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Structural and functional changes in HDL with low grade and chronic inflammation
International Journal of Cardiology 188 (2015) 111–116
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Structural and functional changes in HDL with low grade and chronic inflammation Francis O'Neill a, Meliana Riwanto b, Marietta Charakida a, Sophie Colin d, Jasmin Manz b, Eve McLoughlin a, Tauseef Khan a, Nigel Klein g, Christopher W.M. Kay e, Kalpesh Patel c, Giulia Chinetti d, Bart Staels d, Francesco D'Aiuto c, Ulf Landmesser b, John Deanfield a,f,⁎ a
National Centre for Cardiovascular Prevention and Outcomes (NCCPO), Institute of Cardiovascular Science, University College London, London, UK Cardiology, Cardiovascular Center, University Hospital Zurich, Zurich, Switzerland c Periodontology Unit, Department of Clinical Research, University College London Eastman Dental Institute, London, UK d Université Lille 2, Institut Pasteur de Lille, Inserm UMR1011, EGID, Lille F-59000, France e Institute of Structural & Molecular Biology and London Centre for Nanotechnology, University College London, London, UK f National Institute for Cardiovascular Outcomes Research, University College London, London, UK g Infectious Diseases & Microbiology Unit, Institute of Child Health, University College London, London, UK b
a r t i c l e
i n f o
Article history: Received 13 November 2014 Received in revised form 7 January 2015 Accepted 3 March 2015 Available online 5 March 2015 Keywords: High-density lipoprotein Inflammation Atherosclerosis
a b s t r a c t Objective: HDL functionality has been shown to be impaired in inflammatory conditions, including coronary artery disease. The present study aims to determine the impact of low grade and acute inflammation on HDL function and structure. Approach and results: i) The endothelial protective effects of HDL were compared between 26 periodontal patients and 26 age and sex matched controls by measuring paraoxonase activity in serum and nitric oxide bioavailability and superoxide production in endothelial cells. Paraoxonase activity and nitric oxide bioavailability were reduced, while superoxide production was increased (p b 0.01) in periodontal patients compared to controls. ii) HDL function, including cholesterol efflux and vascular cell adhesion molecule-1 expression, was subsequently measured in the periodontal patients following an inflammatory stimulus. There was an acute deterioration in HDL's endothelial protective function, without change in cholesterol efflux, after 24 h (p b 0.01 for all). These functional changes tracked increases of inflammatory markers and altered HDL composition. Finally, HDL function returned to baseline levels after resolution of inflammation. Conclusion: This study demonstrates that even minor alterations in systemic inflammation can impair the endothelial protective effects of HDL. These functional changes were independent of cholesterol efflux and were associated with remodeling of the HDL proteome. All measures of HDL's endothelial protective functions recovered with resolution of inflammation. These findings suggest that HDL dysfunction may represent a novel mechanism linking inflammation with progression of atheroma. © 2015 Published by Elsevier Ireland Ltd.
1. Introduction Epidemiological studies have shown that high-density lipoprotein (HDL) cholesterol is a strong predictor of cardiovascular (CV) risk, with increments of 1 mg/dL in serum HDL levels being associated with a 3–4% reduction in mortality [1,2]. The traditional mechanism by which HDL exerts its ‘atheroprotective’ effect has been considered to Abbreviations: HDL, high density lipoprotein; CV, cardiovascular; CAD, coronary artery disease; NO, nitric oxide; PD, periodontitis; HAECs, human aortic endothelial cells; TNFα, tumor necrosis factor-alpha; ApoB, apolipoprotein B; SAA, serum amyloid alpha; CRP, Creactive protein; ESR, electron spin resonance; VCAM-1, vascular cell adhesion protein 1. ⁎ Corresponding author at: National Institute for Cardiovascular Outcomes Research, 170 Tottenham Court Rd, London W1T 7HA, UK. E-mail address: j.deanfi[email protected] (J. Deanfield).
http://dx.doi.org/10.1016/j.ijcard.2015.03.058 0167-5273/© 2015 Published by Elsevier Ireland Ltd.
be reverse cholesterol transport from the arterial wall. However, a number of alternative ‘anti-atherogenic’ properties have been ascribed to HDL, including stimulation of endothelial nitric oxide (NO), inhibition of reactive oxygen species as well as anti-thrombotic mechanisms. It may also promote endothelial progenitor cell mediated vascular repair and have a central role in the regulation of the immune system [3–8]. In both experimental and clinical studies, these beneficial properties can be lost and HDL may acquire a ‘pro-inflammatory’ and ‘pro-oxidant’ phenotype, which may be relevant to CV risk. We have reported that HDL, isolated from patients with inflammatory conditions, such as diabetes and anti-phospholipid syndrome, impairs nitric oxide bioavailability and increases superoxide production in cultured endothelial cells [6,9]. In addition, we and others have demonstrated a change in HDL protein cargo in the presence of chronic inflammatory disease
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and coronary artery disease (CAD) [10,11]. These functional changes in HDL may explain the lack of benefit from agents which elevate plasma HDL levels reported in recent large-scale clinical trials. Our group has extensively characterized a novel human inflammatory model in patients with periodontitis (PD) [12]. Using this model, we examined whether chronic and transient changes in inflammation associate with HDL functions including cholesterol efflux properties. 2. Methods and materials 2.1. Study population and protocol Consecutive patients, referred to the Periodontology Unit of the UCL Eastman Dental Institute and Hospital, were invited to participate in a longitudinal study. Only individuals presenting with severe PD, defined as the presence of probing pocket depths of N6 mm and marginal alveolar bone loss of N30% affecting N50% of teeth were recruited as previously described [12]. Patients who had other systemic illness, a history of acute or chronic infection (assessed by the examining clinician), or who were receiving antibiotics or other regular CV medication were excluded. 2.1.1. Study 1 Vascular properties of HDL from 26 patients with severe PD were compared to 26 healthy control subjects selected with comparable age and gender characteristics, and consecutively enrolled by the Blood Donation Service of the University Hospital Zurich, without any CV risk factors (by history, clinical examination, and laboratory tests) or accompanying disorders. 2.1.2. Study 2 26 PD patients underwent treatment with a well characterized inflammatory response [13]. HDL function was studied at baseline, after 24 h and at 6 months following treatment. These time-points were selected as they had previously been shown to correspond to the peak and resolution of the systemic inflammatory response. The study was approved by the joint Eastman and University College Hospitals ethics committee and all patients provided informed consent. 2.2. Anthropometric and biochemical measurements Anthropometric measurements were recorded and body mass index (BMI [kg/m2]) was calculated from weight and height. Blood pressure was measured in triplicate (HEM-705CP, Omron) and the average of the readings was recorded. Blood samples were drawn and processed after an overnight fast and serum and plasma samples were stored at − 70 °C for subsequent analysis. Full blood count, lipid and glucose level measurements were made with standard biochemistry assays and C-reactive protein (CRP) was measured with an immunoturbidimetric, high-sensitivity assay (Tina-quant CRP immunoturbidimetric assay performed on a Cobas Integra analyzer, Roche Diagnostics). Inter and intra assay coefficients of variations for all assays were b 5%. 2.3. HDL measurements 2.3.1. HDL isolation HDL was isolated by sequential ultracentrifugation (d = 1.063– 1.21 g/mL) using solid potassium bromide for density adjustment [14].
BioSpin) with the following instrumental settings: center field (B0) 3455 G; sweep width 80 G; microwave power 39.72 mW; modulation amplitude 10.34 G; sweep time 10.49 s; number of scans 10 [6,15]. Our group has reported has coefficients of variation for intraassay and interassay variability of these measurements as 1.92% and 1.74%, respectively. 2.3.3. ESR measurement of superoxide production The effect of HDL on endothelial cell superoxide production was compared to controls in un-stimulated and tumor necrosis factor (TNF)α-stimulated (5 ng/mL, R&D Systems) HAECs by ESR spectroscopy. Briefly, HAECs were incubated with HDL (50 μg/mL, 60 min, 37 °C), with or without TNFα and re-suspended in Krebs-Hepes buffer (pH 7,4; Noxygen) containing diethyldithiocarbamic acid sodium salt (5 μM, Noxygen) and deferoxamine methanesulfonate salt (25 μM, Noxygen). ESR spectra were recorded after addition of the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH); Noxygen; final concentration (200 μM) using a Bruker e-scan spectrometer (Bruker BioSpin). The ESR instrumental settings were as follows: center field (B0) 3495 G; field sweep width 10 G; microwave frequency 9.75 GHz; microwave power 19.91 mW; magnetic field modulation frequency 86.00 kHz; modulation amplitude 2.60 G; conversion time 10.24 msec; number of x-scans 1020 [6]. The coefficients of variation for intra-assay and interassay variability of these measurements were 5.1% and 12.9%, respectively. 2.3.4. Cholesterol efflux capacity of HDL HDL for measurements of efflux capacity was extracted from serum by ApoB depletion. Briefly, whole serum was incubated for 20 min with a 20% polyethylene glycol (PEG) solution (20% PEG 8000 (sigma P2139) in 200 mM glycine (sigma G8898, pH = 10)). Samples were centrifuged at 1900 G, and the supernatant was collected and stored at 4 °C. Cholesterol efflux capacity was quantified according to a method previously reported [16]. J774 cells were radiolabeled for 24 h in a medium containing 2 μCi of [3H]-cholesterol per mL. Addition of 0.3 mM 8(4-chlorophenylthio)-cyclic AMP for 6 h up-regulated expression of ABCA1. An efflux medium containing 2.8% apolipoprotein B-depleted serum from each individual was added for 4 h. To prevent cholesterol esterification during labeling, equilibration and flux, 2 μg per mL of CP113,818, a acyl-coenzyme A:cholesterol acyltransferase inhibitor was added to all mediums. Efflux capacity was quantified using liquid scintillation to measure radioactive cholesterol effluxed from the cells (medium + intracellular lipids). All assays were performed in duplicate and the final average value normalized against a baseline control for statistical analyses between time-points. 2.3.5. Serum assay of HDL function Serum paraoxonase activity was measured by UV spectrophotometry in a 96-well plate format using paraoxon (Sigma-Aldrich, St Louis, Missouri). Briefly, 50 μg/mL HDL was diluted in a reaction mixture containing 10 mM Tris hydrochloride (pH 8.0), 1 M sodium chloride and 2 Mm calcium chloride. At 24 °C 1.5 mM paraoxon was added to initiate the reaction, and the increase in absorbance at 405 nm was recorded over 30 min. An extinction coefficient (at 405 nm and 24 °C) of 17 000 M−1·cm−1 was used to calculate units of paraoxonase activity [17]. The coefficients of variation for intra-assay and inter-assay variability of these measurements were 7.2% and 13.1%, respectively. 3. Statistics
2.3.2. Electron spin resonance (ESR) measurement of NO bioavailability The effect of HDL from patients and controls (50 μg/mL; 60 min, 37 °C) on endothelial NO production ((human aortic endothelial cells) HAECs; passage 4–7; Lonza Bio Science) was examined by continuouswave electron spin resonance (ESR) spectroscopy using the spinprobe colloid Fe(DETC)2 (Noxygen). ESR spectra of samples frozen in liquid nitrogen were recorded on a Bruker e-scan spectrometer (Bruker
All measures are reported as mean (SD) or median [IQR] for those not normally distributed. Normal distribution was assessed using the Kolmogorov–Smirnov test. Comparisons between the HDL measurements and inflammatory markers were performed using the Pearson correlation coefficient for normally distributed data and the Spearman Rank Correlation method for non-normally distributed data. Comparisons
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between patients and controls were performed using an independent t-test or the Mann Whitney U test for normally and non-normally distributed data respectively. Comparison of HDL function, before and after PD treatment in patients, was performed using repeated-measures analysis of variance to determine differences, with Greenhouse–Geisser correction and using the Friedman test for non-parametric data. For proteomics analysis, a paired t-test was used to analyze differences between baseline and 24 h, and Wilcoxon signed-rank test used for non-normally distributed data. Analysis was performed using STATA v12 analysis software. A two sided p value of b 0.05 was considered to indicate statistical significance. 4. Results 4.1. Comparison of PD patients and controls and impact of chronic inflammation on HDL function PD patients had similar CV risk factors, including lipid levels, smoking status and BMI compared to controls, but CRP levels were statistically significantly higher in PD patients (p = −0.01) (Table 1). Endothelial cells exposed to HDL from the PD patients showed reduced NO bioavailability and increased superoxide production (p b 0.01 for both). Paraoxonase activity was lower in patients with PD (p b 0.01) (Fig. 1). 4.2. Impact of acute inflammation and its resolution on HDL functions There were no statistically significant changes in blood pressure, lipid levels and measures of coagulation following transient inflammatory stimulus. Inflammatory markers including CRP, IL-6 and total white blood cell count were significantly increased 24 h following PD treatment (p b 0.001 and p b 0.01 respectively) (Table 2). No relevant changes in monocyte counts were observed. Apart from HDL dependent cholesterol efflux, all other parameters of HDL function assessed were greatly impaired at 24 h. Endothelial cell NO bioavailability was reduced following stimulation with HDL from patients, compared to baseline (p b 0.01). Endothelial anti-inflammatory activity measured by VCAM-1 expression was also increased (p b 0.01) (Supp. Fig. 1). Furthermore, HDL's anti-oxidant capacity was also impaired with an increase in superoxide production and a reduction in paraoxonase activity (both p b 0.01) (Fig. 2). Quantitative proteomics revealed a statistically significant increase in signal intensities of Complement C3 (C3) (p = 0.03), serum amyloid alpha (SAA) (p = 0.01) and pro-thrombin (p = 0.01) was noted at 24 h (Fig. 3). Inflammatory markers returned to baseline levels 6 months after PD treatment. All HDL functions were normalized including NO Table 1 Baseline characteristics in periodontitis and healthy participants.
Age (years) Smoking status (non-smoker/smoker) BMI (kg/m2) Total Cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) Triglycerides (mmol/L) Glucose (mmol/L) CRP (mg/L)
Healthy (N = 26)
Periodontitis (N = 26)
p-Value
52.90 (4.23) 0/26
51.52 (6.32) 3/26
0.41 0.09
25.83 (2.14) 4.86 (0.71) 1.33 [1.13–1.51] 2.83 (0.88) 1.03 [0.8–1.38] 5.14 (0.62) 1.00 [1.00–1.00]
26.99 (4.03) 5.39 (0.95) 1.50 [1.30–2.00] 3.20 (0.94) 0.90 [0.80–1.55] 4.94 (0.55) 1.6 [1.00–3.55]
0.27 0.05 0.08 0.18 0.46 0.26 0.01
Abbreviations: BMI: body mass index, HDL: high density lipoprotein, LDL: low density lipoprotein, CRP: C-reactive protein. Values expressed as mean (SD) and median [IQR] for non-normally distributed data. Comparisons between periodontitis patients and healthy controls were performed using the independent t-test. Categorical variables were compared using Fisher's exact Chi-squared test. Comparisons for non-parametric measurements were performed using Mann Whitney test. † p b 0.01
Fig. 1. Cross-sectional comparison of HDL between healthy controls and PD. Following isolation, HDL (50 μg/mL) function was assayed using ESR spectroscopy for A) nitric oxide (NO) bioavailability; B) superoxide production (data expressed as percent change versus buffer-treated cells). Serum paraoxonase activity was measured by UV spectrophotometry. Individual data points are shown for each patient. Bars represent mean.
bioavailability, VCAM-1 expression, superoxide production and serum paraoxonase activity (p b 0.01 for all). There was no difference in cholesterol efflux capacity between any time points (Table 3).
5. Discussion This study demonstrates that low grade inflammation can disturb HDL vascular functions and that these can be further aggravated by transient acute inflammation. We show for the first time, that acute changes in HDL function can recover with resolution of inflammation and are paralleled by changes in the HDL proteome. Our findings
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Table 2 Patient clinical characteristics.
BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) CRP (mg/L) Interleukin-6 (pg/mL) LCC (×10−9/L) MC (cells/μL) PLT (109/L) Total cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) Triglycerides (mmol/L) Glucose (mmol/L)
Baseline
Day one
6 months
27.89 (5.20) 128.2 (19.75) 81.26 (12.47) 2.00 [1.00–3.00] 2.15 (5.35) 6.46 (1.53) 6.27 (1.96) 247.56 (52.6) 5.28 (1.10)
N/A 126.28 (17.82) 80.14 (10.85) 6.55 [1.68–12.90]† 5.81 (8.50)† 7.18 (1.84)⁎ 7.03 (2.33) 248.00 (57.41) 5.19 (1.02)
27.79 (5.32) 125.26 (16.49) 79.76 (10.12) 1.30 [0.56–1.30]‡ 2.54 (5.97)‡ 6.21 (1.71)‡ 6.59 (2.01) 251.38 (55.20) 5.15 (0.95)
1.61 (0.41) 3.16 (1.01) 1.14 (0.57) 4.99 (0.49)
1.64 (0.42) 3.06 (0.96) 1.06 (0.41) 5.03 (0.54)
1.63 (0.36) 3.03 (0.84) 1.06 (0.42) 4.79 (0.48)‡,§
Abbreviations: BMI: body mass index, HDL: high density lipoprotein, LDL: low density lipoprotein, CRP: C-reactive protein, SBP: systolic blood pressure, DBP: diastolic blood pressure, LCC: leukocyte count, MC: monocyte count, PLT: platelet count. Values expressed as mean (SD) and median [IQR] for non-normally distributed data. Comparisons between time points were performed using repeated measures ANOVA or Friedman's test for non-normally distributed data. ⁎ p b 0.05 between baseline and day 1. † p b 0.01 between baseline and day 1. ‡ p b 0.001 between day 1 and 6 months. § p b 0.01 between baseline and 6 months.
demonstrate the dynamic nature of the HDL molecule. This may be relevant to its protective role in vascular disease and should be considered when developing drug treatments which aim to increase HDL levels. Over the last 20 years, lowering of LDL cholesterol, particularly with statins has resulted in a major improvement in CV outcomes [18,19]. Nevertheless, there remains a considerable residual risk of CV events and this has prompted interest in strategies to raise HDL cholesterol, based on the consistent association between higher HDL levels and reduced CV risk in population studies of CV risk factors [20]. However, recent clinical trials have failed to demonstrate significant clinical benefit, despite increases in HDL levels [21,22]. A wealth of experimental data suggests HDL is a much more complex particle than previously considered and that the biological functions of HDL may be important. The traditional role of HDL has been to promote reverse cholesterol transport from peripheral tissues, including the arterial vessel wall [23]. Recently, HDL has been shown to have a range of effects on vascular function which may be relevant to atherogenesis and plaque stability [24]. We and others have shown that these can be disturbed in a range of clinical conditions associated with inflammation. These included anti-phospholipid syndrome, diabetes mellitus, chronic kidney disease (CKD) and recently acute coronary syndromes [9,25,26]. In these conditions, HDL from patients had the opposite effect on cultured endothelial cells to that seen with HDL isolated from healthy subjects. Nitric Oxide bioavailability was impaired, super oxide production increased, adhesion molecule expression enhanced and paraoxonase activity decreased. The relationship between these vascular effects and cholesterol efflux, as well as the impact of transient acute inflammation on HDL functions in vivo has not been previously explored. In our current study, we have examined HDL structure and function in patients with a chronic inflammatory condition affecting approximately 10% of the adult population [27]. Our findings support current research demonstrating that HDL function is altered by inflammation but suggests that this may not be confined to the more severe clinical inflammatory conditions previously reported. The fact that a relatively common low-grade systemic disease can derange HDL function suggests that the HDL molecule is very sensitive to inflammation. This is likely to have implications for HDL-targeted therapies. We subsequently tested the effect of transient acute inflammation on HDL function using a periodontal therapy model. The acute inflammatory stimulus further aggravated the already deranged HDL properties, which
Fig. 2. Functional changes in HDL 24 h and 6 months following PD treatment. HDL from PD patients was isolated by sequential ultracentrifugation, and the effects of HDL (50 μg/mL) on endothelial cells was analyzed by ESR spectroscopy analysis measuring A) nitric oxide (NO) bioavailability; B) superoxide production (data expressed as percent change versus buffertreated cells). C) Serum paraoxonase activity was measured by UV spectrophotometry. I bars represent standard error. Data points represent mean of 26 patients.
then returned to baseline levels with resolution of the acute inflammation. These functional changes tracked inflammatory markers and suggest that HDL function can be rapidly altered as a result of inflammation. The link between inflammation and HDL function is further emphasized in our study as there were no changes in other CV risk factors during this period. While this is consistent with previous observations we are first to demonstrate the dynamic nature of the HDL molecule on a range of HDL's endothelial protective functions and show recovery of HDL function [28].
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The disturbances in HDL function which were seen at the peak of inflammation were accompanied by changes in its protein composition. These included increased levels of the acute phase reactants serum amyloid A (SAA) and complement factor C3 (C3). This provides both mechanistic insights into the functional changes of HDL as well as further evidence of the rapid remodeling of HDL during acute inflammation. Interestingly, our proteomic analysis also revealed an increase in prothrombin levels within the HDL particle. This may underlie the reported pro-thrombotic effects of HDL following endotoxin challenge in humans [29]. We were able to follow, for the first time, changes in a range of HDL properties including cholesterol efflux and effects on endothelial cells in culture within the same individuals. We demonstrated a more pronounced impact of acute inflammation on the vascular effects of HDL than on its efflux capacity. The vascular effects of HDL may therefore be more sensitive to inflammation than cholesterol efflux. The relevance of these different properties of HDL may vary at different stages of atherosclerosis and require further study. Our study does present with several limitations. Firstly, we cannot comment on the time course for recovery of HDL function as serial measurements were not made between the peak of inflammation and 6 months. Furthermore, we did not measure the relationship and time course of recovery between structural and functional changes in HDL during this period. This is the subject of on-going research. There were however several advantages with our experimental design, which include a predictable clinical model of increased inflammation and its resolution, without the use of drugs, as well as the measurement of a range of HDL properties (including vascular and cholesterol efflux). In conclusion, the highly dynamic nature of HDL function and its upset by relatively minor inflammatory stimuli, both acute and chronic, may have clinical relevance. Our findings are of particular interest in light of the recent failures of HDL-C raising therapies in prevention clinical trials and underline the need to understand better the properties of HDL, their relationship to CV and the inflammatory phenotype and their response to treatment. While off-target effects and insufficient increase in circulating HDL level may account for the lack of benefit from Torcetrapib, Dalcetrapib and Niacin, the biological function of HDL and the inflammatory status of the population may be of great importance in determining the clinical benefits of HDL elevating therapies [21,30]. Indeed, patients recruited into the Dal-Outcome study were enrolled following an acute coronary syndrome, an event shown drastically to alter vascular properties of HDL. The reversibility of changes in HDL function, which we have demonstrated offers a therapeutic opportunity. This could include treatment of the underlying inflammatory state as well as targeting the HDL particle and its actions. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2015.03.058. Fig. 3. Protein changes in HDL, 24 h after PD treatment. HDL was isolated in the same patients before and after PD treatment, and HDL protein cargo compared using Mass Spectroscopy. A) Complement factor C3, B) serum amyloid alpha 4 (SAA), and C) pro-thrombin were quantified by calculating the area under the curve of extracted ion chromatograms. An average area under the curve for 2 representative spectra is shown for each protein. Bars represent mean and I bars represent standard error.
Funding/support This study was funded by Colgate-Palmolive (09CC25), F. HoffmannLa Roche Ltd (09CC22) and the Leducq Foundation (10CC22). The
Table 3 HDL function.
NO bioavailability SO production PON activity (μmol p-nitrophenol/L/serum/min) VCAM-1 expression (% of TNF-α stimulated cells) Cholesterol efflux (average efflux with normalization)
Baseline
Day one
6 months
5.15 (14.67) −5.8 [−21.30–1.23] 130.05 (21.10) 98.37 (18.93) 0.99 (0.30)
−4.05 (16.85)⁎ 7.56 [0.37–31.57]⁎ 111.63 (26.61)⁎ 115.92 (14.44)⁎
7.00 (19.23)† 4.56 [−36.46–5.21]† 132.75 (26.31)† 78.36 (27.79)† 1.05 (0.31)
0.92 (0.32)
Abbreviations: NO: nitric oxide, SO: superoxide, PON: paraoxonase. Values expressed as mean (SD). The NO bioavailability and SO production were expressed as % inhibition of buffer treated cells. ⁎ p b 0.01 between baseline and day one. † p b 0.01 between day one and 6 months.
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Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Structural and functional changes in HDL with low grade and chronic inflammation Francis O'Neill a, Meliana Riwanto b, Marietta Charakida a, Sophie Colin d, Jasmin Manz b, Eve McLoughlin a, Tauseef Khan a, Nigel Klein g, Christopher W.M. Kay e, Kalpesh Patel c, Giulia Chinetti d, Bart Staels d, Francesco D'Aiuto c, Ulf Landmesser b, John Deanfield a,f,⁎ a
National Centre for Cardiovascular Prevention and Outcomes (NCCPO), Institute of Cardiovascular Science, University College London, London, UK Cardiology, Cardiovascular Center, University Hospital Zurich, Zurich, Switzerland c Periodontology Unit, Department of Clinical Research, University College London Eastman Dental Institute, London, UK d Université Lille 2, Institut Pasteur de Lille, Inserm UMR1011, EGID, Lille F-59000, France e Institute of Structural & Molecular Biology and London Centre for Nanotechnology, University College London, London, UK f National Institute for Cardiovascular Outcomes Research, University College London, London, UK g Infectious Diseases & Microbiology Unit, Institute of Child Health, University College London, London, UK b
a r t i c l e
i n f o
Article history: Received 13 November 2014 Received in revised form 7 January 2015 Accepted 3 March 2015 Available online 5 March 2015 Keywords: High-density lipoprotein Inflammation Atherosclerosis
a b s t r a c t Objective: HDL functionality has been shown to be impaired in inflammatory conditions, including coronary artery disease. The present study aims to determine the impact of low grade and acute inflammation on HDL function and structure. Approach and results: i) The endothelial protective effects of HDL were compared between 26 periodontal patients and 26 age and sex matched controls by measuring paraoxonase activity in serum and nitric oxide bioavailability and superoxide production in endothelial cells. Paraoxonase activity and nitric oxide bioavailability were reduced, while superoxide production was increased (p b 0.01) in periodontal patients compared to controls. ii) HDL function, including cholesterol efflux and vascular cell adhesion molecule-1 expression, was subsequently measured in the periodontal patients following an inflammatory stimulus. There was an acute deterioration in HDL's endothelial protective function, without change in cholesterol efflux, after 24 h (p b 0.01 for all). These functional changes tracked increases of inflammatory markers and altered HDL composition. Finally, HDL function returned to baseline levels after resolution of inflammation. Conclusion: This study demonstrates that even minor alterations in systemic inflammation can impair the endothelial protective effects of HDL. These functional changes were independent of cholesterol efflux and were associated with remodeling of the HDL proteome. All measures of HDL's endothelial protective functions recovered with resolution of inflammation. These findings suggest that HDL dysfunction may represent a novel mechanism linking inflammation with progression of atheroma. © 2015 Published by Elsevier Ireland Ltd.
1. Introduction Epidemiological studies have shown that high-density lipoprotein (HDL) cholesterol is a strong predictor of cardiovascular (CV) risk, with increments of 1 mg/dL in serum HDL levels being associated with a 3–4% reduction in mortality [1,2]. The traditional mechanism by which HDL exerts its ‘atheroprotective’ effect has been considered to Abbreviations: HDL, high density lipoprotein; CV, cardiovascular; CAD, coronary artery disease; NO, nitric oxide; PD, periodontitis; HAECs, human aortic endothelial cells; TNFα, tumor necrosis factor-alpha; ApoB, apolipoprotein B; SAA, serum amyloid alpha; CRP, Creactive protein; ESR, electron spin resonance; VCAM-1, vascular cell adhesion protein 1. ⁎ Corresponding author at: National Institute for Cardiovascular Outcomes Research, 170 Tottenham Court Rd, London W1T 7HA, UK. E-mail address: j.deanfi[email protected] (J. Deanfield).
http://dx.doi.org/10.1016/j.ijcard.2015.03.058 0167-5273/© 2015 Published by Elsevier Ireland Ltd.
be reverse cholesterol transport from the arterial wall. However, a number of alternative ‘anti-atherogenic’ properties have been ascribed to HDL, including stimulation of endothelial nitric oxide (NO), inhibition of reactive oxygen species as well as anti-thrombotic mechanisms. It may also promote endothelial progenitor cell mediated vascular repair and have a central role in the regulation of the immune system [3–8]. In both experimental and clinical studies, these beneficial properties can be lost and HDL may acquire a ‘pro-inflammatory’ and ‘pro-oxidant’ phenotype, which may be relevant to CV risk. We have reported that HDL, isolated from patients with inflammatory conditions, such as diabetes and anti-phospholipid syndrome, impairs nitric oxide bioavailability and increases superoxide production in cultured endothelial cells [6,9]. In addition, we and others have demonstrated a change in HDL protein cargo in the presence of chronic inflammatory disease
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and coronary artery disease (CAD) [10,11]. These functional changes in HDL may explain the lack of benefit from agents which elevate plasma HDL levels reported in recent large-scale clinical trials. Our group has extensively characterized a novel human inflammatory model in patients with periodontitis (PD) [12]. Using this model, we examined whether chronic and transient changes in inflammation associate with HDL functions including cholesterol efflux properties. 2. Methods and materials 2.1. Study population and protocol Consecutive patients, referred to the Periodontology Unit of the UCL Eastman Dental Institute and Hospital, were invited to participate in a longitudinal study. Only individuals presenting with severe PD, defined as the presence of probing pocket depths of N6 mm and marginal alveolar bone loss of N30% affecting N50% of teeth were recruited as previously described [12]. Patients who had other systemic illness, a history of acute or chronic infection (assessed by the examining clinician), or who were receiving antibiotics or other regular CV medication were excluded. 2.1.1. Study 1 Vascular properties of HDL from 26 patients with severe PD were compared to 26 healthy control subjects selected with comparable age and gender characteristics, and consecutively enrolled by the Blood Donation Service of the University Hospital Zurich, without any CV risk factors (by history, clinical examination, and laboratory tests) or accompanying disorders. 2.1.2. Study 2 26 PD patients underwent treatment with a well characterized inflammatory response [13]. HDL function was studied at baseline, after 24 h and at 6 months following treatment. These time-points were selected as they had previously been shown to correspond to the peak and resolution of the systemic inflammatory response. The study was approved by the joint Eastman and University College Hospitals ethics committee and all patients provided informed consent. 2.2. Anthropometric and biochemical measurements Anthropometric measurements were recorded and body mass index (BMI [kg/m2]) was calculated from weight and height. Blood pressure was measured in triplicate (HEM-705CP, Omron) and the average of the readings was recorded. Blood samples were drawn and processed after an overnight fast and serum and plasma samples were stored at − 70 °C for subsequent analysis. Full blood count, lipid and glucose level measurements were made with standard biochemistry assays and C-reactive protein (CRP) was measured with an immunoturbidimetric, high-sensitivity assay (Tina-quant CRP immunoturbidimetric assay performed on a Cobas Integra analyzer, Roche Diagnostics). Inter and intra assay coefficients of variations for all assays were b 5%. 2.3. HDL measurements 2.3.1. HDL isolation HDL was isolated by sequential ultracentrifugation (d = 1.063– 1.21 g/mL) using solid potassium bromide for density adjustment [14].
BioSpin) with the following instrumental settings: center field (B0) 3455 G; sweep width 80 G; microwave power 39.72 mW; modulation amplitude 10.34 G; sweep time 10.49 s; number of scans 10 [6,15]. Our group has reported has coefficients of variation for intraassay and interassay variability of these measurements as 1.92% and 1.74%, respectively. 2.3.3. ESR measurement of superoxide production The effect of HDL on endothelial cell superoxide production was compared to controls in un-stimulated and tumor necrosis factor (TNF)α-stimulated (5 ng/mL, R&D Systems) HAECs by ESR spectroscopy. Briefly, HAECs were incubated with HDL (50 μg/mL, 60 min, 37 °C), with or without TNFα and re-suspended in Krebs-Hepes buffer (pH 7,4; Noxygen) containing diethyldithiocarbamic acid sodium salt (5 μM, Noxygen) and deferoxamine methanesulfonate salt (25 μM, Noxygen). ESR spectra were recorded after addition of the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH); Noxygen; final concentration (200 μM) using a Bruker e-scan spectrometer (Bruker BioSpin). The ESR instrumental settings were as follows: center field (B0) 3495 G; field sweep width 10 G; microwave frequency 9.75 GHz; microwave power 19.91 mW; magnetic field modulation frequency 86.00 kHz; modulation amplitude 2.60 G; conversion time 10.24 msec; number of x-scans 1020 [6]. The coefficients of variation for intra-assay and interassay variability of these measurements were 5.1% and 12.9%, respectively. 2.3.4. Cholesterol efflux capacity of HDL HDL for measurements of efflux capacity was extracted from serum by ApoB depletion. Briefly, whole serum was incubated for 20 min with a 20% polyethylene glycol (PEG) solution (20% PEG 8000 (sigma P2139) in 200 mM glycine (sigma G8898, pH = 10)). Samples were centrifuged at 1900 G, and the supernatant was collected and stored at 4 °C. Cholesterol efflux capacity was quantified according to a method previously reported [16]. J774 cells were radiolabeled for 24 h in a medium containing 2 μCi of [3H]-cholesterol per mL. Addition of 0.3 mM 8(4-chlorophenylthio)-cyclic AMP for 6 h up-regulated expression of ABCA1. An efflux medium containing 2.8% apolipoprotein B-depleted serum from each individual was added for 4 h. To prevent cholesterol esterification during labeling, equilibration and flux, 2 μg per mL of CP113,818, a acyl-coenzyme A:cholesterol acyltransferase inhibitor was added to all mediums. Efflux capacity was quantified using liquid scintillation to measure radioactive cholesterol effluxed from the cells (medium + intracellular lipids). All assays were performed in duplicate and the final average value normalized against a baseline control for statistical analyses between time-points. 2.3.5. Serum assay of HDL function Serum paraoxonase activity was measured by UV spectrophotometry in a 96-well plate format using paraoxon (Sigma-Aldrich, St Louis, Missouri). Briefly, 50 μg/mL HDL was diluted in a reaction mixture containing 10 mM Tris hydrochloride (pH 8.0), 1 M sodium chloride and 2 Mm calcium chloride. At 24 °C 1.5 mM paraoxon was added to initiate the reaction, and the increase in absorbance at 405 nm was recorded over 30 min. An extinction coefficient (at 405 nm and 24 °C) of 17 000 M−1·cm−1 was used to calculate units of paraoxonase activity [17]. The coefficients of variation for intra-assay and inter-assay variability of these measurements were 7.2% and 13.1%, respectively. 3. Statistics
2.3.2. Electron spin resonance (ESR) measurement of NO bioavailability The effect of HDL from patients and controls (50 μg/mL; 60 min, 37 °C) on endothelial NO production ((human aortic endothelial cells) HAECs; passage 4–7; Lonza Bio Science) was examined by continuouswave electron spin resonance (ESR) spectroscopy using the spinprobe colloid Fe(DETC)2 (Noxygen). ESR spectra of samples frozen in liquid nitrogen were recorded on a Bruker e-scan spectrometer (Bruker
All measures are reported as mean (SD) or median [IQR] for those not normally distributed. Normal distribution was assessed using the Kolmogorov–Smirnov test. Comparisons between the HDL measurements and inflammatory markers were performed using the Pearson correlation coefficient for normally distributed data and the Spearman Rank Correlation method for non-normally distributed data. Comparisons
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between patients and controls were performed using an independent t-test or the Mann Whitney U test for normally and non-normally distributed data respectively. Comparison of HDL function, before and after PD treatment in patients, was performed using repeated-measures analysis of variance to determine differences, with Greenhouse–Geisser correction and using the Friedman test for non-parametric data. For proteomics analysis, a paired t-test was used to analyze differences between baseline and 24 h, and Wilcoxon signed-rank test used for non-normally distributed data. Analysis was performed using STATA v12 analysis software. A two sided p value of b 0.05 was considered to indicate statistical significance. 4. Results 4.1. Comparison of PD patients and controls and impact of chronic inflammation on HDL function PD patients had similar CV risk factors, including lipid levels, smoking status and BMI compared to controls, but CRP levels were statistically significantly higher in PD patients (p = −0.01) (Table 1). Endothelial cells exposed to HDL from the PD patients showed reduced NO bioavailability and increased superoxide production (p b 0.01 for both). Paraoxonase activity was lower in patients with PD (p b 0.01) (Fig. 1). 4.2. Impact of acute inflammation and its resolution on HDL functions There were no statistically significant changes in blood pressure, lipid levels and measures of coagulation following transient inflammatory stimulus. Inflammatory markers including CRP, IL-6 and total white blood cell count were significantly increased 24 h following PD treatment (p b 0.001 and p b 0.01 respectively) (Table 2). No relevant changes in monocyte counts were observed. Apart from HDL dependent cholesterol efflux, all other parameters of HDL function assessed were greatly impaired at 24 h. Endothelial cell NO bioavailability was reduced following stimulation with HDL from patients, compared to baseline (p b 0.01). Endothelial anti-inflammatory activity measured by VCAM-1 expression was also increased (p b 0.01) (Supp. Fig. 1). Furthermore, HDL's anti-oxidant capacity was also impaired with an increase in superoxide production and a reduction in paraoxonase activity (both p b 0.01) (Fig. 2). Quantitative proteomics revealed a statistically significant increase in signal intensities of Complement C3 (C3) (p = 0.03), serum amyloid alpha (SAA) (p = 0.01) and pro-thrombin (p = 0.01) was noted at 24 h (Fig. 3). Inflammatory markers returned to baseline levels 6 months after PD treatment. All HDL functions were normalized including NO Table 1 Baseline characteristics in periodontitis and healthy participants.
Age (years) Smoking status (non-smoker/smoker) BMI (kg/m2) Total Cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) Triglycerides (mmol/L) Glucose (mmol/L) CRP (mg/L)
Healthy (N = 26)
Periodontitis (N = 26)
p-Value
52.90 (4.23) 0/26
51.52 (6.32) 3/26
0.41 0.09
25.83 (2.14) 4.86 (0.71) 1.33 [1.13–1.51] 2.83 (0.88) 1.03 [0.8–1.38] 5.14 (0.62) 1.00 [1.00–1.00]
26.99 (4.03) 5.39 (0.95) 1.50 [1.30–2.00] 3.20 (0.94) 0.90 [0.80–1.55] 4.94 (0.55) 1.6 [1.00–3.55]
0.27 0.05 0.08 0.18 0.46 0.26 0.01
Abbreviations: BMI: body mass index, HDL: high density lipoprotein, LDL: low density lipoprotein, CRP: C-reactive protein. Values expressed as mean (SD) and median [IQR] for non-normally distributed data. Comparisons between periodontitis patients and healthy controls were performed using the independent t-test. Categorical variables were compared using Fisher's exact Chi-squared test. Comparisons for non-parametric measurements were performed using Mann Whitney test. † p b 0.01
Fig. 1. Cross-sectional comparison of HDL between healthy controls and PD. Following isolation, HDL (50 μg/mL) function was assayed using ESR spectroscopy for A) nitric oxide (NO) bioavailability; B) superoxide production (data expressed as percent change versus buffer-treated cells). Serum paraoxonase activity was measured by UV spectrophotometry. Individual data points are shown for each patient. Bars represent mean.
bioavailability, VCAM-1 expression, superoxide production and serum paraoxonase activity (p b 0.01 for all). There was no difference in cholesterol efflux capacity between any time points (Table 3).
5. Discussion This study demonstrates that low grade inflammation can disturb HDL vascular functions and that these can be further aggravated by transient acute inflammation. We show for the first time, that acute changes in HDL function can recover with resolution of inflammation and are paralleled by changes in the HDL proteome. Our findings
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Table 2 Patient clinical characteristics.
BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) CRP (mg/L) Interleukin-6 (pg/mL) LCC (×10−9/L) MC (cells/μL) PLT (109/L) Total cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) Triglycerides (mmol/L) Glucose (mmol/L)
Baseline
Day one
6 months
27.89 (5.20) 128.2 (19.75) 81.26 (12.47) 2.00 [1.00–3.00] 2.15 (5.35) 6.46 (1.53) 6.27 (1.96) 247.56 (52.6) 5.28 (1.10)
N/A 126.28 (17.82) 80.14 (10.85) 6.55 [1.68–12.90]† 5.81 (8.50)† 7.18 (1.84)⁎ 7.03 (2.33) 248.00 (57.41) 5.19 (1.02)
27.79 (5.32) 125.26 (16.49) 79.76 (10.12) 1.30 [0.56–1.30]‡ 2.54 (5.97)‡ 6.21 (1.71)‡ 6.59 (2.01) 251.38 (55.20) 5.15 (0.95)
1.61 (0.41) 3.16 (1.01) 1.14 (0.57) 4.99 (0.49)
1.64 (0.42) 3.06 (0.96) 1.06 (0.41) 5.03 (0.54)
1.63 (0.36) 3.03 (0.84) 1.06 (0.42) 4.79 (0.48)‡,§
Abbreviations: BMI: body mass index, HDL: high density lipoprotein, LDL: low density lipoprotein, CRP: C-reactive protein, SBP: systolic blood pressure, DBP: diastolic blood pressure, LCC: leukocyte count, MC: monocyte count, PLT: platelet count. Values expressed as mean (SD) and median [IQR] for non-normally distributed data. Comparisons between time points were performed using repeated measures ANOVA or Friedman's test for non-normally distributed data. ⁎ p b 0.05 between baseline and day 1. † p b 0.01 between baseline and day 1. ‡ p b 0.001 between day 1 and 6 months. § p b 0.01 between baseline and 6 months.
demonstrate the dynamic nature of the HDL molecule. This may be relevant to its protective role in vascular disease and should be considered when developing drug treatments which aim to increase HDL levels. Over the last 20 years, lowering of LDL cholesterol, particularly with statins has resulted in a major improvement in CV outcomes [18,19]. Nevertheless, there remains a considerable residual risk of CV events and this has prompted interest in strategies to raise HDL cholesterol, based on the consistent association between higher HDL levels and reduced CV risk in population studies of CV risk factors [20]. However, recent clinical trials have failed to demonstrate significant clinical benefit, despite increases in HDL levels [21,22]. A wealth of experimental data suggests HDL is a much more complex particle than previously considered and that the biological functions of HDL may be important. The traditional role of HDL has been to promote reverse cholesterol transport from peripheral tissues, including the arterial vessel wall [23]. Recently, HDL has been shown to have a range of effects on vascular function which may be relevant to atherogenesis and plaque stability [24]. We and others have shown that these can be disturbed in a range of clinical conditions associated with inflammation. These included anti-phospholipid syndrome, diabetes mellitus, chronic kidney disease (CKD) and recently acute coronary syndromes [9,25,26]. In these conditions, HDL from patients had the opposite effect on cultured endothelial cells to that seen with HDL isolated from healthy subjects. Nitric Oxide bioavailability was impaired, super oxide production increased, adhesion molecule expression enhanced and paraoxonase activity decreased. The relationship between these vascular effects and cholesterol efflux, as well as the impact of transient acute inflammation on HDL functions in vivo has not been previously explored. In our current study, we have examined HDL structure and function in patients with a chronic inflammatory condition affecting approximately 10% of the adult population [27]. Our findings support current research demonstrating that HDL function is altered by inflammation but suggests that this may not be confined to the more severe clinical inflammatory conditions previously reported. The fact that a relatively common low-grade systemic disease can derange HDL function suggests that the HDL molecule is very sensitive to inflammation. This is likely to have implications for HDL-targeted therapies. We subsequently tested the effect of transient acute inflammation on HDL function using a periodontal therapy model. The acute inflammatory stimulus further aggravated the already deranged HDL properties, which
Fig. 2. Functional changes in HDL 24 h and 6 months following PD treatment. HDL from PD patients was isolated by sequential ultracentrifugation, and the effects of HDL (50 μg/mL) on endothelial cells was analyzed by ESR spectroscopy analysis measuring A) nitric oxide (NO) bioavailability; B) superoxide production (data expressed as percent change versus buffertreated cells). C) Serum paraoxonase activity was measured by UV spectrophotometry. I bars represent standard error. Data points represent mean of 26 patients.
then returned to baseline levels with resolution of the acute inflammation. These functional changes tracked inflammatory markers and suggest that HDL function can be rapidly altered as a result of inflammation. The link between inflammation and HDL function is further emphasized in our study as there were no changes in other CV risk factors during this period. While this is consistent with previous observations we are first to demonstrate the dynamic nature of the HDL molecule on a range of HDL's endothelial protective functions and show recovery of HDL function [28].
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The disturbances in HDL function which were seen at the peak of inflammation were accompanied by changes in its protein composition. These included increased levels of the acute phase reactants serum amyloid A (SAA) and complement factor C3 (C3). This provides both mechanistic insights into the functional changes of HDL as well as further evidence of the rapid remodeling of HDL during acute inflammation. Interestingly, our proteomic analysis also revealed an increase in prothrombin levels within the HDL particle. This may underlie the reported pro-thrombotic effects of HDL following endotoxin challenge in humans [29]. We were able to follow, for the first time, changes in a range of HDL properties including cholesterol efflux and effects on endothelial cells in culture within the same individuals. We demonstrated a more pronounced impact of acute inflammation on the vascular effects of HDL than on its efflux capacity. The vascular effects of HDL may therefore be more sensitive to inflammation than cholesterol efflux. The relevance of these different properties of HDL may vary at different stages of atherosclerosis and require further study. Our study does present with several limitations. Firstly, we cannot comment on the time course for recovery of HDL function as serial measurements were not made between the peak of inflammation and 6 months. Furthermore, we did not measure the relationship and time course of recovery between structural and functional changes in HDL during this period. This is the subject of on-going research. There were however several advantages with our experimental design, which include a predictable clinical model of increased inflammation and its resolution, without the use of drugs, as well as the measurement of a range of HDL properties (including vascular and cholesterol efflux). In conclusion, the highly dynamic nature of HDL function and its upset by relatively minor inflammatory stimuli, both acute and chronic, may have clinical relevance. Our findings are of particular interest in light of the recent failures of HDL-C raising therapies in prevention clinical trials and underline the need to understand better the properties of HDL, their relationship to CV and the inflammatory phenotype and their response to treatment. While off-target effects and insufficient increase in circulating HDL level may account for the lack of benefit from Torcetrapib, Dalcetrapib and Niacin, the biological function of HDL and the inflammatory status of the population may be of great importance in determining the clinical benefits of HDL elevating therapies [21,30]. Indeed, patients recruited into the Dal-Outcome study were enrolled following an acute coronary syndrome, an event shown drastically to alter vascular properties of HDL. The reversibility of changes in HDL function, which we have demonstrated offers a therapeutic opportunity. This could include treatment of the underlying inflammatory state as well as targeting the HDL particle and its actions. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2015.03.058. Fig. 3. Protein changes in HDL, 24 h after PD treatment. HDL was isolated in the same patients before and after PD treatment, and HDL protein cargo compared using Mass Spectroscopy. A) Complement factor C3, B) serum amyloid alpha 4 (SAA), and C) pro-thrombin were quantified by calculating the area under the curve of extracted ion chromatograms. An average area under the curve for 2 representative spectra is shown for each protein. Bars represent mean and I bars represent standard error.
Funding/support This study was funded by Colgate-Palmolive (09CC25), F. HoffmannLa Roche Ltd (09CC22) and the Leducq Foundation (10CC22). The
Table 3 HDL function.
NO bioavailability SO production PON activity (μmol p-nitrophenol/L/serum/min) VCAM-1 expression (% of TNF-α stimulated cells) Cholesterol efflux (average efflux with normalization)
Baseline
Day one
6 months
5.15 (14.67) −5.8 [−21.30–1.23] 130.05 (21.10) 98.37 (18.93) 0.99 (0.30)
−4.05 (16.85)⁎ 7.56 [0.37–31.57]⁎ 111.63 (26.61)⁎ 115.92 (14.44)⁎
7.00 (19.23)† 4.56 [−36.46–5.21]† 132.75 (26.31)† 78.36 (27.79)† 1.05 (0.31)
0.92 (0.32)
Abbreviations: NO: nitric oxide, SO: superoxide, PON: paraoxonase. Values expressed as mean (SD). The NO bioavailability and SO production were expressed as % inhibition of buffer treated cells. ⁎ p b 0.01 between baseline and day one. † p b 0.01 between day one and 6 months.
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