antioxidants Article Oxidative Stress and Inflammatory Markers in Abdominal Aortic Aneurysm David Sánchez-Infantes 1,2 , Meritxell Nus 3,4 , Miquel Navas-Madroñal 5,6 , Joan Fité 7 , Belén Pérez 8 , Antonio J. Barros-Membrilla 9 , Begoña Soto 7 , José Martínez-González 4,6,10 , Mercedes Camacho 4,5,6 , Cristina Rodriguez 4,5,6 , Ziad Mallat 3 and María Galán 4,5,6, * 1 Department of Basic Sciences of Health, Area of Biochemistry and Molecular Biology, University Rey Juan Carlos, 28922 Alcorcón, Spain; dsanchez@igtp.cat 2 Centro de Investigación Biomédica en Red-Fisiopatología de la Obesidad y Nutrición (CIBEROBN), ISCIII, 28029 Madrid, Spain 3 Division of Cardiovascular Medicine, University of Cambridge, Cambridge CB2 0QQ, UK; mn421@cam.ac.uk (M.N.); zm255@medschl.cam.ac.uk (Z.M.) 4 Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), ISCIII, 28029 Madrid, Spain; jose.martinez@iibb.csic.es (J.M.-G.); mcamacho@santpau.cat (M.C.); crodriguezs@santpau.cat (C.R.) 5 Institut de Recerca del Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain; mnavasm7@gmail.com 6 Instituto de Investigación Biomédica Sant Pau (IB Sant Pau), 08025 Barcelona, Spain 7 Servicio de Angiología, Cirugía Vascular y Endovascular, Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain; jfite@santpau.cat (J.F.); bsoto@santpau.cat (B.S.)  8 Faculty of Medicine, Universidad Autónoma de Barcelona, Bellaterra, 08193 Barcelona, Spain;  Belen.Perez@uab.cat 9 Unidad Funcional de Patología de la Aorta (UPA), Servicio de Cardiología, Hospital de la Santa Creu i Sant Citation: Sánchez-Infantes, D.; Nus, M.; Navas-Madroñal, M.; Fité, J.; Pau, 08041 Barcelona, Spain; abarros@santpau.cat 10 Instituto de Investigaciones Biomédicas de Barcelona-Consejo Superior de Investigaciones Pérez, B.; Barros-Membrilla, A.J.; Soto, Científicas (IIBB-CSIC), 08036 Barcelona, Spain B.; Martínez-González, J.; Camacho, * Correspondence: mgalana@santpau.cat; Tel.: +34-935565622 M.; Rodriguez, C.; et al. Oxidative Stress and Inflammatory Markers in Abstract: Abdominal aortic aneurysm (AAA) is increasing due to aging of the population and is a Abdominal Aortic Aneurysm. Antioxidants 2021, 10, 602. https:// major cause of death among the elderly. Ultrasound screening programs are useful in early diagnosis, doi.org/10.3390/antiox10040602 but aneurysm size is not always a good predictor of rupture. Our aim was to analyze the value of circulating molecules related to oxidative stress and inflammation as new biomarkers to assist the Academic Editors: Guillermo Zalba management of AAA. The markers were quantified by ELISA, and their expression in the aneurysmal and María U. Moreno Zulategui wall was studied by real-time PCR and by immunostaining. Correlation analysis of the studied markers with aneurysm diameter and peak wall stress (PWS), obtained by finite element analysis, Received: 22 March 2021 and multivariate regression analysis to assess potential confounding factors were performed. Our Accepted: 12 April 2021 study shows an extensive inflammatory infiltration in the aneurysmal wall, mainly composed by Published: 14 April 2021 T-cells, macrophages and B-cells and altered levels of reactive oxygen species (ROS), IgM, IgG, CD38, GDF15, S100A4 and CD36 in plasma and in the aneurysmal tissue of AAA patients compared with Publisher’s Note: MDPI stays neutral controls. Circulating levels of IgG, CD38 and GDF15 positively correlated with abdominal aortic with regard to jurisdictional claims in diameter, and CD38 was correlated with PWS. Our data show that altered levels of IgG, CD38 and published maps and institutional affil- GDF15 have potential diagnostic value in the assessment of AAA. iations. Keywords: abdominal aorta aneurysm; biomarkers; inflammation; oxidative stress; prognosis Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and Rupture of abdominal aortic aneurysm (AAA) is a life-threatening condition with a conditions of the Creative Commons high mortality rate [1]. The implementation of ultrasound screening programs to identify Attribution (CC BY) license (https:// small AAAs has improved the current diagnosis and management of this condition; how- creativecommons.org/licenses/by/ ever, rates of expansion of AAAs during follow-up vary for intra and inter-patients [2,3]. 4.0/). Although the risk of rupture increases exponentially with maximal anterior–posterior Antioxidants 2021, 10, 602. https://doi.org/10.3390/antiox10040602 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 602 2 of 16 aortic diameter, aneurysm size is not always a good predictor since small aneurysms can lead to rupture and a proportion of large AAA remain stable overtime [4,5]. The identifi- cation of circulating markers of AAA progression will complement the measurement of aortic diameter and will help to improve the identification of patients at risk of aneurysm rupture [4]. Different pathological mechanisms converge on AAA development, such as vascular oxidative stress, inflammation, extracellular matrix (ECM) remodeling, altered cell sig- naling and increased apoptosis in vascular smooth muscle cells (VSMCs) [1,6]. Chronic inflammation plays a central role promoting proteinases-induced tissue destruction and suppressing tissue repair in AAA [7]. Further, the implication of local oxidative stress in the pathogenesis of AAA is also well documented [8–10]. Human AAA tissue is characterized by an early infiltration of innate and adaptive inflammatory cells, which occurs before detectable ECM destruction and aortic diameter expansion [11–13]. This inflammatory en- vironment critically contributes to vascular oxidative stress, which in turn could exacerbate the recruitment of inflammatory cells in a reciprocal feedback loop [13]. In order to identify new circulating biomarkers and determine their potential prog- nostic value in aneurysm progression, we selected a battery of immune-inflammatory and oxidative stress markers, involved in the degenerative events associated with the pathophysiology of AAA. In particular, we focused on the transmembrane enzyme CD38, growth differentiation factor 15 (GDF15), the member of the S100 calcium-binding protein family, S100A4, and the lipid scavenger receptor CD36 proteins that are expressed on the surface of lymphocytes, macrophages, endothelial cells and VSMC. All of them are involved in oxidative stress generation and in inflammatory processes underlying vascular remodeling and contribute to phenotypic changes and cellular apoptosis under different pathological conditions [14–23]. In the current paper, we analyzed vascular expression and circulating levels of IgM, IgG, reactive oxygen species (ROS), CD38, GDF15, S100A4 and CD36 in samples from patients with AAA vs. healthy controls and for the first time, assessed their potential association with two well-established parameters for predicting the risk of rupture, aneurysm diameter and peak wall stress (PWS). 2. Materials and Methods 2.1. Human Samples Human abdominal aneurysmal aortas (n = 80) and blood samples (n = 94) were obtained from patients diagnosed with infrarenal AAA and undergoing open repair or endovascular surgery repair for AAA at the Hospital de la Santa Creu i Sant Pau (HSCSP; Barcelona, Spain). The diagnosis of AAA was confirmed by computed tomography (CT) scan. Patients with AAA and with negative histories of rheumatological, immunological diseases, aortitis or genetic syndromes such as Marfan disease were included in the study. Other exclusion criteria were juxtarenal aneurysms and mycotic aneurysms. The plasma samples were collected prior to anesthesia on the same day of the surgical intervention. Healthy abdominal aortas (n = 15) came from multiorgan donors. The Ethics Committee of the HSCSP approved the use of the discarded human tissue and participation in the study of patients and controls was based upon informed consent. Research was conducted in accordance with the Declaration of Helsinki of 1975. Abdominal aorta segments and plasma samples were obtained according to standard operating procedures and ethical guidelines. No signs of AAA or evidence of atherosclerotic plaques in the abdominal aorta were found in samples of control subjects. For RNA and protein studies part of the tissue samples were collected, snap-frozen and stored at −80 ◦ C and part were fixed and embedded in paraffin for subsequent immunostaining assays. The blood samples from healthy donors were provided by the Banc de Sang i Teixits (BST, Barcelona, Spain) according to the following inclusion criteria: healthy subjects aged 55–65 years, non- hypertensive or with pharmacologically controlled hypertension and without previously reported cardiovascular complications (n = 46). Antioxidants 2021, 10, 602 3 of 16 2.2. ELISA Kits The circulating levels of soluble IgM (ab214568, Abcam, UK), IgG (ab195215), soluble CD38 (KIT10818-1, Sino Biological Inc., Wayne, PA, USA), soluble CD36 (ABE-196-02, Nordic BioSite, Täby, Sweden), S100A4 (CSB-EL020632HU, Cusabio Biotech Co, LTD, Beijing, China) and GDF15 (Quantikine ELISA Human GDF15, DGD150; R&D Systems, Minneapolis, MN, USA), in plasma from patients were measured using commercially available ELISA kits in accordance with the manufacturer’s instructions. 2.3. Total mRNA and Protein Isolation from Tissue To isolate total RNA from human aortic samples, the RNeasy Fibrous Mini Kit (Qiagen, Venlo, The Netherlands) was used following the manufacturer’s instructions and RNA integrity was determined and quantified by a NanoDrop Spectrophotometer (Thermo Scientific). Protein lysates from human aortic samples were homogenized using a Tissue RuptorII (Qiagen) in a RIPA buffer (150 mM NaCl, 1% (v/v) Triton X-100, 0.5% 113 (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 2 mM EDTA and 50 mM Tris-HCl pH 8) following a standard protocol. 2.4. Quantitative Real-Time PCR DNase I-treated total RNA (1 µg) was reverse transcribed into cDNA using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). The mRNA levels were quantified by using specific primers and probes for human CD36 (Hs00169627_m1) and CD38 (Hs01120071_m1) provided by the assay-on-demand system (Applied Biosys- tems). As housekeeping genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs02758991_g1) and β-actin (Hs99999903_m1) were used. Each sample was amplified in duplicate by quantitative RT-PCR in an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) and the same results were obtained after normalization to both housekeeping genes. Relative mRNA levels were determined using the 2−∆∆Ct method. 2.5. Immunostaining and Histology Tissue specimens were formalin fixed and paraffin embedded. Immunostaining assays were performed in human aortic sections as previously described [24]. For immunohisto- chemistry assays sections (5 µm) of aneurysmal aortas and donor abdominal aortas were deparaffinized in xylene, rehydrated in graded ethanol and treated with 0.3% hydrogen peroxide for 30 min to block peroxidase activity. Then, sections were blocked with 10% of fetal bovine serum in PBS containing 0.1% Triton X-100and incubated with antibodies against CD3 (SK202, Dako), CD68 (M0876, Dako), CD19 (F0768, Dako), Neutrophil Elastase (M0752, Dako), IgG (A0423, Dako), CD38 (NBP1-47462), GDF15 (NBP1-81050), S100A4 (NBP1-89402) and CD36 (NB400-144) overnight at 4 ◦ C. After incubation with primary antibodies, three washes with PBS were performed to remove excess of unbound antibody and the slides were incubated with a biotinylated secondary antibody (Vector Laborato- ries, Peterborough, UK) for 1 h. After rinsing 3 times in PBS, standard Vectastain (ABC) avidin-biotin peroxidase complex (Vector Laboratories) was applied to sections for 30 min and after adding the substrate 3,30 -diaminobenzidine (DAB), color was developed. Finally, sections were counterstained with hematoxylin, dehydrated and cleared before mounting. For antigen colocalization studies, after deparaffinization, antigen retrieval and per- meabilization with PBS-0.3% Triton X-100, sections were blocked with PBS containing 5% albumin and 5% FBS for 1 h at room temperature. Double-fluorescence immunostaining was performed by sequentially incubating the sections with mouse polyclonal antibodies against CD206 (ab64693) or CD80 (ab254579) at 4 ◦ C overnight followed by the incubation with a rabbit polyclonal antibody against CD68 (M0876, Dako). After washing the excess of unbound primary antibodies, secondary antibodies (donkey anti-rabbit IgG conjugated to Alexa Fluor 555 and goat anti-mouse IgG conjugated to Alexa Fluor 488, Molecular Probes, Life Technologies) were applied for 1 h at 20–22 ◦ C. Anti-DNA/RNA Damage mouse monoclonal antibody (ab62623) to detect Oxo-8-dG (8-Oxo-7,8-dihydro-20 -deoxyguanosine) Antioxidants 2021, 10, 602 4 of 16 was also used followed by an incubation with a secondary antibody goat antimouse con- jugated to Alex FLuor 488. Finally, the slides were mounted with ProLong Gold antifade reagent with DAPI (Molecular Probes, Life Technologies Co., Eugene, OR, USA). For nega- tive controls the primary antibody was omitted. Imaging of epifluorescent stainings was performed using a Leica DM6000B microscope, and images were analyzed using Adobe Photoshop CS5 and ImageJ (NIH). Results were quantified as a positive cell number per area in independent sections of AAA. 2.6. Reactive Oxygen Species (ROS) Quantification in Plasma 2-hydroxyethidium (2-EOH) was determined in plasma by HPLC with fluorescence detection as described by Laurindo et al. 2008 [25]. Briefly, 25 µL of plasma in the presence of dihydroethidine (DHE, Sigma-Aldrich, St. Louis, MO, USA) 0.8 µM were incubated for 1 h in PBS and centrifuged at 10,000× g for 5 min. The supernatant was injected into the chromatographic system (mobile phase: 65% water with 0.3% Trifluoracetic acid: 35% acetonitrile; flow 1 mL /min; column kromasil C18, 5 µm, 200 × 4.6 mm-Teknokroma Analitica; excitation 510 nm and emission 595 nm). Xanthine/xanthine oxidase (XO) was used to calibrate the signal of O2− and the calibration curve was constructed by comparing the production of 2-EOH and the ratio of XO activity/HE concentration (85–605 nU XO/ng HE). 2-EOH present in the samples was quantified by comparing with the calibration curve based on the reaction xanthine–xanthine oxidase in the presence of DHE (Sigma-Aldrich, St. Louis, MO, USA) following the method described by Michalski et al. 2014 [26]. 2.7. Western Blot Aortic tissue lysates were separated by SDS-PAGE, transferred to the PVDF blotting membrane (Immobilon, Millipore. Merck KGaA, Darmstadt, Germany) and incubated overnight at 4 ◦ C with an antibody against S100A4 acquired from Novus Biologicals (Bio- Techne LD-R&D Systems Europe Ltd., Abingdon, UK). Equal loading of protein in each lane was verified by β-actin (A5441, Sigma Aldrich, St. Louis, MO, USA). 2.8. Finite Element Analysis Finite element analysis was performed by using A4clinics-Research Edition software (VASCOPS Vascular Diagnosis Company, Graz, Austria) on the computed tomography angiography (CTA) of 60 of the AAA patients included in the present study. Only one member of our group (B.S.) performed the analysis to avoid inter-observer errors as previously described [27]. The three dimensional AAA geometry was acquired from routine CTA imaging data. The resultant geometry is subdivided into multiple contiguous elements that form a fine mesh. The lumen, intraluminal thrombus and external wall data were acquired separately and semi-automatically. Manual correction is allowed by the program if some special point is found, such as a penetrating ulcer or some other unusual anatomy. Wall stress computation of the AAA can be obtained after providing the appropriate material properties and components of the aneurysmal wall and results in aneurysm specific wall stress distribution. We additionally analyzed the segment from the infrarenal aorta to the iliac bifurcation. Among the biomechanical variables that were determined, PWS (the maximal stress on the surface of the AAA wall based on aneurysm shape, diameter and blood pressure values) was the variable chosen for this study. 2.9. Statistical Analysis Results are expressed as mean ± SEM of the number (n) of samples indicated in the figure legends. Demographic and clinic characteristics of the human individuals were expressed as mean ± SD. Normal distribution of the variables was determined by Shapiro– Wilk test. When data followed a normal distribution, differences between two groups were assessed using the Student’s t test (two-tailed). When normality failed, the Mann– Whitney rank sum test was applied to compare two groups. To study the association between variables following a normal distribution, Pearson product-moment correlation Antioxidants 2021, 10, 602 5 of 16 coefficient was used whereas Spearman’s rank-order correlation was applied to determine the association between variables that are not. To assess potential confounding factors (age, sex, hypertension, DM and smoking), multivariate logistic regression analysis was performed after log-transforming non-normal distributed variables using the statistical software package R-3.2.2 (www.r-project.org (accessed on 22 March 2021)). The results were expressed as an odd ratio (OR) and 95% confidence intervals (CIs). Data analysis was carried out using GraphPad Prism version 8.4.2 software (La Jolla, CA, USA). Values of p ≤ 0.05 were considered significant. 3. Results 3.1. Immune Infiltrate and Immunoglobulins in Human AAA We evaluated the immune cell content in aneurysmal tissue from our AAA patient cohort. Table 1 shows demographic data of the patients and donors included in this study. The majority of the immune cells present in aneurysmal wall were T-cells (CD3), macrophages (CD68) and B cells (CD19), and to a lesser extent neutrophils (neutrophil elastase) as shown in Figure 1A–D. Table 1. Demographics of individuals included in the study. mRNA Plasma AAA Normal Aorta AAA Blood Donors N 80 15 94 46 Age (years) 70 ± 6.2 63 ± 11.5 71.4 ± 6.5 59.9 ± 5.3 * Women (%, N) 5 (4) 20 (3) 4.3 (4) 10.9 (5) Aortic diameter (mm) 63.5 ± 1.4 - 63.9 ± 1.4 - Dyslipidemia (%, N) 56.3 (45) 26.7 (4) 63.8 (60) - HTN (%, N) 81.25 (66) 40 (6) 77.6 (73) 19.6 (9) Diabetes (%, N) 21.25 (17) 33.3 (5) 19.1 (18) 23.91 (11) Smokers/ex-smokers (%, N) 81.25 (66) 40 (6) 80.1 (76) 45.7 (21) PWS (kPa) 222 ± 74.8 - 222 ± 74.8 - TC (mM) - - 3.9 ± 1.2 5.6 ± 0.8 HDL-C (mM) - - 1 ± 0.3 1.5 ± 0.3 LDL-C (mM) - - 2.3 ± 0.9 3.5 ± 0.7 VLDL-C (mM) - - 0.6 ± 0.3 0.6 ± 0.3 Triglycerides (mM) - - 1.3 ± 0.7 1.4 ± 0.7 Nominal variables are presented as %. Continuous variables are presented as mean ± SD. Due to the nature of normal aorta and plasma samples from donors, some of the clinical characteristics are not always recorded and infra-evaluation of them is probable. HTN, chronic hypertension; PWS, peak wall stress; TC: total cholesterol; HDL-C: high-density lipoprotein-cholesterol; LDL-C: low density lipoprotein-cholesterol. * p < 0.05 AAA vs. Donors. Recent studies have shown that there are distinct macrophage subsets with different functionalities in human and experimental AAA tissues [9,28,29]. Aneurysmal tissue from AAA patients exhibited markers of both classical M1 (CD68+ CD80+) macrophages and alternatively activated M2 (CD68+ CD206+) macrophages (Supplementary Figure S1A,B) in the immune infiltrate. On another hand, we found that circulating IgM levels were significantly decreased, whereas IgG levels were increased in AAA patients compared to controls (Figure 2A,B). Confounding factors had no impact on the difference between circulating levels of IgGs or IgMs found in patients with AAA in comparison with those found in control subjects (Table S1). When IgG was measured in situ in paraffin-embedded tissue, a higher number of positively stained cells located in the adventitia was observed in stark contrast to that observed in healthy aortas (Figure 2C). Antioxidants Antioxidants2021, 2021,10, 10,x 602 FOR PEER REVIEW 6 of 6 of1516 Figure 1. Immune infiltrate in AAA. (A–D) Representative images of immunostaining assays performed in abdominal aorta Figure 1. from sections Immune AAA infiltrate patientsinand AAA. (A–D) donors Representative targeting images of s), CD3 (T-lymphocyte immunostaining assays CD19 CD68 (macrophages), performed in abdominal (B-lymphocytes) and aorta sections from AAA patients and donors targeting CD3 (T-lymphocyte s), CD68 (macrophages), CD19 (B-lympho- elastase (Neutrophils), respectively (n = 10; Scale bars: 50 µm). AAA (-) indicates negative control for immunohistochemistry. cytes) and elastase (Neutrophils), respectively (n = 10; Scale bars: 50 μm). AAA (-) indicates negative control for immuno- Arrows indicate the positively stained cells. histochemistry. Arrows indicate the positively stained cells. controls (Figure 2A,B). Confounding factors had no impact on the difference between cir culating levels of IgGs or IgMs found in patients with AAA in comparison with those found in control subjects (Table S1). When IgG was measured in situ in paraffin-embed Antioxidants 2021, 10, 602 ded tissue, a higher number of positively stained cells located in the adventitia 7 of 16 was ob served in stark contrast to that observed in healthy aortas (Figure 2C). Figure 2. Figure Circulating levels levels 2. Circulating of IgM andand of IgM IgGIgGare arealtered inAAA altered in AAA patients. patients. (A,B)(A,B) Circulating Circulating levels oflevels IgM andofIgG, IgMrespectively and IgG, respec- tively in AAA in AAA(n(n== 94) vs.healthy 94) vs. healthy donors donors (n =(C) (n = 46). 46). (C) Representative Representative images of immunostaining images of immunostaining assays performedassays performed in in abdominal abdominal aorta aorta sections sections from donors from donors and AAAand AAA patients patients targeting targeting IgGs IgGs (n = 10; scale (n 100 bars: = 10; scale µm). AAAbars: 100 μm). (-) indicates AAA negative (-) indicates control for immunohistochemistry. Arrows indicate the positively stained cells. negative control for immunohistochemistry. Arrows indicate the positively stained cells. 3.2. Vascular and Circulating Levels of Oxidative Stress and Inflammation-Related Biomarkers in 3.2. Vascular and Circulating Levels of Oxidative Stress and Inflammation-Related Biomarkers AAA Patients in AAA Patients When ROS plasma levels were tested in our cohort, we observed a 2.5-fold increase in AAA patients When compared ROS plasma to controls levels were (Figure tested 3A) andcohort, in our this difference remainedasignificant we observed 2.5-fold increas after adjusting for confounding factors (Table S1). This was accompanied by an increase in in AAA patients compared to controls (Figure 3A) 0and this difference remained significan the number of positive cells for 8-Oxo-7,8-dihydro-2 -deoxyguanosine (a marker of DNA after damage adjustingduefor confounding to ROS) factors in aneurysmal (Table tissues S1). This compared wasaortas to donor accompanied by an increase (Figure 3B,C). in the number CD38 is of positive expressed on cells for 8-Oxo-7,8-dihydro-2′-deoxyguanosine the surface of lymphocytes, macrophages and neutrophils, (aand marker o DNAitdamage dueinto is involved ROS) their in aneurysmal activation, tissues proliferation and compared to donor aortas homing by interaction (Figure with CD31 in 3B,C) endothelial cells and in the regulation of the innate immune response [14,17,18]. It has been recently involved in the production of O2− and in the modulation of Nrf2 activity in VSMC [30]. Hematopoietic and vascular cells also express CD36 receptor in response to oxidized lipids [23] and CD36 signaling downregulates key antioxidant factors, leading to the accumulation of ROS [31]. CD38 and CD36 mRNA levels were upregulated in the aneurysmal vascular wall compared to healthy abdominal aortas (Figure 4A, Supple- it is involved in their activation, proliferation and homing by interaction with CD31 in endothelial cells and in the regulation of the innate immune response [14,17,18]. It has been recently involved in the production of O2− and in the modulation of Nrf2 activity in VSMC [30]. Hematopoietic and vascular cells also express CD36 receptor in response to Antioxidants 2021, 10, 602 oxidized lipids [23] and CD36 signaling downregulates key antioxidant factors, leading 8 of 16 to the accumulation of ROS [31]. CD38 and CD36 mRNA levels were upregulated in the aneurysmal vascular wall compared to healthy abdominal aortas (Figure 4A, Supplemen- tary Figure S2A). Immunohistochemistry revealed that CD38 and CD36 were barely de- mentary Figure S2A). Immunohistochemistry revealed that CD38 and CD36 were barely tectable in donor aortas, but in aneurysmal tissues CD38 was markedly found in the media detectable in donor aortas, but in aneurysmal tissues CD38 was markedly found in the and the adventitia layers (Figure 4D) whereas CD36 was mainly observed in the media media and the adventitia layers (Figure 4D) whereas CD36 was mainly observed in the (Supplementary Figure S2B). Regarding plasma levels, we found a 1.7-fold increase of media (Supplementary Figure S2B). Regarding plasma levels, we found a 1.7-fold increase CD38 inofAAACD38patients in AAAcompared to controls patients compared (Figure 4B) to controls that4B) (Figure remained significant that remained after after significant adjusting for confounding factors (Table S1). However, the circulating levels of soluble adjusting for confounding factors (Table S1). However, the circulating levels of soluble CD36, which CD36,were whichbarely weredetected or undetected barely detected in mostinofmost or undetected the analyzed samples, of the analyzed were were samples, similar in both groups (Supplementary Figure S2C). similar in both groups (Supplementary Figure S2C). A B p < 0.0001 C Donor AAA 8-OH-G lumen 8-OH-G media media adventitia Figure 3. Reactive oxygen species (ROS) circulating levels are increased in AAA patients. (A) Quantification of ROS plasma levels in AAA (n = 94) vs. healthy donors (n = 46). (B) Histograms showing the quantification of the number of positive cells per aortic area (10×). Results are expressed as mean ± SEM. (C) Representative images of immunostaining assays performed in abdominal aorta sections from donors and AAA patients targeting 8-oxo-DG positive stained cells. GDF15 is a stress-induced cytokine, produced and secreted by endothelial cells, mac- rophages, VSMC and cardiac myocytes in response to ischemia, proinflammatory stimuli or oxidative stress [19,20]. Plasma GDF15 levels were increased by 3-fold in AAA patients compared to controls (Figure 4C) and this difference remained significant after adjusting Antioxidants 2021, 10, 602 for confounding factors (Table S1). GDF15 was slightly expressed in the media9 layer of 16 of aneurysmal aortas, whereas no expression was detected in healthy vessels (Figure 4E). Figure 4. CD38 and GDF15 expression and circulating levels are increased in AAA patients. (A) Human abdominal aortic mRNA levels of CD38 measured by quantitative real-time PCR and normalized to β-actin in healthy donors (n = 15) and patients (AAA) (n = 80). (B) CD38 plasma levels in AAA (n = 94) vs. healthy donors (n = 46); (C) plasma levels of GDF15 in AAA (n = 94) vs. healthy donors (n = 46). (D) Representative images of immunostaining assays performed in abdominal aorta sections from donors and AAA patients targeting CD38 (n = 10; scale bars: 100 µm). (E) Representative images of immunostaining assays performed in abdominal aorta sections targeting GDF15 (n = 0; scale bars: 100 µm). AAA (-) indicates negative control for immunohistochemistry. Arrows indicate the positively stained cells. GDF15 is a stress-induced cytokine, produced and secreted by endothelial cells, macrophages, VSMC and cardiac myocytes in response to ischemia, proinflammatory Figure 4. CD38 and GDF15 expression and circulating levels are increased in AAA patients. (A) Human abdominal aortic mRNA levels Antioxidants of CD38 2021, 10, 602 measured by quantitative real-time PCR and normalized to β-actin in healthy donors (n = 15)10and of 16 patients (AAA) (n = 80). (B) CD38 plasma levels in AAA (n = 94) vs. healthy donors (n = 46); (C) plasma levels of GDF15 in AAA (n = 94) vs. healthy donors (n = 46). (D) Representative images of immunostaining assays performed in abdominal aorta sections from donors and AAA patients targeting CD38 (n = 10; scale bars: 100 μm). (E) Representative images of immunostaining assays performedstimuliin or abdominal oxidativeaorta sections stress [19,20].targeting Plasma GDF15 GDF15 (n = 0; scale levels werebars: 100 μm). increased AAA (-) by 3-fold in indi- AAA cates negative control for immunohistochemistry. patients compared toArrows controls indicate (Figurethe 4C)positively and this stained cells. difference remained significant after ad- justing for confounding factors (Table S1). GDF15 was slightly expressed in the media layer S100A4 is aaortas, of aneurysmal potentwhereas trigger noof inflammatory expression wasprocesses detected inbyhealthy inducing the (Figure vessels release 4E). of cyto- S100A4 is a potent trigger of inflammatory processes by inducing the kines, growth factors and ROS production under different pathological conditions [22,32]. release of cy- tokines, growth Circulating S100A4factors wasand ROS production significantly under different decreased in AAApathological conditionsto patients compared [22,32]. controls Circulating S100A4 was significantly decreased in AAA patients compared (Figure 5A) and this difference remained significant after adjusting for every confounding to controls (Figure 5A) and this difference remained significant after adjusting for every confounding factor (Table S1). This was supported by a decreased S100A4 expression in the aneurysmal factor (Table S1). This was supported by a decreased S100A4 expression in the aneurysmal wall compared to healthy abdominal aortas (Figure 5B,C). wall compared to healthy abdominal aortas (Figure 5B,C). Figure 5. S100A4 Figure is decreased 5. S100A4 is decreased inin AAA AAApatients patientscompared to healthy compared to healthydonors. donors.(A) (A)Plasma Plasma levels levels of of S100A4 S100A4 in AAA in AAA (n =(n 94)= 94) vs. healthy vs. healthy donors (n = 46); (B) representative Western blot analysis of S100A4 in protein lysates of abdominal aortas fromfrom donors (n = 46); (B) representative Western blot analysis of S100A4 in protein lysates of abdominal aortas AAA AAApatients and patients anddonors donors(AAA: (AAA: nn ==15 15and anddonors: donors: n =n10); = 10); (C) representative (C) representative imagesimages of immunostaining of immunostaining assays per- assays performed formed in abdominal aorta sections from donors and AAA patients targeting S100A4 (n = 10; scale bars: 100 μm). AAA (- in abdominal aorta sections from donors and AAA patients targeting S100A4 (n = 10; scale bars: 100 µm). AAA (-) indicates ) indicates negative control for immunohistochemistry. Arrows indicate the positively stained cells. negative control for immunohistochemistry. Arrows indicate the positively stained cells. 3.3. Correlation of Biomarker Levels and Preoperative Aortic Diameter 3.3. Correlation of Biomarker Levels and Preoperative Aortic Diameter Positive Positive correlations between correlations between IgG, IgG, GDF15GDF15 or levels or CD38 CD38andlevels and preoperative preoperative abdominal ab- dominal aortic diameter were found by correlation analysis (Figure 6A–C) whereas no aortic diameter were found by correlation analysis (Figure 6A–C) whereas no correlations correlations between thebetween diameterthe diameter and andROS circulating circulating or S100A4ROS or S100A4 levels levels were found were (data notfound (data shown). Antioxidants 2021, 10, x FOR PEER REVIEW 11 of 15 Antioxidants 2021, 10, 602 11 of 16 not shown). When correlations between each biomarker and PWS were analyzed, only a When correlations between each biomarker and PWS were analyzed, only a positive positive correlation correlation withlevels with CD38 CD38 levels was was found found (Figure (Figure 6D). All the6D). All the associations established establishedwere associa- tions were maintained after adjustment maintained after adjustment for age. for age. Figure 6. IgG, CD38 and GDF15 circulating levels positively correlate with the AAA diameter whereas only CD38 correlates Figure 6. IgG, CD38 and GDF15 circulating levels positively correlate with the AAA diameter whereas only CD38 corre- with PWS. (A) Graphs showing the correlation analysis between IgG plasma levels and AAA diameter (n = 90) and (B,C) lates with PWS. (A) Graphs showing the correlation analysis between IgG plasma levels and AAA diameter (n = 90) and the correlation analysis between CD38 plasma levels and AAA diameter (n = 90) or PWS values in AAA patients (n = 58). (B,C) the correlation analysis between CD38 plasma levels and AAA diameter (n = 90) or PWS values in AAA patients (n (D) Graph showing the correlation analysis between GDF15 plasma levels and AAA diameter (n = 90). The r and p-values = 58). (D) Graph showing the correlation analysis between GDF15 plasma levels and AAA diameter (n = 90). The r and p- are obtained by performing the Spearman or the Pearson correlation coefficient test. Results are expressed as mean ± SEM. values are obtained by performing the Spearman or the Pearson correlation coefficient test. Results are expressed as mean ± SEM. 4. Discussion The identification of novel circulating markers of AAA is an active focus of inves- 4. Discussion tigation. In the present study, we aimed to determine the value of circulating levels of The identification ofand immune-inflammatory novel circulating oxidative markersmarkers stress-related of AAAtoisassist an active focus of investi- AAA management. gation. In the showed Our results present higher study,IgG, we aimed to determine ROS, CD38, CD36 andthe valueexpression GDF15 of circulating levels of im- in aneurysmal mune-inflammatory andand tissue of AAA patients, oxidative stress-related higher circulating markers levels of to assist soluble IgG, ROS, AAA CD38 andmanagement. GDF15 Our results showed compared higher to controls. IgG, ROS, In contrast, IgM CD38, CD36were and S100A4 andfound GDF15 expression decreased in AAAin aneurysmal patients while tissue ofnoAAA differences wereand patients, found in soluble higher CD36 levels circulating levelsbetween AAA and of soluble IgG,controls. ROS, CD38When and GDF15 compared to controls. In contrast, IgM and S100A4 were found decreased in AAA patients while no differences were found in soluble CD36 levels between AAA and con- trols. When we studied the association between circulating levels of all the altered mole- cules and the preoperative diameter of the abdominal aorta of AAA patients, only IgG, Antioxidants 2021, 10, 602 12 of 16 we studied the association between circulating levels of all the altered molecules and the preoperative diameter of the abdominal aorta of AAA patients, only IgG, CD38 and GDF15 showed a significant correlation independently of age and of cardiovascular risk factors such as hypertension or diabetes. PWS, evaluated using computer modeling by finite ele- ment analysis, is a useful parameter for predicting the risk of rupture [33,34]. Interestingly, when we studied the association of the different circulating molecules with the PWS values, only CD38 was positively correlated. According to our data, circulating IgM, ROS and S100A4 did not correlate either with the diameter or with PWS and since CD36 circulating levels are barely detected, no correlations were studied. However, we cannot underestimate their role in the pathogenesis of AAA. In fact, human and animal studies have confirmed that oxidative stress is well associated with AAA development [13,35–37]; whereas other authors have previously reported CD36 overexpression in aneurysmal aortas [38], and found a positive correlation between the amount of CD36-labeled platelets in the blood of AAA patients and aneurysm diameter [38,39]. Regarding S100A4, circulating levels and tissue expression were strikingly decreased in both plasma and aortic samples from AAA patients compared to controls. Since immune-histochemical studies in donor aortas localize S100A4 expression mainly in VSMC, its decrease in AAA could be due to the well-reported loss of VSMC in this disease. On another hand, despite a previous study reported an increase of S100A4 levels in tissue and in serum of patients with thoracic aortic aneurysm [40], it could be due to the different mechanisms underlying the pathogenesis of abdominal and thoracic aneurysms development. In our AAA patient cohort the circulating levels of IgM were decreased, of note in other cardiovascular diseases IgM have been described as protective [41–43], so further studies are needed in order to clarify if this will be the case in AAA. On the contrary, we found an increase in IgG levels both systemically and in the aneurysmal tissue, potentially indicating an activation of long-lived plasma B cells [44] and of B cells residing in the aneurysmal tissue respectively [8,10]. Immunoglobulins are deposited on AAA tissue and mostly localized to adventitial cells suggesting that they may impact on diverse cell types in the aneurysmal wall [45]. Accordingly, we found increased deposits of IgG and a high number of B-cells in aneurysmal tissue together with T-lymphocytes and macrophages, which are most of the cells detected in the inflammatory infiltrate of the aneurysmal wall. The main characteristic of M1 macrophages is the production of proteolytic enzymes and proinflammatory cytokines. In contrast to M1, M2 macrophages participate in the anti-inflammatory response and are involved in ECM remodeling and tissue repair [28,29]. Interestingly, the number of M2 macrophages was higher in AAA to that number of M1-like macrophages indicating that the first ones are quite abundant in advanced AAA disease. This is in agreement with previous studies where a higher M2 macrophages cellularity in aneurysmal tissue has been reported [46]. Further, Sharma et al. recently demonstrated that facilitating the recruitment of M2-like macrophages promoted formation of AAA counter- acting the concept that M2 macrophages are beneficial in AAA [47]. Interestingly, blocking oxidative metabolism drives the macrophages toward an M1-like state and impairs the development of an M2-like phenotype. Thus, we hypothesized that the high concentration of ROS in diseased tissue and blood found in our AAA patients cohort might influence the M1/M2 polarization ratio as previously described [48,49]. We additionally found that the circulating levels of other biomarkers such as the transmembrane enzyme CD38 and GDF15 were increased in plasma of AAA patients. Moreover, CD38 was highly expressed in aortic aneurysm tissue. In agreement, a recent study has proposed the implication of CD38 signaling as a proinflammatory intermediate in AAA [50]. Due to the CD38 relationship with the modulation of immune response, aging and oxidative damage in vascular cells [51], we believe that the exacerbated expres- sion of CD38 found in the aneurysmal arterial wall might contribute to maintain chronic inflammation, ROS generation and VSMC apoptosis during AAA development. Further studies are needed to clarify the mechanisms by which CD38 is involved in this disease. Antioxidants 2021, 10, 602 13 of 16 Furthermore, accumulating evidence suggests that circulating GDF-15 concentrations are elevated and serve as independent prognostic biomarker in a wide spectrum of cardiovas- cular diseases, including ischemic heart disease, heart failure, atherosclerosis, hypertrophic cardiomyopathy and as a predictor of first-ever stroke in hypertensive patients [52–55]. Finally, we would like to remark the limitations of the work with human samples. The availability of aortic specimens from multi-organ donors was limited by the age and sex requirements. Moreover, it was not possible to obtain blood samples from these donors and for this reason, healthy abdominal aorta and blood samples taken as controls came from two different groups of individuals. Regarding AAA patients data, we included in Table 1 those parameters available in their clinical reports, and the average of PWS values calculated with the finite element analysis from the available CTAs (n = 60) of the studied cohort. 5. Conclusions In summary, our study shows the increase of vascular and circulating levels of IgG, ROS, CD38 and GDF15 in patients with AAA that could contribute to the chronic inflamma- tory state of this disease. Most importantly, our findings suggest the potential usefulness of the circulating levels of IgG, CD38 and GDF15 to assist AAA management in conjunction with aneurysm diameter and/or PWS during the follow-up of these patients. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antiox10040602/s1, Figure S1: Presence of M1 and M2 infiltrated macrophages in AAA tissue, Figure S2: CD36 protein is increased in aneurysmal tissue but not in the circulation; Table S1: Logistic regression analysis. Author Contributions: M.G. conceived, designed and supervised the whole study. D.S.-I., M.N.-M., B.P., B.S. and M.G. performed experiments, analyzed and interpreted data. J.F., A.J.B.-M., M.C. and M.G. were responsible for the clinical aspects of the study, and participated in sample collection and analysis. M.N., J.M.-G. and Z.M. conceived specific experiments, revised and carried out results interpretation. M.G., D.S.-I. and C.R. drafted the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by grants from the Spanish Ministerio de Economía y Compet- itividad (MINECO)-Instituto de Salud Carlos III (ISCIII) (grants PI17/01837 to M.G.; PI17/01455 to D.S. and PI18/00919 to C.R. and M.C.) and by CIBER on Cardiovascular Diseases (CIBERCV, CB16/11/00257), an initiative from ISCIII. The study was co-founded by Fondo Europeo de Desar- rollo Regional (FEDER), a way to make Europe M.N. is supported by BHF (PG17/73/33251). Institutional Review Board Statement: The use of human discarded tissue was performed in accor- dance with the Declaration of Helsinki of 1975, revised in 2013 and approved by the HSCSP Ethics Committee (IIBSP-AAA-2016-12). Informed Consent Statement: A written informed consent was obtained from all patients or le- gal representatives. Data Availability Statement: The data used to support the findings of this study is available from the corresponding author upon request. 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