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Decreased Tissue Levels of Cyclophilin A, a Cyclosporine A Target and Phospho-ERK1/2 in Simvastatin Patients with Abdominal Aortic Aneurysm

Open ArchivePublished:April 03, 2013DOI:https://doi.org/10.1016/j.ejvs.2013.02.015

      Background

      Cyclophilin A (CyPA), a cyclosporine A-binding protein, influences abdominal aortic aneurysm (AAA) formation and the ERK1/2 signalling pathway in animal and in vitro studies. Statins decrease CyPA in smooth muscle cells although their influence on CyPA in human AAA is unknown.

      Material and methods

      The study was performed on AAA wall-tissue samples obtained from 30 simvastatin-treated and 15 non-statin patients (2:1 case to control). The patients were matched by age, sex and AAA diameter. We investigated the gene expression of CyPA, its receptor extracellular matrix metalloproteinase inducer (EMMPRIN) by real-time RT-PCR. CyPA and EMMPRIN protein level and phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured by Western blot.

      Results

      The AAA wall tissue from simvastatin-treated patients had significantly lower CyPA gene expression and protein levels (P = 0.0018, P = 0.0083, respectively). Furthermore, phosphorylation of ERK1 and ERK2 was markedly suppressed in the simvastatin group (P = 0.0002, P = 0.0027, respectively). However, simvastatin did not influence EMMPRIN gene and protein expression.

      Conclusion

      Simvastatin-treated patients with AAA exert lower CyPA messenger RNA (mRNA), as well as CyPA intracellular protein levels and a decreased amount of phospho-ERK1/2. Thus, the interference with signalling pathways leading to CyPA formation and ERK1/2 activation reveals a new anti-inflammatory role of statins in AAA.

      Keywords

      In this paper we propose a new anti-inflammatory role for simvastatin in human abdominal aortic aneurysm (AAA) wall tissue. Simvastatin interferes with cyclophilin A (CyPA) formation and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation. CyPA, a cyclosporine A-binding protein, influences AAA formation and the ERK1/2 signalling pathway in animal and in vitro studies and CyPA-deficient mice are resistant to aneurysm formation. Statins decrease CyPA in smooth muscle cells but their influence on CyPA in human AAA is unknown. Therefore, investigating the statins in cardiovascular disease (CVD), such as AAA, may reveal new therapeutic effects of those drugs.
      Abdominal aortic aneurysms (AAAs) are an important health issue in the elderly, affecting approximately 10% of individuals over 65.
      • Alcorn H.G.
      • Wolfson Jr., S.K.
      • Sutton-Tyrrell K.
      • Kuller L.H.
      • O'Leary D.
      Risk factors for abdominal aortic aneurysms in older adults enrolled in The Cardiovascular Health Study.
      In Western countries AAA may cause as much as 2% of all deaths.
      • van der Vliet J.A.
      • Boll A.P.
      Abdominal aortic aneurysm.
      At our unit 1198 patients with AAAs have been treated within the last 12 years.
      From the morphological point of view, the AAA is considered as a dilatative form of atherosclerosis that is critically connected with inflammation, oxidative stress and degradation of the cellular matrix.
      • Satoh K.
      • Nigro P.
      • Matoba T.
      • O'Dell M.R.
      • Cui Z.
      • Shi X.
      • et al.
      Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms.
      • Demyanets S.
      • Konya V.
      • Kastl S.P.
      • Kaun C.
      • Rauscher S.
      • Niessner A.
      Interleukin-33 induces expression of adhesion molecules and inflammatory activation in human endothelial cells and in human atherosclerotic plaques.
      Overproduction of reactive oxygen species (ROS) is regarded as an important driver that lies upstream of the inflammatory cascade. Some mediators, which are induced in response to ROS, were named secreted oxidative stress-induced factors (SOXFs). Cyclophilin A (CyPA), a chaperone that binds cyclosporine A, is highlighted as a major SOXF.
      • Handschumacher R.E.
      • Harding M.W.
      • Rice J.
      • Drugge R.J.
      • Speicher D.W.
      Cyclophilin: a specific cytosolic binding protein for cyclosporin A.
      • Jin Z.G.
      • Melaragno M.G.
      • Liao D.F.
      • Yan C.
      • Haendeler J.
      • Suh Y.A.
      • et al.
      Cyclophilin A is a secreted growth factor induced by oxidative stress.
      • Libby P.
      • Ridker P.M.
      Novel inflammatory markers of coronary risk: theory versus practice.
      Elevated levels of CyPA have been reported in advanced atherosclerosis lesions
      • Satoh K.
      • Nigro P.
      • Matoba T.
      • O'Dell M.R.
      • Cui Z.
      • Shi X.
      • et al.
      Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms.
      and up-regulation of intracellular and extracellular CyPA occurs during AAA formation in mice.
      • Satoh K.
      • Matoba T.
      • Suzuki J.
      • O'Dell M.R.
      • Nigro P.
      • Cui Z.
      • et al.
      Cyclophilin A mediates vascular remodeling by promoting inflammation and vascular smooth muscle cell proliferation.
      Furthermore, excessive free radical generation during ischaemia/reperfusion
      • Huk I.
      • Nanobashvili J.
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      • Punz A.
      • Mueller M.
      • Afkhampour K.
      • et al.
      L-arginine treatment alters the kinetics of nitric oxide and superoxide release and reduces ischemia/reperfusion injury in skeletal muscle.
      leads to increased CyPA production by leucocytes and macrophages.
      • Seizer P.
      • Ochmann C.
      • Schönberger T.
      • Zach S.
      • Rose M.
      • Borst O.
      • et al.
      Disrupting the EMMPRIN (CD147)-cyclophilin A interaction reduces infarct size and preserves systolic function after myocardial ischemia and reperfusion.
      Further, increased CyPA protein levels are observed in polymorphonuclear neutrophils (PMNs) of AAA patients.
      • Ramos-Mozo P.
      • Madrigal-Matute J.
      • Martinez-Pinna R.
      • Blanco-Colio L.M.
      • Lopez J.A.
      • Camafeita E.
      • et al.
      Proteomic analysis of polymorphonuclear neutrophils identifies catalase as a novel biomarker of abdominal aortic aneurysm: potential implication of oxidative stress in abdominal aortic aneurysm progression.
      CyPA wields its action when binding to the extracellular matrix metalloproteinase inducer (EMMPRIN) receptor localised on vascular smooth muscle cells (VSMCs).
      • Guo H.
      • Majmudar G.
      • Jensen T.C.
      • Biswas C.
      • Toole B.P.
      • Gordon M.K.
      Characterization of the gene for human EMMPRIN, a tumor cell surface inducer of matrix metalloproteinases.
      Recently Chen et al.
      • Chen X.F.
      • Wang J.A.
      • Hou J.
      • Gui C.
      • Tang L.J.
      • Chen X.Q.
      • et al.
      Extracellular matrix metalloproteinase inducer (EMMPRIN) is present in smooth muscle cells of human aneurysmal aorta and is induced by angiotensin II in vitro.
      demonstrated that EMMPRIN is strongly expressed in human AAA lesions. The overexpression of EMMPRIN leads to activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2). The cascade of EMMPRIN–ERK–nuclear factor-κB (NF-κB) is speculated to be the main signalling pathway for CyPA in monocytes/macrophages.
      • Yuan W.
      • Ge H.
      • He B.
      Pro-inflammatory activities induced by CyPA-EMMPRIN interaction in monocytes.
      In our recent study we presented decreased activity of the NF-κB signalling pathway and lowered level of ROS and tumour necrosis factor (TNF)-α in AAA tissue from patients treated with simvastatin.
      • Piechota-Polanczyk A.
      • Goraca A.
      • Demyanets S.
      • Mittlboeck M.
      • Domenig C.
      • Neumayer C.
      • et al.
      Simvastatin decreases free radicals formation in the human abdominal aortic aneurysm wall via NF-κB.
      Simvastatin belongs to hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors which, among others, influence aneurysm formation.
      • Forsdahl S.H.
      • Singh K.
      • Solberg S.
      • Jacobsen B.K.
      Risk factors for abdominal aortic aneurysms: a 7-year prospective study: the Tromsø Study, 1994–2001.
      • Takagi H.
      • Matsui M.
      • Umemoto T.
      A meta-analysis of clinical studies of statins for prevention of abdominal aortic aneurysm expansion.
      Suzuki et al.
      • Suzuki J.
      • Jin Z.G.
      • Meoli D.F.
      • Matoba T.
      • Berk B.C.
      Cyclophilin A is secreted by a vesicular pathway in vascular smooth muscle cells.
      indicate that simvastatin decreases ROS-mediated CyPA release from VSMC. Additionally, simvastatin inhibits angiotensin II-mediated stimulation of ERK1/2 phosphorylation in VSMC.
      • Tristano A.G.
      • Castejon A.M.
      • Castro A.
      • Cubeddu L.X.
      Effects of statin treatment and withdrawal on angiotensin II-induced phosphorylation of p38 MAPK and ERK1/2 in cultured vascular smooth muscle cells.
      However, the role of statins in intracellular CyPA level and their influence on the ERK1/2 pathway in human AAA tissue remains elusive.
      Therefore, the aim of the study was to assess the influence of simvastatin on cyclophilin A levels in AAA wall tissue. Next, we wanted to verify if the ERK1/2 signalling pathway is modulated by simvastatin in AAA wall tissue.

      Material and methods

      Patients

      The study was performed on 45 patients undergoing open AAA repair between September 2009 and December 2011 at our institution according to our previous study.
      • Piechota-Polanczyk A.
      • Goraca A.
      • Demyanets S.
      • Mittlboeck M.
      • Domenig C.
      • Neumayer C.
      • et al.
      Simvastatin decreases free radicals formation in the human abdominal aortic aneurysm wall via NF-κB.
      Briefly, exclusion criteria were the intake of statins other than simvastatin, non-steroidal anti-inflammatory drugs except aspirin in the medication list, chronic diseases such as liver disease, inflammatory disease, malignant disease, recreational drugs' intake and alcohol abuse. After written informed consent, patient data were prospectively collected and aneurysm wall tissue was harvested during aneurysm repair for retrospective analysis. Patients were matched in a 2 (simvastatin) to 1 (non-statin) ratio, respectively, by age, gender and AAA diameter. Fifteen patients without statin medication (12 men, 3 women) were incorporated into the control group and 30 patients who had simvastatin (24 men, 6 women) in their medical history (20–40 mg daily dosage) for a minimum of 6 months were included in the study as the simvastatin group. The AAA diameter was measured with preoperative computed tomography angiography (CTA).
      Coronary artery disease (CAD) was defined by a history of angina pectoris or myocardial infarction. Cerebrovascular disease (CVAD) was defined by a history of transient ischaemic attack, stroke, carotid artery stenting or surgery, respectively. Cardiac insufficiency was defined by a global ejection fraction of <50% in echocardiography; all patients were without cardiac symptoms at the time of the surgery. Peripheral artery disease was defined by symptomatic claudication and a corresponding finding in the CTA at the level of the iliac and/or femoro-popliteal vessels.
      Hypertension was defined by the intake of antihypertensive and/or a repeatedly elevated blood pressure exceeding 140 over 90 mmHg. Type 2 diabetes was defined by the intake of antidiabetics or requirement of insulin. Dyslipidaemia was defined as recommended in the European Society of Cardiology/European Atherosclerosis Society (ESC/EAS) guidelines for the management of dyslipidaemia.
      • Reiner Z.
      • Catapano A.L.
      • De Backer G.
      • Graham I.
      • Taskinen M.R.
      • Wiklund O.
      • et al.
      ESC/EAS Guidelines for the management of dyslipidaemias: the Task Force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS).
      Smoking (y/n) pertains to nicotine consumption within the last 3 years.
      The study was approved by the local research Ethics Committee (EC 294/2009).

      Tissue harvesting

      After aortic cross-clamping and longitudinal incision of the aneurysm, the thrombus (present in 39 of 45 patients) was removed and about 3 cm2 of the aneurysm sac was excised at the site of its maximum diameter. Aneurysm samples were immediately frozen in liquid nitrogen and stored at −80 °C. For subsequent analysis aneurysm tissue was processed on ice. The aneurysm wall was divided into 50-mg pieces and rinsed with ice-cold saline to eliminate the liquid components, such as blood and residual thrombi.

      Western blot analyses of CyPA, EMMPRIN and ERK1/2

      Equal protein amounts of tissue extracts were separated by sodium dodecyl sulphate-polyacryalamide gel electrophoresis (SDS-PAGE), and CyPA, EMMPRIN, p42, phospho-42, p44 and phospho-44 were assessed by Western blotting using the respective rabbit anti-human monoclonal antibodies followed by horseradish peroxidase (HRP)-conjugated donkey secondary antibodies (Abcam, Cambridge, UK and Cell Signalling, Danvers, MA, USA, respectively). Signal intensity was quantified using an Imagine Master VDS (Bio-Rad Laboratories Inc., Hercules, CA, USA) and normalised to β-tubulin. Assays were performed twice with tissue samples from different donors.

      Real-time polymerase chain reaction for CyPA and EMMPRIN

      Frozen tissue was homogenised using a ball mill (Retsch, Haan, Germany), and mRNA was isolated using the High Pure RNA Tissue Kit (Roche, Basel, Switzerland). Reverse transcription was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche). Real-time polymerase chain reaction (PCR) was performed using the LightCycler® TaqMan® Master (Roche) according to the manufacturer's instructions. Primers were designed using the Roche Universal ProbeLibrary Assay Design Centre: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward primer: 5′-AGCCACATCGCTCAGACAC-3′, reverse primer: 5′-GCCCAATACGACCAAATCC-3′), CypA (forward primer 5′-GTCAACCCCACCGTGTTCTTC-3′, reverse primer: 5′-TTTCTGCTGTCTTTGGGACCTTG-3′) and EMMPRIN (forward primer. 5′-GGGAGAGTACTCCTGCGTCTT-3′, reverse primer: 5′-ACTTCACAGCCTTCACTCTGG-3′). The amplification conditions consisted of an initial incubation at 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 s, 63 °C for 20 s and 72 °C for 6 s and a final cooling to 40 °C. Data were analysed using LightCycler Software Version 3.5 (Roche).

      Statistical analysis

      Continuous demographic and biochemical data are presented as median, minimum and maximum, and demographic categorical data are described with absolute frequencies and percentages. Data are 2:1 matched in the simvastatin and non-statin groups. A generalised linear model (binomial, logit) with an exchangeable correlation matrix was used to analyse matched binary outcome data. A linear mixed model with a compound symmetry variance–covariance matrix was used to analyse matched continuous outcome data. In the case of skew residuals a logarithmic transformation usually led to normally distributed errors.
      All P-values are two-sided and P ≤ 0.05 was considered significant. Statistical analyses were performed by the software package SAS (Version 9.3; SAS Institute Inc., Cary, NC, USA) and the software package Statistical Package for the Social Sciences (SPSS) (SPSS 17.0, Chicago, IL, USA) was used for graphics.

      Results

      Demographic data

      In Table 1, the characteristics for non-statin and simvastatin-treated patients are shown. The two groups were comparable in age, aneurysm diameter, co-morbidities and risk factors. The median aneurysm diameter was 57 mm (48–102 mm) for the non-statin and 55 mm (48–120 mm) for the simvastatin patients. The simvastatin group had markedly lower body mass index (P = 0.025) and better lipid profile with significantly decreased total cholesterol and low density lipoprotein (LDL) cholesterol (P = 0.002 and P = 0.003, respectively). We found no differences in C-reactive protein (CRP), fibrinogen, creatinine and leucocyte levels among the two groups (all P ≥ 0.05).
      Table 1Patient demographics.
      Non-statin patients (n = 15)Simvastatin patients (n = 30)P
      Age (years), median (range)68 (50–73)67 (55–80)0.186
      Sex (male)12 (80%)24 (80%)1.000
      AAA diameter (mm)57 (48–102)55 (48–120)0.439
      Body mass index, mean (range)27.55 (21.95–37.55)25.89 (21.56–31.25)0.025
      Coronary artery disease3 (20%)9 (30%)0.479
      Cerebrovascular artery disease10 (67%)15 (50%)0.161
      Peripheral artery disease4 (27%)9 (30%)0.803
      Cardiac insufficiency1 (7%)8 (23%)0.083
      Hypertension15 (100%)28 (93%)0.317
      Type 2 diabetes4 (27%)8 (27%)1.000
      Smoking12 (80%)22 (73%)0.617
      Cholesterol [mg/dl], median (range)
      The measurements were made in serum samples.
      240 (143–323)199 (110–264)0.002
      LDL [mg/dl], median (range)
      The measurements were made in serum samples.
      164.4 (79.2–218)112.5 (47–218)0.003
      HDL [mg/dl], median (range)
      The measurements were made in serum samples.
      45.0 (36–68)48.0 (29–75)0.477
      CRP [mg/dl], median (range)
      The measurements were made in serum samples.
      0.43 (0.03–7.6)0.43 (0.06–3.0)0.291
      Fibrinogen [mg/dl], median (range)
      The measurements were made in serum samples.
      407 (280–594)359 (240–549)0.102
      Leucocytes [mln/ml], median (range)
      The measurements were made in serum samples.
      8.15 (5.5–12)8.0 (5.09–13.0)0.473
      Creatinine [mg/dl], median (range)
      The measurements were made in serum samples.
      1.05 (0.75–1.44)0.99 (0.76–4.0)0.907
      Data are presented as frequencies or median (minimum–maximum). Statistical significance for binary variables was assessed using generalised linear models, while metric values were analysed using linear mixed regression models.
      a The measurements were made in serum samples.

      Simvastatin decreases CyPA gene expression and protein level in human AAA wall explants

      The tissue gene expression of CyPA from AAA patients treated with simvastatin was significantly lower than in the non-statin group (P = 0.0018, Fig. 1(A)). Similarly, the intracellular CyPA protein level was markedly decreased in the simvastatin group when compared to the non-statin group (P = 0.0083, Fig. 1(B) and (C)). However, there was no difference in the tissue gene expression and the protein level of EMMPRIN in the two examined groups (P = 0.6495 and P = 0.408, respectively; Fig. 2(A) and (B)). Fig. 2(C) shows a representative Western blot including three simvastatin and two non-statin patients.
      Figure thumbnail gr1
      Figure 1Simvastatin treatment suppressed CyPA mRNA expression and CyPA protein level in human AAA wall tissue. (A) CyPA mRNA expression. (B) Densitometrical quantification of CyPA protein level (mean values ± SD). (C) Representative Western blot (n = 2 and n = 3 for AAA tissue wall from the non-statin and simvastatin patients, respectively).
      Figure thumbnail gr2
      Figure 2Simvastatin treatment had no influence on EMMPRIN mRNA expression and protein level in human AAA wall tissue. (A) EMMPRIN gene expression. (B) Densitometrical quantification of EMMPRIN protein level (mean values ± SD). (C) Representative Western blot (n = 2 and n = 3 for AAA tissue wall from the non-statin and simvastatin patients, respectively).

      Simvastatin reduces pERK1/2 in human AAA wall explants

      In Fig. 3(A) the densitometric analysis comparing 30 simvastatin and 15 non-statin patients is shown (2:1 case to control). The amounts of phospho-p42/p42 (pERK1) and phospho-p44/p-44 (pERK2) were significantly lower in AAA tissues from simvastatin patients when compared to the non-statin group (P = 0.0002 and P = 0.0027, respectively) (Fig. 3(A) and (B)). Fig. 3(C) shows a representative Western blot including three simvastatin and two non-statin patients.
      Figure thumbnail gr3
      Figure 3Simvastatin treatment inhibited p-ERK1 (A) and p-ERK2 (B) activation in human AAA wall samples. (C) Representative Western blot (n = 2 and n = 3 for AAA tissue wall from the non-statin and simvastatin patients, respectively). Data are present as the ratio of phosphorylated form to total form (fold) over tubulin and represents mean values ± SD.

      Discussion

      In the present study we showed that AAA wall tissue from patients treated with simvastatin has lower gene expression and intracellular concentration of CyPA. Moreover, simvastatin-treated subjects with AAA had lower activity of the ERK1/2 signalling pathway compared to age-, sex- and AAA diameter-matched non-statin patients. However, we observed no difference in the gene expression and the protein level of EMMPRIN between the simvastatin and non-statin AAA wall tissue.
      AAA development is a multifactorial process that depends to a great extent on macrophage-derived matrix metalloproteinase-9 (MMP-9) and VSMC-derived MMP-2.
      • Longo G.M.
      • Xiong W.
      • Greiner T.C.
      • Zhao Y.
      • Fiotti N.
      • Baxter B.T.
      Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms.
      Statins were shown to interfere with AAA expansion; however their role remains controversial. Recently, Takagi et al.
      • Takagi H.
      • Matsui M.
      • Umemoto T.
      A meta-analysis of clinical studies of statins for prevention of abdominal aortic aneurysm expansion.
      indicated that statin therapy might be effective in prevention of the growth of small AAA, while Karrowni et al.
      • Karrowni W.
      • Dughman S.
      • Hajj G.P.
      • Miller Jr., F.J.
      Statin therapy reduces growth of abdominal aortic aneurysms.
      showed no correlation. Further, controversies have arisen around statin influence on VSMC proliferation which number is decreased in the media of human AAA tissue.
      • Allaire E.
      • Schneider F.
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      New insight in aetiopathogenesis of aortic diseases.
      Here some studies highlight the anti-proliferative role of statins in the neointima while others described their proliferative action under hyperglycaemia.
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      • Lu H.
      • Huang J.
      • Guan Y.
      • Sun H.
      Simvastatin exerts favourable effects on neointimal formation in a mouse model of vein graft.
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      • Kim M.
      • et al.
      Potential role of HMG CoA reductase inhibitor on oxidative stress induced by advanced glycation endproducts in vascular smooth muscle cells of diabetic vasculopathy.
      Yet it is not clear whether the induction of apoptosis in VSMC by statins is beneficial or detrimental. Nevertheless, it is apparent that a short course of cyclosporine A, a CyPA-binding drug, stabilises the diameter of formed AAA and increases VSMC content in an animal model.
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      • Gervais M.
      • et al.
      Long term stabilization of expanding aortic aneurysms by a short course of cyclosporine A through transforming growth factor-beta induction.
      It was previously demonstrated that AAA tissue had a higher concentration of CyPA than the healthy aorta;
      • Satoh K.
      • Nigro P.
      • Matoba T.
      • O'Dell M.R.
      • Cui Z.
      • Shi X.
      • et al.
      Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms.
      • Yurchenko V.
      • Zybarth G.
      • O'Connor M.
      • Dai W.W.
      • Franchin G.
      • Hao T.
      • et al.
      Active site residues of cyclophilin A are crucial for its signalling activity via CD147.
      increased CyPA levels were found in PMNs in AAA patients.
      • Ramos-Mozo P.
      • Madrigal-Matute J.
      • Martinez-Pinna R.
      • Blanco-Colio L.M.
      • Lopez J.A.
      • Camafeita E.
      • et al.
      Proteomic analysis of polymorphonuclear neutrophils identifies catalase as a novel biomarker of abdominal aortic aneurysm: potential implication of oxidative stress in abdominal aortic aneurysm progression.
      Therefore, drugs that influence CyPA concentration may impact AAA formation. Statins whose pleiotropic actions include a decrease in the gene expression and protein concentration of MMP-3, MMP-9 and TNF-α and lead to an increase in antioxidant enzyme activity in human AAA tissue may play an important role.
      • Piechota-Polanczyk A.
      • Goraca A.
      • Demyanets S.
      • Mittlboeck M.
      • Domenig C.
      • Neumayer C.
      • et al.
      Simvastatin decreases free radicals formation in the human abdominal aortic aneurysm wall via NF-κB.
      • Wilson W.R.
      • Evans J.
      • Bell P.R.
      • Thompson M.M.
      HMG-CoA reductase inhibitors (statins) decrease MMP-3 and MMP-9 concentrations in abdominal aortic aneurysms.
      • Hurks R.
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      • Vink A.
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      • Schoneveld A.
      • Kerver M.
      • et al.
      Different effects of commonly prescribed statins on abdominal aortic aneurysm wall biology.
      Here we present evidence that simvastatin treatment in patients with AAA significantly decreased CyPA in AAA wall on the gene and protein levels. Our results are in line with the study of Suzuki et al.,
      • Suzuki J.
      • Jin Z.G.
      • Meoli D.F.
      • Matoba T.
      • Berk B.C.
      Cyclophilin A is secreted by a vesicular pathway in vascular smooth muscle cells.
      who suggested that simvastatin inhibits vesicular secretion of CyPA in VSMC probably by inhibiting isoprenylation of small guanosine triphosphatases (GTPases).
      • Suzuki J.
      • Jin Z.G.
      • Meoli D.F.
      • Matoba T.
      • Berk B.C.
      Cyclophilin A is secreted by a vesicular pathway in vascular smooth muscle cells.
      However, further studies are required to investigate the mechanism of simvastatin-induced decrease of CyPA in human AAA wall in detail.
      In cells CyPA exerts its action when it binds to the EMMPRIN receptor,
      • Seizer P.
      • Schönberger T.
      • Schött M.
      • Lang M.R.
      • Langer H.F.
      • Bigalke B.
      • et al.
      EMMPRIN and its ligand cyclophilin A regulate MT1-MMP, MMP-9 and M-CSF during foam cell formation.
      which is highly expressed in the diseased aortic wall of AAA patients.
      • Chen X.F.
      • Wang J.A.
      • Hou J.
      • Gui C.
      • Tang L.J.
      • Chen X.Q.
      • et al.
      Extracellular matrix metalloproteinase inducer (EMMPRIN) is present in smooth muscle cells of human aneurysmal aorta and is induced by angiotensin II in vitro.
      So far there are limited data about the influence of statins on EMMPRIN tissue expression. Abe et al.
      • Abe N.
      • Osanai T.
      • Fujiwara T.
      • Kameda K.
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      • Okumura K.
      C-reactive protein-induced upregulation of extracellular matrix metalloproteinase inducer in macrophages: inhibitory effect of fluvastatin.
      indicated that fluvastatin influences EMMPRIN expression in macrophages probably via its antioxidant properties. However, our study showed no difference in the gene expression and protein level of EMMPRIN in simvastatin and non-statin AAA wall tissue. This may be explained by the occurrence of different cell types such as VSMC, endothelial cells and immune cells in aneurysm tissue.
      AAA tissue is characterised by up-regulated activity of different signalling transcription pathways including NF-κB and activator protein-1 (AP-1).
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      The pathophysiology of abdominal aortic aneurysm growth: corresponding and discordant inflammatory and proteolytic processes in abdominal aortic and popliteal artery aneurysms.
      Lately Ghosh et al.
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      The role of extracellular signal-related kinase during abdominal aortic aneurysm formation.
      reported on increased activity of ERK1/2 in human AAA tissue and reduced AAA formation after ERK1/2 inhibition in animal models. Our results show a decreased amount of pERK1/2 in AAA wall tissue in simvastatin-treated patients compared to the non-statin group. ERK-mediated cell signals are essential for cellular proliferation, differentiation and survival, and improper activation of ERK1/2 is associated with immunological disorders.
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      Both ERK isoforms are ubiquitously expressed in almost all mammalian tissues, with ERK-2 levels generally greater than ERK-1 levels. Recently, Bahmed et al.
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      • Holliday M.
      • Redzic J.
      • Ciobanu M.
      • Zhang F.
      • et al.
      Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B.
      indicated that extracellular CyPA stimulates ERK1/2 phosphorylation in cancer cells and Jin et al.
      • Jin Z.G.
      • Lungu A.O.
      • Xie L.
      • Wang M.
      • Wong C.
      • Berk B.C.
      Cyclophilin A is a proinflammatory cytokine that activates endothelial cells.
      showed that CyPA mediates phosphorylation of ERK1/2 and IkBα of NF-κB in human endothelial cells. Therefore, decreased CyPA levels in the simvastatin group observed in our study may be causative for the blunted activity of ERK1/2. This corresponds to the report by Tristano et al.
      • Tristano A.G.
      • Castejon A.M.
      • Castro A.
      • Cubeddu L.X.
      Effects of statin treatment and withdrawal on angiotensin II-induced phosphorylation of p38 MAPK and ERK1/2 in cultured vascular smooth muscle cells.
      that the phosphorylation of ERK1/2 is inhibited by simvastatin in VSMC. Simvastatin also reduced c-Raf and Ras expression, the components of mitogen-activated protein kinase (MAPK) pathway, in VSMC during high glucose conditions.
      • Chan K.C.
      • Wu C.H.
      • Huang C.N.
      • Lan K.P.
      • Chang W.C.
      • Wang C.J.
      Simvastatin inhibits glucose-stimulated vascular smooth muscle cell migration involving increased expression of RhoB and a block of Ras/Akt signal.
      Inhibition of phosphorylation of ERK1/2 by statins may be potentially dangerous regarding preservation of VSMC. It was indicated that simvastatin inhibits angiotensin II-mediated stimulation of ERK1/2 but simvastatin withdrawal escalates angiotensin II-mediated effect, therefore leading to VSMC degradation.
      • Castejon A.M.
      • Zollner E.
      • Tristano A.G.
      • Cubeddu L.X.
      Upregulation of angiotensin II-AT1 receptors during statin withdrawal in vascular smooth muscle cells.
      Nevertheless, targeting the ERK1/2 pathway with drugs that inhibit either the expression CyPA or phosphorylation of ERK1/2 isoforms could be a potential therapeutic approach to prevent AAA formation as well as progression.

      Conclusion

      Our study demonstrates that simvastatin treatment reduces cyclophilin A gene expression and protein concentration in human AAA wall tissue as compared to the non-statin patients. Moreover, simvastatin inhibits phosphorylation of the ERK1/2 signalling pathway. Thus, decreased CyPA concentration in AAA tissue by simvastatin may influence ERK1/2 inhibition suggesting a yet-unknown anti-inflammatory action of statins in human abdominal aortic aneurysms.

      Acknowledgements

      The study was supported by grant number 181110 from the Medical University of Vienna, Department of Surgery.

      Conflict of Interest

      None.

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