Male Zucker rats with the MetS (MSZR, fa/fa; n = 18) and their age-matched male lean Zucker rat controls (LZR, FA/-; n = 18) were obtained from the breeding colony (animal facility, Faculty of Medicine, University of Lorraine, France). The animals were maintained at a constant temperature of 22–24°C, with a 12 h light-dark cycle (light beginning at 8 a.m.) and given free access to water and standard chow (A04, Scientific Animal Food and Engineering advance, Augy, France). The metabolic status of MSZR and LZR has been published previously (Sloboda et al., 2012).

Eighty weeks of age corresponds to 5 weeks before the mean maximum life span of rats from our local breeding colony.

This study was carried out in accordance with recommendations of the Animal Ethics Committee of the Institut National de la Santé et de la Recherche Médicale and conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. The protocols were approved by the Animal Ethics Committee of the Institut National de la Santé et de la Recherche Médicale.

Rats were anesthetized with isoflurane and whole blood was collected via a carotid catheter into syringes containing one-tenth the volume of 0.106 M sodium citrate. Platelet count was determined with an automatic cell counter (Micros 60 ABX model, Montpellier, France). Blood was centrifuged at 190 g for 10 min at room temperature to obtain platelet-rich plasma (PRP) and then at 1,750 g for 10 min to obtain platelet-poor plasma. PRP was adjusted to 200 × 10⁹ platelets/l by addition of autologous platelet-poor plasma and used for platelet aggregation and thrombin generation. Platelet-free plasma (PFP) was obtained by centrifugation of platelet-poor plasma at 13,000 g for 30 min at 4°C, and frozen at −80°C.

Artery cryo-sections were collected in the cross-sectional orientation and used subsequently for in situ gelatin zymography. The descending thoracic aorta was embedded in Optimal Cutting Temperature (OCT) medium and frozen using iso-pentane pre-cooled in liquid N₂ and stored at −80°C until cryo-sectioning. Cryo-sections were cut at a thickness of 5 μm and mounted onto glass slides (Leica, Milton Keynes, UK) and stored at −80°C until use.

The descending thoracic aorta was excised from rats after isoflurane anesthesia (4.5% in 1.5 l/min dioxygen) and exsanguination. VSMCs were isolated as described previously (Ait Aissa et al., 2015). VSMCs were grown in DMEM/F12 supplemented with 10% fetal bovine serum (Lonza, Basel, Switzerland). For thrombin generation assays, VSMCs at passages 3–5 were seeded (7,500 cells/well) in 96-well tissue culture flat-bottom plates (MICROTEST™96), grown to subconfluence and washed with HBS before use.

Blood was centrifuged at 190 g for 4 min followed by 70 s at 1,900 g at room temperature to obtain PRP and then platelets were sedimented by centrifugation at 5,000 g for 4 min. Platelets were re-suspended in Tyrode buffer (5 mM Hepes, 137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO₃, 0.4 mM NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, 5.5 mM glucose, pH 7.3). Platelet aggregation was measured by turbidimetry at 37°C under stirred conditions. PRP or washed platelets were adjusted to 200 × 10⁹ platelets/l and were stimulated by 5 μg/ml collagen or 5 μM ADP (SD Innovation, Frouard, France). Aggregation was followed for 10 min using a TA-8V aggregometer (SD Innovation).

Calibrated automated thrombinography (CAT) in PRP or PFP was performed in a microtiter plate fluorometer (Fluoroskan Ascent, ThermoLabsystems, Helsinki, Finland) using a dedicated software program (Thrombinoscope BV, Maastricht, The Netherlands) as reported previously (Regnault et al., 2004). All reagents were used at half the ordinary volume as follows: 40 μl PRP or PFP, 10 μl of 5 pM recombinant human tissue factor (TF) (Dade Behring, Marburg, Germany) and phospholipid vesicles (PV) consisted of phosphatidylcholine-serine-ethanolamine (PC/PS/PE) 60/20/20 mole% at a final concentration of 4 μM equivalent PS, 10 μl fluorogenic substrate and calcium. PV were replaced by buffer in PRP and VSMC experiments. Round-bottom 96-well Greiner blue plates were used for PFP and PRP, and MICROTEST™96 plates for VSMC monolayers. Thrombin generation curves were recorded in triplicate. Thrombin generation was monitored also following supplementing PFP with adiponectin or leptin (BioVision, San Francisco, USA), with fibrinogen (Sigma-Aldrich, St Louis, USA), or with palmitic acid or linoleic acid (Sigma-Aldrich).

Prothrombin and FVIII were measured in PFP samples diluted 1:40–80 in factor diluent (Instrumentation Laboratory, Le Pré Saint Gervais, France). For each assay 50 μl of diluted sample were added to 50 μl of human prothrombin-deficient plasma (Siemens Healthcare Diagnostics SAS, Saint-Denis, France) or FVIII deficient plasma (Dade Behring, Deerfield, USA). After 1 min of incubation at 37°C in a KC10 coagulometer, coagulation was started by addition of 80 μl of Thromborel® S. Calibration curves were generated using the reference plasma Unicalibrator (Diagnostica Stago, Asnières, France). Fibrinogen was measured in PFP samples diluted 1:10–20 in Owren–Koller buffer (Diagnostica Stago, Asnières, France). Unicalibrator was used to generate calibration curves. After 4 min of incubation at 37°C in a KC10 coagulometer, coagulation was started by addition of 100 μl of Fibriquik (Biomérieux-Trinity Biotech, Bray, Ireland). Antithrombin levels were measured with the Coamatic® antithrombin test kit from Chromogenix, and TAT with the Enzygnost® TAT micro (Instumentation Laboratory). TF and TF pathway inhibitor (TFPI) activities were measured in PFP using the Actichrome® tissue factor and Actichrome® TFPI activity assay respectively (American Diagnostica, Stamford, CT). PAI-1 levels were measured with the rat PAI-1 total antigen ELISA kit from Innovative Research, Inc. IL-13 and IL-1β concentrations were measured with the IL-13 and IL-1 beta rat ELISA kits from Invitrogen. MMP-9 levels were measured with the Quantikine rat total MMP-9 immunoassay from R&D Systems. VCAM-1 was assessed with the rat VCAM-1 ELISA kit from Elabscience.

PFP (20 μl) was diluted by addition of 40 μl buffer containing 5 pM recombinant TF, PV at 4 μM equivalent PS, 5 nM rabbit thrombomodulin (TM) (American Diagnostica, Greenwich, USA) and 4 μg/ml recombinant human tissue Plasminogen Activator (tPA) Actilyse® (Boehringer Ingelheim, Ingelheim am Rhein, Germany). Clot formation was initiated by addition of 10 μl of 100 mM CaCl₂. To monitor clot lysis, absorbance was read kinetically at 405 nm using a microplate reader. To standardize the figure, for each sample basal optical density (OD) after lysis was subtracted from each point of the curve. Half lysis time was defined as the time required to reach half-maximal variation in OD.

The thrombin generation assay was performed in order to generate fibrin for fixation using the same TF and PV concentrations as in the CAT experiments. This was done using plasma on paper disks and a Rhodamine substrate was used (Ninivaggi et al., 2012). Immediately after thrombin generation was finished (50 min for each run), the mineral oil was removed from the well and a solution of glutaraldehyde (grade I) in phosphate buffered saline (PBS) (Sorensen's PBS, pH 7.2) was applied. This was put at room temperature for 1 h and then kept at 4°C overnight. The samples were then washed 5 times with PBS and a secondary fixation was performed in OsO₄ (1%) in sodium cacodylate (200 nM, pH 7.4) for 1 h at RT. The samples were then dehydrated with increasing concentrations of ethanol each during 3 min (30, 50, 70, 90, 100%) and the last step (100%) was performed three times. Further dehydration was accomplished by a hexamethyldisilazane (HMDS)/ethanol solution (1:1) for 3 min and HMDS for 10 min. The samples were removed from the wells and left to dry. In order to visualize the samples with a Phenom G2Pro scanning electron microscopy (SEM) (Phenom-World, Eindhoven, the Netherlands), they were put on stubs using carbon tabs and coated with gold.

For each sample, 3–5 pictures were analyzed. Fiber thickness was measured using ImageJ software (version 1.48v). For each picture 100 measurements were performed. The density of the fibers was calculated from the pictures by counting the number of fibers that crossed a line of 26.8 μm (Konings et al., 2011).

The Rat Cytokine Array Panel A (Cat # ARY008) from R&D system (Minneapolis, MN) was used to probe cytokines in PFP from MSZR and LZR by following the procedures recommended by the manufacturer. Bound antibodies were detected by chemiluminescence using the Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA). This was performed once with a plasma pool from 5 to 6 animals to reduce inter-animal variability in each group.

The chromogenic assay measuring the phospholipid-related procoagulant activity (PPA) in VSMCs was performed as described previously for plasma (Wagenvoord et al., 1994; Membre et al., 2008). VSMCs cultured in 96 well plates were washed and 50 μl of 50 mM Tris, 175 mM NaCl, pH 7.9 (TBS) containing 2 g/l bovine serum albumin (BSA) were added as well as 50 μl of activated factor X (1.2 nM), activated factor V (2.4 nM), CaCl₂ (15 mM) and 50 μl of bovine prothrombin (6 μM) plus Z-Gly-Gly-Arg-AMC substrate (1.25 mM) in 20 mM HEPES pH 7.5 containing 60 g/l BSA. The plate was placed in the Fluoroskan Ascent fluorometer and allowed to warm up to 37°C for 5 min before kinetic readings were taken over 10 min. Phospholipid concentration was estimated from the initial rate of thrombin formation by reference to a standard curve constructed with PV, and expressed as PS equivalents.

Cell extracts were obtained by lysing VSMCs in complete Lysis-M buffer (Roche Diagnostics Corporation, Basel, Switzerland). Detergent-soluble fractions were retained, and protein concentrations in samples were determined using a Bradford protein assay (Bio-Rad, Hercule, USA). Lysates containing 30 μg of protein were electrophoresed on polyacrylamide gels (8% gel), transferred to Hybond-C nitrocellulose membranes (transblot turbo, Bio-Rad, Hercule, USA) and blotted with the following antibodies: α-smooth muscle actin (αSMA), 4/1,000 (Sigma-Aldrich), smooth muscle myosin heavy chain (SM-MHC), 1/1,000 (Abcam; Cambridge, UK); smoothelin, 1/500 (Santa Cruz Biotechnology, USA); integrin α_v, 1/1,000 (Santa Cruz Biotechnology, Dallas Texas); integrin β₃ 1/500 (Merck Millipore, Billerica, USA) and tubulin, 2/1,000 (Sigma-Aldrich). After rinsing, incubation with a secondary rabbit antibody 1/1,000 (α_v, β₃, smoothelin, SM-MHC, Sigma-Aldrich) and mouse antibody 1/1000 (αSMA, tubulin, Sigma-Aldrich). Reactions were visualized by the ECL Western Blot Detection Kit (Bio-Rad, Hercule, USA) after incubation with peroxidase conjugates 1/2000 (GE Healthcare, Little Chalfont, UK). Tubulin was used as loading control and the protein expression was normalized to tubulin.

In situ gelatin zymography was performed to determine the gelatinase activity across the aortic wall using DQ-gelatin (Life Technologies, Paisley, UK) as described previously (Mook et al., 2003). Fluorescein isothiocyanate (FITC, 1/110), and 4′,6-diamidino-2-phenylindole (DAPI, 1/150) filters were used to visualize the degree of gelatinase activity and the localisation of nuclear tissue by fluorescence microscopy using a x20 optical objective (Keynece, Osaka, Japan). Analysis of average fluorescence was performed for three 20 μm thick profile lines across 3 arterial wall regions for each sample.

VSMCs from LZR or MSZR (passage 4–6) were seeded (50,000 cells/well) in 6-well culture plates in DMEM/F-12 supplemented with 10% fetal bovine serum (life technology Thermo Fisher Scientific, Waltham, USA). Cells were grown to subconfluence and after 16 h in serum-free medium, cells were washed with PBS (Sigma-Aldrich), the medium was changed and cells were incubated for 4, 8, or 20 h at 37°C. Conditioned media were then removed and centrifuged at 500 g for 10 min at room temperature and used for the determination of MMP-2 secretion.

Conditioned media were analyzed for gelatin degradation by electrophoresis under non-reducing conditions on a 10% polyacrylamide-SDS gel containing 0.1% gelatin. Gels were washed for 1 h at room temperature in a 2% triton X-100 solution and incubated overnight at 37°C in 50 mM Tris–HCl/10 mM CaCl₂ (pH 7.6) buffer.

Gels were stained in a 0.1% coomassie Blue (G250)/45% methanol/10% acetic acid solution and de-stained in a 10% acetic acid/20% methanol solution. White lysis strips, indicative of gelatinolytic activity, were revealed and scanned (Fujifilm LAS 4000, Life sciences, Branford, USA). Densitometric analysis was made using MultiGauge software (Fuji, Tokyo, Japan). Fetal bovine serum diluted at 1% in serum free medium was used as a positive control.

Results are presented as mean ± standard error of the mean. Data were analyzed by a one-way or two-way ANOVA, followed by a Fisher's test for multiple comparisons to evaluate the influence of age and strain and their interaction on the different variables. In the case of SEM measurements, the differences in fiber thickness were analyzed using the Mann Whitney U-test.

Platelet count in blood was increased in MSZR at both ages compared to the same aged LZR (Table 1). Platelet aggregation using washed platelets and collagen as a strong agonist was not significantly modified as shown by the mean maximum aggregation (Figure 1A). For platelet aggregation in PRP using ADP, mean maximum aggregation was increased in 80 week-old MSZR and LZR compared to 25 week-old controls (Figure 1B). The F1+2 fragment was analyzed to evaluate the in vivo reactivity of the coagulation system. The amount of F1+2 fragment was increased in 25 week-old MSZR compared to the same aged LZR (Table 1). Thrombin generation measurement was performed as an integrative in vitro phenotype of coagulation. Adult and very old MSZR had a significantly increased endogenous thrombin potential (ETP) compared to same aged LZR. The other thrombin generation parameters (lag time, peak, and velocity) were not changed significantly except for the time to peak which was increased in obese at both ages (Table 1; Figure 1C). The ratio of thrombin generation in PFP and PRP compared to 25 week-old LZR was made to evaluate the platelet reactivity impact on thrombin generation. Interestingly, thrombin generation was more increased in PRP from MSZR at 25 week of age compared to 80 week-old rats (Figure 1D). The coagulation parameters, TF, TFPI, prothrombin and fibrinogen, were all increased in MSZR compared to LZR at both ages. TFPI was decreased and fibrinogen was increased with age in MSZR and prothrombin was increased with age in LZR. FVIII was increased significantly with age and MetS in 80 week-old MSZR. Antithrombin measurements showed no modification in MSZR and LZR rats (Table 1). Fibrin clots were characterized by SEM. Computerised analysis of the SEM images showed a decrease of fibrin fiber thickness in MSZR compared to LZR at both ages while fiber density was only increased in 80 week-old LZR (Figures 1E-G). Circulating levels of PAI-1 were increased in both 80 week-old LZR and MSZR (Figure 1H). In a fibrinolysis test (Figure 1I), half-time lysis was increased in MSZR compared to LZR at both ages and aging significantly increased half-time lysis in both groups (Figure 1J). Maximal lysis speed was not modified (Figure 1K).

Fibrinogen concentration was correlated highly to ETP (r = 0.069) and supplementing plasma with exogenous fibrinogen at concentrations that agreed with the changes between MSZR and LZR gradually increased ETP (Figures 2A,B). The 1.2-fold increase in ETP with the 2.5 mg/mL concentration is consistent with the 1.4 increase in plasma fibrinogen in MSZR. We have then tested the effects of addition of exogenous leptin, adiponectin, linoleic acid, and palmitic acid to PFP at concentrations selected to encompass the range previously reported for each molecule in MSZR (Sloboda et al., 2012; Godin et al., 2013). Addition of leptin or adiponectin elicited similar concentration-dependent changes in ETP whatever the group of rat. The two adipokines had opposite effects on thrombin generation, leptin increased ETP whereas adiponectin decreased it (Figures 2C,D). The two lower concentrations of added linoleic acid (0.75 and 1.5 mg/mL) had clear procoagulant effects whereas the higher concentration (3 mg/mL) was less effective in increasing thrombin generation (Figures 2E,F). There was a significant increase in thrombin generation for all added concentrations of palmitic acid whatever the group of rat. The results show an additive effect of FFAs on MSZR plasma.

To explore inflammation in our model we performed a plasma cytokine array of 27 cytokines in order to provide qualitative data that will subsequently be used to quantify cytokines known likely to promote prothrombotic phenotypes (Figure 3). Panel A presents pictures of the cytokine array membranes. A 50% variation between two groups was chosen as a threshold to classify cytokines into four groups. The first group of five cytokines showed no modifications (Figure 3B), a second group of eight cytokines were increased with MetS (Figure 3C), a third group of three cytokines were increased with aging (Figure 3D) and a last group of 11 cytokines were increased with both MetS and aging (Figure 3E). The highest variation between 25 week-old MSZR and LZR was found for IL-1β (>3,000% variation) and the highest variation between 80 and 25 week-old rats was observed for IL-13 (>400% variation). ELISAs performed with individual rat PFP for IL-1β and IL-13 showed an increase of these cytokine levels in LZR and MSZR with age (Figures 3F,G). IL-13 was increased also in 80 week-old MSZR compared to same aged LZR.

To explore the contribution of VSMCs, thrombin generation was measured at the surface of cultured VSMCs isolated from LZR and MSZR. Thrombin generation with PFP from LZR and MSZR was always increased at the surface of MSZR VSMCs compared to LZR VSMCs. Remarkably, addition of palmitic acid in LZR VSMCs increased thrombin generation to the level of MSZR independently of the PFP used (Figure 4A). MSZR VSMCs displayed increased procoagulant phospholipids at their surface compared to LZR VSMCs (Figure 4B). Integrin subunit α_v was increased in MSZR compared to LSZ VSMCs while the β₃ subunit was not modified. VSMC differentiation markers α-SMA, SM-MHC, and smoothelin, interestingly, were all decreased in MSRZ VSMCs compared to LZR VSMCs (Figures 4C,D). Thus, in situ gelatin zymography was performed to explore MMP activity through gelatinase activity (Figure 4E). Figure 4E shows representative photographs of in situ gelatin zymography in aorta, gelatinase activity is in green. Mean gelatinase activity in the aortic wall was increased in 25 and 80 week-old MSZR compared to age matched LZR aortas (Figure 4F). However, age did not modulate gelatinase activity. At the cellular level MSZR VSMCs displayed increased MMP-2 secretion compared to LZR VSMCs (Figures 4G,H). Circulating levels of MMP-9 were increased in 80 week-old MSZR whereas VCAM-1 was increased in 25 week-old MSZR compared to same aged LZR and in 80 week-old LZR (Figures 4I,J).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.