Abdominal aortic aneurysm (AAA) is a permanent dilation of the aorta due to excessive proteolytic, oxidative and inflammatory injury of the aortic wall. We aimed to identify novel mediators involved in AAA pathophysiology, which could lead to novel therapeutic approaches. For that purpose, plasma from four AAA patients and four controls were analysed by a label-free proteomic approach. Among identified proteins, paraoxonase-1 (PON1) was decreased in plasma of AAA patients compared with controls, which was further validated in a bigger cohort of samples by ELISA. The phenylesterase enzymatic activity of PON1 was also decreased in serum of AAA patients compared with controls. To address the potential role of PON1 as a mediator of AAA, experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and human transgenic PON1 (HuTgPON1) mice. Similar to humans, PON1 activity was also decreased in serum of elastase-induced AAA mice compared with healthy mice. Interestingly, overexpression of PON1 was accompanied by smaller aortic dilation and higher elastin and vascular smooth muscle cell (VSMC) content in the AAA of HuTgPON1 compared with WT mice. Moreover, HuTgPON1 mice display decreased oxidative stress and apoptosis, as well as macrophage infiltration and monocyte chemoattractant protein-1 (MCP1) expression, in elastase-induced AAA. In conclusion, decreased circulating PON1 activity is associated with human and experimental AAA. PON1 overexpression in mice protects against AAA progression by reducing oxidative stress, apoptosis and inflammation, suggesting that strategies aimed at increasing PON1 activity could prevent AAA.

CLINICAL PERSPECTIVES

  • Abdominal aortic aneurysm (AAA) has no specific therapy except surgery for those patients with aortic diameter>5.5 cm. It is important to find mediators that could lead to novel therapies to prevent AAA.

  • Paraoxonase-1 (PON1) concentration and activity are decreased in AAA. PON1 overexpression decreased experimental AAA formation and contributed to vessel structure preservation by decreasing oxidative stress, apoptosis and the inflammatory response

  • PON1 is a potential therapeutic target for prevention of AAA development and rupture

INTRODUCTION

Abdominal aortic aneurism (AAA) is defined as a permanent dilation of the aorta diameter by more than 3 cm and/or 50% of the initial artery diameter. AAA occurs in up to 9% of adults older than 65 years of age, causing approximately 1–2% of male deaths in Western countries [1]. Diameter growth until aorta rupture is characterized by a non-linear behaviour, making difficult the prognosis of the disease [2]. Despite the origin of AAA still being poorly understood, proteolysis, oxidative stress, smooth muscle cell apoptosis and aortic wall inflammation are key mechanisms implicated in the formation and progression of AAA [3]. Nowadays, surgery is the only therapy recommended for asymptomatic patients with aorta diameter >5.5 cm, and, adding to the fact that there is no specific pharmacological treatment, it is imperative to find novel biomarkers for AAA.

Proteomics is a rapidly expanding research platform that allows the identification of potential biomarkers and provides essential information of different physiological–pathological scenarios. There have been several proteomics studies comparing plasma of control and AAA subjects by gel-based (e.g. 2-DE (2D electrophoresis), 2D-DIGE (2D difference gel electrophoresis)) or gel-free (e.g. LC–MS) techniques [48]. In the present study, we performed a label-free proteomic analysis of depleted plasma. Label-free proteomics is becoming more used due to the simplicity of its working scheme and to the advances in both MS instrumentation and bioinformatics tools. Following this approach, we identified, among other proteins, a differential expression of paraoxonase-1 (PON1) in plasma of AAA patients. PON1 hydrolyses a broad spectrum of organophosphate substrates and aromatic carboxylic acid esters, lipoprotein-associated peroxides and lactones. PON1 is mainly synthesized in the liver and, in circulation, is associated with high-density lipoprotein (HDL). However, PON1 is not a fixed component of HDL since the enzyme could also exert its protective functions outside the lipoprotein environment. It has been demonstrated that HDL transfers PON1 to cell membranes to improve cellular resistance to oxidative stress [9,10]. Previous studies supported a role for PON1 in atheroprotection, through its ability to prevent lipid oxidation and limit atherosclerotic lesion development; moreover, low PON1 activity has been associated with different cardiovascular pathologies [1113]. One previous study showed a significant association between AAA and PON1 haplotype [14]. However, the role of PON1 in AAA has not been previously addressed.

In the present study, we analysed the potential association between the concentration and activity of PON1 and AAA presence in humans. Moreover, through the established elastase-induced AAA model, we have investigated the role of PON1 in the mechanisms underlying AAA development.

MATERIALS AND METHODS

Blood samples

Venous blood samples were collected into EDTA or serum-separating tubes. Samples tubes were centrifuged for 20 min at 2500 g. Then, plasma and serum were divided into aliquots and immediately frozen at −80°C. For proteomic study, plasma from four male patients with an infrarenal AAA (aortic size >3 cm) was collected and from four male controls with non-dilated infrarenal aortas (aortic size <3 cm, confirmed with abdominal ultrasound) was obtained from a screening programme undertaken in our area of care. For analysis of PON1 concentration, a cohort of plasma samples from AAA patients (aortic size >3 cm, n=52) and controls (aortic size <3 cm, n=29) was obtained from Hospital Galdakao (Supplementary Table S1). For analysis of PON1 activity, a second cohort of serum samples from AAA patients (aortic size >3 cm, n=58) and controls (aortic size <3 cm, n=60) were obtained from the biobank of IIS-FJD (Supplementary Table S2).

Hypertension was defined as systolic blood pressure >140 mmHg and/or diastolic pressure ≥90 mmHg measured during the examination, after the participant had been sitting for at least 30 min, or if the participant was already taking hypotensive medication. A patient was considered diabetic if he was under treatment (supervised diet, hypoglycaemic oral medication, insulin) or we found basal glycaemia >120 mg/dl and/or HbA1c ≥ 6.5%. Hypercholesterolemia was defined as total basal cholesterol levels ≥200 mg/dl, low-density lipoprotein (LDL) levels ≥100 mg/dl or the patients were receiving specific medication or a supervised diet. Cardiac disease included coronary heart disease, valvular disease, cardiomyopathy and arrhythmia. Smoking was quantified as active smoking subjects if they were smoking at the moment of the inclusion in the study or non-smoking subjects if they were ex-smoking subjects or they have never smoked.

The study protocols conformed to the ethical guidelines of the Declaration of Helsinki as reflected in a priori approval by the Spanish centre's Research and Ethics Committees, and informed consent from the patients and the controls for their inclusion in the study was obtained in all cases.

Plasma depletion

The 14 most abundant proteins present in human plasma samples were removed using a Multiple Affinity Removal System Column (MARS14, Agilent Technologies), based on affinity interactions [8]. The targeted high-abundant proteins are specifically retained when the crude sample passes through the column. The low-abundant proteins in the flow-through fractions were then analysed by proteomic analysis. A flow rate of 1 ml/min was used to elute the bound proteins. The flow-through fractions (four fractions per sample) were dialysed with ammonium bicarbonate to buffer exchange, and then lyophilized.

LC–MS/MS analysis

Tryptic peptides were on line injected on to a C18 reversed-phase (RP) nano-column (100 mm internal diameter and 12 cm length, Mediterranea Sea, Teknokroma) and analysed in a continuous acetonitrile gradient consisting of 0–50% B in 90 min, 50–90% B in 1 min (B=95% acetonitrile, 0.5% acetic acid). A flow rate of 300 nl/min was used to elute peptides from the RP nano-column to an emitter nanospray needle for real-time ionization and peptide fragmentation on an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher).

Database searching

Tandem mass spectra were extracted by Proteome Discoverer v1.0 software (Thermo Fisher). Charge state deconvolution and deisotoping were not performed. For protein identification, fragmentation spectra were searched against a curated subset of a human database (human_ref.fasta; 2003, April; 39 414 entries) using Sequest (Thermo Fisher Scientific; version 1.0.43.2) and X-Tandem (The GPM, thegpm.org; version 2007.01.01.1) engines. Sequest and X-Tandem were searched allowing two missed trypsin cleavages and a tolerance of 15 p.p.m. or 0.8 Da was set for full MS or MS/MS spectra searches respectively. Methane thiosulfate alkylation of cysteine residues, oxidation of methionine and phosphorylation of serine and threonine residues were allowed as variable modifications.

Criteria for protein identification

Scaffold v.3.00.02 software (Proteome Software) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 80% probability and protein identification was accepted if they could be established at greater than 80% probability and contained at least one identified peptide, as specified by the Peptide Prophet [15] and Protein Prophet [16] algorithms respectively. Proteins containing similar peptides, which could not be differentiated based on MS/MS analysis alone, were grouped to satisfy the principles of parsimony.

Assessment of relative protein abundance

Given that the number of spectral counts of a protein can be used as a measure of its abundance [17], the β-binomial distribution was used to test the significance of differential protein abundances expressed in spectral counts (as provided by Scaffold software) by means of a publicly available software package [18]. Differences with a P-value < 0.05 were considered significant. Spectral count data were normalized using quartile normalization [19] to calculate the corresponding average fold change ratios.

Quantification of PON1

Soluble concentration of PON1 in human plasma samples was measured with a commercial ELISA kit (SK00141-01, Aviscera Biosciences) following the manufacturer's instructions.

PON1 activity

Arylesterase activity was measured using phenylacetate as the substrate in human and mice serum samples. Initial rates of hydrolysis were determined spectrophotometrically at 275 nm every 15 s for 2 min. The assay mixture included 2 μl of serum, 1 mM phenylacetate and 1 mM CaCl2 in 50 mM Tris/HCl, pH 8.0. The ε270 for the reaction is 1310 M−1 cm−1. One unit of arylesterase activity is equal to 1 mmol of phenylacetate hydrolysed per min per ml [20].

Immunohistochemistry (human)

AAA tissue samples were collected from patients undergoing surgery at IIS-FJD (IIS-FJD ethical committee number PIC 31/2011). Control aortas were sampled from dead organ donors with the authorization of the French Biomedicine Agency (PFS 09-007). These control aortic samples were macroscopically normal and devoid of early atheromatous lesions. AAA (thrombus and wall) and control aortic wall tissue samples were fixed in 3.7% paraformaldehyde, embedded in paraffin and sectioned at 6 μm.

Sections were deparaffinized and hydrated. Anti-PON1 was used as primary antibody (ab24261; Abcam) and irrelevant immunoglobulin (Dako) was used at the same concentration in order to assess non-specific staining. Sections were then counterstained with haematoxilin.

Experimental model

Human transgenic PON1 (HuTgPON1) mice [provided by Dr Michael Aviram (The Lipid Research Laboratory, Technion Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Rambam Medical Center, Haifa, Israel)] and wild-type (WT) counterparts were used. Twelve-week-old male (HuTgPON1 [n=12] and WT [n=12]) mice underwent aortic perfusion with 0.411 unit/ml type I porcine pancreatic elastase (E1250; Sigma) to produce experimental AAA, as previously described [21,22]. On day 14 post-surgery, all mice were anaesthetized with a mixture of ketamine and xylazine (100 nsmg/kg and 10 mg/kg of body weight respectively) and killed by cervical dislocation. Aortic tissue samples were obtained for histological analysis. AAA was defined as aortic diameter expansion ≥100% of that before perfusion [21]. An additional cohort of elatase-infused WT mice (n=10) and untreated healthy WT mice (n=10) were subjected to serum isolation for PON1 activity determination.

All mice were maintained under barrier conditions. Water and normal laboratory diet were available ad libitum. The Ethics Review Board of our institute approved all animal procedures, and the project was authorized by the IIS-FJD-Universidad Autónoma de Madrid (CEI 59-1036-A061) and by the Spanish Authority governing animal experimentation, the Comunidad Autónoma de Madrid (registered approval letter 10/008932.9/15). The investigation was carried out conform the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

Immunohistochemistry (experimental model)

AAA samples were embedded in paraffin and cross-sectioned in 5-μm-thick pieces. Immunohistochemistry analysis was performed in all animals included in each group using as primary antibodies the macrophage marker anti-CD68 (ab53444; Abcam), the chemokine anti-monocyte chemoattractant protein-1 (MCP1, sc-1785, Santa Cruz Biotechnology), the vascular smooth muscle cell (VSMC) marker anti-α-actin (Clone 1A4; Sigma), the oxidative marker anti-8-hydroxyguanosine (8-OHdG, ab10802; Abcam) and the apoptosis marker anti-cleaved poly(ADP-ribose) polymerase (PARP, ab32064; Abcam). The secondary antibodies and ABComplex were added and sections were stained with 3,30-diaminobenzidine (DAB) or 3-Amino-9-ethylcarbazole (AEC), counterstained with hematoxylin and mounted in DPX or in aqueous medium, respectively. Computer-assisted morphometric analysis was performed with the Image-Pro Plus software (version 1.0 for Windows). The threshold setting for area measurement was equal for all images. Samples from each animal were examined in a blinded manner. Results were expressed as the percentage positive area compared with total area of macrophages, MCP1 and 8-OHdG and as positive cells per square millimetre of whole lesion or medial layer of cleaved-PARP.

Elastin degradation and VSMC paucity grade

Verhoeff–van Gieson stain was performed using Elastic Stain Kit (Sigma–Aldrich, HT25A-1KT) according to the manufacturer's protocol. Elastin fragmentation and VSMC content were graded as described previously [23]. Briefly, elastin preservation was graded as followed: grade 1, intact well-organized elastin laminae; grade 2, elastic laminae with some interruptions and breaks; grade 3, elastic laminae with multiple interruptions and breaks; and grade 4, severe elastin fragmentation or loss. VSMC content in the tunica media was graded using the following key: grade 1, intact VSMCs; grade 2, minimal abnormalities; grade 3, loss of few VSMCs; and grade 4, loss of VSMCs in prolonged areas of the tunica media. Thus, the higher grade indicates poorer VSMC preservation.

Statistical analysis

Data are expressed as means±S.E.M. Between-group comparisons were assessed for categorical variables with the χ2 test and for numerical variables by Student's t test (human) or Mann–Whitney non-parametric test (experimental model). PON1 concentration and/or activity were transformed to a categorical variable by dividing their levels below or above the media. Multivariable logistic regression analysis including only variables that were statistically significant in the univariate analysis was performed to assess predictors of the presence of AAA. Also, 95% confidence intervals (CI) were calculated for each comparison. A value of 0.05 was considered to be statistically significant. SPSS 15.0 was used as statistical software.

RESULTS

Identification of PON1 as a differentially expressed protein in plasma of AAA patients

Plasma samples from AAA patients (n=4) and controls (n=4) were depleted of the 14 most abundant proteins and compared by spectral counting using β-binomial distribution. Considering proteins quantified in at least two comparatives, we found ten proteins differentially expressed, seven decreased and three increased, in AAA compared with control samples (Figure 1). Among them, we were especially interested in PON1, which has not been previously related to AAA. PON1 showed a significant 2.5-fold decrease in AAA plasma samples compared with controls (P<0.05).

Differential proteins found in plasma from AAA patients compared with control subjects by a label-free-based proteomic approach

Figure 1
Differential proteins found in plasma from AAA patients compared with control subjects by a label-free-based proteomic approach

(A) Volcano plot of identified proteins from plasma of AAA patients and controls [threshold limits (fold change, FC >1.5, P-value < 0.05) for up- or down-regulated proteins]. (B) Table with the identified proteins (1) protein code according to SwissProt Database (2), protein fold change (positive FC denotes increase in expression in control group whereas negative FC denotes decrease in expression in control group), and (3) P-value associated with protein fold change. E=×10 to the power indicated.

Figure 1
Differential proteins found in plasma from AAA patients compared with control subjects by a label-free-based proteomic approach

(A) Volcano plot of identified proteins from plasma of AAA patients and controls [threshold limits (fold change, FC >1.5, P-value < 0.05) for up- or down-regulated proteins]. (B) Table with the identified proteins (1) protein code according to SwissProt Database (2), protein fold change (positive FC denotes increase in expression in control group whereas negative FC denotes decrease in expression in control group), and (3) P-value associated with protein fold change. E=×10 to the power indicated.

Decreased systemic PON1 levels and activity in AAA patients

In order to validate the differential expression observed for PON1 in the proteomic study, we performed an ELISA analysis in a bigger cohort of plasma from AAA patients (n=52) and controls (n=29). Clinical characteristics of the subjects are shown in Supplementary Table S1. Confirming the proteomic results, PON1 concentration was lower in AAA patients compared with control subjects (369.9±25.1 compared with 467.7±30.4 ng/ml, P<0.05) (Figure 2A). Supplementary Table S3 shows the univariate and multivariate regression logistic analysis to assess predictors of the presence of AAA. Smoking, hypertension and PON1 concentration below the media were independent predictors for the presence of AAA.

Systemic PON1 levels and activity in human AAA

Figure 2
Systemic PON1 levels and activity in human AAA

(A) PON1 levels were measured in plasma of 29 control subjects and 52 patients with AAA (*P<0.05). (B) PON1 activity was measured in serum of 60 control subjects and 58 patients with AAA (*P<0.005).

Figure 2
Systemic PON1 levels and activity in human AAA

(A) PON1 levels were measured in plasma of 29 control subjects and 52 patients with AAA (*P<0.05). (B) PON1 activity was measured in serum of 60 control subjects and 58 patients with AAA (*P<0.005).

Since PON1 enzymatic activity must be determined in serum, serum samples were obtained from another cohort of AAA patients [n=58] and controls subjects [n=60]. Clinical characteristics are shown in Supplementary Table S2. Decreased PON1 activity was observed in AAA patients as compared with controls subjects (39.1±1.7 compared with 47.1±2.1 units/ml, P<0.001) (Figure 2B). Multivariable logistic regression analysis showed that older age and PON1 activity below the media were independent predictors for the presence of AAA (Supplementary Table S4).

PON1 is present in AAA tissue

After showing that PON1 was decreased in blood of AAA patients, we analysed its expression in human AAA tissue. As observed in Figure 3, strong PON1 immunostaining was mainly detected in extracellular areas of the thrombus, suggesting its potential trapping from the circulation. In contrast, faint PON1 staining was shown in the wall of AAA and, to a lesser extent, in the healthy wall.

Tissue PON1 levels in human AAA

Figure 3
Tissue PON1 levels in human AAA

Representative images of PON1 immunostaining of human AAA thrombus (right, magnification ×20), pathological media and healthy wall.

Figure 3
Tissue PON1 levels in human AAA

Representative images of PON1 immunostaining of human AAA thrombus (right, magnification ×20), pathological media and healthy wall.

PON1 transgenic mice display decreased aortic dilatation and growth in the elastase-induced AAA experimental model

After showing decreased serum PON1 activity in human AAA, we studied PON1 activity in serum from the elastase-perfused experimental model of AAA. Similarly, we observed that PON1 activity was lower in AAA mice (n=10) as compared with control (n=10) healthy mice (47.7± 2.6 compared with 58.6±3.9 units/ml; P<0.05) (Figure 4A).

PON1 overexpression decreased aortic dilation in the elastase-perfused AAA experimental model

Figure 4
PON1 overexpression decreased aortic dilation in the elastase-perfused AAA experimental model

(A) PON1 activity was measured in ten control WT mice and ten AAA WT mice (*P<0.05). (B) Representative Masson's trichrome staining in AAA lesions from WT and HuTgPON1 mice at 14 days postperfusion. Aortic diameter increase (percentage) at day 14 postperfusion with elastase in WT mice (n=12) and HuTgPON1 mice (n=12) (*P<0.01).

Figure 4
PON1 overexpression decreased aortic dilation in the elastase-perfused AAA experimental model

(A) PON1 activity was measured in ten control WT mice and ten AAA WT mice (*P<0.05). (B) Representative Masson's trichrome staining in AAA lesions from WT and HuTgPON1 mice at 14 days postperfusion. Aortic diameter increase (percentage) at day 14 postperfusion with elastase in WT mice (n=12) and HuTgPON1 mice (n=12) (*P<0.01).

Because we observed that PON1 activity was decreased in experimental AAA induced by elastase perfusion in WT mice, we performed the AAA experimental model by elastase aortic perfusion in HuTgPON1 to investigate the potential role of PON1 as a mediator of AAA. Analysis at 14 days postperfusion showed that HuTgPON1 mice have a significant attenuation of aortic diameter increase in compared with WT mice (60.5±9.3 compared with 106.1±9.1% of increase; P<0.01) (Figure 4B).

Two main characteristics of aneurysmal medial injury are elastin degradation and VSMC loss. In order to analyse whether PON1 overexpression protects from elastic degradation we performed a Verhoeff–van Gieson staining. Elastic preservation is graded from intact elastic layer (grade 1) to highly damaged or lost (grade 4). We observed that elastic fibres were more preserved in HuTgPON1 as compared with control mice (2.8±0.3 compared with 3.8±0.1 grade; P<0.01) (Figure 5A).

PON1 overexpression prevented elastin degradation, VSMC loss and apoptosis in AAA

Figure 5
PON1 overexpression prevented elastin degradation, VSMC loss and apoptosis in AAA

(A) Representative Verhoeff–van Gieson staining showing elastin degradation grading keys (four grades). Quantification of medial elastin degradation in WT (n=12) and HuTgPON1 (n=12) mice (*P<0.01). (B) Representative staining of α-actin (red) in AAA lesions from WT and HuTgPON1 mice at 14 days postperfusion. Quantification of VSMC preservation by grading (higher scores indicate loss of VSMCs) in WT (n=12) and HuTgPON1 (n=12) mice at 14 days postperfusion (*P<0.05). (C) Representative cleaved PARP staining at 14 days postperfusion. Quantification of cleaved-PARP+ cells per mm2 in wall (media + adventitia) or media layer of AAA from WT (n=10) and HuTgPON1 (n=8) mice (*P<0.01).

Figure 5
PON1 overexpression prevented elastin degradation, VSMC loss and apoptosis in AAA

(A) Representative Verhoeff–van Gieson staining showing elastin degradation grading keys (four grades). Quantification of medial elastin degradation in WT (n=12) and HuTgPON1 (n=12) mice (*P<0.01). (B) Representative staining of α-actin (red) in AAA lesions from WT and HuTgPON1 mice at 14 days postperfusion. Quantification of VSMC preservation by grading (higher scores indicate loss of VSMCs) in WT (n=12) and HuTgPON1 (n=12) mice at 14 days postperfusion (*P<0.05). (C) Representative cleaved PARP staining at 14 days postperfusion. Quantification of cleaved-PARP+ cells per mm2 in wall (media + adventitia) or media layer of AAA from WT (n=10) and HuTgPON1 (n=8) mice (*P<0.01).

Medial smooth muscle cell density is decreased in AAA tissues [24]. Immunohistochemical analysis of α-actin demonstrated that VSMC loss in HuTgPON1 mice was milder than in WT mice (2.7±0.3 compared with 3.6±0.1 grade; P<0.05) (Figure 5B). Apoptosis may contribute to the loss of medial VSMCs. In order to evaluate vascular apoptosis, cleaved PARP immunostaining was performed in AAA tissues. We observed a significant decrease in the apoptotic marker PARP in HuTgPON1 mice as compared with control mice either in the wall (media + adventitia) (86.4±18.9 compared with 370.4±79.5 positive cell number/mm2; P<0.01) or in the media (257.9±72.6 compared with 507.1±78.4 positive cell number/mm2; P<0.01) (Figure 5C).

PON1 overexpression decrease oxidative stress and inflammation in experimental AAA

The antioxidant properties of PON1 are widely recognized. In order to analyse whether PON1 protects the vessel against oxidant damage, we performed 8-OHdG immunostaining in transgenic and control mice. HuTgPON1 mice presented less oxidative 8-OHdG staining as compared with the WT control mice (2.1±0.8 compared with 3.9±0.4% positive area; P<0.05) (Figure 6A).

PON1 overexpression reduced oxidative stress and inflammation in AAA

Figure 6
PON1 overexpression reduced oxidative stress and inflammation in AAA

(A) Quantification of positive staining of 8-OHdG in AAA lesions from WT (n=11) and HuTgPON1 (n=7) mice (*P<0.05) and representative 8-OHdG staining at 14 days postperfusion. (B) Quantification of positive staining of CD68 in AAA lesions from WT (n=9) and HuTgPON1 (n=7) mice (*P<0.05) and representative CD68 staining at 14 days postperfusion. (C) Quantification of positive staining of MCP1 in AAA lesion from WT (n=10) and HuTgPON1 (n=8) mice (*P<0.05) and representative MCP1 staining at 14 days postperfusion.

Figure 6
PON1 overexpression reduced oxidative stress and inflammation in AAA

(A) Quantification of positive staining of 8-OHdG in AAA lesions from WT (n=11) and HuTgPON1 (n=7) mice (*P<0.05) and representative 8-OHdG staining at 14 days postperfusion. (B) Quantification of positive staining of CD68 in AAA lesions from WT (n=9) and HuTgPON1 (n=7) mice (*P<0.05) and representative CD68 staining at 14 days postperfusion. (C) Quantification of positive staining of MCP1 in AAA lesion from WT (n=10) and HuTgPON1 (n=8) mice (*P<0.05) and representative MCP1 staining at 14 days postperfusion.

It has been also clearly demonstrated that PON1 display multiple anti-inflammatory actions [25]. For that reason, inflammatory cell infiltrate was measured in AAA sections from HuTgPON1 and WT control mice. First, we observed a decrease in macrophage CD68 content in HuTgPON1 mice as compared with control mice (2.2±0.8 compared with 9.9±2.8% positive area; P<0.05) (Figure 6B). Additionally, the chemokine MCP1 was also decreased in HuTgPON1 mice compared with the control group (5.5±1.1 compared with 11.8±2.1% positive area; P<0.05) (Figure 6C). Moreover, in HuTgPON1 mice, T-cell content and neutrophil infiltration was lower than in control mice, but these results did not achieve statistical significance (P=0.1 for both; results not shown). All of these data showed a decrease in different pathological mechanisms of AAA, such as oxidative stress and inflammation in AAA lesions of HuTgPON1 mice.

DISCUSSION

In the present study, we have shown that circulating PON1 concentration and, more importantly, PON1 activity are diminished in AAA patients. Moreover, overexpression of PON1 protects against AAA progression in an experimental model induced by elastase in mice. Furthermore, we have shown that PON1 could contribute to vessel structure preservation by decreasing oxidative stress and the inflammatory response.

The application of proteomics techniques in the field of complex diseases has emerged as a powerful tool for revealing new proteins and signalling pathways implicated in the development of pathologies. Different proteomic approaches have been previously applied to plasma of AAA [26]. In a previous study performed in depleted plasma of AAA patients by iTRAQ (isobaric tags for relative and absolute quantification) labelling and LC–MS/MS [8], several proteins identified in the present study were also observed, such as complement component C9, lumican and gelsolin. However, in this first study applying a label-free proteomic approach in depleted plasma of AAA patients and controls, we were able to identify PON1, which has not been previously associated with AAA. Moreover, a significant decrease in PON1 in plasma from AAA patients as compared with controls was observed, which was further validated in a bigger cohort of plasma from AAA patients and controls. Moreover, as PON1 is an enzyme, we analysed its phenylesterase activity in serum samples. We consistently found that PON1 activity was lower in serum from AAA patients compared with control subjects, in agreement with previous studies in other cardiovascular pathologies [1113]. When we analysed potential confounding factors such as smoking or hypertension that could influence the negative association of PON1 concentration and/or activity with AAA presence, we observed that PON1 concentration and/or activity remained significantly associated with AAA. Therefore, our data demonstrate a decreased systemic PON1 concentration or activity associated with AAA presence suggesting the potential usefulness of PON1 as a biomarker of AAA.

PON1 is found in many tissues, as well as in blood, where it is associated with HDL. AAA patients have low HDL cholesterol levels and a reduced number of small HDL particles [8,27]. Moreover, HDL cholesterol levels have been inversely associated with AAA thrombus volume [28]. We previously showed that high levels of apolipoprotein A1 (ApoA1), the main constitutive protein of HDL, are present in AAA thrombi [8]. Similarly, we have observed a high extracellular staining of PON1 in AAA thrombi, whereas almost no staining was shown in healthy arteries. In agreement, increased PON1 in atherosclerotic plaques of peripheral artery disease (PAD) patients has been observed compared with healthy arteries [29]. Similarly, PON1 staining is not detected in the medial layer of healthy arteries in rats. In contrast, when rats were subjected to a balloonization procedure to remove the endothelium (similar to the situation of AAA thrombus were no endothelium is present), PON1 was detected in the medial layer [10]. Thus, our results suggest that the decrease in PON1 concentration and/or activity could be due, at least in part, to the retention of PON1 in AAA thrombi. In the other hand, serum PON1 activity has been inversely correlated with systemic levels of oxidative stress markers in cardiovascular diseases [30,31]. In AAA patients, blood levels of malondialdehyde and MPO were significantly increased [27,32], whereas catalase levels and activity were decreased [33]. Thus, the observed decrease in PON1 activity could reflect the increase in oxidative stress in AAA patients. On the whole, whether the decreased PON1 concentration and/or activity are bystanders of HDL retention/high oxidative stress or mediators of AAA cannot be deciphered.

High PON1 activity has been associated with vascular protection, whereas low PON1 activity is related to different pathological conditions [34]. When we analysed PON1 activity in the elastase-induced AAA experimental model, we observed a decrease in PON1 activity in AAA mice compared with healthy mice, similar to the data observed in humans. In order to address the potential role of PON1 as a mediator of AAA, we performed an experimental model of elastase-induced AAA comparing WT mice with HuTgPON1 mice. Interestingly, the increase in PON1 activity in HuTgPON1 mice led to lower aortic diameter dilation induced by elastase, when compared with WT mice. In agreement, PON1 overexpression in apolipoprotein E (ApoE)-knockout (KO) mice displayed smaller atherosclerotic lesions as compared with WT mice [35]. Aneurysmal dilation is associated with elastin degradation and VSMC depletion in the aortic media. Elastin layers are one of the main structural components of the aorta. Similarly, VSMCs are the main producers of extracellular matrix and also give mechanical support to the vessel wall. Consequently, degradation of elastin layers and loss of VSMCs are involved in the pathological remodelling taking place in AAA. In this respect, the observed decrease in aortic diameter in AAA of HuTgPON1 mice was accompanied by more elastic lamina conservation and VSMC content. On the whole, we can confidently conclude that PON1 overexpression impaired aneurysm formation and expansion that is associated with lower elastin degradation and VSMC loss from the wall.

PON1 protective effects in the arterial wall have been mainly related to its role as an antioxidant. Oxidative stress is a main determinant of chronic pathological remodelling of AAA, partially linked to the presence of red blood cells and leucocytes within tissue and to the imbalance between pro/anti-oxidant molecules [36]. In this respect, it has been shown that superoxide production was increased in AAA segments compared with non-dilated aortic specimens [37]. In the other hand, manganese-dependent superoxide dismutase activity in human diseased aorta was 65% of that of controls [38]. Similarly, in experimental models of AAA, increased haem-derived iron, leucocyte-derived oxidants, pro-oxidant enzymes, reactive oxygen species (ROS) and lipid oxidation products have been observed [39,40]. In this respect, we have also observed an increase in the marker of oxidative stress 8-OHdG in our mouse model of elastase-induced experimental AAA in WT mice, which was reduced in HuTgPON1 mice. Antioxidant systems are crucial for tissues to detoxify free radical species and protect organisms against oxidative stress. The antioxidant properties of PON1 activity have been associated with the hydrolysis of lipid peroxidation products [41]. Moreover, it has been demonstrated that PON1 participates in the capacity of HDL to stimulate endothelial NO production and to exert NO-dependent endothelial vasculoprotective effects [42,43]. Our data are in agreement with previous studies showing decreased aortic dilation and growth by different antioxidant systems in AAA experimental models [4448]. However, oxidative stress can mediate other mechanisms in pathological vascular remodelling, such as inflammation. For example, PON1 attenuates the oxidized LDL induced MCP1 production by endothelial cells [49]. Moreover, previous data showed decreased MCP1 expression in atherosclerotic plaques of HuTgPON1/ApoE-KO mice, potentially associated with a decrease in lipid hidroperoxide levels [35]. In this regard, we observed decreased MCP1 expression and macrophage infiltration in AAA tissue from HuTgPON1 mice compared with WT mice after elastase perfusion. However, PON1 also inhibits monocyte-to-macrophage differentiation [50,51] so the reduction in macrophage number by PON1 overexpression could be due to a decrease in either MCP1 synthesis and/or in the differentiation of monocytes in the aortic wall of elastase-induced AAA. On the whole, PON1 could prevent the chronic pathological remodelling of the arterial wall in AAA through PON1 antioxidant and anti-inflammatory properties.

In conclusion, this is the first study exploring the role of PON1 in human and experimental AAA. PON1 protects against AAA development preserving VSMC density and elastin structure through a decrease in oxidative stress, apoptosis and inflammatory cell accumulation. These promising results propose PON1 as a new potential therapeutic target in the treatment of AAA.

AUTHOR CONTRIBUTION

Elena Burillo performed the analysis of PON1 concentration and activity in human samples, as well as immunohistochemical analysis in human and animal tissues and contributed to the writing of the paper before submission. Carlos Tarin performed animal experiments and edited the paper before submission. Monica-Maria Torres-Fonseca, Carlos-Ernesto Fernandez-García and Diego Martinez-Lopez helped with the animal experiments and participated in the immunohistochemistry. Roxana Martinez-Pinna, Emilio Camafeita and Juan Antonio Lopez performed the proteomic studies. Patricia Llamas-Granda performed the histological preparations and participated in the immunohistochemistry. Melina Vega de Ceniga recruited the human samples and edited the paper before submission. Michael Aviram provided the PON1 animals and participated in the design of the study. Jesus Egido and Luis-Miguel Blanco-Colio edited and contributed to the discussion of the paper. Jose-Luis Martín-Ventura conceived, designed and co-ordinated the research plan and wrote/edited the paper.

We are grateful to Dr Jean-Baptiste Michel (INSERM, Paris, France) for kindly supplying human healthy aortic samples.

FUNDING

This work was supported by the Spanish Ministry of Economy and Competitiveness MICINN [grant number SAF2013/42525]; the Fondo de Investigaciones Sanitarias Instituto de Salud Carlos III (ISCIII)-Fondo Europeo de Desarrollo Regional (FEDER) [grant numbers Redes RIC RD12/0042/00038, Biobancos RD09/0076/00101 and CA12/00371]; the Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas; and the Fundación Renal Íñigo Álvarez de Toledo (FRIAT).

Abbreviations

     
  • AAA

    abdominal aortic aneurysm

  •  
  • ApoE

    apolipoprotein E

  •  
  • HDL

    high-density lipoprotein

  •  
  • HuTgPON1

    human transgenic PON1

  •  
  • KO

    knockout

  •  
  • LDL

    low-density lipoprotein

  •  
  • MCP1

    monocyte chemoattractant protein-1

  •  
  • 8-OHdG

    8-hydroxyguanosine

  •  
  • PARP

    poly(ADP-ribose) polymerase

  •  
  • PON1

    paraoxonase-1

  •  
  • RP

    reversed-phase

  •  
  • VSMC

    vascular smooth muscle cell

  •  
  • WT

    wild-type

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Supplementary data