Free Radical Biology and Medicine 174 (2021) 171–181 Contents lists available at ScienceDirect Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed Malondialdehyde-modified HDL particles elicit a specific IgG response in abdominal aortic aneurysm Javier Rodríguez-Carrio a, 1, Isabel Cerro-Pardo b, 1, Jes S. Lindholt c, Elena Bonzon-Kulichenko d, e, Diego Martínez-López b, Raquel Roldán-Montero b, Joan-Carles Escolà-Gil f, g, Jean-Baptiste Michel h, Luis Miguel Blanco-Colio b, e, Jesús Vázquez d, e, Ana Suárez a, 1, José Luis Martín-Ventura b, e, *, 1 a Area of Immunology, University of Oviedo, Instituto de Salud Del Principado de Asturias (ISPA), Oviedo, Asturias, Spain b IIS-Fundación Jiménez-Díaz, Madrid, Spain c Department of Cardiothoracic and Vascular Surgery, Odense University Hospital, Odense, Denmark d Laboratorio de Proteómica Cardiovascular, CNIC, Madrid, Spain e CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain f Institut de Investigació Biomédica Sant Pau, Spain g CIBER de Diabetes y Enfermedades Metabólicas (CIBERDEM), Barcelona, Spain h Inserm U1148, Paris, France A R T I C L E I N F O A B S T R A C T Keywords: High Density Lipoprotein (HDL) plays a protective role in abdominal aortic aneurysm (AAA); however, recent HDL findings suggest that oxidative modifications could lead to dysfunctional HDL in AAA. This study aimed at testing MDA the effect of oxidized HDL on aortic lesions and humoral immune responses in a mouse model of AAA induced by Oxidative stress elastase, and evaluating whether antibodies against modified HDL can be found in AAA patients. Immune response Antibodies HDL particles were oxidized with malondialdehyde (HDL-MDA) and the changes were studied by biochemical Abdominal aortic aneurysm and proteomics approaches. Experimental AAA was induced in mice by elastase perfusion and then mice were treated with HDL-MDA, HDL or vehicle for 14 days. Aortic lesions were studied by histomorphometric analysis. Levels of anti-HDL-MDA IgG antibodies were measured by an in-house immunoassay in the mouse model, in human tissue-supernatants and in plasma samples from the VIVA cohort. HDL oxidation with MDA was confirmed by enhanced susceptibility to diene formation. Proteomics demon­ strated the presence of MDA adducts on Lysine residues of HDL proteins, mainly ApoA-I. MDA-modification of HDL abrogated the protective effect of HDL on cultured endothelial cells as well as on AAA dilation in mice. Exposure to HDL-MDA elicited an anti-HDL-MDA IgG response in mice. Anti-HDL-MDA were also detected in tissue-conditioned media from AAA patients, mainly in intraluminal thrombus. Higher plasma levels of anti-HDL- MDA IgG antibodies were found in AAA patients compared to controls. Anti-HDL-MDA levels were associated with smoking and were independent predictors of overall mortality in AAA patients. Overall, MDA-oxidized HDL trigger a specific humoral immune response in mice. Besides, antibodies against HDL-MDA can be detected in tissue and plasma of AAA patients, suggesting its potential use as surrogate stable biomarkers of oxidative stress in AAA. 1. Introduction in most of the cases and accounts for 150,000 to 200,000 deaths per year around the globe [3]. Thoracic endovascular aortic repair (TEVAR) and Abdominal aortic aneurysm (AAA) is a vascular disease hallmarked open surgical repair are generally accepted as efficient and safe surgical by a permanent and chronic dilation of the abdominal aorta [1,2]. methods, but they are only applicable to the late stage of the disease. Although AAA progression is mostly asymptomatic, AAA rupture is fatal Therefore, in the absence of specific therapeutic approaches, especially * Corresponding author. Vascular Research Lab, IIS-Fundación Jiménez Díaz, Autónoma University, Av. Reyes Católicos 2, 28040, Madrid, Spain. E-mail address: jlmartin@fjd.es (J.L. Martín-Ventura). 1 Equal contribution of both first and senior authors. https://doi.org/10.1016/j.freeradbiomed.2021.08.004 Received 27 May 2021; Received in revised form 22 July 2021; Accepted 4 August 2021 Available online 6 August 2021 0891-5849/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J. Rodríguez-Carrio et al. Free Radical Biology and Medicine 174 (2021) 171–181 for the early phase, a better characterization of pathogenic mechanisms (Roche Diagnostics). underlying AAA progression, as well as potential biomarkers, is imper­ MDA (0.5 M) was obtained from malonaldehyde-bis-dimethylacetal ative to improve clinical management and patient stratification. (Sigma) by acid hydrolysis [25]: 88 μl of Risk factors correlated to AAA onset and progression include hy­ malonaldehyde-bis-dimethylacetal were incubated with 12 μl 4 N HCl pertension, smoking, and obesity, among others [1]. Recent genetic and 400 μl distilled water at 37 ◦ C for 10 min pH was adjusted to 7.4 analysis suggests that lipids play a role in AAA etiology [3–6]. Several adding 1 N NaOH and measuring with a universal pH indicator. Distilled epidemiological studies have shown a connection between decreased water was added to reach a final volume of 1 ml. MDA-HDL was pre­ High-Density Lipoprotein Cholesterol (HDLc) levels and AAA progres­ pared by incubating HDL with 0.5 M MDA at 37 ◦ C during 3 h at a sion [7–9]. These results were supported by experimental animal models constant ratio of 100 μl/mg of HDL. Unbound MDA was eliminated using reporting that raising HDLc levels can cause abrogation of AAA initia­ PD-10 desalting columns (GE Healthcare). MDA-HDL was concentrated tion and progression [10,11]. However, these findings were confronted with a centrifugal filter of 30 KDa (Centricon Merck Millipore). Con­ with clinical trials reporting no clinical benefit of HDLc-raising agents centration of MDA-HDL was measured using the measurement of ApoAI on cardiovascular (CV) patients [12–14]. These findings seem to suggest as reported above. that the association between HDLc and cardiovascular diseases is more HDL susceptibility to copper-induced lipid oxidation was determined complex than initially considered. On the one hand, HDLc levels may not by monitoring the formation of conjugated dienes at 234 nm at 37 ◦ C in a be as relevant on themselves as previously conceived, and functional BioTek Synergy HT spectrophotometer (BioTek, Agilent). Oxidation was properties of HDL particles should be included into the equation. In this started by adding 2.5 μmol/L CuSO4 in wells each containing HDL (100 respect, the quality of HDL is the relevant factor for its vasculoprotective μg/ml of apoA1), and the maximum slope during the propagation phase activities [15]. Cardiovascular protective functions of HDL include of the kinetics was calculated. reverse cholesterol transport (RCT), as well as antioxidant, anti-inflammatory, anti-apoptotic and antithrombotic effects. Regarding 2.2. Proteomics AAA, we previously demonstrated that systemic paraoxonase-1 activity, a surrogate marker of HDL (antioxidant) functionality, is decreased in Samples (200 μg of total proteins of MDA-HDL or vehicle-treated- AAA patients [16]. Moreover, we have shown that AAA patients display HDL) were digested overnight at 37 ◦ C in FASP filters with trypsin impaired HDL cholesterol efflux capacity, the first step of RCT [17]. We (Promega, Madison, WI, USA) at an 40:1 protein:trypsin (w/w) ratio in have recently demonstrated that HDLs are sensitive to oxidation in the 50 mM ammonium bicarbonate, pH 8.8, according to manufacter’s in­ AAA milieu, mainly in the intraluminal thrombus (ILT), which is rich in structions. The resulting peptides were desalted on C18 Oasis cartridges neutrophils and red blood cells. Moreover, those HDL-oxidized forms in (Waters Corporation, Milford, MA, USA) using 50% acetonitrile (ACN) plasma were independently linked to AAA presence [18]. Importantly, (v/v) in 0.1% trifluoroacetic acid (v/v) as eluent, and vacuum dried. The oxidized HDL was associated with impaired HDL functionality [18]. peptides were then subjected to isobaric TMT labelling following man­ Previous studies from our group have reported the presence of an­ ufacturer’s instructions. The TMT10-plex experiment was composed by tibodies against HDL particles in AAA patients, which were associated 4 biological replicates coming from MDA-treated HDL, 4 from vehicle- with HDLc levels and clinical features [19], hence proposing a role for treated HDL, and two channels reserved for internal standard (I.S.) antibodies against lipoproteins in AAA. Similar results were observed in samples. The I.S. was created by pooling the four samples from the other conditions [20–22], thus suggesting that antibodies against lipo­ vehicle-treated-HDL and was used as reference to express relative proteins may help to understand the crosstalk among lipids, inflamma­ quantification values. Peptides were fractionated using the high pH tion and clinical outcomes. Taking into account that oxidation can lead reversed-phase peptide fractionation kit (Thermo Fisher Scientific) ac­ to modifications in proteins and lipids, it is conceivable that antibodies cording to manufacter’s instructions. Peptide fractions were analyzed directed against HDL oxidized forms could be also detected in AAA. In using an Easy nano-flow HPLC system (Thermo Fisher Scientific, Bre­ fact, antibodies against malondialdehyde (MDA)-modified LDL have men, Germany) coupled via a nanoelectrospray ion source (Thermo been reported [23] but whether oxidized HDL may represent an anti­ Fisher Scientific) to an Orbitrap Fusion mass spectrometer (Thermo genic target remains unknown. Therefore, the present study is aimed at Fisher Scientific). C18-based reverse phase separation was used with a 2- (i) testing the effect of HDL-MDA on aortic lesions and immune re­ cm trap column and a 50-cm analytical column (EASY column, Thermo sponses in the elastase-induced AAA mouse model and (ii) assessing Fisher Scientific). Peptides were loaded in buffer A (0.1% formic acid (v/ whether anti-HDL-MDA antibodies can be detected in tissue and plasma v)) and eluted with a 240-min linear gradient of buffer B (80% aceto­ samples from AAA patients. nitrile, 0.1% formic acid (v/v)) at 200 nL/min. Mass spectra were ac­ quired in a data-dependent manner, with an automatic switch between 2. Material and methods MS and MS/MS using a top-speed acquisition method. MS spectra were acquired in the Orbitrap analyzer with a mass range of 400–1500 m/z 2.1. HDL isolation and oxidation with MDA and 120,000 resolution. HCD fragmentation was performed at 35 normalized collision energy and MS/MS spectra were analyzed at HDL was isolated from EDTA plasma samples of healthy volunteers 30,000 resolution in the Orbitrap. Data analysis was performed with by ultracentrifugation as described [24]. Plasma density was adjusted to Xcalibur 2.2 (Thermo Scientific). ρ = 1.063 g/ml with KBr and the sample was overlaid with KBr saline Peptide identification from MS/MS spectra was performed by open solution (ρ 1.063 g/ml). Samples were ultracentrifuged at 53,000 rpm search on Comet-PTM [26] against a human database, build by for 18 h at 4 ◦ C in a TLA-100 rotor (Beckman). After centrifugation, the concatenating the human reference proteome database (Uni­ upper, apolipoprotein-B-containing lipoproteins, fraction was recovered ProtKB/Swiss-Prot July 2014, 40555 sequences), with the inverted and stored. The bottom fraction, containing whole HDL, was adjusted to database constructed from the same target database using DecoyPYrat 1.25 g/ml with KBr and overlaid with KBr saline solution (ρ = 1.22 [27]. Search parameters were: full trypsin digestion with 2 missed g/ml). The HDL-containing fraction was then ultracentrifuged at 63,000 cleavages, fixed modifications Cys carbamidomethylation (57.021464 rpm for 24 h at 4 ◦ C. After this step, HDLs (top layer) were recovered as a Da) and TMT labeling at N-terminal end and Lys (229.162932 Da). single band and were extensively rinsed with saline and concentrated Precursor mass tolerance was set to 500 Da and fragment mass tolerance using a centrifugal concentrating device (cutoff 10 kDa). All HDL sam­ at 0.03 Da; precursor charge range was set to 2–4; and 3 was the ples were desalted by centrifugation and 3 washes with saline. ApoA-I maximum fragment charge. Only y- and b-ions were used for scoring. concentration in HDL was determined by an immunoturbidimetric The FDR (<1%) of PTM identification was controlled at the global, local assay, using a commercial kit adapted to a COBAS 501c autoanalyzer and peak levels by SHIFTS [26]. Quantitative information was extracted 172 J. Rodríguez-Carrio et al. Free Radical Biology and Medicine 174 (2021) 171–181 from MS/MS spectra of TMT-labeled peptides using Proteome Discov­ of animals used for scientific purposes. erer 2.1 (Thermo Scientific). Modified peptides and protein quantifica­ Twelve-week-old mice were anesthetized by 2% isofluorane inhala­ tion was performed using the Generic Integration Algorithm [28,29] on tional anaesthesia and a horizontal laparotomy was performed. Using a the basis of the WSPP model [30] with some modifications [26]. Sig­ surgical stereomicroscope, the abdominal aorta was separated from the nificant abundance changes of modified peptides across the different level of the left renal vein to the bifurcation and temporarily ligated samples were detected by Student’s t-test. An in-house developed script between the renal and iliac arteries. An aortotomy was created with a was used for the semisupervised annotation of peptide modifications as 30-gauge needle and the aorta was exsanguinated. A PE-26 polyethylene described [26]. For simplicity, artifactual modifications introduced tube was introduced through the aortotomy, and the aorta was infused during sample preparation were not considered in further analysis. Met for 5 min at 100 mmHg with type I porcine pancreatic elastase (specific mono-oxidation, or Asn, Gln and alkylated Cys deamidation, were not activity 6 U/mg protein; E1250; Sigma Chemical). The aortotomy was considered as post-translationally modifications (PTMs). Proteomics then repaired, the ligation was eliminated, and the restoration of blood data have been deposited at the Proteome Xchange [31] Consortium via flow visually confirmed. Incisions were closed and the mice housed the PRIDE partner repository with the dataset identifier PXD025322. under standard conditions. Experimental groups were: elastase-infused WT mice treated i.v. with saline (WT, n = 9); elastase-infused WT 2.3. In vitro studies mice treated i.v. with HDL (HDL, 400 μg every day, n = 8) and elastase- infused WT mice treated i.v. with MDA-HDL (MDA-HDL, 400 μg every Human aortic endothelial cells (HAECs) were isolated from entire day, n = 9). On day 14 post-surgery, all mice were anesthetized with a arterial segments from aortae of patients undergoing cardiac surgery as mixture of ketamine/xylazine (100 mg/kg and 10 mg/kg body weight, described [32]. HAECs were incubated in DMEM/F-12 medium with respectively) and euthanized by cervical dislocation. Lipid and lipo­ 0.1% Collagenase I (Gibco, Thermo Fisher Scientific, UK) overnight. protein levels were measured in plasma using commercial kits adapted After incubation, the reaction was stopped and the ECs were scraped off to a COBAS 501c autoanalyzer (Roche Diagnostics). the vessel walls, centrifuged at 500 g for 5 min at room temperature (RT) and resuspended in DMEM/F-12 complete medium (DMEM/F-12 + 100 2.6. Histomorphometry and immunohistochemistry U/ml penicillin +100 μg/ml streptavidin +2 mM L-glutamine + 2.5 μg/ml Amphotericin B + 100 μg/ml heparin +30 μg/ml ECGF + 20% Mouse aortic samples were embedded in paraffin. Serial sections (4 FBS). After 2–5 days of expansion, the HAECs were detached from the μm) of aortas were cut for histomorphometry. Histomorphometry was culture plates using 0.025% trypsin-EDTA and isolated through positive performed on Masson trichrome-stained histological sections as previ­ selection using 2.5 g/ml mouse anti-human CD31 antibody (BD ously described [16]. Aortic diameter increase was obtained after Biosciences-EU) followed by 2⋅106 beads/ml magnetic beads coated normalization to normal aorta diameter before surgery. AAA was with anti-mouse IgG (Invitrogen, Thermo Fisher Scientific, UK). Both defined as aortic diameter expansion 100% of that before perfusion incubation steps were made for 30 min at 4 ◦ C. The cell suspensions were [34]. Elastin fragmentation were graded from grade 1 (intact then passed through a column placed in a magnet, ensuring the isolation well-organized elastin laminae) to grade 4, severe elastin fragmentation of HAECs. The isolated cells were resuspended in complete medium and or loss. For immunohistometry, primary Ab was CD68 (Ab53444; cultured on 0.5% gelatin-coated plates. Once in confluence, primary cell Abcam) and donkey anti-rat HRP (Jackson) was used as secondary cultures were FBS 0.5% starved for 24 h before the experiments were antibody. HRP was then added and sections were stained with DAB performed. All experiments were performed during passages 3–7. Cells substrate-chromogen (DAKO) and counterstained with haematoxylin. were preincubated with HDL and HDL-MDA (0.1 mg/ml for both) for 24 Computer-assisted morphometric analysis was performed with the h and then stimulated with TNF-alfa (50 ng/ml) for 4 additional h. After Image-Pro Plus software (version 1.0 for Windows) as described [16]. that, cells were pelleted and resuspended for RNA analysis. The threshold setting for area measurement was equal for all images. The study was approved by the Research Ethics Committee at Hos­ Samples from each animal were examined in a blinded manner. Results pital Universitario Fundacion Jimenez Diaz (approval number PIC076- of CD68 were expressed as % positive area versus total area. 18_FJD). Informed consent was obtained from all patients. 2.7. Tissue-conditioned media 2.4. Real time PCR Tissue-conditioned media were obtained as previously reported Lysates from HAECs were resuspended in TRIzol, and total RNA was [35]. Briefly, tissue samples from diseased (AAA) aortas were obtained purified. Duplicate samples were quantified by determining absorbance during surgical repair and dissected into intraluminal thrombus at 260 nm, and Real-Time PCR was performed as described [33]. The (AAA-ILT) and arterial wall (AAA-wall). Healthy aortas were collected expression of target genes was normalized to housekeeping transcript from brain-deceased organ donors and included abdominal and thoracic (GADPH). The TaqMan probes (Hs00174575 m1 CCL5, Hs00234140 m1 aortas. Tissue sections were cut into small pieces and incubated in RPMI CCL2, IL1b Hs01555410_m and Hs02786624_g1 GAPDH) were pur­ 1640 medium (at 6 ml/g of wet tissue) for 24 h at 37 ◦ C. Then, cultures chased from Applied Biosystems and optimized according to the man­ were centrifuged (3000 g, 10 min, 20 ◦ C) and supernatants containing ufacturer’s protocol. proteins released from the tissue samples (tissue-conditioned media) were collected and frozen at − 80 ◦ C until analyzed. Procedures were 2.5. Animal model approved by the local Institutional Review Board (INSERM Institutional Review Board: RESAA and AMETHYST studies, CPP Paris-Cochin n◦ Wild type (WT) control mice (C57BL6/J) were purchased from The 2095, 1930 and 1931; French Biomedicine Agency: PFS 09-007, BBMRI Jackson Laboratory (Bar Harbor). All animals were housed in isolation network, BB-0033-00029) and patient informed consent was obtained. rooms in the animal facility of our institute. Water and chow-fed diet was available ad libitum. The Ethics Review Board of our institute 2.8. AAA patients and controls approved all animal procedures, and the project was authorized by the IIS-FJD-Universidad Autónoma de Madrid (CEI 59-1036-A061) and by Plasma samples were obtained from individuals participating in the the Spanish Authority governing animal experimentation, the Comuni­ VIVA study (ClinicalTrials.gov NCT00662480), a population-based dad Autónoma de Madrid (registered approval letter 10/008932.9/15). image-screening trial for AAA in men aged between 64 and 74 years All animal procedures were performed in accordance with the guidelines old from Denmark. of Directive 2010/63/EU of the European Parliament on the protection The study recruitment protocol has been reported in detail elsewhere 173 J. Rodríguez-Carrio et al. Free Radical Biology and Medicine 174 (2021) 171–181 [36,37]. In brief, 50,156 male adults were randomized between October phosphatase-conjugated anti-human IgG or anti-mouse IgG (both 2008 and October 2010 to receive either an invitation for vascular 1:1000) (Immunostep, Spain) was added for 1 h. Lastly, p-nitro­ screening or participate as control subjects. The study protocol was phenylphosphate (Sigma) in diethanolamine buffer (pH 9.8) was added performed in compliance with the Declaration of Helsinki and received and absorbance at 405 nm was recorded. For each measurement, the approval from the Institutional Review Board from the Mid Region of absorbance in the control half was substracted to that of the test half. For Denmark (M20080028). All participants gave written informed consent plasma samples, IgG anti-HDL-MDA Arbitrary Units (AU) were calcu­ prior enrolment. lated according to the standard curves. Intra- and inter-assay variations Study participants underwent an ankle-brachial index (ABI) mea­ for our assay were 6.5% and 11.2%, respectively. Standard curves using surement and an ultrasound scan of the abdominal aorta. The ABI was pooled sera were used for anti-HDL-MDA measurements in plasma from calculated as the mean of the two-recorded ankle arterial blood pres­ patients. Due to the lack of a standard material, internal controls were sures divided by the brachial systolic blood pressure. The presence of used instead for anti-HDL-MDA measurements in mouse plasma and peripheral arterial disease (PAD) was defined by an ABI <0.90 or >1.4. tissue-conditioned supernatants. For mouse plasma samples, plasma was Questionnaires regarding lifestyle parameters, medical pre-existing diluted 1:50 and the raw absorbances were analyzed. For conditions and smoking status were administered by trained nurses. tissue-conditioned media, samples were assayed undiluted and the raw Study participants were followed-up for up to 5 years and information absorbances were analyzed. about death was obtained from the Civil Registration System. No pa­ Total IgG plasma levels were quantified by conventional ELISA tients experiencing AAA rupture were registered in our cohort. techniques and AU values obtained from the anti-HDL-MDA and anti- A final cohort consisting on 427 AAA patients and 139 HC was HDL ELISAs were corrected using total IgG levels (anti-HDL-MDA/IgG analyzed (Table 1). Blood samples were obtained by venipuncture. and anti-HDL/IgG ratios). Plasma samples were immediately processed and stored at − 80 ◦ C until analyses. 2.10. Statistical analyses Variables were checked for normal distribution using graphical 2.9. Quantification of IgG anti-HDL-MDA and anti-HDL antibodies methods and the Shapiro–Wilk test. Residuals were assessed in histo­ grams and p-p plots. Continuous variables were summarized as median IgG anti-HDL antibodies were measured as previously reported [20]. (interquartile range) or mean ± standard deviation (SD) depending on IgG anti-HDL-MDA antibodies were measured by means of an in-house the distribution of the data. Categorical variables were expressed as n immunoassay. ELISA plates (Maxisorp, Nunc) were coated with either (%). 20 μg/ml of HDL-MDA (test half) or HDL-cholesterol (unfractioned HDL Differences across groups were assessed by Student’s t tests, Mann- isolated from human plasma, Sigma) in 70% ethanol (control half) Whitney U test, ANOVA or Kruskal-Wallis tests, as appropriate. Dunn overnight at 4 ◦ C. Plates were then blocked with blocking solution (PBS correction for multiple comparisons tests was performed when more containing 1% BSA (Sigma)) for 1 h at room temperature and washed than 2 groups were analyzed. Levels of IgG anti-HDL-MDA were log- with PBS. Then, 1:50-diluted plasma samples and standard curves from transformed in order to being entered in parametric and multivariate patients’ pooled sera (diluted 1:16 to 1:512) (in PBS containing 0.1% tests. Independent associations between AAA prevalence and IgG anti- BSA) were incubated in both plate halves for 2 h at room temperature. HDL-MDA levels were assessed by logistic regression analyses with ad­ Plates were then washed twice with TBS and alkaline justments for confounders. Selection of confounders was performed for each model by identifying those variables associated with the outcome, Table 1 dependent variable with a p-value below 0.1 in univariate analyses as Demographical and clinical parameters of the study participants (VIVA previously described [38,39]. study). Variables were summarized as mean ± SD or n(%). Differences between A p-value<0.050 was considered as statistically significant. Data groups were assessed by χ2 or unpaired t tests for categorical or continuous were analyzed by SPSS 23.0 for Windows (IBM). variables, respectively. AAA (n = Healthy controls (n = p-value 3. Results 427) 139) Age 69.5 ± 2.8 69.2 ± 2.9 0.321 3.1. Effect of HDL-MDA on aortic lesions and immune responses in the Sex, n male 427 (100) 139 (100) – elastase-induced AAA mouse model BMI, kg/cm2 27.3 ± 3.6 24.7 ± 3.3 0.003 ABI 0.8 ± 0.2 1.1 ± 0.1 0.004 Aortic size, mm 39.5 ± 11.8 18.2 ± 2.8 <0.001 As we recently showed that oxidation of HDL leads to an impaired AAA repair, n(%) 171 (40.0) 0 (0.0) <0.001 HDL activity [18], we tested the functional impact of the oxidative Death, n(%) 95 (22.2) 16 (139) 0.006 modification of HDL by MDA on AAA. We firstly characterized the effect CV risk factors, n(%) of MDA treatment on diene formation in HDL. MDA-oxidized HDL Current smoking 182 (42.6) 24 (17.3) <0.001 particles exhibited a higher diene formation compared to their naïve Hypertension 230 (53.7) 62 (44.9) 0.070 Diabetes 49 (11.5) 23 (16.7) 0.115 counterparts (HDL-MDA: 8.99 ± 1.08 vs HDL: 5.85 ± 0.91) (Fig. 1A). We History of CV events, n(%) next applied a novel mass spectrometry-based technology for the unbi­ Former CVD 90 (20.8) 19 (13.7) 0.062 ased characterization and quantification of PTMs [26], which we have Former PAD 6 (1.4) 1 (0.7) 0.525 applied to biomedical research [40], including characterization of HDL Former angina 43 (10.1) 10 (7.2) 0.312 Former AMI 24 (5.6) 3 (2.2) 0.096 on the context of AAA [18]. We observed that the majority of PTMs Former stroke 17 [4] 2 (1.4) 0.148 induced by MDA treatment were MDA adducts on Lys residues (Fig. 1B). Treatments, n(%) Out of the 22 ApoA1 Lys sites, 18 were detected, and from those, 15 were Use of aspirin 192 (45.0) 36 (25.9) <0.001 found to be modified by MDA (Fig. 1C). MDA also produced kynurenin Use of ACE inhibitors 116 (27.2) 30 (21.6) 0.184 oxidation on Trp residues and other additional PTMs, although in much Use of b-blockers 123 (28.8) 33 (23.7) 0.250 Use of calcium 106 (24.8) 24 (17.3) 0.067 lower abundance. antagonist We then analyzed the effect of MDA-treated HDL particles in vitro Use of statins 219 (51.3) 48 (34.5) 0.001 (human aortic endothelial cells, HAEC) and in vivo (elastase-induced Use of glucocorticoids 37 (8.7) 10 (7.2) 0.574 AAA lesions in mice). In HAEC, HDL prevented TNF-induced IL-1b, CCL- Use of bronchodilator 45 (10.6) 12 (8.6) 0.500 2 and CCL-5 mRNA expression, while this protective anti-inflammatory 174 J. Rodríguez-Carrio et al. Free Radical Biology and Medicine 174 (2021) 171–181 Fig. 1. MDA-induced modifications in human HDLs. (A) Susceptibility to oxidation of HDL particles (with or without MDA) was induced by adding 2.5 μM CuSO4 and conjugated dienes formation monitored at 234 nm. Data are expressed as the absorbance increase per minute during the phase of maximal slope. Bars indicate 25th, median and 75th percentiles. Differences between HDL-MDA (orange, n = 9) and native HDL (green, n = 8) were assessed by T tests. ***p < 0.001. (B) An absolute abundance-proportional graph of the post-translationally modified peptides (PTMs) (with more than 2 quantified spectra) significantly increased in MDA- treated HDLs as compared to the vehicle-treated ones, is shown [n = 4, t-test, p < 0.050]. The adscription of the type of modification and the modified residue to HDL proteins is depicted. The abundance of PTMs was assessed by spectral counting. (C) Heat-maps showing changes in ApoA1 peptide abundance produced by MDA treatment (in units of standard deviation, zpq), whose magnitude is shaded according to the color scale at the bottom. For simplicity, the figure only includes non- modified or MDA-modified peptide forms quantified with more than 5 spectra. The number of scans used to quantify each peptide (PSM) as well as the ApoA1 (P02647-1) residue numbering of the N-terminal residue of each peptide sequence is shown. Note that a minor proportion of peptides containing MDA-modified Lys (MDA) are also detected in other forms where they have an additional TMT labeling moiety (MDA1) or are unlabeled (MDA2) in other amino groups. NM: non- modified; MDA: malondialdehyde; UK: unknown; KYN: Kynurenine; HEX: hexose; SAS: single amino acid substitution; Ox and diox: addition of one or two oxy­ gens, respectively. effect was lost when HAEC were treated with HDL-MDA (Fig. 2a). In protective effects of HDL on aortic dilation in an elastase-induced AAA addition, treatment with HDL decreased aortic dilation induced by model. Moreover, exposure to HDL-MDA particles triggered a specific elastase in mice, in agreement with previous studies in other AAA anti-HDL-MDA response in mice, which was different to that of HDL- models [10,11]. In contrast, MDA-oxidized HDL did not prevent prompted. elastase-induced AAA dilation (HDL-MDA: 228.2 ± 29.98% vs HDL: 201.3 ± 29.04% and control: 255.5 ± 38.58%) (Fig. 2B and C). Neither treatment (HDL or HDL-MDA) lead to significant changes in plasma lipid 3.2. IgG Anti-HDL-MDA levels in tissue-conditioned media and plasma of and lipoprotein levels, elastin or macrophage content (Supplementary AAA patients Fig. 1). Then, we aimed to evaluate whether MDA modifications on HDL Next, we assessed whether IgG anti-HDL-MDA antibodies could be elicited a specific immune response in mice. To this end, the presence of found in human samples obtained from patients at the local (tissue) and anti-HDL-MDA IgG was tested in plasma samples obtained from animals systemic (plasma) level. First, the levels of IgG anti-HDL-MDA were treated with HDL-MDA, HDL or vehicle (control). Our results revealed analyzed in tissue-conditioned media samples from AAA patients and that animals treated with HDL-MDA mounted an anti-HDL-MDA specific controls (Supplementary Table 1). Interestingly, the levels of IgG anti- response (HDL-MDA: 0.74 ± 0.25 vs controls: 0.55 ± 0.17). This HDL-MDA antibodies were found to be elevated in ILT samples response was absent when HDL was administered (HDL: 0.27 ± 0.10) compared to those obtained from the AAA wall or control aortic tissues (Fig. 3A), which in turn prompted a specific anti-HDL response (HDL: (AAA-ILT: 0.28 ± 0.22 vs AAA-wall: 0.13 ± 0.27 and HC: 0.06 ± 0.07) 0.56 ± 0.21 vs controls: 0.07 ± 0.05 and HDL-MDA: 0.18 ± 0.11) (Fig. 4A). Of note, differences were noted between anti-HDL-MDA and (Fig. 3B). Furthermore, adjustment by total IgG levels allowed us to anti-HDL, showing increased anti-HDL levels in the AAA wall (AAA-ILT: confirm that this effect was not attributed to differences in the produc­ 0.08 ± 0.10 vs AAA-wall: 0.15 ± 0.13 and HC: 0.05 ± 0.07) (Fig. 4B), in tion of total IgG levels (Fig. 3C). line with previous findings [19]. Again, a potential effect of differences All these results suggest that oxidative modification of HDL with in total IgG production was ruled out (Fig. 4C). IgG anti-HDL-MDA levels MDA, mostly related to MDA adducts on Lys residues, impaired the were not correlated with age in any of the groups (AAA-ILT: r = 0.018, p = 0.919, AAA-wall: r = 0.129, p = 0.454, and controls: r = 0.036, p = 175 J. Rodríguez-Carrio et al. Free Radical Biology and Medicine 174 (2021) 171–181 Fig. 2. Effect of HDL-MDA treatment in vitro and in vivo. The effect of HDL-MDA in vitro on endothelial cells stimulated with TNFa was evaluated by measuring the gene expression of IL-1b, CCL2 and CCL5 (A). Bars indicate mean and standard error of the means. Results are expressed as fold change vs basal. Differences were assessed by T-test. The effect of HDL-MDA treatment in an elastase-induced AAA mouse model (B, representative images) was evaluated by measuring aortic diameter (C). Bars indicate 25th, median and 75th percentiles. Differences among mice treated with HDL-MDA (orange), HDL (green) or vehicle (control) (blue) were assessed by one-way ANOVA and Dunn tests for multiple comparisons. The p-values from multiple comparisons are indicated in the graphs as follows: *p < 0.050, **p < 0.010. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 0.779). A similar picture was observed for IgG anti-HDL levels (all p > hallmarked by elevated IgG anti-HDL-MDA plasma levels, irrespective of 0.050). Furthermore, these antibodies were not associated with sex in traditional risk factors. Anti-HDL-MDA antibodies are independent any of the study groups (all p≫0.050). No differences between origins predictors of overall mortality. (abdominal vs thoracic) were observed for anti-HDL-MDA (p = 0.241) or anti-HDL (p = 0.321) levels in control samples. Excluding samples from 4. Discussion thoracic donors did not change these findings (data not shown). Finally, we aimed to assess whether anti-HDL-MDA antibodies could Despite the vast evidence supporting an association between low be detected in plasma samples from AAA patients. Then, levels of IgG HDLc levels and AAA, this notion has been challenged by recent find­ anti-HDL-MDA were measured in AAA patients and controls from the ings, hence pointing to a more complex scenario. Oxidative stress and VIVA study (Table 1). AAA patients exhibited higher IgG anti-HDL-MDA immune responses are central mechanisms in AAA pathogenesis. How­ levels compared to age-matched controls (AAA: 39.53 ± 23.63 vs HC: ever, how lipoproteins interplay with these processes in AAA is poorly 28.73 ± 19.01 AU) (Fig. 5A). These differences were not explained by characterized. In the present study, we reported that oxidized HDL total IgG levels (Fig. 5B). IgG anti-HDL-MDA levels were associated with particles not only were associated with a loss of its protective functions current smoking habit (40.00 ± 25.50 vs 35.89 ± 21.18 AU, p = 0.040), in vivo to counteract AAA progression, but also triggered a specific whereas no associations were observed for other risks factors including adaptive immune response. To the best of our knowledge, the findings hypertension (p = 0.906), diabetes (p = 0.986), history of previous CV herein reported demonstrate, for the first time, the involvement of anti- events (p = 0.120) nor with treatments (all p > 0.050). Moreover, HDL-MDA response in AAA, thereby pointing to a connection among multivariate regression analysis adjusted for confounders revealed that oxidative stress, HDL dysfunction and immune response. anti-HDL-MDA antibodies were independent predictors of AAA Oxidative stress has been proposed to contribute to AAA, although (Table 2). Lastly, associations between anti-HDL-MDA and clinical the exact mediators are not completely elucidated [41]. In addition to outcomes in AAA were analyzed. No differences were found between causing direct cell injury, DNA and protein damage, free radicals are AAA patients undergoing surgical repair during follow-up and their non- known to prompt lipid peroxidation [1,2,42]. Certain molecules such as repair counterparts (38.77 ± 22.23 vs 39.43 ± 23.77, p = 0.776). MDA are able to enhance oxidative stress via lipid peroxidation [43]. Interestingly, IgG anti-HDL-MDA levels were associated with overall Previous findings from our group have documented that oxidized HDL mortality in AAA patients in both univariate and multivariate analyses was associated with impaired activity [18]. In the present study, we (Table 3). further expand these findings. We first show that in our conditions, MDA In sum, these results confirm that IgG anti-HDL-MDA are enriched in induces oxidative modifications of HDL in vitro, predominating the the ILT of AAA samples, thus suggesting an enrichment of oxidized generation of MDA adducts on Lys residues, which were identified in the epitopes in this location compared to the vascular wall. AAA patients are most abundant HDL protein components, among them ApoA-I. Among 176 J. Rodríguez-Carrio et al. Free Radical Biology and Medicine 174 (2021) 171–181 Fig. 3. Anti-HDL-MDA responses in elastased-induced AAA mouse model. IgG anti-HDL-MDA (A) and IgG anti-HDL (B) levels (measured as absorbance) were quantified at day 14 in plasma samples from mice treated with HDL-MDA (orange, n = 9), HDL (green, n = 8) or vehicle (control) (blue, n = 9). (C) Anti-HDL-MDA and anti-HDL levels were corrected for the total IgG levels and ratios were analyzed. Bars indicate 25th, median and 75th percentiles. Differences across groups were assessed by Kruskal-Wallis tests and Dunn tests for multiple comparisons. The p-values from multiple comparisons are indicated in the graphs as follows: **p < 0.010, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) other functions, ApoA-I contributes to the inhibition of lipid hydroper­ analyses on AAA tissue-conditioned media showed diverging patterns at oxides accumulation in HDL, while lipid hydroperoxides in HDL could the local level. Whereas anti-HDL-MDA are increased in the ILT, their impair a variety of HDL’s vasculoprotective functions by oxidizing anti-HDL counterparts were found to be elevated in the AAA wall. The ApoA-I via a reactive aldehyde-mediated reaction [44]. In addition, it latter observation is in line with previous findings from our group in a was previously shown that modifying ApoA-I by MDA, but not by an proof-of-concept study [19]. Importantly, the ILT is an oxidative envi­ array of other reactive carbonyls, blocks cholesterol efflux [45]. We then ronment, mostly due to the ferrous iron released by trapped erythrocytes demonstrate that MDA-induced modifications abrogate the vasculo­ and MPO-derived reactive oxygen species (ROS) released by activated protective functions of native HDL in HAEC and in the elastase-induced neutrophils [54]. In this respect, we previously shown increased levels mouse model of AAA. In vitro, we have shown that HDL prevented the of oxidized lipids and proteins in the ILT [55]. Specifically, ApoA-I, the overexpression of inflammatory mediators such as IL-1b, CCL-2 and main component of HDL, co-localized with MDA in ILT, suggesting the CCL-5, while this protective effect was lost by HDL-MDA treatment. In oxidation of HDL in AAA ILT [18]. Moreover, an enhanced proteolytic vivo, HDL prevented elastase-induced AAA dilation, while HDL-MDA activity characterizes the ILT [1], which could generate new epitopes. did not. The mechanisms underlying this protective effect was not All these notions may explain the higher anti-HDL-MDA levels observed related to changes in plasma lipid and lipoprotein levels, elastin pres­ at the ILT compared to the AAA wall, since oxidized epitopes are highly ervation and/or macrophage infiltration. Thus, further studies are enriched in the latter, thus provoking an enhanced anti-HDL-MDA needed to completely understand the beneficial effect of HDL in vivo. depot. Elevated anti-HDL responses at the AAA wall may be caused by Moreover, we have found that HDL-MDA is able to elicit a specific im­ lipoprotein retention and local immune response in the adventitial ter­ mune humoral response in both mouse and AAA patients. Oxidative tiary lymphoid organs [56]. Moreover, it must be noted that whereas the stress is known to cause changes in proteins and lipids that can not only anti-HDL-MDA response should be regarded as ‘common’ immune generate new epitopes either by prompting oxidation-specific epitopes response towards a new antigen, the anti-HDL response represents an (OSE) (neo-antigens) [46], but also by inducing conformational changes autoimmune phenomenon, whose origin in human patients cannot be which can expose additional epitopes [47,48]. Interestingly, smoking established in the present study. All these findings uncover a functional has been associated with higher oxidative stress in aneurysmal diseases and pathophysiological diversity of humoral responses in AAA. [49,50] and other conditions [51]. Similarly, smoking has been pro­ Taken together, our findings shed new light on the role of adaptive posed to induce protein modifications that trigger specific humoral re­ responses in the pathogenesis of AAA. Early studies have demonstrated sponses [52,53], which in turn participate in disease pathogenesis in an overt B-cell infiltration in AAA lesions from patients and animal other conditions. These lines of evidence are in accordance with our models [57,58]. In fact, experimental B-cell depletion has been associ­ findings, which point to a similar pathogenic circuit in the framework of ated with smaller AAA lesions [59], and enhanced IgG deposition have AAA, antibodies against HDL-MDA being a missing link among oxidative been reported in arterial wall and thrombi in AAA [58], although the stress, HDL dysfunction and immune responses. relevance of the latter remains unclear. Our findings demonstrate that Of note, our study revealed that immune response against oxidized an important, multifaceted humoral response against modified lipo­ HDL is different and independent from anti-HDL response in the context proteins hallmarks AAA. It is tempting to speculate that these antibodies of AAA. Although both can be detected in serum in AAA patients, our can further contribute to tissue damage and remodeling once deposited, 177 J. Rodríguez-Carrio et al. Free Radical Biology and Medicine 174 (2021) 171–181 Fig. 4. Anti-HDL-MDA responses in tissue-conditioned media. IgG anti-HDL-MDA (A) and IgG anti-HDL (B) levels (measured as absorbance) were quantified in tissue-conditioned media samples obtained from thrombus (AAA-ILT, n = 82) and media layer (AAA-wall, n = 84) samples from AAA patients as well as from aortas obtained from healthy controls (HC, n = 83). Bars indicate medians, whereas whiskers represent 25th and 75th percentiles. Differences across groups were assessed by Kruskal-Wallis tests and Dunn tests for multiple comparisons. The p-values from multiple comparisons are indicated in the graphs as follows: **p < 0.010, ***p < 0.001, ****p < 0.0001. Fig. 5. IgG anti-HDL-MDA antibodies in AAA patients. IgG anti-HDL-MDA plasma levels measured as AU (A) or normalized after total IgG correction (B) in AAA patients (n = 427) and healthy controls (n = 139). Bars indicate 25th, median and 75th percentiles. Differences were assessed by Mann-Whitney U tests. **p < 0.010, ***p < 0.001. due to antigen recognition or as immune-complexes. The activation of Our experimental approach combining a pre-clinical model of AAA complement fragments, mainly in the ILT of AAA, supports this notion and human samples from AAA patients allowed us to suggest that anti- [60]. However, although the findings herein reported are mostly sup­ HDL-MDA emergence is a consequence of previous exposure to HDL- portive of a detrimental effect of anti-HDL-MDA antibodies, potential MDA. This antibody response was not generated upon treatment with protective actions may be also hypothesized. By virtue of their highly native HDL particles. Actually, our method allows us to establish that all specific binding capacity, anti-HDL-MDA antibodies could also act by the anti-HDL-MDA response arise from oxidized epitopes, thus excluding blocking and scavenging the circulating HDL-MDA particles, hence any potential effect of the native HDL particle (and thus, of potential counteracting the negative effects of the latter. As potential double-edge inter-species reactivity). Therefore, anti-HDL-MDA antibodies can be swords, to what extent detrimental effects outweigh potential beneficial surrogate readouts of HDL-MDA exposure. In fact, MDA plasma levels roles in different scenarios must be established. Thus, further research is are increased in AAA patients and are associated with higher risk of AAA needed to determine their net effect in AAA progression along disease rupture [61,62]. However, measuring oxidative stress status is chal­ course. lenging from a technical point of view and difficult to translate into 178 J. Rodríguez-Carrio et al. Free Radical Biology and Medicine 174 (2021) 171–181 Table 2 4.1. Strengths and limitations Association between IgG anti-HDL-MDA levels and AAA by univariate and multivariate regression analyses. The association between IgG anti-HDL-MDA This study has important strengths that should be remarked, such as levels and AAA presence was analyzed in the cohort presented in Table 1. the use of a large, prospective cohort of AAA patients, and the simul­ OR 95% CI p-value taneous assessment in an animal, pre-clinical model. Although a po­ Univariate model tential effect of heterologous lipoproteins in immunity may be Anti-HDL-MDA, per unit 1.16 1.006–1.026 0.001 hypothesized, infused, native lipoproteins were observed to maintain a Multivariate model functional activity and our protocol (dosages, time schemes and Anti-HDL-MDA, per unit 1.015 1.003–1.026 0.012 administration pathway) can be characterized as tolerogenic. Moreover, Current smoking, yes 2.372 1.369–4.110 0.002 Lowest ABI, per unit 0.000 0.000–0.004 <0.001 our experimental procedure allowed to distinguish the signal produced Former CVD, yes 0.777 0.381–1.581 0.486 by oxidized epitopes compared to that of the native lipoprotein, hence Use of aspirin, yes 1.945 1.078–3.510 0.027 ruling out a potential confounding effect. The combined analyses of the Use of statins, yes 1.274 0.716–2.172 0.435 systemic (plasma) and local (tissue supernatants) from AAA patients further strengthen our results. However, as we do not have paired data in tissue and plasma of the same patients, we could not ascertain in this Table 3 observational study the potential mechanism(s) behind the observed Association between IgG anti-HDL-MDA levels and overall mortality by association of anti-HDL-MDA with AAA presence. Regarding informa­ univariate and multivariate regression analyses. The association between tion bias, follow-up data were based upon a nation-wide registry where IgG anti-HDL-MDA levels and overall mortality was analyzed in AAA patients reporting is mandatory for reimbursement causing a close to 100% from the cohort presented in Table 1. follow-up, and validation studies of the registries have shown a high HR 95% CI p-value internal validity. Consequently, information bias is unlikely. Finally, we Univariate model used a systematic approach to identify confounders, but in the end, re­ Anti-HDL-MDA, per unit 1.008 1.001–1.016 0.023 sidual confounding by nature is always a risk in observational studies. Multivariate model In conclusion, MDA-modified HDL particles elicit a specific humoral Anti-HDL-MDA, per unit 1.009 1.002–1.017 0.016 response and antibodies directed against HDL-MDA can be detected in Current smoking, yes 1.170 0.770–1.777 0.462 Lowest ABI, per unit 0.237 0.084–0.673 0.007 AAA patients, both in the systemic and local compartments. Further­ Former CVD, yes 1.212 0.700–2.097 0.493 more, anti-HDL-MDA levels were associated with overall mortality in Use of aspirin, yes 1.175 0.408–1.094 0.511 AAA patients. These findings expand the previous knowledge on the Use of statins, yes 0.668 0.408–1.094 0.109 contribution of humoral adaptive responses in AAA and point to a connection with oxidized HDL as a potential trigger. clinical routine. Regarding free MDA, thiobarbituric acid reactive sub­ Funding stances (TBARS) assay is the method of choice but with limited speci­ ficity. Further methods have been developed in order to improve the This study was funded by the Spanish MINECO (SAF2016-80843-R, specificity of MDA measurement, such as HPLC combined with UV, fluorescence detection or reversed-phase HPLC coupled with fluores­ PID2019-106814RB-I00 and PGC2018-097019-B-I00), CAM (Com­ plemento II-CM, S2017/BMD-3673), Fondo de Investigaciones Sani­ cence detection [63]. However, most of these methods lack of broad availability. Conversely, the determination of anti-HDL-MDA levels is a tarias ISCIII-FEDER (PI19/00128, Biobancos RD09/0076/00101, PI16/ 00113 and PT17/0019/0003-ISCIII-SGEFI/ERDF, ProteoRed), and relatively simple and feasible approach, which can indirectly account for the HDL-MDA burden, and thus lipid peroxidation, in AAA patients. The FEDER “Una manera de hacer Europa”. CIBERCV and CIBERDEM are Instituto de Salud Carlos III projects. The work leading to these results detection of autoantibodies against known lipo-oxidation adducts, namely MDA, malondialdehyde-acetaldehyde (MAA), MDA-LDL, and has also received funding from “la Caixa” Banking Foundation under the project code HR17-00247. MAA-LDL, has been used to evaluate their potential as biomarkers of CV diseases [64,65]. In this respect, circulating levels of anti-MAA anti­ Author contributions bodies were elevated in patients with AAA [66]. More importantly, an IgG anti-HDL-MDA response would imply a certain and sustained Conceptualization, JRC, AS, JLMV; methodology, JRC, JSL, AS, exposure to HDL-MDA, which is more likely to have a biological effect, whereas the exact significance of a single assessment with elevated ROS JLMV; formal analysis, JSL; resources, JRC, ICP, JSL, EBB, DML, RRM, JCEG, JBM, LMBC, JV, AS, JLMV; writing—original draft preparation, or oxidative stress status may be difficult to evaluate. Capturing the exposure to lipid peroxidation and oxidative stress can be of interest for JRC, ICP, AS, JLMV; writing—review and editing, JRC, ICP, JSL, EBB, DML, RRM, JCEG, JBM, LMBC, JV, AS, JLMV; funding acquisition, the clinical setting. In fact, oxidative stress has been reported as a pre­ JCEG, AS, JLMV. dictor of overall (all-cause) mortality in a number of conditions [67–69], in part due to its widespread involvement in pathology. This is in line Declaration of competing interest with our findings of anti-HDL-MDA as predictors of overall mortality in AAA. Therefore, the quantification of anti-HDL-MDA levels may have The authors declared no conflicts of interest and no financial in­ potential applications in the decision-taking process in order to guide terests or competing interests. Funders have no role in study conception preventive and/or therapeutic measures to counteract the oxidative and design, data analysis and interpretation or decision to publish. stress-induced traits. However, clinical validation and appraisal of their added value are needed. Overall, our findings align with the idea that Appendix A. Supplementary data the association between HDL and AAA is more complex than initially proposed. Then, additional features such as lipoprotein functionality, Supplementary data to this article can be found online at https://doi. lipoprotein quality [70] or the presence of (auto)antibodies should be considered as well, hence moving from a conventional lipid org/10.1016/j.freeradbiomed.2021.08.004. levels-centered model to a much more sophisticated approach that ac­ counts for their actual biological relevance. 179 J. Rodríguez-Carrio et al. Free Radical Biology and Medicine 174 (2021) 171–181 References inflammation and cardiovascular disease in rheumatoid arthritis patients, Transl. Res. 166 (2015) 529–539, https://doi.org/10.1016/j.trsl.2015.07.004. [21] J. Rodríguez-Carrio, L. Mozo, P. López, E. Nikiphorou, A. Suárez, Anti-high-density [1] Abdominal aortic aneurysms, Nat. Rev. Dis. Prim. 4 (2018) 35, https://doi.org/ lipoprotein antibodies and antioxidant dysfunction in immune-driven diseases, 10.1038/s41572-018-0036-1. Front. Med. 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