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Enhanced atherogenesis and altered high density lipoprotein in patients with Crohn's disease

Open AccessPublished:September 22, 2007DOI:https://doi.org/10.1194/jlr.M700176-JLR200
      A chronic inflammatory state is a risk factor for accelerated atherogenesis. The aim of our study was to explore whether Crohn's disease (CD), characterized by recurrent inflammatory episodes, is also associated with accelerated atherogenesis. In 60 CD patients and 122 matched controls, carotid intima media thickness (IMT), a validated marker for the burden and progression of atherosclerosis, was assessed ultrasonographically. Additional subgroup analyses, including plasma levels of acute phase reactants and HDL protein profiling, were performed in 11 consecutive patients with CD in remission, 10 patients with active CD, and 15 healthy controls. Carotid IMT in patients with CD was increased compared with healthy volunteers: 0.71 (0.17) versus 0.59 (0.14) mm (P < 0.0001), respectively. In the subgroup analysis, HDL levels in controls and patients in remission were identical [(1.45 (0.48) and 1.40 (0.46) mmol/l; P = 0.797], whereas HDL during exacerbation was profoundly reduced: 1.02 (0.33) (P = 0.022). HDL from patients with active CD and CD patients in remission was characterized by a reduced ability to attenuate oxidation compared with controls (P = 0.008 and P = 0.024 respectively). Patients with CD have increased IMT compared with matched controls, indicative of accelerated atherogenesis. The changes during CD exacerbation in terms of HDL concentration and composition imply a role for impaired HDL protection in these patients.
      In the last decade, it has become increasingly clear that inflammation plays a pivotal role in the pathogenesis of atherosclerosis. Leukocytes can adhere to activated endothelium and transmigrate to the subendothelial space. Subsequently, immune cell activation leads to plaque progression and eventually plaque rupture, resulting in atherothrombotic disease (
      • Libby P.
      Inflammation in atherosclerosis.
      ,
      • Ross R.
      Atherosclerosis—an inflammatory disease.
      ). Conversely, systemic inflammation itself has been suggested to promote the atherosclerotic process (
      • Ross R.
      Atherosclerosis—an inflammatory disease.
      ). Indeed, in several chronic inflammatory disorders, such as systemic lupus erythematosus (SLE) (
      • van Leuven S.I.
      • Kastelein J.J.
      • Hayden M.R.
      • d'Cruz D.
      • Hughes G.R.
      • Stroes E.S.
      Cardiovascular disease in systemic lupus erythematosus: has the time for action come?.
      ), rheumatoid arthritis (RA) (
      • Sattar N.
      • McCarey D.W.
      • Capell H.
      • McInness I.B.
      Explaining how “high-grade” systemic inflammation accelerates vascular risk in rheumatoid arthritis.
      ), human immunodeficiency virus (
      • van Leuven S.I.
      • Sankatsing R.R.
      • Vermeulen J.N.
      • Kastelein J.J.
      • Reiss P.
      • Stroes E.S.
      Atherosclerotic vascular disease in HIV: it is not just antiretroviral therapy that hurts the heart!.
      ), and even periodontitis (
      • Pussinen P.J.
      • Nyyssonen K.
      • Alfthan G.
      Serum antibody levels to Actinobacillus actinomycetemcomitans predict the risk for coronary heart disease.
      ), systemic inflammation has been linked to enhanced atherogenesis, illustrated by an increased incidence of cardiovascular disease (CVD). Several mechanisms by which a systemic inflammatory state can accelerate the atherosclerotic process have been suggested. Cytokine-mediated damaging of the endothelium, immune cell activation, and activation of the coagulation cascade have all been implicated. In addition, inflammation can also induce changes in lipoprotein metabolism. Particularly, inflammation can decrease HDL concentrations as well as qualitatively affect HDL (
      • Khovidhunkit W.
      • Kim M.S.
      • Memon R.A.
      • Shigenaga J.K.
      • Moser A.H.
      • Feingold K.R.
      • Grunfeld C.
      Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanisms and consequences to the host.
      ). During systemic inflammation, specific enzyme and protein components of HDL, contributing to HDL's antiatherogenic potential, are modified, thereby impeding its antiatherogenic functions, and may even render it proatherogenic (
      • Navab M.
      • Anantharamaiah G.M.
      • Fogelman A.M.
      The role of high-density lipoprotein in inflammation.
      ). In several chronic inflammatory disorders, dyslipidemic changes have been linked to enhanced atherogenesis. The current exploratory study was designed to evaluate whether Crohn's disease (CD) is associated with an increased progression of the atherosclerotic process and whether inflammatory exacerbations are associated with alterations in HDL metabolism.

      MATERIALS AND METHODS

      Patients

      CD patients were recruited at the outpatient inflammatory bowel disease clinic at the Academic Medical Center in Amsterdam. During study visits, disease activity was assessed using the Harvey Bradshaw index (HBI). The HBI is a research tool composed of clinical parameters (general well-being, abdominal pain, number of liquid stools per day, abdominal mass, and complications) used to quantify the symptoms of patients. Patients with HBI ⩾ 4 were considered to have active CD, and patients with HBI < 4, were considered to be in remission. Blood samples were collected for C-reactive protein (CRP) and lipid profiling. CRP measurements were used for the second criteria defining active CD and CD in remission: CRP ⩾ 10 mg/l and CRP < 10 mg/l, respectively. All study patients were asked to participate in ultrasound carotid intima media thickness (IMT) measurements. Healthy controls matched for age and gender were recruited at the Department of Vascular Medicine and participated in the analysis of lipid profiles and IMT measurements. Patients gave written informed consent, and the study was approved by the local Medical Ethical Committee.

      Ultrasound measurements of the carotid IMT

      As described elsewhere, IMT measurements allow for the investigation of arterial wall morphology and can describe the status as well as present and future CVD risk noninvasively (
      • de Groot E.
      • van Leuven S.I.
      • de Graaf A.
      • Bots M.L.
      • Kastelein J.J.P.
      Ultrasound measurements of carotid arterial walls and the progression and regression of atherosclerosis.
      ). In summary, B-mode ultrasound carotid IMT measurements depict the intima media complex of carotid arterial walls. As shown in prospective epidemiological studies, a modest increase of IMT substantially increases the relative risk for myocardial infarction and stroke. IMT measurements have also shown the benefit of cholesterol-lowering and antihypertensive agents. IMT is an accepted validated surrogate marker for the status of atherosclerosis and present and future atherosclerotic disease risk (
      • O'Leary D.H.
      • Polak J.F.
      • Kronmal R.A.
      • Manolio T.A.
      • Burke G.L.
      • Wolfson Jr., S.K.
      Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults.
      ).

      Laboratory measurements

      All blood samples were immediately stored at −80°C and thawed once before measurement. CRP was measured using a commercial high-sensitivity CRP kit for human CRP (hsCRP; HemoIL, Lexington, MA) according to the manufacturer's protocol. The minimum detectable CRP concentration of the assay was 2.0 mg/l. Samples with CRP concentrations > 8 mg/l were diluted using CRP diluent. Serum amyloid A (SAA) analysis was carried out using a commercially available Human SAA ELISA kit from Anogen (Mississauga, Ontario, Canada) according to the manufacturer's protocol, and apolipoprotein A-I (apoA-I) and apoB were determined by nephelometric immunochemistry (Beckman).

      Lipoprotein composition

      Total cholesterol distribution among the lipoproteins was measured by fast-phase liquid chromatography. In brief, the system contained a PU-980 ternary pump with an LG-980-02 linear degasser and a UV-975 ultraviolet/visible light detector (Jasco, Tokyo, Japan). An extra P-50 pump (Pharmacia Biotech, Uppsala, Sweden) was used for in-line enzymatic reagent (Biomerieux, Marcy l'Etoile, France) addition at 0.1 ml/min. Plasma lipoprotein separations were performed using a Superose 6 HR 10/30 column (Pharmacia Biotech) with TBS, pH 7.4, as eluent at a flow rate of 0.31 ml/min. Total cholesterol was determined quantitatively using the PAP 250 cholesterol enzymatic method (Biomerieux, Le Fontanille, France). Computer analyses of the chromatograms for quantitative peak integration of the lipoproteins were carried out using Borwin Chromatographic software, version 1.23 (JMBS Developments, Le Fontanil, France).

      Sample preparation

      HDL protein profiling was carried out as described previously (
      • Levels J.H.
      • Bleijlevens B.
      • Rezaee F.
      • Aerts J.M.
      • Meijers J.C.
      SELDI-TOF mass spectrometry of high-density lipoprotein.
      ). For coating of antibodies, a 5 μl mixture containing 2.8 nM anti-apoA-I monoclonal antibodies, 3 μM ethylenediamine, and 0.1 M Na2SO4 was added per spot of a PS-20 protein chip, and covalent binding of antibodies through primary amine-epoxide chemistry was achieved by incubating the chip in a humid chamber overnight at 4°C. Excess antibody was removed by one wash with distilled water, and subsequently, free amine binding places were blocked by incubating the chip for 30 min at room temperature with 1 M Tris buffer (pH 8.0).
      For HDL capture, after mounting the PS-20 protein chip(s) in a 96-well bioprocessor, 100 μl of diluted plasma aliquots (diluted 1:2 with TBS buffer: 50 mM Tris, pH 7.4, and 150 mM NaCl) was applied onto spot and allowed to bind for 2 h at room temperature on a horizontal shaker. The protein chips were washed four times with TBS for 10 min, followed by a 5 min TBS-Tween (0.005%) rinse unless indicated otherwise. A final wash step with HEPES solution (5 mM) was carried out to remove the excess salt. All spots were allowed to dry, and subsequently, 1.2 μl of sinapinic acid (10 mg/ml) in a 50:49.9:0.1% acetonitrile-water-trifluoric acid mix was applied on each spot. All chips were air-dried and stored at room temperature in the dark.

      Surface-enhanced laser desorption/ionization time-of-flight analysis (SELDI-TOF)

      Analysis was carried out using a PBS IIc protein chip reader (Ciphergen Biosystems, Fremont, CA) using an automated data collection protocol within the Protein-Chip Software (version 3.1). Data were collected up to 200 kDa. Laser intensity was set in a range from 190 to 220 arbitrary units, and the focus mass was set to 28 kDa specific for the anti-apoA-I capture. Measurement of the spectra was performed with an average of ∼100 shots at 13 positions per SELDI spot. Calibration was done using a protein calibration chip (Ciphergen). Spectra were normalized on total ion current. Detected peaks having a signal-to-noise ratio > 5 were recognized as significant peaks.

      HDL antioxidant score

      The assay was performed as described previously with slight modifications (
      • Navab M.
      • Hama S.Y.
      • Hough G.P.
      • Subbanagounder G.
      • Reddy S.T.
      • Fogelman A.M.
      A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids.
      ) using historical controls. Briefly, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) is a proinflammatory phospholipid that triggers vascular inflammation processes. The HDL OxPAPC assay measures the potential of plasma-derived HDL to reduce/inactivate previously (air) oxidized phospholipids. HDL for this assay is isolated from plasma using dextran sulfate-coated magnetic beads. These beads precipitate apoB-containing lipoproteins. The cholesterol content of the HDL-containing supernatant is determined. HDL cholesterol (25 μM final concentration) is added to a reaction mixture that contains OxPAPC. Adding 2‘,7‘-dichlorofluorescein, a fluorochrome, to the reaction mixture produces a fluorescent signal dependent on the concentration of OxPAPC. Reduction of OxPAPC as a result of preincubation with HDL results in a loss of fluorescence, the readout parameter of this assay. As such, HDL with an antioxidant score > 1 is considered proinflammatory, whereas a score of <1 is considered anti-inflammatory.

      Statistical analysis

      SPSS statistical program 11.0.1(SPSS, Inc., Chicago, IL) was used. Standard descriptive and comparative analyses were undertaken. Results are expressed as means ± SD. Student's t-test for unpaired data was used to compare CD patients with healthy volunteers. Linear regression analyses were performed to asses the relationships between CRP, SAA, HDL, apoA-I, and apoB. One-way ANOVA tests were used to investigate differences between lipid, lipoprotein, and acute-phase protein values. P < 0.05 was considered statistically significant.

      RESULTS

      Characteristics of the study groups

      A total of 60 CD patients participated in this study, of which 12 suffered from active disease and 48 were in clinical remission (Table 1). In total, 122 healthy controls were included in the reference group. No differences were observed in smoking behavior or the presence of hypertension or diabetes mellitus between the three groups.
      TABLE 1Baseline characteristics of the study subjects
      CharacteristicsPatients with Active CD (n = 12)Patients with CD in Remission (n = 48)Patients with CD (n = 60)Healthy Controls (n = 122)
      Age, years34.4 ± 9.044.4 ± 12.942.4 ± 12.840.5 ± 16.4
      Male gender, n (%)7 (58.3)20 (41.7)27 (45.0)55 (45.1)
      Body mass index (kg/m2)25.624.1 ± 3.924.4 ± 3.925.0 ± 8.1
      Hypertension, n (%)1 (8.3)4 (8.3)5 (8.3)7 (5.7)
      Diabetes mellitus, n (%)0 (0)0 (0)0 (0)0 (0)
      Smoking, n (%)4 (33.3)9 (18.8)13 (21.7)22 (18.0)
      Harvey Bradshaw index8.1 ± 3.21.3 ± 0.72.7 ± 4.8NA
      Total cholesterol, mmol/l3.79 ± 0.894.73 ± 1.114.54 ± 1.125.04 ± 0.99
      LDL cholesterol, mmol/l2.46 ± 0.912.62 ± 0.972.59 ± 0.962.99 ± 0.81
      HDL cholesterol, mmol/l1.01 ± 0.301.66 ± 0.431.53 ± 0.481.47 ± 0.53
      Triglycerides, mmol/l0.70 ± 0.880.98 ± 0.540.92 ± 0.621.34 ± 1.25
      High-sensitivity CRP, mg/l94.5 ± 108.32.7 ± 2.521.0 ± 59.71.9 ± 1.7
      IMT, mm0.62 ± 0.130.73 ± 0.170.71 ± 0.170.59 ± 0.14
      Values are given as means ± SD. CD, Crohn's disease; IMT, intima media thickness.

      IMT measurements

      IMT measurements were performed in 60 CD patients and 122 healthy controls. Both groups exhibited a wide age range (Table 1); nevertheless, the IMT of CD patients (0.71 ± 0.17 mm) was increased compared with that of healthy controls (0.59 ± 0.14 mm) (P < 0.001). The estimated IMT increase in CD patients and control subjects with age is graphically displayed in Fig. 1 . The regression line for CD differed significantly from that of controls (P < 0.05).
      Figure thumbnail gr1
      Fig. 1Carotid intima media thickness (cIMT) and age in Crohn's disease (CD) patients and healthy controls.

      Lipid profiles

      Additional analyses were performed in the first consecutive 11 patients with CD in remission, 10 patients with active CD, and 15 healthy controls (Table 2). Mean HDL concentrations were higher in controls and patients in remission compared with patients with active CD (P = 0.022 and P = 0.043, respectively). HDL concentrations did not differ between controls and patients in remission. Reduced VLDL concentrations were found in active patients compared with controls and patients in remission (P = 0.019 and P = 0.028, respectively). Concentrations of LDL did not differ among the groups. With respect to apolipoproteins, controls and patients in remission had significantly higher serum apoA-I compared with CD patients with active disease (Table 2), whereas apoB levels were similar in all groups.
      TABLE 2Lipid values, acute-phase proteins, and apolipoproteins of consecutive subjects in a subgroup analysis
      ParameterControls (n = 15)Active CD Patients (n = 10)CD Patients in Remission (n = 11)
      HDL, mmol/l1.45 ± 0.481.02 ± 0.33
      P < 0.05 compared with controls.
      ,
      P < 0.05 compared with patients in remission.
      1.40 ± 0.46
      VLDL, mmol/l0.37 ± 0.200.18 ± 0.13a,b0.37 ± 0.22
      LDL, mmol/l2.64 ± 0.662.57 ± 0.962.54 ± 0.70
      CRP, mg/l0.8 ± 0.4108.8 ± 113.8
      P < 0.005 compared with controls.
      ,
      P < 0.005 compared with patients in remission.
      2.6 ± 2.7
      Serum amyloid A, mg/l2.6 ± 1.4530.8 ± 655.3c,d25.1 ± 61.3
      Apolipoprotein A-I, g/l1.48 ± 0.271.11 ± 0.27c,d1.52 ± 0.22
      Apolipoprotein B, g/l0.87 ± 0.210.88 ± 0.260.90 ± 0.27
      Values are given as means ± SD.
      a P < 0.05 compared with controls.
      b P < 0.05 compared with patients in remission.
      c P < 0.005 compared with controls.
      d P < 0.005 compared with patients in remission.

      Acute-phase proteins

      Mean CRP concentrations were increased significantly in patients with active CD compared with patients in remission and controls (Table 2). A linear regression analysis (log-transformed) showed that CRP and HDL were correlated (r2 = 0.24, P = 0.002). Another acute-phase protein, SAA, was not significantly different between controls and patients in remission (P = 0.870), whereas SAA concentrations in active CD were increased significantly compared with those in controls and patients in remission (Table 2).

      Surface-enhanced laser desorption/ionization time-of-flight analyses

      After SELDI-time-of-flight (TOF) analyses, protein spectra from HDL were obtained. Within each of the groups, all spectra showed virtually similar profiles. Comparing the fingerprints of the healthy controls and CD in remission group with those of active CD patients, statistically significant deviations were observed in relative intensity on five different markers: 3,602, 4,634, 11,695, 13,843, and 14,106 m/z (Fig. 2). No statistically significant differences between the HDL fingerprints from healthy controls and patients in remission were seen.
      Figure thumbnail gr2
      Fig. 2A representative example of HDL spectra in Gelview of patients with CD and controls. Depicted are the spectra in the m/z mass range between 3,000 and 5,000 Da (panel 1), 5,000 and 10,000 Da (panel 2), and 10,000 and 15,000 Da (panel 3). Within the dotted boxes, the statistically significant deviations after Bonferroni correction among active CD patients (A), CD patients in remission (R), and controls (C) are seen. All spectra were normalized on total ion current.

      Antioxidant potential of HDL

      The antioxidative indices of HDL of controls and patients with CD, active and in remission, are displayed in Fig. 3 . The antioxidative capacity of HDL did not differ between CD patients in remission and those with active disease (P = 0.53). HDL isolated from patients with active CD but also from CD patients in remission was characterized by a reduced ability to attenuate oxidation compared with that of controls (P = 0.008 and P = 0.024, respectively).
      Figure thumbnail gr3
      Fig. 3The antioxidative index of HDL isolated from controls, patients with CD in remission, and patients with active CD.

      DISCUSSION

      In the current exploratory analysis, we show that CD is associated with an acceleration of the atherosclerotic process, as illustrated by an increased carotid IMT in CD patients compared with healthy controls. In addition, CD patients were characterized during an inflammatory exacerbation by profoundly decreased levels of HDL combined with biochemical changes of the HDL particle. These data suggest that early detection of atherosclerosis and subsequent cardiovascular prevention in patients with CD might be warranted.

      IMT in CD

      Arterial wall thickness can be measured as a continuous variable from childhood into old age and can be used in patients as well as controls. Consequently, carotid IMT measurements are used on a broad scale to assess risk in individuals with an increased cardiovascular risk, such as patients with chronic inflammatory disease such as SLE and RA and those with familial hypercholesterolemia. In the latter group, IMT measurements have also been used to evaluate the effectiveness of various therapeutic interventions. As such, IMT is now a validated surrogate marker for the assessment of atherosclerotic vascular disease (
      • de Groot E.
      • van Leuven S.I.
      • de Graaf A.
      • Bots M.L.
      • Kastelein J.J.P.
      Ultrasound measurements of carotid arterial walls and the progression and regression of atherosclerosis.
      ,
      • O'Leary D.H.
      • Polak J.F.
      • Kronmal R.A.
      • Manolio T.A.
      • Burke G.L.
      • Wolfson Jr., S.K.
      Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults.
      ). In the current study, we also assessed the progression of the atherosclerotic process in CD by measuring IMT. Indeed, IMT was increased significantly in patients with CD compared with healthy controls. Because of the plethora of risk factors that are of influence on IMT in CD, a CVD risk assessment based on IMT data from other populations at risk in a parallel risk assessment is premature. It must be noted, however, that the average IMT increase of 0.12 mm in CD patients and 0.59 mm in unaffected individuals is similar to the estimated IMT increase in other patient groups characterized by a proatherogenic state. This strongly implies that CD patients are at increased CVD risk. Long-term follow-up studies investigating the causality of inflammatory, dyslipidemic, and other causes of accelerated atherosclerosis in these patients, as well as clinical trials to evaluate preventive drug therapy, are warranted.

      CVD in CD

      For up to 70 years, it has been known that inflammatory bowel disease is associated with venous but also arterial thrombosis (
      • Bargen J.A.
      • Barker N.W.
      Extensive arterial and venous thrombosis complicating chronic ulcerative colitis.
      ,
      • Silverstein A.
      • Present D.H.
      Cerebrovascular occlusions in relatively young patients with regional enteritis.
      ,
      • Schneiderman J.H.
      • Sharpe J.A.
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      Cerebral and retinal vascular complications of inflammatory bowel disease.
      ,
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      ,
      • Prior A.
      • Strang F.A.
      • Whorwell P.J.
      Internal carotid artery occlusion in association with Crohn's disease.
      ,
      • Halliday C.E.
      • Farthing M.J.
      Arterial thrombosis in Crohn's disease.
      ,
      • Novotny D.A.
      • Rubin R.J.
      • Slezak F.A.
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      Arterial thromboembolic complications of inflammatory bowel disease.
      ,
      • Hahn T.L.
      • Dalsing M.C.
      • Sawchuk A.P.
      • Cikrit D.F.
      • Lalka S.G.
      Primary aortic mural thrombus: presentation and treatment.
      ,
      • Novacek G.
      • Haumer M.
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      • Miehsler W.
      • Polterauer P.
      • Vogelsang H.
      Aortic mural thrombi in patients with inflammatory bowel disease: report of two cases and review of the literature.
      ). The incidence ranges from 1.2% to 6.1% according to different studies and up to 39% in autopsy studies (
      • Danese S.
      • Semeraro S.
      • Papa A.
      • Roberto I.
      • Scaldaferri F.
      • Fedeli G.
      • Gasbarrini G.
      • Gasbarrini A.
      Extraintestinal manifestations in inflammatory bowel disease.
      ). The same risk factors that have been suggested to underlie this atherothrombotic state in CD are also risk factors for atherosclerosis, such as hyperhomocysteinemia (
      • Cattaneo M.
      • Vecchi M.
      • Zighetti M.L.
      • Saibeni S.
      • Martinelli I.
      • Omodei P.
      • Mannucci P.M.
      • de Franchis R.
      High prevalence of hyperhomocysteinemia in patients with inflammatory bowel disease: a pathogenic link with thromboembolic complications?.
      ), antiphospholipid antibodies (
      • Vianna J.L.
      • D'Cruz D.P.
      • Khamashta M.A.
      • Asherson R.A.
      • Hughes G.R.
      Anticardiolipin antibodies in a patient with Crohn's disease and thrombosis.
      ), and a procoagulant state (
      • Vrij A.A.
      • Rijken J.
      • van Wersch J.W.
      • Stockbrugger R.W.
      Coagulation and fibrinolysis in inflammatory bowel disease and in giant cell arteritis.
      ). Interestingly, atherosclerosis and CD also share a common pathway in the CD40/CD40L system (
      • Danese S.
      • Fiocchi C.
      Atherosclerosis and inflammatory bowel disease: sharing a common pathogenic pathway?.
      ), and although circulating activated platelets underlie a procoagulant milieu in CD (
      • Danese S.
      • Motte Cd Cde L.
      • Fiocchi C.
      Platelets in inflammatory bowel disease: clinical, pathogenic, and therapeutic implications.
      ), they have also been shown to exacerbate atherosclerosis (
      • Huo Y.
      • Schober A.
      • Forlow S.B.
      • Smith D.F.
      • Hyman M.C.
      • Jung S.
      • Littman D.R.
      • Weber C.
      • Ley K.
      Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E.
      ). Furthermore, systemic inflammation has emerged as a causal factor for accelerated atherogenesis in inflammatory disease states such as SLE and RA (
      • van Leuven S.I.
      • Kastelein J.J.
      • D'Cruz D.P.
      • Hughes G.R.
      • Stroes E.S.
      Atherogenesis in rheumatology.
      ,
      • van Leuven S.I.
      • Franssen R.
      • Kastelein J.J.
      • Levi M.
      • Stroes E.S.
      • Tak P.P.
      Systemic inflammation as a risk factor for atherothrombosis.
      ). Recently, evidence for such an association has emerged in other chronic inflammatory disorders as well. Indeed, several reports have also suggested that CD is associated with premature atherosclerosis (
      • Levy P.J.
      • Tabares A.H.
      • Olin J.W.
      Lower extremity arterial occlusions in young patients with Crohn's colitis and premature atherosclerosis: report of six cases.
      ,
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      • Philippeau F.
      • Salzmann M.
      • Trouillas P.
      Large-artery stroke in a young patient with Crohn's disease. Role of vitamin B6 deficiency-induced hyperhomocysteinemia.
      ,
      • Nuutinen H.
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      • Farkkila M.
      • Seppala K.
      Association of ulcerative colitis and ischemic heart disease (Abstract).
      ,
      • Nuutinen H.
      • Reunanen M.
      • Farkkila M.
      • Seppala K.
      Association of Crohn's disease and ischemic heart disease (Abstract).
      ,
      • Mutlu B.
      • Ermeydan C.M.
      • Enc F.
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      • Demirkol O.
      • Bayrak F.
      • Basaran Y.
      Acute myocardial infarction in a young woman with severe ulcerative colitis.
      ) In addition, our findings of accelerated atherosclerosis correspond well with several other studies that showed evidence of subclinical atherosclerosis in inflammatory bowel disease by demonstrating IMT thickening (
      • Papa A.
      • Santoliquido A.
      • Danese S.
      • Covino M.
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      • Urgesi R.
      • Grillo A.
      • Guglielmo S.
      • Tondi P.
      • Guidi L.
      • et al.
      Increased carotid intima-media thickness in patients with inflammatory bowel disease.
      ,
      • Papa A.
      • Danese S.
      • Urgesi R.
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      • Guglielmo S.
      • Roberto I.
      • Bonizzi M.
      • Guidi L.
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      • Santoliquido A.
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      Early atherosclerosis in patients with inflammatory bowel disease.
      ) or endothelial dysfunction (
      • Schinzari F.
      • Armuzzi A.
      • De Pascalis B.
      • Mores N.
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      • Melina D.
      • Cardillo C.
      Tumor necrosis factor-alpha antagonism improves endothelial dysfunction in patients with Crohn's disease.
      ). Although numbers on cardiovascular morbidity are lacking, cardiovascular mortality does not appear to be increased in patients with CD. However, there is significant disparity in the reported mortality rates in CD, ranging from 30% lower than expected to 70% higher than expected (
      • Loftus Jr, E.V.
      Crohn's disease: why the disparity in mortality?.
      ). Most of the patients in these cohorts were identified retrospectively and were diagnosed before improved medical treatment became available. In a more recent study, the mortality risk of CD patients was increased significantly, and a standardized mortality ratio for cardiovascular mortality was 1.49 (
      • Wolters F.L.
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      Crohn's disease: increased mortality 10 years after diagnosis in a Europe-wide population based cohort.
      ). Long-term follow-up studies are required to resolve these issues.

      HDL and CD

      The outcome of the atherosclerotic process is determined by the balance between proatherogenic and antiatherogenic stimuli. HDL is among the most powerful endogenous mediators in atheroprotection, which is illustrated by the strong inverse relationship between HDL levels and the incidence of CVD (
      • Kannel W.B.
      High-density lipoproteins: epidemiologic profile and risks of coronary artery disease.
      ,
      • Gordon T.
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      • Dawber T.R.
      High density lipoprotein as a protective factor against coronary heart disease. The Framingham Study.
      ). Via the reverse cholesterol transport pathway, HDL can transport cholesterol from peripheral tissues, such as the arterial wall, back to the liver. Several additional properties of HDL contribute to its antiatherogenic potential. First, HDL exerts various anti-inflammatory effects and can reduce vascular inflammation in atherogenesis. For instance, HDL reduces the endothelial expression of adhesion molecules and chemokines, thereby reducing the recruitment of leukocytes to the subendothelial space (
      • Barter P.J.
      • Nicholls S.
      • Rye K.A.
      • Anantharamaiah G.M.
      • Navab M.
      • Fogelman A.M.
      Antiinflammatory properties of HDL.
      ). Oxidative modification of lipoproteins plays a pivotal role in atherogenesis. HDL has potent antioxidant properties and can reduce oxidative stress by a transport mechanism that binds oxidant molecules and carries anti-oxidative enzymes as well (
      • Barter P.J.
      • Nicholls S.
      • Rye K.A.
      • Anantharamaiah G.M.
      • Navab M.
      • Fogelman A.M.
      Antiinflammatory properties of HDL.
      ). Consistent with inflammation, there is increased oxidative stress in CD patients, resulting in increased lipid peroxidation (
      • Aghdassi E.
      • Wendland B.E.
      • Steinhart A.H.
      • Wolman S.L.
      • Jeejeebhoy K.
      • Allard J.P.
      Antioxidant vitamin supplementation in Crohn's disease decreases oxidative stress. A randomized controlled trial.
      ). This will stimulate atherogenesis in patients with CD but can be neutralized by HDL. Third, HDL has several antithrombotic properties that are of interest because CD is associated with a procoagulant state, illustrated by an increased risk of venous thromboembolism. Thus, distinct changes in HDL are likely to contribute to the proatherogenic state in CD, particularly during exacerbation of the disease.
      With regard to the effects of HDL beyond its role in reverse cholesterol transport, it has been advocated that changes in HDL's functional characteristics may provide information on its vasculoprotective effects over and beyond merely focusing on HDL levels. In agreement with this, “dysfunctional” HDL particles have been demonstrated in patients with overt CVD (
      • Navab M.
      • Anantharamaiah G.M.
      • Reddy S.T.
      • Van Lenten B.J.
      • Fogelman A.M.
      Mechanisms of disease: proatherogenic HDL—an evolving field.
      ). Interestingly, HDL from patients with active CD, without CVD, was characterized by a higher anti-oxidative score compared with healthy volunteers, indicating an attenuated antiatherogenic potential (
      • Navab M.
      • Anantharamaiah G.M.
      • Reddy S.T.
      • Van Lenten B.J.
      • Fogelman A.M.
      Mechanisms of disease: proatherogenic HDL—an evolving field.
      ). The fact that there was still a higher antioxidative score of HDL in CD patients in remission compared with controls may imply that even in remission the low-grade inflammatory state has an impact on HDL quality, in spite of the normalization of HDL levels. These findings bear close resemblance to those reported in patients with SLE and RA, in whom proinflammatory changes in HDL have been observed (
      • McMahon M.
      • Grossman J.
      • FitzGerald J.
      • Dahlin-Lee E.
      • Wallace D.J.
      • Thong B.Y.
      • Badsha H.
      • Kalunian K.
      • Charles C.
      • Navab M.
      • et al.
      Proinflammatory high-density lipoprotein as a biomarker for atherosclerosis in patients with systemic lupus erythematosus and rheumatoid arthritis.
      ). In agreement, Navab et al. (
      • Navab M.
      • Berliner J.A.
      • Subbanagounder G.
      • Hama S.
      • Lusis A.J.
      • Castellani L.W.
      • Reddy S.T.
      • Shih D.
      • Shi W.
      • Watson A.D.
      • et al.
      HDL and the inflammatory response induced by LDL-derived oxidized phospholipids.
      ) have elegantly demonstrated that during acute-phase reactions the concentration of “protective” proteins on the HDL particle actually decreases significantly. Overall, our findings imply that during CD exacerbation, the loss of HDL's atheroprotective effects, combined with the decrease in HDL levels, may contribute to accelerated atherogenesis.
      Simultaneously, HDL protein profiling has emerged as a promising tool to unravel the biochemical composition of HDL (
      • Levels J.H.
      • Bleijlevens B.
      • Rezaee F.
      • Aerts J.M.
      • Meijers J.C.
      SELDI-TOF mass spectrometry of high-density lipoprotein.
      ,
      • Rezaee F.
      • Casetta B.
      • Levels J.H.
      • Speijer D.
      • Meijers J.C.
      Proteomic analysis of high-density lipoprotein.
      ,
      • Vaisar T.
      • Pennathur S.
      • Green P.S.
      • Gharib S.A.
      • Hoofnagle A.N.
      • Cheung M.C.
      • Byun J.
      • Vuletic S.
      • Kassim S.
      • Singh P.
      • et al.
      Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.
      ), with which functional characteristics are intertwined. In addition to showing diminished levels of HDL during an inflammatory exacerbation of CD, using SELDI-TOF analysis we were able to show alterations in HDL composition. Interestingly, it has been suggested that HDL during an inflammatory episode can lose its protective properties and can even enhance atherogenesis (
      • Navab M.
      • Anantharamaiah G.M.
      • Fogelman A.M.
      The role of high-density lipoprotein in inflammation.
      ). In the present study, we observed changes in the biochemical composition of the HDL particle. Even though SELDI-TOF mass spectrometry is not a direct approach for the identification of proteins, the 11,695 marker can be identified as SAA. This has already been confirmed in previous reports using SELDI-TOF combined with MALDI-TOF mass spectrometry for the identification of this specific marker (
      • Le L.
      • Chi K.
      • Tyldesley S.
      • Flibotte S.
      • Diamond D.L.
      • Kuzyk M.A.
      • Sadar M.D.
      Identification of serum amyloid A as a biomarker to distinguish prostate cancer patients with bone lesions.
      ,
      • Tolson J.
      • Bogumil R.
      • Brunst E.
      • Beck H.
      • Elsner R.
      • Humeny A.
      • Kratzin H.
      • Deeg M.
      • Kuczyk M.
      • Mueller G.A.
      • et al.
      Serum protein profiling by SELDI mass spectrometry: detection of multiple variants of serum amyloid alpha in renal cancer patients.
      ,
      • Pruzanski W.
      • Stefanski E.
      • de Beer F.C.
      • de Beer M.C.
      • Ravandi A.
      • Kuksis A.
      Comparative analysis of lipid composition of normal and acute-phase high density lipoproteins.
      ). The acute-phase response in patients with active CD, characterized by increased SAA levels, thus seems to underlie an increased presence of SAA within the HDL particle. In fact, it was recently shown that proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6, which are increased in patients with CD, induce SAA expression in hepatocytes (
      • Han C.Y.
      • Chiba T.
      • Campbell J.S.
      • Fausto N.
      • Chaisson M.
      • Orasanu G.
      • Plutzky J.
      • Chait A.
      Reciprocal and coordinate regulation of serum amyloid A versus apolipoprotein A-I and paraoxonase-1 by inflammation in murine hepatocytes.
      ). It is already known that SAA has the capacity to replace apoA-I in the HDL particle, which renders them less protective (
      • Van Lenten B.J.
      • Hama S.Y.
      • de Beer F.C.
      • Stafforini D.M.
      • McIntyre T.M.
      • Prescott S.M.
      • La Du B.N.
      • Fogelman A.M.
      • Navab M.
      Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response: loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures.
      ,
      • Steinmetz A.
      • Hocke G.
      • Saile R.
      • Puchois P.
      • Fruchart J.C.
      Influence of serum amyloid-A on cholesterol esterification in human plasma.
      ).
      In conclusion, these data suggest that atherogenesis is enhanced in patients with CD. Changes in HDL concentration as well as in HDL's compositional and functional characteristics during exacerbations of CD imply that attenuation of this antiatherogenic mediator during these exacerbations might contribute to the progression of atherogenesis. The present findings call for consideration of the implementation of CVD detection and prevention in patients with CD.

      Acknowledgments

      The authors thank Boris Bleijlevens for his useful suggestions with regard to the SELDI-TOF ms analysis and Alinda Schimmel for her excellent technical assistance.

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