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Fenofibrate Increases High-Density Lipoprotein and Sphingosine 1 Phosphate Concentrations Limiting Abdominal Aortic Aneurysm Progression in a Mouse Model

      There are currently no acceptable treatments to limit progression of abdominal aortic aneurysm (AAA). Increased serum concentrations of high-density lipoprotein (HDL) are associated with reduced risk of developing an AAA. The present study aimed to assess the effects of fenofibrate on aortic dilatation in a mouse model of AAA. Male low-density lipoprotein receptor-deficient (Ldlr−/−) mice were maintained on a high-fat diet for 3 weeks followed by 6 weeks of oral administration of vehicle or fenofibrate. From 14 to 18 weeks of age, all mice were infused with angiotensin II (AngII). At 18 weeks of age, blood and aortas were collected for assessment of serum lipoproteins, aortic pathology, aortic Akt1 and endothelial nitric oxide synthase (eNOS) activities, immune cell infiltration, eNOS and inducible NOS (iNOS) expression, sphingosine 1 phosphate (S1P) receptor status, and apoptosis. Mice receiving fenofibrate had reduced suprarenal aortic diameter, reduced aortic arch Sudan IV staining, higher serum HDL levels, increased serum S1P concentrations, and increased aortic Akt1 and eNOS activities compared with control mice. Macrophages, T lymphocytes, and apoptotic cells were less evident and eNOS, iNOS, and S1P receptors 1 and 3 were up-regulated in aortas from mice receiving fenofibrate. The present findings suggest that fenofibrate antagonizes AngII-induced AAA and atherosclerosis by up-regulating serum HDL and S1P levels, with associated activation of NO-producing enzymes and reduction of aortic inflammation.
      Ultrasound screening programs identify abdominal aortic aneurysms (AAA) in 2% to 5% of men aged over 65 years.
      Chichester Aneurysm Screening Group
      Viborg Aneurysm Screening Study; Western Australian Abdominal Aortic Aneurysm Program; Multicentre Aneurysm Screening Study: a comparative study of the prevalence of abdominal aortic aneurysms in the United Kingdom, Denmark, and Australia.
      Most of these AAAs measure <55 mm in maximum diameter; under current management strategies, these patients are treated conservatively to monitor AAA expansion.
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      Two-year outcomes after conventional or endovascular repair of abdominal aortic aneurysms.
      EVAR trial participants
      Endovascular aneurysm repair and outcome in patients unfit for open repair of abdominal aortic aneurysm (EVAR trial 2): randomised controlled trial.
      Approximately 50% of AAAs <50 mm eventually expand to such a size that surgical intervention is required.
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      The absence of effective AAA regressive medication complicates disease management, although a number of medications are being evaluated in ongoing trials to address this shortfall.
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      Current status of medical management for abdominal aortic aneurysm.
      Given that cardiovascular events, such as myocardial infarction and stroke, are the most common cause of death in patients with small AAAs, an ideal medication would reduce both AAA progression and atherothrombotic events in these patients.
      United Kingdom Small Aneurysm Trial Participants
      Long-term outcomes of immediate repair compared with surveillance of small abdominal aortic aneurysms.
      Reports suggest that high-density lipoprotein (HDL) is protective for AAA, although the underlying mechanisms are not clear.
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      Screening study of abdominal aortic aneurysm in a general population: lipid parameters.
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      Risk factors for abdominal aortic aneurysms in older adults enrolled in the Cardiovascular Health Study.
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      Prevalence of and risk factors for abdominal aortic aneurysms in a population-based study: the Tromsø Study.
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      Soluble adhesion molecules, endothelial markers and atherosclerosis risk factors in abdominal aortic aneurysm: a comparison with claudicants and healthy controls.
      Reverse cholesterol transport is considered the major reason for the antiatherogenic properties of HDL,
      • Assmann G.
      • Gotto Jr, A.M.
      HDL cholesterol and protective factors in atherosclerosis.
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      Molecular regulation of HDL metabolism and function: implications for novel therapies.
      although HDL is also implicated in antiatherogenic and anti-inflammatory actions independent of changes in cholesterol metabolism.
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      • Gotto Jr, A.M.
      HDL cholesterol and protective factors in atherosclerosis.
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      • Fogelman A.M.
      Antiinflammatory properties of HDL.
      One pathway implicated in the beneficial effects of HDL is mediated through the bioactive lipid sphingosine 1 phosphate (S1P).
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      • Levkau B.
      HDL and its sphingosine-1-phosphate content in cardioprotection.
      S1P acts through specific G-protein coupled receptors, triggering multiple second messenger systems, including the serine/threonine protein kinase Akt,
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      • Peters S.L.
      Sphingosine kinase-dependent activation of endothelial nitric oxide synthase by angiotensin II.
      and inhibiting proinflammatory responses in endothelial cells (ECs) and vascular smooth muscle cells (SMCs).
      • Karliner J.S.
      Sphingosine kinase and sphingosine 1-phosphate in cardioprotection.
      Akt influences many cell signaling pathways that regulate inflammation, cell survival, apoptosis, and endothelial nitric oxide synthase (eNOS), all of which may be relevant to the pathogenesis of AAA and atheroma. S1P stimulates eNOS activity through Akt/phosphoinositide 3-kinase-dependent and calcium-dependent pathways, resulting in the subsequent production of NO.
      • Levine Y.C.
      • Li G.K.
      • Michel T.
      Agonist-modulated regulation of AMP-activated protein kinase (AMPK) in endothelial cells Evidence for an AMPK -> Rac1 -> Akt -> endothelial nitric-oxide synthase pathway.
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      • Itoh H.
      • Kurose H.
      • Murakami M.
      • Okajima F.
      Mechanism and role of high density lipoprotein-induced activation of AMP-activated protein kinase in endothelial cells.
      Randomized trials have suggested that fenofibrate raises serum HDL by approximately 10%, accompanied by a modest reduction in cardiovascular events in patients with type 2 diabetes mellitus.
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      • Gebski V.J.
      • Scott R.S.
      • Keech A.C.
      Fenofibrate Intervention and Event Lowering in Diabetes Study investigators
      Effects of fenofibrate on renal function in patients with type 2 diabetes mellitus: the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) Study.
      The effects of fibrates on human AAA have not been assessed. Fibrates raise HDL in some but not all mouse models.
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      • Palomer X.
      • Roglans N.
      • Rotllan N.
      • Fievet C.
      • Tailleux A.
      • Julve J.
      • Laguna J.C.
      • Blanco-Vaca F.
      • Escolà-Gil J.C.
      Paradoxical exacerbation of combined hyperlipidemia in human apolipoprotein A-II transgenic mice treated with fenofibrate.
      • Duez H.
      • Chao Y.S.
      • Hernandez M.
      • Torpier G.
      • Poulain P.
      • Mundt S.
      • Mallat Z.
      • Teissier E.
      • Burton C.A.
      • Tedgui A.
      • Fruchart J.C.
      • Fiévet C.
      • Wright S.D.
      • Staels B.
      Reduction of atherosclerosis by the peroxisome proliferator-activated receptor alpha agonist fenofibrate in mice.
      The present study evaluated the effect of fenofibrate in limiting aortic dilatation and atherosclerosis progression induced by angiotensin II (AngII) infusion in a low-density lipoprotein receptor-deficient mouse model, Ldlr−/−.

      Materials and Methods

      Animals

      Male Ldlr−/− mice (C57BL/6J background; n = 31) from the Jackson Laboratory (Bar Harbor, ME) were fed a high-fat and high-cholesterol diet (1.25% cholesterol and 45% ghee, SF08-043; Specialty Feeds, Perth, Australia) from 9 weeks of age. At 12 weeks of age, mice were randomly allocated to receive vehicle (0.1% carboxymethylcellulose; n = 16) or fenofibrate (100 mg/kg per day; n = 15) in their drinking water for 6 weeks. At 14 weeks of age, all mice were anesthetized by intraperitoneal injection of ketamine (150 mg/kg) and xylazine (10 mg/kg), and miniosmotic pumps (ALZET model 2004; BioScientific, Gymea, Australia) were implanted subcutaneously to deliver 1 μg/kg per minute of AngII. Mice were sacrificed at 18 weeks of age by CO2 asphyxiation; unless otherwise indicated, all analyses were performed on animals receiving 4 weeks of AngII treatment in addition to vehicle control or fenofibrate. All animal protocols were according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (7th edition, 2004; available at http://www.nhmrc.gov.au/guidelines/publications/ea16). Institutional ethics approval was obtained from James Cook University (Ethics number A1341) before commencement of the study.

      Ultrasound Measurement of Abdominal Aortic Diameter

      Measurement of abdominal aortic diameter was performed using ultrasound immediately before AngII infusion (14 weeks of age) and weekly for 4 weeks (15, 16, 17 and 18 weeks). Mice were immobilized with intraperitoneal injections of ketamine (40 mg/kg) and xylazine (4 mg/kg). Ultrasound was performed in B-mode using a MyLab 70 VETXV platform (Esaote, Genoa, Italy) with an LA435 linear transducer (Esaote) at an operating frequency of 10 MHz,
      • Golledge J.
      • Cullen B.
      • Moran C.
      • Rush C.
      Efficacy of simvastatin in reducing aortic dilatation in mouse models of abdominal aortic aneurysm.
      to generate sagittal images of the suprarenal aorta. The maximum suprarenal aortic (SRA) diameter was measured at peak systole using the caliper measurement feature. In a study of interobserver repeatability performed on 22 mice, there was a good interobserver reproducibility [coefficient of repeatability = 0.92, 95% confidence interval (CI) = 0.883–0.946, average coefficient of variation (CV) = 9.5%]. All measurements were collected by two observers (S.M.K. and J.V.M.) blinded to the treatment groups.

      Aorta Morphometry

      Mouse aortas were harvested from their origin at the left ventricle to the iliac bifurcation, placed on a black background, and digitally photographed. Maximum diameter of the aortic arch, thoracic aorta, and the suprarenal and infrarenal aorta was determined using computer-aided analysis (Scion Image version 4.0.3.2; Scion Corporation, Frederick, MD).
      • Rush C.
      • Nyara M.
      • Moxon J.V.
      • Trollope A.
      • Cullen B.
      • Golledge J.
      Whole genome expression analysis within the angiotensin II-apolipoprotein E deficient mouse model of abdominal aortic aneurysm.
      In a preliminary study (n = 27), we established that these measurements could be repeated with good intraobserver reproducibility (coefficient of repeatability = 0.98, 95% CI = 0.975–0.982, and CV = 4%).
      • Rush C.
      • Nyara M.
      • Moxon J.V.
      • Trollope A.
      • Cullen B.
      • Golledge J.
      Whole genome expression analysis within the angiotensin II-apolipoprotein E deficient mouse model of abdominal aortic aneurysm.
      After being photographed, aortic arch segments were cleaned from adherent tissue, divided into aortic arch, thoracic, suprarenal, and infrarenal segments, and then processed for various analyses.

      Atherosclerosis Quantification

      The region from the origin of aorta at the aortic valve to the origin of the left subclavian artery was considered the aortic arch and was processed for Sudan IV staining. Aortic arches were longitudinally opened and fixed overnight in 10% neutrally buffered formalin. Sections were transferred to 70% ethanol and stained with Sudan IV (0.1% Sudan IV dissolved in equal parts acetone and 70% ethanol) to identify intimal atherosclerotic plaque, as described previously.
      • Golledge J.
      • Cullen B.
      • Rush C.
      • Moran C.S.
      • Secomb E.
      • Wood F.
      • Daugherty A.
      • Campbell J.H.
      • Norman P.E.
      Peroxisome proliferator-activated receptor ligands reduce aortic dilatation in a mouse model of aortic aneurysm.

      Analysis of Serum Lipids and S1P

      Blood was collected from mice at 14 weeks of age into serum collection tubes (BD Diagnostic Systems, Sparks, MD) by tail bleeds and at termination by cardiac puncture. Serum was recovered by centrifugation at 6400 × g for 10 minutes at room temperature and was stored at −80°C for later lipid assessment. Total cholesterol, triglycerides (TG), and HDL cholesterol were quantified using COBAS Integra tests (Roche Diagnostics, Indianapolis, IN). LDL values were calculated from total cholesterol, TG, and HDL values as LDL = (total cholesterol − TG − HDL)/2.2, as described previously.
      • Golledge J.
      • Cullen B.
      • Moran C.
      • Rush C.
      Efficacy of simvastatin in reducing aortic dilatation in mouse models of abdominal aortic aneurysm.
      Serum S1P level was analyzed using a S1P competitive ELISA, according to the manufacturer's recommendations (Echelon Biosciences, Salt Lake City, UT).

      Measurement of Aortic Akt1 and eNOS

      At sacrifice, a portion of the SRA and the infrarenal aorta (IRA) was snap-frozen for later assessment. Protein was extracted by homogenizing in protein extraction buffer containing Complete Mini Protease Inhibitor tablets (Roche Diagnostics). Protein concentrations were determined using Bradford reagent (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's recommendations. Detection of phosphorylated and total aortic Akt1 and eNOS activity was estimated by Western blotting. Proteins (25 μg) were loaded onto a 7.5% SDS polyacrylamide gel. After electrophoresis (100 V, 90 minutes), separated proteins were transferred (15 mA, 60 minutes) to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories). Membranes were blocked with 5% nonfat milk (GE Healthcare, Piscataway, NJ) for 120 minutes at room temperature and then were incubated overnight with the following primary antibodies at 4°C: anti-Akt1 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-Akt1 (Ser473; 1:1000; Santa Cruz Biotechnology), anti-eNOS (1:1000; Cell Signaling Technology, Danvers, MA), and anti-phospho-eNOS (Ser1177; 1:1000; Cell Signaling Technology). Anti-rabbit horseradish-peroxidase-conjugated IgG (1:1000; DakoCytomation, Carpinteria, CA) or anti-mouse horseradish-peroxidase-conjugated IgG (1:1000; DakoCytomation) were used as secondary antibodies. Blots were developed using an ECL advanced Western blotting detection kit (GE Healthcare); staining bands were quantified using Quantity One (version 4.6.7) analysis software (Bio-Rad Laboratories). Blots were stripped and reprobed for β-actin (1:2000; Cell Signaling Technology) to ensure equal protein loading between replicates.

      Histology

      Five representative SRAs from each group were selected using a random number generator and processed for H&E staining.

      Immunohistochemistry

      SRA segments from six mice from each group were selected using a random number generator. Serial cryostat sections (7 μm thick) were cut from each SRA segment and processed for IHC, as described previously.
      • Golledge J.
      • Cullen B.
      • Rush C.
      • Moran C.S.
      • Secomb E.
      • Wood F.
      • Daugherty A.
      • Campbell J.H.
      • Norman P.E.
      Peroxisome proliferator-activated receptor ligands reduce aortic dilatation in a mouse model of aortic aneurysm.

      TUNEL Assay

      To localize cells undergoing nuclear DNA fragmentation, in situ terminal TUNEL assay was performed using an in situ apoptosis kit (Calbiochem; EMD Millipore, San Diego, CA). Briefly, SRA segments from six mice from each group were selected using a random number generator and serial cryostat sections were taken. The 7-μm-thick cryostat sections were fixed in 70% methanol, washed with PBS, and incubated with proteinase K (20 μg/mL) for 20 minutes. Terminal deoxynucleotidyl transferase, which catalyzes a template-independent addition of deoxynucleotide to 3′-OH ends of DNA, was used to incorporate digoxigenin-conjugated dUTP to the ends of DNA fragments in situ. The TUNEL signal was then detected with a peroxidase-conjugated anti-digoxigenin antibody and developed with fluorescein isothiocyanate. Sections were mounted using Fluorescein-FragEL (Calbiochem) mounting medium (EMD Millipore) and visualized using a standard fluorescein filter (465 to 495 nm) on a Zeiss microscope fitted with a CCD camera (Carl Zeiss MicroImaging, Göttingen, Germany; Diagnostic Instruments, Sterling Heights, MI). Fluorescent digital images were captured to a microcomputer supported with Zeiss AxioVision version 4.8.2 software. Positive and negative control slides were processed at the same time. Apoptotic cells were identified by nuclear fluorescein isothiocyanate (green) staining and the distinctive morphological appearance associated with cell shrinkage and chromatin condensation (apoptotic cells) or cytoplasmic fragments with or without condensed chromatin (apoptotic bodies). The sections were examined at ×400 magnification. Fluorescein isothiocyanate-positive apoptotic cells were scored in five fields and reported in terms of percentage of positive cells among the total cells analyzed. All histological evaluations were done in a blinded fashion.

      Cell Culture

      ECs were harvested from Ldlr−/− mice aortas under sterile conditions, as described previously.
      • Sobczak M.
      • Dargatz J.
      • Chrzanowska-Wodnicka M.
      Isolation and culture of pulmonary endothelial cells from neonatal mice.
      Aortas (n = 6) were harvested from 12-week-old male Ldlr−/− mice and SRA segments were combined from two mice for further processing, thereby yielding three pools of ECs. SRA tissue was first cut into pieces, digested with collagenase/dispase solution (Roche Applied Science, Indianapolis, IN), and dispersed mechanically into single-cell suspension. ECs were purified from cell suspensions using positive selection with anti-PECAM-1 antibody (BD Pharmingen, San Diego, CA) conjugated to Dynabeads (Invitrogen-Life Technologies, Carlsbad, CA) using a magnetic-activated cell separation system (MiniMACS; Miltenyi Biotec, Auburn, CA). Purified cells were pooled and cultured on gelatin-coated tissue culture dishes with Clonetics EGM-2 EC growth medium (Lonza, Walkersville, MD) until they became confluent. Cells were further purified using Dynabeads coupled to anti-ICAM-2 antibody (BD Pharmingen). Positively selected ECs obtained by magnetic-activated cell separation were allowed to grow to confluency, and their endothelial phenotype was confirmed using immunofluorescence staining with anti-PECAM-1 antibody (BD Pharmingen).
      Purified aortic ECs obtained from Ldlr−/− mice were incubated in the presence of 0, 0.1, 1, 100, or 1000 nmol/L S1P (S9666; Sigma-Aldrich, Sydney, Australia; St. Louis, MO) and vehicle control. Cytosolic protein extracts were collected and quantitated by Bradford reagent (Bio-Rad Laboratories) according to the manufacturer's recommendations. Protein (25 μg) was processed by SDS-PAGE and analyzed by Western blotting as described above.

      Statistical Analyses

      Quantitative outcomes were not normally distributed; nonparametric tests were therefore used. Continuous numbers are presented as median and interquartile range (IQR) and were compared between groups using Mann-Whitney U-test and Kruskal-Wallis test. Differences were considered to be statistically significant at P < 0.05. All statistical analysis was performed using SPSS software (PASW statistics 18; IBM, Armonk, NY).

      Results

      Fenofibrate Reduces Suprarenal Aortic Dilatation in Response to AngII

      No significant difference in maximum SRA diameter was observed between mice receiving vehicle (median aortic diameter = 1.2 mm, IQR = 1.1–1.3) or fenofibrate (median aortic diameter = 1.15 mm, IQR = 1.1–1.37) before AngII infusion (P = 0.64). After infusion of AngII for 28 days, SRA diameter was significantly smaller in mice receiving fenofibrate, compared with vehicle controls, when assessed by both ultrasound and morphometry (Figure 1, A–C, and Table 1). The maximum diameter of the aortic arch segment was also smaller in mice receiving fenofibrate (Figure 1C).
      Figure thumbnail gr1
      Figure 1Influence of fenofibrate on aortic dilatation induced by AngII in Ldlr−/− mice. A: Ultrasound data showing maximum suprarenal aortic diameter of Ldlr−/− mice infused with AngII and receiving vehicle control or fenofibrate. Shown are means ± SEM. B: Box plot showing maximum suprarenal and infrarenal aortic diameters measured by ultrasound in mice receiving vehicle control or fenofibrate after 4 weeks of AngII infusion (18 weeks of age). Fenofibrate significantly inhibited suprarenal aortic expansion (P = 0.0003). C: Box plots comparing aortic morphometry of mice receiving vehicle control or fenofibrate after 4 weeks of AngII infusion. Fenofibrate inhibited expansion of the aortic arch (P = 0.006) and suprarenal aorta (P = 0.0004) induced by AngII. D: Box plots comparing aortic arch Sudan IV staining area in mice receiving vehicle control or fenofibrate after 4 weeks of AngII infusion. Fenofibrate significantly reduced aortic arch Sudan IV staining area (P < 0.0001). Box plots indicate median and IQR.
      Table 1Comparison of Outcomes in Mice Receiving Fenofibrate and Vehicle Control at Completion of Study
      GroupsVehicle controlFenofibrateP value
      Sample sizen = 16n = 15
      US-SRA (mm)1.40 (1.40–1.55)1.20 (1.10–1.30)0.0003
      Morphometry-SRA (mm)1.24 (1.13–1.34)1.05 (0.92–1.55)0.0004
      Aortic arch Sudan IV staining (%)30.34 (26.80–39.87)6.41 (4.28–10.70)0.0001
      Serum HDL (mg/dL)286.50 (245.30–322.50)429.50 (373.5–547.80)0.0003
      Serum TG (mg/dL)544.50 (209.60–830.40)253.20 (127.60–341.40)0.044
      Serum LDL (mg/dL)407.8 (327.20–442)329.50 (265.30–391.70)0.360
      Serum total cholesterol (mg/dL)1700 (1443–1866)1468 (1291–1832)0.291
      Serum S1P (μmol/L)1.53 (1.34–1.56)1.64 (1.57–1.83)0.028
      p-Akt1:Akt1 ratio within SRA0.38 (0.31−0.49)0.94 (0.73–1.08)0.028
      p-eNOS:eNOS ratio within SRA0.67 (0.58–0.90)1.84 (1.21–1.95)0.028
      Data are expressed as median and interquartile range.
      The SRA aortic diameter was measured at the completion of 4 weeks of AngII infusion.
      HDL, high density lipoprotein; LDL, low density lipoprotein; SRA, suprarenal aorta; TG, triglycerides; US, ultrasound.

      Fenofibrate Reduces Aortic Arch Sudan IV Staining Area

      Aortic arch Sudan IV staining area was significantly lower in mice receiving fenofibrate, compared with vehicle controls (P < 0.0001) (Table 1; Figure 1D).

      Fenofibrate Increases Serum HDL and S1P Concentrations

      At the completion of the study, serum HDL concentration was significantly higher in mice receiving fenofibrate, compared with vehicle control (P = 0.0003) (Table 1). In addition, the concentration of serum TG was significantly lower in mice receiving fenofibrate than in those allocated vehicle control (P = 0.044) (Figure 2A and Table 1). Total cholesterol and LDL concentrations were not significantly different in the two groups of mice (Table 1). Serum S1P concentration was observed to increase between 14 and 18 weeks in mice assigned to fenofibrate administration; by the end of the study, it was significantly higher than in mice allocated vehicle control (P = 0.028) (Figure 2B and Table 1).
      Figure thumbnail gr2
      Figure 2Influence of fenofibrate on serum lipids in AngII-infused Ldlr−/− mice. A: Box plots comparing serum concentrations of lipoproteins in mice receiving vehicle control or fenofibrate after 4 weeks of AngII infusion. Fenofibrate induced an increase in serum HDL (P = 0.0003) and a reduction in serum TG levels (P = 0.044). Serum total cholesterol and LDL levels were not influenced. B: Box plots comparing serum concentrations of S1P in mice receiving vehicle control or fenofibrate after 4 weeks of AngII infusion. S1P levels were higher in mice receiving fenofibrate, compared with vehicle control (P = 0.028) at 18 weeks and in the fenofibrate group at 18 weeks compared with 14 weeks (P = 0.0014).

      Fenofibrate Up-Regulates Aortic Akt1 and eNOS

      Akt1 and eNOS expression in mouse SRA and IRA were assessed by Western blotting. Stained protein bands for total Akt1, phospho-Akt1, total eNOS, and phospho-eNOS were identified at the expected molecular weights of 60, 62, 140, and 140 kDa, respectively (Figure 3, A and B). Treatment with fenofibrate before AngII infusion caused an approximately twofold increase in phospho-Akt1 expression (P = 0.028; n = 4) but not total Akt1 protein expression in the SRA, relative to the vehicle control group (Figure 3, A and C). Similarly, approximately a twofold increase in phospho-eNOS (Ser1177) but not total eNOS protein expression was observed in the SRA of fenofibrate allocated mice, compared with controls (P = 0.028; n = 4) (Figure 3, B and D). Western blotting suggested that fenofibrate administration had no significant effect on the activation of Akt1 and eNOS within the IRA (Figure 3, E–H).
      Figure thumbnail gr3
      Figure 3Fenofibrate stimulates an increase in Akt1 and eNOS activity in the suprarenal aorta of AngII-infused Ldlr−/− mice. A: Representative blots of phospho-Akt1 (p-Akt1) and total Akt1 protein expression in the suprarenal aorta of AngII-infused mice. B: Representative blots of phospho-eNOS (p-eNOS) and total eNOS protein expression in the suprarenal aorta of AngII-infused mice. C: Fenofibrate increased Akt1 activity by approximately twofold compared with the vehicle control group (*P = 0.028; n = 4). D: Fenofibrate increased eNOS activity by approximately twofold compared with the vehicle control group (*P = 0.028; n = 4). E: Representative Western blots of phospho-Akt1 and total Akt1 using protein from the infrarenal aorta of AngII-infused mice. F: Representative Western blots of phospho-eNOS and total eNOS using protein from the infrarenal aorta of AngII-infused mice. G: Akt1 activity was similar in fenofibrate and vehicle control groups (P = 0.246; n = 6). H: eNOS activity was similar in fenofibrate and vehicle control groups (P = 0.484; n = 6). NS, nonsignificant.

      Effects of Fenofibrate on the Histology of the SRA

      Histopathological examination of AngII-induced AAA revealed increased thickening of the abdominal aortic wall and pronounced inflammatory cell infiltration (Figure 4, A–D). Infiltrating inflammatory cells were detected in the tunica adventitia and tunica media of the suprarenal aorta harvested from the mice receiving vehicle control. The aortic elastic laminae were disrupted in mice receiving vehicle control (Figure 4, A and B); these mice also showed medial degeneration and adventitial thickening. In mice receiving fenofibrate, there was a marked decrease in the adventitial inflammatory cell infiltrate, and the endothelial layer was generally intact (Figure 4, C and D).
      Figure thumbnail gr4
      Figure 4Representative photomicrographs of suprarenal aorta sections from AngII-infused mice receiving vehicle control or fenofibrate. Histological assessment of suprarenal aortic sections stained by H&E from mice receiving vehicle control (A and B) and fenofibrate (C and D). Adventitial inflammation and thickening was less marked in the mice receiving fenofibrate. Scale bars: 100 μm (A and C); 20 μm (B and C).

      Fenofibrate Reduces Aortic Inflammation

      Immunostaining area for macrophages (CD68) was significantly reduced in the SRA of mice receiving fenofibrate, compared with animals receiving vehicle control [median staining area (MSA) = 1.57% versus 8.6%, IQR = 0.78–3.72 versus 4.20–16.62] (P = 0.010; n = 6) (Figure 5, A–C). Similarly, immunostaining area for T lymphocytes (CD3) was also lower in the SRA of mice receiving fenofibrate (MSA = 3.30%, IQR = 0.53–4.77) than in control animals (MSA = 13.30%, IQR = 11.04–16.41) (P = 0.002; n = 6) (Figure 5, D–F).
      Figure thumbnail gr5
      Figure 5Representative photomicrographs of macrophage (CD68) and T lymphocyte (CD3) staining in sections of suprarenal aorta from AngII-infused Ldlr−/− mice receiving vehicle control or fenofibrate. Examples of immunostaining for macrophages (A and B) and T lymphocytes (D–E) from 18-week-old male Ldlr−/− mice that received 4 weeks AngII infusion plus vehicle control (A and D) or fenofibrate (B and E). The lumen is indicated by an asterisk. Quantification of macrophage (C) and T lymphocyte (F) infiltration (n = 6 mice per group). Six harvested suprarenal aortic segments were randomly selected from each group for immunostaining for macrophages (CD68) and T lymphocytes (CD3). Staining area was estimated by computer-aided analysis. Box plots indicate median and interquartile range of staining areas from mice for each group. A significantly smaller staining area for macrophages (P = 0.010) and T lymphocytes (P = 0.002) was found in mice receiving fenofibrate. Scale bar = 20 μm.

      Fenofibrate Up-Regulates Aortic eNOS, iNOS, S1P-3R, and S1P-1R Expression

      To gain better understanding of which aortic cells were influenced by fenofibrate administration, IHC was performed to localize the studied proteins within the SRA. The IHC findings suggested that eNOS was mainly expressed in intimal ECs and up-regulated in response to fenofibrate (MSA = 14.87%, IQR = 9.55–20.73 in the fenofibrate group; MSA = 6.85%, IQR = 3.33–7.51 in the vehicle control group) (P = 0.028; n = 6) (Figure 6, A–C). Expression of iNOS was abundant throughout the tunica media and adventitia and was up-regulated in response to fenofibrate (MSA = 54.04%, IQR = 39.77–59.67 in the fenofibrate group; MSA = 14.37%, IQR = 3.828–25.37 in the vehicle control group) (P = 0.019; n = 6) (Figure 6, D–F). The staining pattern suggested that medial SMCs and adventitial cells were the major source of iNOS.
      Figure thumbnail gr6
      Figure 6Representative photomicrographs of sections of suprarenal aorta from AngII-infused Ldlr−/− mice receiving vehicle control or fenofibrate show immunostaining for eNOS (A and B), iNOS (D and E), S1P-3R (G and H), and S1P-1R (J and K) from 18-week-old male Ldlr−/− mice that received 4 weeks AngII infusion plus vehicle control (A, D, G, and J) or fenofibrate (B, E, H, and K). The lumen is indicated by an asterisk. Intimal ECs are indicated by arrowheads. For quantification of eNOS (C), iNOS (F), S1P-3R (EDG-3) (I), and S1P-1R (EDG-1) (L), six harvested suprarenal aortic segments were randomly selected from each group for immunostaining. Staining area was estimated by computer-aided analysis. Box plots indicate median and interquartile range of staining areas. In mice receiving fenofibrate, significant up-regulation in staining areas was found for eNOS (P = 0.028), iNOS (P = 0.019), S1P-3R (P = 0.007), and S1P-1R (P = 0.028). n = 6 mice per group. Scale bar = 20 μm.
      Staining for S1P-R1 and S1P-R3 was localized mainly in the adventitia of the SRA. Both SIP-R3 (fenofibrate: MSA = 46.47%, IQR = 34.91–58.68; vehicle control: MSA = 15.33%, IQR = 9.73–28.07) (P = 0.007; n = 6) (Figure 6, G–I) and S1P-R1 (fenofibrate: MSA = 57.12% and IQR = 40.93–65.99; vehicle control: MSA = 19.12%, IQR = 15.77–29.58) (P = 0.028; n = 6) (Figure 6, J–L) were up-regulated in response to fenofibrate.

      Fenofibrate Reduces Apoptosis

      TUNEL staining suggested that apoptotic cells were more numerous in the SRA media and adventitia in mice receiving vehicle control than in mice receiving fenofibrate (Figure 7, A and B). TUNEL staining area decreased from a median of 17.50% (IQR = 4.75–28.0) to 3% (IQR = 0.0–4.0) (P = 0.021; n = 6) (Figure 7C).
      Figure thumbnail gr7
      Figure 7TUNEL staining. A and B: Representative photomicrographs of TUNEL staining in cryostat sections of suprarenal aorta from Ldlr−/− mice, recovered after infusion with AngII plus vehicle control (A) and AngII plus fenofibrate (B) and stained for the presence of apoptotic cells by TUNEL (green) and counterstained with DAPI (blue). The elastic lamina of the vessels shows a prominent background staining. The lumen is indicated by an asterisk. C: The percentage of TUNEL-positive cells was determined by counting the number of TUNEL-positive cells in five high-power fields per cross section from each group. Box plots indicate median and interquartile range of percentage of positive cells. The staining area of TUNEL-positive cells was significantly smaller in mice receiving fenofibrate (P = 0.021; n = 6 mice per group). Scale bar = 20 μm.

      S1P Up-Regulates eNOS Activity in Ldlr−/− ECs

      To examine eNOS activation by S1P, we incubated mouse ECs with increasing doses of S1P in vitro. Incubation of ECs with increasing concentrations of S1P (0, 0.1, 1, 100, and 1000 nmol/L) appeared to up-regulate eNOS activation, although this trend did not reach statistical significance (P = 0.25) (Figure 8).
      Figure thumbnail gr8
      Figure 8S1P stimulates an increase in eNOS activity in ECs derived from Ldlr−/− mice. A: Representative blots of phospho-eNOS and total eNOS, using protein derived from primary aortic ECs from Ldlr−/− mice cultured with vehicle control (VC) or different concentrations of S1P for 4 hours. Cytosolic protein extracts were collected and analyzed by SDS-PAGE for phospho-eNOS and total eNOS. B: Densitometry data are expressed as means ± SEM from studies performed using ECs from 6 mice. A dose-dependent increase in eNOS phosphorylation was demonstrated, although this trend was not statistically significant (P = 0.25).

      Discussion

      Previous studies suggest that the PPAR-α ligand fenofibrate has a number of beneficial effects in preclinical models of vascular disease, including reducing inflammation and limiting fibrosis.
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      Immunomodulator FTY720 Induces eNOS-dependent arterial vasodilatation via the lysophospholipid receptor S1P3.
      In dose-response studies in vitro, we found that S1P appeared to increase eNOS activity in aortic ECs derived from Ldlr−/− mice. Our findings suggest that the NO system was augmented in the aortas of mice receiving fenofibrate, an effect that was probably induced by HDL-bound-S1P. We propose that concomitant activation of S1P-3R and S1P-1R receptors exerts protective effects on cells of the intima, media, and adventitia via activation of the NO system and thereby attenuates inflammation (Figure 9). The present study does not rule out other mechanisms that may also contribute to the effect of fenofibrate we observed, such as stimulation of PPAR-α.
      Figure thumbnail gr9
      Figure 9Proposed mechanisms by which fenofibrate inhibits AngII-induced AAA and atherosclerosis. Fenofibrate limits suprarenal aortic expansion in AngII-infused Ldlr−/− mice by a variety of mechanisms: i) fenofibrate increases serum HDL and reduces triglyceride concentrations; ii) fenofibrate increases serum S1P concentrations; iii) fenofibrate up-regulates aortic S1P-1R and S1P-3R expression; iv) increased S1P activation of S1P-1R and S1P-3R promotes Akt1 phosphorylation, which in turn leads to up-regulation and activation of eNOS and iNOS, all of which may result in increased NO levels. Together, mechanisms 1 through 4 attenuate inflammation and apoptosis, thus restoring the balance between matrix synthesis and degradation favoring tissue repair. The schematic here shows adventitial inflammation. Dark balls, thrombus; light balls, foam cells.
      To our knowledge, this is the first study assessing the effect of fenofibrate administration on S1P receptors. Statins (pitavastatin and simvastatin) have been shown to enhance HDL-induced eNOS activation in bovine aortic ECs and HUVECs, which is mediated in part through S1P-R1 up-regulation.
      • Kimura T.
      • Mogi C.
      • Tomura H.
      • Kuwabara A.
      • Im D.S.
      • Sato K.
      • Kurose H.
      • Murakami M.
      • Okajima F.
      Induction of scavenger receptor class B type I is critical for simvastatin enhancement of high-density lipoprotein-induced anti-inflammatory actions in endothelial cells.
      • Hughes J.E.
      • Srinivasan S.
      • Lynch K.R.
      • Proia R.L.
      • Ferdek P.
      • Hedrick C.C.
      Sphingosine-1-phosphate induces an antiinflammatory phenotype in macrophages.
      Simvastatin potentiates the HDL- and S1P-induced cyclooxygenase 2 (COX-2) up-regulation by stimulating S1P-3R expression in human VSMCs.
      • González-Díez M.
      • Rodríguez C.
      • Badimon L.
      • Martínez-González J.
      Prostacyclin induction by high-density lipoprotein (HDL) in vascular smooth muscle cells depends on sphingosine 1-phosphate receptors: effect of simvastatin.
      Furthermore, previous reports in knockout mouse models support our present observations that the vasoprotective effect of HDL-S1P complex may be elicited through S1P-3R or S1P-1R. Studies in these murine models suggest that S1P stimulates vasorelaxation similar to native HDL and that this vasorelaxing effect is absent in mice deficient in eNOS.
      • Nofer J.R.
      • van der Giet M.
      • Tölle M.
      • Wolinska I.
      • von Wnuck Lipinski K.
      • Baba H.A.
      • Tietge U.J.
      • Gödecke A.
      • Ishii I.
      • Kleuser B.
      • Schäfers M.
      • Fobker M.
      • Zidek W.
      • Assmann G.
      • Chun J.
      • Levkau B.
      HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3.
      The vasodilatory effect of HDL was inhibited by 57% in S1P-3R-deficient mice, compared with wild-type mice, whereas the vasodilation induced by S1P was completely abolished in S1P-3R-deficient mice.
      • Nofer J.R.
      • van der Giet M.
      • Tölle M.
      • Wolinska I.
      • von Wnuck Lipinski K.
      • Baba H.A.
      • Tietge U.J.
      • Gödecke A.
      • Ishii I.
      • Kleuser B.
      • Schäfers M.
      • Fobker M.
      • Zidek W.
      • Assmann G.
      • Chun J.
      • Levkau B.
      HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3.
      Studies in ECs from S1P-3R-deficient mice indicated that Akt phosphorylation and Ca2+ increase in response to HDL and lysophospholipids were severely reduced, supporting the role of SIP-3R in NO release in the vasculature. Overall, our findings suggest that fenofibrate acts to up-regulate SIP-mediated mechanisms, including those relying on NO and Akt. Further studies in individual S1P receptor-deficient mouse models are needed to more completely assess the effects of fenofibrate on S1P-dependent responses.
      Apoptosis has been suggested to play an important role in altering the structural integrity of the aorta during AAA formation.
      • López-Candales A.
      • Holmes D.R.
      • Liao S.
      • Scott M.J.
      • Wickline S.A.
      • Thompson R.W.
      Decreased vascular smooth muscle cell density in medial degeneration of human abdominal aortic aneurysms.
      • Rowe V.L.
      • Stevens S.L.
      • Reddick T.T.
      • Freeman M.B.
      • Donnell R.
      • Carroll R.C.
      • Goldman M.H.
      Vascular smooth muscle cell apoptosis in aneurysmal, occlusive, and normal human aortas.
      Depletion of medial vascular SMCs by apoptosis eliminates a cell population capable of synthesizing matrix proteins, thus potentially contributing to AAA formation.
      • Daugherty A.
      • Manning M.W.
      • Cassis L.A.
      Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.
      Increased TUNEL-positive apoptotic bodies within the aortic media has been reported previously in human AAA.
      • Satta J.
      • Mennander A.
      • Soini Y.
      Increased medial TUNEL-positive staining associated with apoptotic bodies is linked to smooth muscle cell diminution during evolution of abdominal aortic aneurysms.
      In the present study, we observed reduced apoptosis in SRA of mice receiving fenofibrate. It was shown previously that fenofibrate reduced palmitate-induced apoptosis in neonatal mouse cardiomyocytes by enhancing PPAR-α expression.
      • Kong J.Y.
      • Rabkin S.W.
      Reduction of palmitate-induced cardiac apoptosis by fenofibrate.
      Recent reports also suggest that fenofibrate decreased aldosterone-induced apoptosis in adult rat ventricular myocytes by inhibiting JNK phosphorylation and down-regulating proapoptotic mediators, such as Bax and caspase 3.
      • Irukayama-Tomobe Y.
      • Miyauchi T.
      • Sakai S.
      • Kasuya Y.
      • Ogata T.
      • Takanashi M.
      • Iemitsu M.
      • Sudo T.
      • Goto K.
      • Yamaguchi I.
      Endothelin-1-induced cardiac hypertrophy is inhibited by activation of peroxisome proliferator-activated receptor-alpha partly via blockade of c-Jun NH2-terminal kinase pathway.
      • De Silva D.S.
      • Wilson R.M.
      • Hutchinson C.
      • Ip P.C.
      • Garcia A.G.
      • Lancel S.
      • Ito M.
      • Pimentel D.R.
      • Sam F.
      Fenofibrate inhibits aldosterone-induced apoptosis in adult rat ventricular myocytes via stress-activated kinase-dependent mechanisms.
      Overall, our findings suggest that fenofibrate inhibits both inflammatory and proapoptotic pathways, thereby restoring the balance between matrix synthesis and degradation favoring tissue repair in AAA.
      In conclusion, in the present study fenofibrate inhibited aortic dilatation and atheroma progression, which was associated with reduced aortic inflammation and apoptosis, most likely secondary to up-regulation of Akt1 and NOS (Figure 9). We propose that activation of the S1P pathway promotes the NO pathway, leading to reduced aortic inflammation and medial damage. Our findings suggest that fenofibrate is a potential candidate to be studied in human trials to assess its efficacy in limiting progression of small AAAs.

      Acknowledgment

      We thank Surabhi Khosla for technical support.

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