomarker of cardiovascular diseases. Crit Rev Clin Lab Sci. 2015;52: 43. Wiśniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample prepa-
70–85. doi: 10.3109/10408363.2014.992063 ration method for proteome analysis. Nat Methods. 2009;6:359–362. doi:
25. Senders ML, Que X, Cho YS, Yeang C, Groenen H, Fay F, 10.1038/nmeth.1322
Calcagno C, Meerwaldt AE, Green S, Miu P, et al. PET/MR imaging of malo- 44. Chong J, Soufan O, Li C, Caraus I, Li S, Bourque G, Wishart DS, Xia J. Metabo-
ndialdehyde-acetaldehyde epitopes with a human antibody detects clini- Analyst 4.0: towards more transparent and integrative metabolomics analysis.
cally relevant atherothrombosis. J Am Coll Cardiol. 2018;71:321–335. doi: Nucleic Acids Res. 2018;46:W486–W494. doi: 10.1093/nar/gky310
10.1016/[Link].2017.11.036 45. Sevastianova K, Santos A, Kotronen A, Hakkarainen A, Makkonen J,
26. Christensen JJ, Narverud I, Ruuth M, Heier M, Matti J, Ulven SM, Silander K, Peltonen M, Romeo S, Lundbom J, Lundbom N, et al. Effect
Bogsrud MP, Kovanen PT, Halvorsen B, Oda M. Children with familial of short-term carbohydrate overfeeding and long-term weight loss on

Arterioscler Thromb Vasc Biol. 2021;41:2823–2836. DOI: 10.1161/ATVBAHA.120.315766 November 2021   2835
 Ruuth et al Diet and Functional Properties of Lipoproteins

liver fat in overweight humans. Am J Clin Nutr. 2012;96:727–734. doi: thickenings followed by macrophage infiltration. Arterioscler Thromb Vasc
CLINICAL AND POPULATION

10.3945/ajcn.112.038695 Biol. 2007;27:1159–1165. doi: 10.1161/ATVBAHA.106.134080

46. Forouhi NG, Krauss RM, Taubes G, Willett W. Dietary fat and cardiometa- 59. Williams KJ, Tabas I. The response-to-retention hypothesis of early ath-
bolic health: evidence, controversies, and consensus for guidance. BMJ. erogenesis. Arterioscler Thromb Vasc Biol. 1995;15:551–561. doi:
STUDIES - AL

2018;361:k2139. doi: 10.1136/bmj.k2139 10.1161/[Link].15.5.551

47. Borén J, Chapman MJ, Krauss RM, Packard CJ, Bentzon JF, Binder 60. Manninen S, Lankinen M, Erkkilä A, Nguyen SD, Ruuth M, de Mello V,
CJ, Daemen MJ, Demer LL, Hegele RA, Nicholls SJ, et al. Low-density Öörni K, Schwab U. The effect of intakes of fish and Camelina sativa oil
lipoproteins cause atherosclerotic cardiovascular disease: pathophysi- on atherogenic and anti-atherogenic functions of LDL and HDL parti-
ological, genetic, and therapeutic insights: a consensus statement from cles: a randomized controlled trial. Atherosclerosis. 2019;281:56–61. doi:
the European Atherosclerosis Society Consensus Panel. Eur Heart J. 10.1016/[Link].2018.12.017
2020;41:2313–2330. doi: 10.1093/eurheartj/ehz962 61. Jones PJ, MacKay DS, Senanayake VK, Pu S, Jenkins DJ, Connelly PW,
48. Laufs U, Weingärtner O. Pathological phenotypes of LDL particles. Eur Lamarche B, Couture P, Kris-Etherton PM, West SG, et al. High-oleic
Heart J. 2018;39:2574–2576. doi: 10.1093/eurheartj/ehy387 canola oil consumption enriches LDL particle cholesteryl oleate con-
49. Oörni K, Kovanen PT. PLA2-V: a real player in atherogenesis. Arte- tent and reduces LDL proteoglycan binding in humans. Atherosclerosis.
rioscler Thromb Vasc Biol. 2007;27:445–447. doi: 10.1161/[Link]. 2015;238:231–238. doi: 10.1016/[Link].2014.12.010
[Link] 62. Nestel PJ, Pomeroy SE, Sasahara T, Yamashita T, Liang YL, Dart AM,
50. Grosheva I, Haka AS, Qin C, Pierini LM, Maxfield FR. Aggregated LDL in Jennings GL, Abbey M, Cameron JD. Arterial compliance in obese sub-
contact with macrophages induces local increases in free cholesterol lev- jects is improved with dietary plant n-3 fatty acid from flaxseed oil despite
els that regulate local actin polymerization. Arterioscler Thromb Vasc Biol. increased LDL oxidizability. Arterioscler Thromb Vasc Biol. 1997;17:1163–
2009;29:1615–1621. doi: 10.1161/ATVBAHA.109.191882 1170. doi: 10.1161/[Link].17.6.1163
51. Costales P, Fuentes-Prior P, Castellano J, Revuelta-Lopez E, Corral- 63. Whitman SC, Fish JR, Rand ML, Rogers KA. n-3 fatty acid incorporation into
Rodríguez MÁ, Nasarre L, Badimon L, Llorente-Cortes V. K Domain CR9 LDL particles renders them more susceptible to oxidation in vitro but not
of low density lipoprotein (LDL) receptor-related protein 1 (LRP1) is criti- necessarily more atherogenic in vivo. Arterioscler Thromb. 1994;14:1170–
cal for aggregated LDL-induced foam cell formation from human vascu- 1176. doi: 10.1161/[Link].14.7.1170
lar smooth muscle cells. J Biol Chem. 2015;290:14852–14865. doi: 64. De Caterina R, Liao JK, Libby P. Fatty acid modulation of endothe-
10.1074/jbc.M115.638361 lial activation. Am J Clin Nutr. 2000;71(suppl 1):213S–223S. doi:
52. Abbas A, Aukrust P, Russell D, Krohg-Sørensen K, Almås T, Bundgaard D, 10.1093/ajcn/71.1.213S
Bjerkeli V, Sagen EL, Michelsen AE, Dahl TB, et al. Matrix metalloproteinase 65. Virtanen JK, Mursu J, Voutilainen S, Tuomainen TP. The associations of
7 is associated with symptomatic lesions and adverse events in patients serum n-6 polyunsaturated fatty acids with serum C-reactive protein in men:
with carotid atherosclerosis. PLoS One. 2014;9:e84935. doi: 10.1371/ the Kuopio Ischaemic Heart Disease Risk Factor Study. Eur J Clin Nutr.
[Link].0084935 2018;72:342–348. doi: 10.1038/s41430-017-0009-6
53. Ruuth M, Äikäs L, Tigistu-Sahle F, Käkelä R, Lindholm H, Simonen P, 66. Rosqvist F, Iggman D, Kullberg J, Cedernaes J, Johansson HE, Larsson A,
Kovanen PT, Gylling H, Öörni K. Plant stanol esters reduce LDL (low- Johansson L, Ahlström H, Arner P, Dahlman I, et al. Overfeeding poly-
density lipoprotein) aggregation by altering LDL surface lipids: the blood unsaturated and saturated fat causes distinct effects on liver and vis-
flow Randomized Intervention Study. Arterioscler Thromb Vasc Biol. ceral fat accumulation in humans. Diabetes. 2014;63:2356–2368. doi:
2020;40:2310–2321. doi: 10.1161/ATVBAHA.120.314329 10.2337/db13-1622
54. Martínez-Bujidos M, Rull A, González-Cura B, Pérez-Cuéllar M, Montoliu- 67. Rosqvist F, Kullberg J, Ståhlman M, Cedernaes J, Heurling K, Johansson HE,
Gaya L, Villegas S, Ordóñez-Llanos J, Sánchez-Quesada JL. Clusterin/ Iggman D, Wilking H, Larsson A, Eriksson O, et al. Overeating saturated
apolipoprotein J binds to aggregated LDL in human plasma and plays a fat promotes fatty liver and ceramides compared with polyunsaturated fat:
protective role against LDL aggregation. FASEB J. 2015;29:1688–1700. a randomized trial. J Clin Endocrinol Metab. 2019;104:6207–6219. doi:
doi: 10.1096/fj.14-264036 10.1210/jc.2019-00160
55. Schwarz M, Spath L, Lux CA, Paprotka K, Torzewski M, Dersch K, 68. Piepoli MF, Hoes AW, Agewall S, Albus C, Brotons C, Catapano AL,
Koch-Brandt C, Husmann M, Bhakdi S. Potential protective role of apo- Cooney MT, Corrà U, Cosyns B, Deaton C, et al; ESC Scientific Document
protein J (clusterin) in atherogenesis: binding to enzymatically modified Group. 2016 European Guidelines on cardiovascular disease prevention in
low-density lipoprotein reduces fatty acid-mediated cytotoxicity. Thromb clinical practice: The Sixth Joint Task Force of the European Society of Car-
Haemost. 2008;100:110–118. doi: 10.1160/TH07-12-0737 diology and Other Societies on Cardiovascular Disease Prevention in Clini-
56. Merrilees MJ, Beaumont B. Structural heterogeneity of the diffuse intimal cal Practice (constituted by representatives of 10 societies and by invited
thickening and correlation with distribution of TGF-beta 1. J Vasc Res. experts)Developed with the special contribution of the European Associa-
1993;30:293–302. doi: 10.1159/000159008 tion for Cardiovascular Prevention & Rehabilitation (EACPR). Eur Heart J.
57. O’Brien KD, Olin KL, Alpers CE, Chiu W, Ferguson M, Hudkins K, Wight 2016;37:2315–2381. doi: 10.1093/eurheartj/ehw106
TN, Chait A. Comparison of apolipoprotein and proteoglycan deposits in 69. Sacks FM, Lichtenstein AH, Wu JHY, Appel LJ, Creager MA, Kris-Etherton PM,
human coronary atherosclerotic plaques: colocalization of biglycan with apo- Miller M, Rimm EB, Rudel LL, Robinson JG, et al; American Heart Asso-
lipoproteins. Circulation. 1998;98:519–527. doi: 10.1161/[Link].98.6.519 ciation. Dietary fats and cardiovascular disease: a presidential advisory
58. Nakashima Y, Fujii H, Sumiyoshi S, Wight TN, Sueishi K. Early human from the American Heart Association. Circulation. 2017;136:e1–e23. doi:
atherosclerosis: accumulation of lipid and proteoglycans in intimal 10.1161/CIR.0000000000000510

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