Atherosclerosis 240 (2015) 73e79
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Vascular effects of oxysterols and oxyphytosterols in apoE / mice €rtner a, b, *, Constanze Husche f, Hans F. Scho € tt f, Timo Speer c, Oliver Weinga b d € hm , Charlotte M. Miller , Florence McCarthy d, Jogchum Plat e, Michael Bo Dieter Lütjohann f, 1, Ulrich Laufs b, 1 €t Oldenburg, Oldenburg, Abteilung für Kardiologie, Klinikum Oldenburg, European Medical School Oldenburg-Groningen, Carl von Ossietzky Universita Germany b €tsklinikum des Saarlandes, Homburg/Saar, Germany Klinik für Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universita c €tsklinikum des Saarlandes, Homburg/Saar, Germany Klinik für Innere Medizin IV, Klinik für Nephrologie und Hypertensiologie, Universita d Department of Chemistry and ABCRF, University College Cork, Western Road, Cork, Ireland e Department of Human Biology, Maastricht University, Maastricht, The Netherlands f Institute for Clinical Chemistry and Clinical Pharmacology, University Clinics Bonn, Germany a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 October 2014 Received in revised form 12 January 2015 Accepted 14 February 2015 Available online 26 February 2015
Objectives: The aim of our study was to investigate vascular effects of oxysterols and oxyphytosterols on reactive oxygen species (ROS), endothelial progenitor cells, endothelial function and atherogenesis. Methods: Male apoE/mice were treated with cholesterol, sitosterol, 7-ßeOHecholesterol, 7-ßeOH esitosterol, or cyclodextrin by daily intraperitoneal application. The respective concentrations in the plasma and in the arterial wall were determined by gas chromatography-flame ionization or mass spectrometry. ROS production was assessed by electron-spin resonance spectroscopy in the aorta, endothelial function of aortic rings and atherosclerosis in the aortic sinus was quantitated after 4 weeks. Results: Compared to vehicle, there was no difference in plasma cholesterol levels and arterial wall concentrations after i.p. application of cholesterol. 7-ßeOHecholesterol concentrations were increased in the plasma (33.7 ± 31.5 vs. 574.57.2 ± 244.92 ng/ml) but not in the arterial wall (60.1 ± 60.1 vs. 59.3 ± 18.2 ng/ mg). Sitosterol (3.39 ± 0.96 vs. 8.16 ± 4.11 mg/dL; 0.08 ± 0.04 vs. 0.16 ± 0.07 mg/mg, respectively) and 7ßeOHesitosterol concentrations (405.1 ± 151.8 vs. 7497 ± 3223 ng/ml; 0.24 ± 0.13 vs. 16.82 ± 11.58 ng/mg, respectively) increased in the plasma and in the aorta. The i.p-application of the non-oxidized cholesterol or sitosterol did not induce an increase of plasma oxysterols or oxyphytosterols concentrations. Oxidative stress in the aorta was increased in 7-ßeOHesitosterol treated mice, but not in mice treated with cholesterol, sitosterol, or 7-ßeOHecholesterol. Moreover, cholesterol, sitosterol, 7-ßeOHecholesterol, and 7-ßeOHesitosterol did not affect endothelial-dependent vasodilation, or early atherosclerosis. Conclusion: Increased oxyphytosterol concentrations in plasma and arterial wall were associated with increased ROS production in aortic tissue, but did not affect endothelial progenitor cells, endothelial function, or early atherosclerosis. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sterols Oxysterols Phytosterols Oxyphytosterols Endothelial function Atherosclerosis
Hypercholesterolemia is a major risk factor for the development of cardiovascular diseases [1]. High dietary intake of cholesterol
Abbreviations: EPC, endothelial progenitor cells; ESR, electron-spin resonance; i.p., intraperitoneal; MNC, mononuclear cells; PSE, plant sterol esters; ROS, reactive oxygen species. * Corresponding author. Abteilung für Kardiologie, Klinikum Oldenburg, Euro€t Rahel pean Medical School Oldenburg-Groningen, Carl von Ossietzky Universita Straus-Strasse 10, 26133 Oldenburg Germany. E-mail address: [email protected] (O. Weing€ artner). 1 UL and DL contributed equally to the manuscript. http://dx.doi.org/10.1016/j.atherosclerosis.2015.02.032 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
together with sedentary habits have been identified as major contributors to atherosclerosis [2]. The latter has long been considered a cholesterol storage disease; however, today atherosclerosis is considered a more complex disease in which both innate and adaptive inflammatory mechanisms as well as interactions between the arterial wall and blood components play a role [3]. Supplementation of foods with plant sterols has been suggested to prevent atherosclerosis because of their cholesterol-lowering effect [4]. Similar to cholesterol, plant sterols can be oxidized and can make an important contribution to the pro-atherogenic effects
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of both cholesterol and cholesterol oxidation oxides in relation to inflammatory disease onset and the development of plaques [5]. Several lines of evidence suggest that oxysterols (oxidized cholesterol) are atherogenic and play a role in the pathogenesis of cardiovascular disease. The presence of one or more unsaturated bonds also makes plant sterols susceptible to oxidation. Only small amounts of oxidized plant sterols (oxyphytosterols) can be found in the diet [6]; nevertheless, relatively high concentrations of oxyphytosterols are present in the serum of sitosterolemia patients and smaller amounts in the plasma of healthy individuals [7e9]. Thus, higher plasma plant sterol concentrations may translate into higher plasma oxyphytosterol concentrations. Intervention trials found conflicting results whether the consumption of a plant sterolenriched margarine for 4 weeks increases plasma 7ßeOHecampesterol concentrations in healthy individuals [10,12]. Differences with regard to the post-prandial state, the genetic background and different duration of the wash out periods may explain the heterogenicity of the data, however, additional research is needed to address the relation of specific diets with plasma oxyphytosterol concentrations. The factors related to the oxidative behavior of plant sterols are unknown; however, it has been suggested for cholesterol that patients characterized by oxidative stress, such as type 2 diabetics and patients with stable coronary artery disease have increased oxysterol concentrations [11], [12]. Since the exact factors predisposing for plant sterol oxidation or oxyphytosterol formation are unknown, it is difficult to experimentally show the effects of endogenously formed oxyphytosterols on vascular wall characteristics and atherosclerotic lesion formation. Moreover, oxyphytosterol concentrations due to endogenous formation are usually rather low which makes long-term interventions necessary. Another approach is to feed oxyphytosterols, as has been done for oxysterols [13]. So far, there have been only limited data regarding the effects of an oxyphytosterol-enriched diet on atherogenesis, which was however suggestive for atherogenic effects and incorporation of oxyphytosterols in the lesion [14]. However, also in this case serum concentrations are low due to the low percent absorption of oxyphytosterol from the diet. Tomoyori et al. showed that oxyphytosterols are indeed absorbed from the diet in low amounts followed by transport to the lymph and accumulation in the serum, liver, and aorta [15]. To overcome these problems, in the present study we therefore synthesised specifically highly purified sitosterol and its respective 7b-hydroxylated metabolite and investigated the i.p. application route on different vascular phenotypes such as oxidative stress (ROS production) in the aorta, endothelial progenitor cells, and on endothelial function and atherogenesis using male apoE/mice. 1. Methods
unless otherwise stated. The starting materials cholesterol (>95%) and stigmasterol (>95%) were both purchased from Sigma Aldrich. b-Sitosterol (4) was produced in 4 steps (in 40% yield) from Stigmasterol (3) and was converted to 7b-OH b-Sitosterol (6) in 4 steps via acetate formation, allylic oxidation, hydrolysis of the acetate, and stereoselective borohydride-mediated ketone reduction. 7bOH Cholesterol (2) was synthesized in 4 steps from cholesterol (1) via a similar method. For further information refer to Supplementary material. All sterols were diluted in sodium chloride with 30% cyclodextrin and applicated intraperitoneally 1 mg per day per mouse. 1.3. 7-b-Hydroxy-b-Sitosterol (6) mp 137e139 C (from EtOAc/hexane) (Found: C, 79.30; H, 11.43. Calc. for C29H50O2.(½H2O): C, 79.21; H, 11.69%). ymax/cm1 3400, 2959, 2871, 1465, 1384, 1056; dH 0.70e2.32 (47H, m), 3.51e3.58 (1H, m, 3a-H), 3.85 (1H, bd, J ¼ 2.6, H-7), 5.29 (1H, bs, H-6); dC (75.5 MHz) 11.82 (CH3), 11.98 (CH3), 18.84 (CH3), 19.03 (CH3), 19.16 (CH3), 19.81 (CH3), 21.09 (CH2), 23.08 (CH2), 26.13 (CH2), 26.39 (CH2), 28.55 (CH2), 29.16 (CH), 31.59 (CH2), 33.99 (CH2), 36.10 (CH), 36.46 (quaternary C), 36.95 (CH2), 39.57 (CH2), 40.93 (CH), 41.74 (CH2), 42.94 (quaternary C), 45.86 (CH), 48.28 (CH), 55.39 (CH), 55.97 (CH), 71.44 (CH), 73.36 (CH), 125.46 (CH), 143.47 (quaternary C); m/z (ESIþ): 431 [(M þ H)þ]. 1.4. 7-b-Hydroxy-Cholesterol (2) mp 182e183 C (from EtOAc/hexane) ymax/cm1 3400, 2959, 2871, 1465, 1384, 1056; dH 0.70e2.37 (43H, m), 3.50e3.60 (1H, m, 3a-H), 3.85 (1H, bd, J ¼ 2.6, H-7), 5.29 (1H, bs, H-6); dC (75.5 MHz) 11.83 (CH3), 18.78 (CH3), 19.17 (CH3), 21.08 (CH2), 22.57 (CH3), 22.84 (CH3), 23.84 (CH2), 26.39 (CH2), 28.03 (CH), 28.56 (CH2), 31.57 (CH2), 35.74 (CH), 36.20 (CH2), 36.44 (quaternary C), 36.94 (CH2), 39.50 (CH2), 39.56 (CH2), 40.90 (CH), 41.73 (CH2), 42.93 (quaternary C), 48.25 (CH), 55.44 (CH), 55.95 (CH), 71.44 (CH), 73.36 (CH), 125.44 (CH), 143.48 (quaternary C); m/z (ESIþ): 403 [(M þ H)þ]. 1.5. Plasma and arterial wall concentrations of sterols, phytosterols, and their respective oxides After 4 weeks, blood samples were drawn from the abdominal vena cava and the descending aorta was excised on sacrifice. Blood samples were centrifuged immediately and plasma and arterial wall tissue were stored at e 70 C. The sterol content of plasma and arterial wall tissue samples was analyzed by gas liquid chromatography-mass spectrometry with epicoprostanol as the internal standard, the amount of 7beOHecholesterol and 7beOHesitosterol using deuterium labeled internal standards as described previously [12,13,29].
1.1. Animals and diets 1.6. Reactive oxygen species in aortic tissue and the spleen Male apoE/(C57/Bl6 genetic background) mice, 8e12 weeks of age, weighing 20e25 g were purchased from Charles River, Sulzfeld, Germany. The 50 apoE/ mice were randomized to 5 treatment groups, n ¼ 10 per group, treated for 4 weeks. All groups were fed a “Western-type” diet (40 kcal% butterfat, 0.15% (w/w) cholesterol). Animal experiments were performed in accordance with the German animal protection law. Over the 4 week study period mice had ad libitum access to water and chow. Diets were prepared by the SNIFF Company (Soest, Germany). 1.2. Cholesterol, oxysterols, plant sterols, and oxyphytosterols All commercial reagents were used without further purification
The production of ROS in aortic tissue was analyzed by ESR spectroscopy as described previously [16]. Briefly, aortic rings were incubated in Krebs HEPES buffer containing 25 mmol/L deferoxamine (Noxygen) and 55 mmol/L diethyldithiocarbamic acid (DETC, Noxygen) together with the spin probe 1-hydroxy-3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH, 500 mM) for 1 h at 37 C. Afterwards, supernatants were immediately analyzed by ESR spectroscopy. ESR spectra were recorded using a Bruker e-scan spectrometer (Bruker Biospin) with the following settings: center field, 3484.5 g; microwave power, 18.11 mW; modulation amplitude, 2.3 G; sweep time, 5.24 s; field sweep, 16 G. Results were normalized to the dry weight of the aortic rings or the
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cell count. 1.7. Isolation, culture, and functional assessment of endothelial progenitor cells To select for endothelial progenitor cells (EPC), mononuclear cells (MNC) were isolated from spleen homogenates from control mice and each treatment group by Ficoll density gradient centrifugation (Biocoll Separating Solution; Biochrom, Heidelberg, Germany) and cultured on fibronectin-coated culture dishes in endothelial basal medium (Lonza, Wuppertal, Germany) with supplements (1 mg/mL hydrocortisone, 3 mg/mL bovine brain extract, 30 mg/mL gentamicin, 50 mg/mL amphotericin B, 10 mg/mL human endothelial growth factor, and 20% fetal calf serum). EPC were characterized by the expression of surface markers such as Sca-1 and vascular endothelial growth factor receptor2 (VEGFR2; corresponding to human CD34 and KDR respectively). Mouse blood and bone marrow were processed for FACS analysis. The viable lymphocyte population was incubated with Sca-1-flourescein isothiocyanate (FITC; E13e161.7; BD Pharming, Heidelberg, Germany) and VEGFR2 (Flk-1) (Avas12a1, BD Pharming) antibodies conjugated with the corresponding phycoerythrin (PE)- labeled secondary antibody (Sigma-Adrich, Taufkirchen, Germany), Isotypeidentical antibodies (lgG2ak FITC and PE, BD Pharmingen) served as controls in every experiment (BectoneDickinson, Heidelberg, Germany). FACS analysis was performed immediately after the staining using a FACSCalibur instrument (BectoneDickinson) and Cell Quest software version 6.0 (BD Biosciences, Heidelberg, Germany). The migratory capacity of the EPC was determined in modified Boyden chamber assays [17]. For each animal, 4 106 MNC were plated on fibronectin-coated six-well culture dishes and cultured for 4 days. Culture medium was removed, cells were harvested and suspended in EBM without supplements and counted under a light microscope, followed by transfer of 1 105 cells to a migration chamber (HTS Fluoroblock, 8 mm pore size, BD Biosciences). The chambers were then placed in a 24-well plate containing EBM without supplements and 100 ng$mL1 SDF-1 to induce migration (R&D Systems). After incubation at 37 C for 24 h, the filters were carefully washed, cells fixed and incubated with diLDL as described above. DiLDL-positive CAC that had migrated to the lower surface of the filter in response to SDF-1 were quantified using fluorescence microscopy (400 magnification) by an observer blinded to the study as described previously [17]. 1.8. Endothelial function After excision of the descending aorta, the vessel was immersed in chilled buffer (pH 7.4) containing NaCl, 118.0 mmol/L; CaCl2, 2.5 mmol/L; KCl, 4.73 mmol/L; MgCl2, 1.2 mmol/L; KH2PO4, 1.2 mmol/L; NaHCO3, 25 mmol/L; Na EDTA, 0.026 mmol/L; and D(þ) glucose, 5.5 mmol/L. Adventitial tissue was carefully removed, and 2 mm rings were mounted in organ baths filled with the above described buffer (37 C; continuously aerated with 95% O2/5% CO2) and were attached to a force transducer, and isometric tension was recorded. The vessel segments were gradually stretched over 60 min to a resting tension of 10 mN, which was maintained throughout the experiment, and were allowed to equilibrate for another 30 min. Drugs were added in increasing concentrations to obtain cumulative concentrationeresponse curves: KCl, 20 and 40 mmol/L; phenylephrine, 1 nmol to 10 mmol/L; carbachol, 10 nmol to 100 mmol/L; and nitroglycerin 1 nmol/L to 10 mmol/L. The drug concentration was increased when vasoconstriction or relaxation was completed. Drugs were washed out before the next substance was added, as described previously [18].
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1.9. Analysis of atherosclerotic lesions Hearts of ApoE/ mice with the ascending aorta were embedded in Tissue Tek O.C.T. (Miles), snap frozen, and stored at 80 C. Samples were sectioned on a Leica cryostat (10 mm), starting at the apex and progressing through the aortic valve into the ascending aorta, and were placed on slides. At least 25 consecutive sections per animal were used for analysis. For the detection of atherosclerotic lesions, the sections were fixed with 3.7% formaldehyde for 1 h, rinsed with deionized water, stained with oil red O working solution (0.5%) for 30 min, and then rinsed again. For morphometric analysis, additional hematoxylin staining was performed according to standard protocols. Picrosirius red solution was used to stain collagen fibers. All sections were examined under a Nikon E 600 microscope. Lucia Measurement Version 4.6 software was used to measure the area of histological sections. Two observers independently and blindly performed all measurements and graded standard and immunostaining as described [19]. 1.10. Statistics Data are reported as the mean ± SEM. Differences between 2 experimental groups were tested by 2-tailed Student t tests or by analysis of variance, followed by the application of Bonferroni test when means from 3 or more groups were compared. For quantification of correlations Pearson's correlations coefficient was used. Pvalues less than 0.05 were considered statistically significant. All statistical tests were performed using SPSS software. 2. Results 2.1. Effects on plasma concentrations of cholesterol, plant sterols, and their respective oxides Five groups of apoE/mice were each fed western-type diets for 4 weeks and then cyclodextrin (control) or the respective sterols were delivered by i.p. application. The average cholesterol level in control animals was 417.2 ± 122.1 mg/dL and did not significantly change after the i.p. application of cholesterol (366.3 ± 60.4 mg/dL). Similarly, the i.p.-application of sitosterol (294.0 ± 85.3 mg/dL), 7ßeOHecholesterol (357.7 ± 93.8 mg/dL) or 7-ßeOHesitosterol (450.7 ± 119.1 mg/dL) did not significantly alter plasma cholesterol levels (Fig. 1A). The i.p.-application of sitosterol (8.16 ± 4.11 vs. 3.39 ± 0.96 mg/dL) (Fig. 1B), 7-ßeOHecholesterol (574.57.2 ± 244.92 vs. 33.7 ± 31.5 ng/ml) (Fig. 1C), and 7ßeOHesitosterol (7497 ± 3223 vs. 405.1 ± 151.8 ng/ml) (Fig. 1D) increased the respective plasma levels compared with controls. Importantly, the i.p-application of the non-oxidized cholesterol or sitosterol did not induce an increase in plasma oxysterol (Fig. 1C) or oxyphytosterol (Fig. 1D) concentrations, respectively, indicative for absence of endogenous substrate oxidation. 2.2. Effects on arterial tissue wall concentrations of sterols, phytosterols, and their respective oxides The average cholesterol concentration in the arterial wall in control animals was 5.86 ± 1.93 mg/mg and did not change after the i.p. application of cholesterol (4.14 ± 0.78 mg/mg, Fig. 2A). The concentrations of sitosterol (0.16 ± 0.07 vs. 0.08 ± 0.04 mg/mg) (Fig. 2B) and 7-ßeOHesitosterol (16.82 ± 11.58 vs. 0.24 ± 0.13 ng/ mg) (Fig. 2C) in the arterial wall increased after i.p. application. However, despite the observed increase in plasma concentrations, i.p. application of 7-ßeOHecholesterol had no effect on the respective arterial wall concentration (59.3 ± 18.2 vs.
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Fig. 1. Effects of i.p. application of cholesterol, sitosterol, 7-betaeOHecholesterol, and 7-betaeOHesitosterol on serum levels of cholesterol (A), sitosterol (B), 7betaeOHecholesterol (C), and 7-betaeOHesitosterol (D). *P < 0.05 for sitosterol vs. control; #P, 0.05 for 7-betaeOHecholesterol vs. control; &P, 0.05 for 7-betaeOHesitosterol vs. control.
60.1 ± 60.1 ng/mg). 2.3. Effects on reactive oxygen species in the aortic wall Electron-spin resonance (ESR) spectroscopy demonstrated that oxidative stress (ROS production) in the aorta was increased only in 7-ßeOHesitosterol treated mice (157.7 ± 48.9% compared to control), but not in mice treated with cholesterol (91.9 ± 67.5%), sitosterol (106.4 ± 12.5%), or 7-ßeOHecholesterol (109.0 ± 52.1%) (Fig. 3). 2.4. Effects on endothelial progenitor cell quantification and migration The EPC number and function were determined by DiLDL-lectin staining and a modified Boyden chamber assay. The EPC number did not show a significant difference compared to control animals in any of the four experimental groups (control: 58 ± 22; sitosterol: 52 ± 13; 7-ßeOHecholesterol: 52 ± 28; 7-ßeOHesitosterol: 54 ± 16 cells/microscopic field). There was no difference in the migratory activity of EPCs compared to control animals (control: 41 ± 19; cholesterol: 62 ± 58; sitosterol: 35 ± 26; 7ßeOHecholesterol: 34 ± 16; 7-ßeOHesitosterol: 50 ± 30 migrated EPCs per 105). 2.5. Effects on endothelial dysfunction Despite the changes in plasma and aortic tissue sterol concentrations, the functional analysis of aortic rings showed no difference in endothelial-dependent vasorelaxation after i.p. application of
any of the four experimental sterols compared to the control group. Moreover, endothelial-independent vasorelaxation in response to nitroglycerin was equal in all groups (Fig. 4A and B). 2.6. Effects on atherosclerotic lesion development After 4 weeks of i.p. application of specific sterols, the mice were sacrificed, the aorta was excised, and the aortic lesion area was quantified by histomorphometric analysis. Early atherosclerotic lesion formation was similar between controls (17.2 ± 8.5) and sitosterol treated mice (17.0 ± 9.5%) and showed no difference after i.p. application of cholesterol (14.5 ± 9.1%), 7-ßeOHecholesterol (7.9 ± 4.5%) and 7-ßeOHesitosterol (10.1 ± 6.4%) (Fig. 5A and B). 3. Discussion In this study, we investigated the effects of oxidized cholesterol and oxidized sitosterol in mice. The major novel finding in this study is that the i.p. application of plant sterols or their oxidation products result in elevated plasma levels and increases sterol concentrations in the arterial wall. Interestingly, application of nonoxidized cholesterol or sitosterol increases their respective plasma concentrations but does not give rise to increased oxysterol or oxyphytosterol concentrations suggesting that endogenous oxidation does not play a major role in this context. Finally, only the elevation of 7-betaeOHesitosterol resulted in increased ROS production in the arterial wall, but this did not affect endothelial cell quantification, endothelial cell migration, endothelial function, and early atherosclerotic lesion development. Our group previously observed that a diet supplemented with
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Fig. 2. Effects of i.p.-application of cholesterol (A), sitosterol (B), 7-betaeOHecholesterol (C), and 7-betaeOHesitosterol (D) on arterial wall concentrations. *P < 0.05 for sitosterol vs. control; #P, 0.05 for 7-betaeOHesitosterol vs. control.
Fig. 3. Effects of i.p.-application of cholesterol, sitosterol, 7-betaeOHecholesterol, and 7-betaeOHesitosterol on oxidative stress (ROS). *P < 0.05 for 7-betaeOHesitosterol vs. control.
plant sterols (equivalent to a commercially available spread) induced endothelial dysfunction and led to an increase in ischemic stroke size in wild-type-mice [19,20]. Moreover, we observed that inhibition of cholesterol absorption by diet supplementation with plant sterols was associated with twice the amount of atherosclerotic lesion formation compared with ezetimibe treatment (a drug that reduces both plasma cholesterol and plant sterol levels), despite similar plasma cholesterol levels in apoE/mice [19]. Thus, our previous studies identified a positive correlation between plasma plant sterol concentrations, impaired endothelial function, and the extent of atherosclerotic lesions. In this study, however, there was no negative effect on endothelial function or early atherosclerotic lesion
development. It can be speculated that bypassing the intestine through i.p. application might exert different effects without a negative impact on the vasculature. However, sitosterol concentrations in the plasma in the current model were increased 2-fold, which is comparable to our previous studies [19,21]. Theoretically it is possible that the lipoprotein fraction responsible for transport of plant sterols through the circulation is different after feeding and i.p-application. Moreover, it is important to take into account that lipoprotein transport and plant sterol transport in apoE/ mice differs from the human situation. In the experimental model plant sterols are injected i.p. and the details of their route of entering the circulation are not fully known compared to diet-derived plant sterols that enter the circulation via chylomicrons. Lipoprotein transport and plant sterol transport in apoE/mice differs from the human situation. However, the potential impact related to the route of entrance is independent from the mouse model used but might cause differences in outcome in humans after dietary consumption. However, since this was not evaluated in our experiments, this needs to be studied in more detail in future studies. Another difference to the first study is the shorter time period of sterol exposure. In our previous studies, we investigated dietary plant sterol exposure up to six months [19]. Over this extended period of time, control mice developed atherosclerotic lesions that comprised more than 50% of the lumen in the aortic sinus in apoE/mice. In the current study, a short term 4-week sterol exposure in apoE/mice resulted in only a 17% atherosclerotic lesion area; thus, only the development of early atherosclerotic lesions could be investigated. Data from clinical studies in which a possible pro-atherogenic effect of phytosterols was assessed underscore the hypothesis that time is a crucial factor in the
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Fig. 4. Effects of i.p. application of cholesterol, sitosterol, 7-betaeOHecholesterol, and 7-betaeOHesitosterol on endothelial function. The functional performance of isolated aortic segments was assessed in organ chamber experiments. (A) Endothelium-dependent vasorelaxation induced by carbachol and (B) endothelial-independent vasorelaxation induced by nitroglycerin (NTG), expressed as a percent of the maximal phenylephrine-induced vasoconstriction. Values are mean ± SEM (n ¼ 10 per group).
Fig. 5. Atherosclerotic lesion formation in the aortic sinus of apoE/mice after i.p. application of cholesterol, sitosterol, 7-betaeOHecholesterol, and 7-betaeOHesitosterol for 4 weeks. (A) Representative examples (oil-red-O). (B) Values are mean ± SEM.
assessment. In a young, healthy cohort with low cardiovascular risk and an average age of 40 years, there was no relation between cardiovascular risk and plant sterol plasma levels [22]; however, there appears to be a positive relation between plant sterol plasma levels and an increased cardiovascular risk in older populations with a higher cardiovascular risk and a longer lifetime exposure to circulating plant sterols [19,23,24]. Finally, in the current study, the effects specifically for this study synthesized highly purified cholesterol, sitosterol, and their respective oxides were in focus. In the previous experimental studies, the dietary supplement included a variety of phytosterols and oxyphytosterols that resembled what is currently available for human consumption. Therefore, it might be speculated that other plant sterols and their oxides not tested in the current experiment exert vascular effects. It seems appropriate to further investigate also other plant sterols and their respective oxides over a longer period of time in this model. Our group previously showed that diet supplementation with plant sterols in humans leads to increased plant sterol
concentrations in plasma and aortic valve cusps [20]. Moreover, it has been demonstrated that phytosterols cross the bloodebrain barrier and are incorporated in the central nervous system and various other organ systems such as the liver in both wild-type and apoE/mice [20,25]. Therefore, another aim of this study was to determine whether a short time period of plant sterol exposure results in an increase of plant sterol concentrations in the arterial wall. Results of this study demonstrate for the first time that plant sterols are quickly incorporated in the arterial wall after only a 4week time period in apoE/mice. Recently we investigated in a clinical study in patients with severe aortic stenosis the relationships of phytosterols and oxyphytosterols in plasma and in aortic valve cusps [26]. Interestingly, in this clinical study the plant sterol sitosterol and its oxide 7-ßeOHesitosterol showed only a weak correlation between plasma and aortic valve cusps. The results of the current experimental findings, however, demonstrate a strong correlation for 7-b-OH sitosterol between plasma and the arterial wall. In our opinion these divergent results are due to different pathophysiological processes in atherosclerotic vascular disease and the
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evolution of aortic valve stenosis. Supposedly, the major calcification of cardiovascular tissue in aortic valve stenosis leads to differences in sterol composition compared to atherosclerotic lesion formation. These data are not in line with a previously published study, in which the authors did not find increased phytosterol concentrations in the arterial wall after phytosterol enriched diet supplementation in apoE/mice [27].This study has been criticized [28] and is not in line with our previous findings in clinical studies [19]. Interestingly, only the i.p. application of 7-ßeOHestitosterol resulted in increased plasma levels, a 70-fold increase of vascular sterol deposition, and also increased oxidative stress in the vessel wall. Sitosterol and 7-ßeOHecholesterol did not affect arterial wall ROS production after a 4-week period. A possible explanation is that 7-ßeOHesitosterol cannot be broken down in the vessel wall and leads to a more pronounced sterol deposition. However the increased oxidative stress did not translate into functional consequences like endothelial function and aortic lesion development. Future studies will have to confirm these findings. We also could not detect any influence of the i.p. application of sterols on endothelial progenitor cell quantification and migration. As argued above, this could be due to the sterols investigated or the short time period of the experiment. Future studies will have to elucidate this finding in more detail. In summary, the i.p. application of cholesterol, sitosterol, and their oxides has no effect on plasma cholesterol concentrations in apoE/mice. After i.p. application of sitosterol and 7ßeOHecholesterol, mice show increased plasma levels of the injected sterols. Application of sitosterol and 7-ßeOHesitosterol increased arterial wall deposition and leads to a 70-fold increase of 7-ßeOHesitosterol in the arterial wall, which increased oxidative stress in the arterial wall, however without functional consequences on endothelial function and aortic lesion development. Finally, and probably most importantly, the i.p-application of the non-oxidized sitosterol did not induce an increase in plasma oxyphytosterol concentrations indicative for absence of endogenous substrate oxidation. Conflicts of interest Dieter Lütjohann and Oliver Weing€ artner received funding for analytical contract work by Unilever, Vlaardingen, The Netherlands. €rtner and Ulrich Laufs received lecture fees and Oliver Weinga consulting fees from Merck. Acknowledgments We thank Ellen Becker, Jennifer Kieffer, Silvia Friedrichs and Simone J€ ager for excellent technical assistance. This study was funded by the Universit€ at des Saarlandes and in part by the Dutch Organization for Scientific Research (NOW TOP grant No. 91208006). Sterol and oxysterol analytical work was supported from the EUFP7 project Lipididiet (FP7/2007-2013) under grant agreement n 211696. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2015.02.032. References [1] N.J. Wald, M.R. Law, Serum cholesterol and ischaemic heart disease, Atherosclerosis 118 (Suppl. S1eS5) (1995). [2] A.H. Lichtenstein, L.J. Appel, M. Brands, et al., Summary of American heart association diet and lifestyle recommendations revision 2006, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 2186e2191.
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[3] R. Ross, Atherosclerosis e an inflammatory disease, N. Engl. J. Med. 340 (1999) 115e126. [4] T.A. Miettinen, P. Puska, H. Gylling, H. Vanhanen, E. Vartiainen, Reduction of serum cholesterol with sitostanol-ester margarine in a mildly hypercholesterolemic population, N. Engl. J. Med. 333 (1995) 1308e1312. [5] L. Alemany, R. Barbera, A. Allegria, J.M. Lapparra, Plant sterols from food in inflammation and risk of cardiovascular disease: a real threat, Food Chem. Toxicol. 69 (2014) 140e149. [6] L. Normen, M. Johnsson, H. Andersson, Y. van Gameren, P. Dutta, Plant sterols in vegetables and fruits commonly consumed in Sweden, Eur. J. Nutr. 38 (1999) 84e89. [7] J. Plat, H. Brzezinka, D. Lütjohann, et al., Oxidized plant sterols in human serum and lipid infusions as measured by combined gas-liquid chromatography-mass spctrometry, J. Lipid Res. 42 (2001) 2030e2038. [8] A. Grandgirard, L. Martine, L. Demaison, et al., Oxyphytosterols are present in plasma of healthy human subjects, Br. J. 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Rapp, et al., Oxidized cholesterol in the diet accelerates the development of aortic atherosclerosis in cholesterol-fed rabbits, Arterioscler. Thromb. Vasc. Biol. 18 (1998) 977e983. [14] J. Plat, E. Theuwissen, C. Husche, D. Lütjohann, M.J. Gijbels, M. Jeurissen, R. Shiri-Sverdov, Mensink RP. van der Made, Oxidised plant sterols as well as oxycholesterol increase the proportion of severe atheroscleroitc lesions in female LDL receptorþ/ mice, Br. J. Nutr. 111 (2014) 64e70. [15] H. Tomoyori, Y. Kawata, T. Higuchi, et al., Phytosterol oxidation products are absorbed in the intestinal lymphatics in rats but do not accelerate atherosclerosis in apoliporpotein E-deficient mice, J. Nutr. 134 (2004) 1690e1696. €hli, M. Razavi, [16] T. Speer, F.O. Owala, E.W. Holy, S. Zewinger, F.L. Frenzel, B.E. Sta S. Triems, H. Cvija, L. Roher, S. Seiler, G.H. Heine, J. Jankowski, G.G. Camici, A. Akhmedov, D. Fliser, T.F. Lüscher, F.C. 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Laufs, Alterations in cholesterol homeostasis are H.J. Sch€ afers, M. Bo associated with coronary artery disease in patients with aortic stenosis, Coron. Artery Dis. 20 (2009) 376e382. [25] A.A. Saheed, G. Genove, T. Li, D. Lütjohann, et al., Effects od a disrupted bood-brain barrier on cholesterol homeostasis in the brain, J. Biol. Chem. 289 (2014) 23712e23722, http://dx.doi.org/10.1074/jbc.M114556159 online before print. €tt, A. Luister, C. Husche, et al., The relationsships of phytosterol and [26] H.F. Scho oxyphytosterols in plasma and aortic valve cusps in patients with severe aortic stenosis, Biochem. Biophys. Res. Commun. 446 (2014) 805e810. [27] R.P. Bombo, M.S. Alfonso, R.M. Machado, M.S. Lavrador, V.S. Nunes, E.R. Quintao, M. Koike, S. Catanozi, C.J. Lin, E.R. Nakandakare, A.M. Lottenberg, Dietary phytosterol does not accumulate in the arterial wall and prevents atherosclerosis of LDLr-KO mice, Atherosclerosis 231 (2013) 442e447. € tt, J. Plat, et al., Dietary phytosterol does not accumulate in [28] D. Lütjohann, H.F. Scho the arterial wall and prevents atherosclerosis of LDLr-KO mice, Invited commentary on the paper published by Bombo, Atherosclerosis 233 (2014) 157e159. €rtner, I. Bjo €rkhem, D. Lütjohann, Intestinal cholesterol absorption [29] O. Weinga and cardiovascular risk, J. Am. Coll. Cardiol. 63 (2014) 695e696.
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Vascular effects of oxysterols and oxyphytosterols in apoE / mice €rtner a, b, *, Constanze Husche f, Hans F. Scho € tt f, Timo Speer c, Oliver Weinga b d € hm , Charlotte M. Miller , Florence McCarthy d, Jogchum Plat e, Michael Bo Dieter Lütjohann f, 1, Ulrich Laufs b, 1 €t Oldenburg, Oldenburg, Abteilung für Kardiologie, Klinikum Oldenburg, European Medical School Oldenburg-Groningen, Carl von Ossietzky Universita Germany b €tsklinikum des Saarlandes, Homburg/Saar, Germany Klinik für Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universita c €tsklinikum des Saarlandes, Homburg/Saar, Germany Klinik für Innere Medizin IV, Klinik für Nephrologie und Hypertensiologie, Universita d Department of Chemistry and ABCRF, University College Cork, Western Road, Cork, Ireland e Department of Human Biology, Maastricht University, Maastricht, The Netherlands f Institute for Clinical Chemistry and Clinical Pharmacology, University Clinics Bonn, Germany a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 October 2014 Received in revised form 12 January 2015 Accepted 14 February 2015 Available online 26 February 2015
Objectives: The aim of our study was to investigate vascular effects of oxysterols and oxyphytosterols on reactive oxygen species (ROS), endothelial progenitor cells, endothelial function and atherogenesis. Methods: Male apoE/mice were treated with cholesterol, sitosterol, 7-ßeOHecholesterol, 7-ßeOH esitosterol, or cyclodextrin by daily intraperitoneal application. The respective concentrations in the plasma and in the arterial wall were determined by gas chromatography-flame ionization or mass spectrometry. ROS production was assessed by electron-spin resonance spectroscopy in the aorta, endothelial function of aortic rings and atherosclerosis in the aortic sinus was quantitated after 4 weeks. Results: Compared to vehicle, there was no difference in plasma cholesterol levels and arterial wall concentrations after i.p. application of cholesterol. 7-ßeOHecholesterol concentrations were increased in the plasma (33.7 ± 31.5 vs. 574.57.2 ± 244.92 ng/ml) but not in the arterial wall (60.1 ± 60.1 vs. 59.3 ± 18.2 ng/ mg). Sitosterol (3.39 ± 0.96 vs. 8.16 ± 4.11 mg/dL; 0.08 ± 0.04 vs. 0.16 ± 0.07 mg/mg, respectively) and 7ßeOHesitosterol concentrations (405.1 ± 151.8 vs. 7497 ± 3223 ng/ml; 0.24 ± 0.13 vs. 16.82 ± 11.58 ng/mg, respectively) increased in the plasma and in the aorta. The i.p-application of the non-oxidized cholesterol or sitosterol did not induce an increase of plasma oxysterols or oxyphytosterols concentrations. Oxidative stress in the aorta was increased in 7-ßeOHesitosterol treated mice, but not in mice treated with cholesterol, sitosterol, or 7-ßeOHecholesterol. Moreover, cholesterol, sitosterol, 7-ßeOHecholesterol, and 7-ßeOHesitosterol did not affect endothelial-dependent vasodilation, or early atherosclerosis. Conclusion: Increased oxyphytosterol concentrations in plasma and arterial wall were associated with increased ROS production in aortic tissue, but did not affect endothelial progenitor cells, endothelial function, or early atherosclerosis. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sterols Oxysterols Phytosterols Oxyphytosterols Endothelial function Atherosclerosis
Hypercholesterolemia is a major risk factor for the development of cardiovascular diseases [1]. High dietary intake of cholesterol
Abbreviations: EPC, endothelial progenitor cells; ESR, electron-spin resonance; i.p., intraperitoneal; MNC, mononuclear cells; PSE, plant sterol esters; ROS, reactive oxygen species. * Corresponding author. Abteilung für Kardiologie, Klinikum Oldenburg, Euro€t Rahel pean Medical School Oldenburg-Groningen, Carl von Ossietzky Universita Straus-Strasse 10, 26133 Oldenburg Germany. E-mail address: [email protected] (O. Weing€ artner). 1 UL and DL contributed equally to the manuscript. http://dx.doi.org/10.1016/j.atherosclerosis.2015.02.032 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
together with sedentary habits have been identified as major contributors to atherosclerosis [2]. The latter has long been considered a cholesterol storage disease; however, today atherosclerosis is considered a more complex disease in which both innate and adaptive inflammatory mechanisms as well as interactions between the arterial wall and blood components play a role [3]. Supplementation of foods with plant sterols has been suggested to prevent atherosclerosis because of their cholesterol-lowering effect [4]. Similar to cholesterol, plant sterols can be oxidized and can make an important contribution to the pro-atherogenic effects
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of both cholesterol and cholesterol oxidation oxides in relation to inflammatory disease onset and the development of plaques [5]. Several lines of evidence suggest that oxysterols (oxidized cholesterol) are atherogenic and play a role in the pathogenesis of cardiovascular disease. The presence of one or more unsaturated bonds also makes plant sterols susceptible to oxidation. Only small amounts of oxidized plant sterols (oxyphytosterols) can be found in the diet [6]; nevertheless, relatively high concentrations of oxyphytosterols are present in the serum of sitosterolemia patients and smaller amounts in the plasma of healthy individuals [7e9]. Thus, higher plasma plant sterol concentrations may translate into higher plasma oxyphytosterol concentrations. Intervention trials found conflicting results whether the consumption of a plant sterolenriched margarine for 4 weeks increases plasma 7ßeOHecampesterol concentrations in healthy individuals [10,12]. Differences with regard to the post-prandial state, the genetic background and different duration of the wash out periods may explain the heterogenicity of the data, however, additional research is needed to address the relation of specific diets with plasma oxyphytosterol concentrations. The factors related to the oxidative behavior of plant sterols are unknown; however, it has been suggested for cholesterol that patients characterized by oxidative stress, such as type 2 diabetics and patients with stable coronary artery disease have increased oxysterol concentrations [11], [12]. Since the exact factors predisposing for plant sterol oxidation or oxyphytosterol formation are unknown, it is difficult to experimentally show the effects of endogenously formed oxyphytosterols on vascular wall characteristics and atherosclerotic lesion formation. Moreover, oxyphytosterol concentrations due to endogenous formation are usually rather low which makes long-term interventions necessary. Another approach is to feed oxyphytosterols, as has been done for oxysterols [13]. So far, there have been only limited data regarding the effects of an oxyphytosterol-enriched diet on atherogenesis, which was however suggestive for atherogenic effects and incorporation of oxyphytosterols in the lesion [14]. However, also in this case serum concentrations are low due to the low percent absorption of oxyphytosterol from the diet. Tomoyori et al. showed that oxyphytosterols are indeed absorbed from the diet in low amounts followed by transport to the lymph and accumulation in the serum, liver, and aorta [15]. To overcome these problems, in the present study we therefore synthesised specifically highly purified sitosterol and its respective 7b-hydroxylated metabolite and investigated the i.p. application route on different vascular phenotypes such as oxidative stress (ROS production) in the aorta, endothelial progenitor cells, and on endothelial function and atherogenesis using male apoE/mice. 1. Methods
unless otherwise stated. The starting materials cholesterol (>95%) and stigmasterol (>95%) were both purchased from Sigma Aldrich. b-Sitosterol (4) was produced in 4 steps (in 40% yield) from Stigmasterol (3) and was converted to 7b-OH b-Sitosterol (6) in 4 steps via acetate formation, allylic oxidation, hydrolysis of the acetate, and stereoselective borohydride-mediated ketone reduction. 7bOH Cholesterol (2) was synthesized in 4 steps from cholesterol (1) via a similar method. For further information refer to Supplementary material. All sterols were diluted in sodium chloride with 30% cyclodextrin and applicated intraperitoneally 1 mg per day per mouse. 1.3. 7-b-Hydroxy-b-Sitosterol (6) mp 137e139 C (from EtOAc/hexane) (Found: C, 79.30; H, 11.43. Calc. for C29H50O2.(½H2O): C, 79.21; H, 11.69%). ymax/cm1 3400, 2959, 2871, 1465, 1384, 1056; dH 0.70e2.32 (47H, m), 3.51e3.58 (1H, m, 3a-H), 3.85 (1H, bd, J ¼ 2.6, H-7), 5.29 (1H, bs, H-6); dC (75.5 MHz) 11.82 (CH3), 11.98 (CH3), 18.84 (CH3), 19.03 (CH3), 19.16 (CH3), 19.81 (CH3), 21.09 (CH2), 23.08 (CH2), 26.13 (CH2), 26.39 (CH2), 28.55 (CH2), 29.16 (CH), 31.59 (CH2), 33.99 (CH2), 36.10 (CH), 36.46 (quaternary C), 36.95 (CH2), 39.57 (CH2), 40.93 (CH), 41.74 (CH2), 42.94 (quaternary C), 45.86 (CH), 48.28 (CH), 55.39 (CH), 55.97 (CH), 71.44 (CH), 73.36 (CH), 125.46 (CH), 143.47 (quaternary C); m/z (ESIþ): 431 [(M þ H)þ]. 1.4. 7-b-Hydroxy-Cholesterol (2) mp 182e183 C (from EtOAc/hexane) ymax/cm1 3400, 2959, 2871, 1465, 1384, 1056; dH 0.70e2.37 (43H, m), 3.50e3.60 (1H, m, 3a-H), 3.85 (1H, bd, J ¼ 2.6, H-7), 5.29 (1H, bs, H-6); dC (75.5 MHz) 11.83 (CH3), 18.78 (CH3), 19.17 (CH3), 21.08 (CH2), 22.57 (CH3), 22.84 (CH3), 23.84 (CH2), 26.39 (CH2), 28.03 (CH), 28.56 (CH2), 31.57 (CH2), 35.74 (CH), 36.20 (CH2), 36.44 (quaternary C), 36.94 (CH2), 39.50 (CH2), 39.56 (CH2), 40.90 (CH), 41.73 (CH2), 42.93 (quaternary C), 48.25 (CH), 55.44 (CH), 55.95 (CH), 71.44 (CH), 73.36 (CH), 125.44 (CH), 143.48 (quaternary C); m/z (ESIþ): 403 [(M þ H)þ]. 1.5. Plasma and arterial wall concentrations of sterols, phytosterols, and their respective oxides After 4 weeks, blood samples were drawn from the abdominal vena cava and the descending aorta was excised on sacrifice. Blood samples were centrifuged immediately and plasma and arterial wall tissue were stored at e 70 C. The sterol content of plasma and arterial wall tissue samples was analyzed by gas liquid chromatography-mass spectrometry with epicoprostanol as the internal standard, the amount of 7beOHecholesterol and 7beOHesitosterol using deuterium labeled internal standards as described previously [12,13,29].
1.1. Animals and diets 1.6. Reactive oxygen species in aortic tissue and the spleen Male apoE/(C57/Bl6 genetic background) mice, 8e12 weeks of age, weighing 20e25 g were purchased from Charles River, Sulzfeld, Germany. The 50 apoE/ mice were randomized to 5 treatment groups, n ¼ 10 per group, treated for 4 weeks. All groups were fed a “Western-type” diet (40 kcal% butterfat, 0.15% (w/w) cholesterol). Animal experiments were performed in accordance with the German animal protection law. Over the 4 week study period mice had ad libitum access to water and chow. Diets were prepared by the SNIFF Company (Soest, Germany). 1.2. Cholesterol, oxysterols, plant sterols, and oxyphytosterols All commercial reagents were used without further purification
The production of ROS in aortic tissue was analyzed by ESR spectroscopy as described previously [16]. Briefly, aortic rings were incubated in Krebs HEPES buffer containing 25 mmol/L deferoxamine (Noxygen) and 55 mmol/L diethyldithiocarbamic acid (DETC, Noxygen) together with the spin probe 1-hydroxy-3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH, 500 mM) for 1 h at 37 C. Afterwards, supernatants were immediately analyzed by ESR spectroscopy. ESR spectra were recorded using a Bruker e-scan spectrometer (Bruker Biospin) with the following settings: center field, 3484.5 g; microwave power, 18.11 mW; modulation amplitude, 2.3 G; sweep time, 5.24 s; field sweep, 16 G. Results were normalized to the dry weight of the aortic rings or the
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cell count. 1.7. Isolation, culture, and functional assessment of endothelial progenitor cells To select for endothelial progenitor cells (EPC), mononuclear cells (MNC) were isolated from spleen homogenates from control mice and each treatment group by Ficoll density gradient centrifugation (Biocoll Separating Solution; Biochrom, Heidelberg, Germany) and cultured on fibronectin-coated culture dishes in endothelial basal medium (Lonza, Wuppertal, Germany) with supplements (1 mg/mL hydrocortisone, 3 mg/mL bovine brain extract, 30 mg/mL gentamicin, 50 mg/mL amphotericin B, 10 mg/mL human endothelial growth factor, and 20% fetal calf serum). EPC were characterized by the expression of surface markers such as Sca-1 and vascular endothelial growth factor receptor2 (VEGFR2; corresponding to human CD34 and KDR respectively). Mouse blood and bone marrow were processed for FACS analysis. The viable lymphocyte population was incubated with Sca-1-flourescein isothiocyanate (FITC; E13e161.7; BD Pharming, Heidelberg, Germany) and VEGFR2 (Flk-1) (Avas12a1, BD Pharming) antibodies conjugated with the corresponding phycoerythrin (PE)- labeled secondary antibody (Sigma-Adrich, Taufkirchen, Germany), Isotypeidentical antibodies (lgG2ak FITC and PE, BD Pharmingen) served as controls in every experiment (BectoneDickinson, Heidelberg, Germany). FACS analysis was performed immediately after the staining using a FACSCalibur instrument (BectoneDickinson) and Cell Quest software version 6.0 (BD Biosciences, Heidelberg, Germany). The migratory capacity of the EPC was determined in modified Boyden chamber assays [17]. For each animal, 4 106 MNC were plated on fibronectin-coated six-well culture dishes and cultured for 4 days. Culture medium was removed, cells were harvested and suspended in EBM without supplements and counted under a light microscope, followed by transfer of 1 105 cells to a migration chamber (HTS Fluoroblock, 8 mm pore size, BD Biosciences). The chambers were then placed in a 24-well plate containing EBM without supplements and 100 ng$mL1 SDF-1 to induce migration (R&D Systems). After incubation at 37 C for 24 h, the filters were carefully washed, cells fixed and incubated with diLDL as described above. DiLDL-positive CAC that had migrated to the lower surface of the filter in response to SDF-1 were quantified using fluorescence microscopy (400 magnification) by an observer blinded to the study as described previously [17]. 1.8. Endothelial function After excision of the descending aorta, the vessel was immersed in chilled buffer (pH 7.4) containing NaCl, 118.0 mmol/L; CaCl2, 2.5 mmol/L; KCl, 4.73 mmol/L; MgCl2, 1.2 mmol/L; KH2PO4, 1.2 mmol/L; NaHCO3, 25 mmol/L; Na EDTA, 0.026 mmol/L; and D(þ) glucose, 5.5 mmol/L. Adventitial tissue was carefully removed, and 2 mm rings were mounted in organ baths filled with the above described buffer (37 C; continuously aerated with 95% O2/5% CO2) and were attached to a force transducer, and isometric tension was recorded. The vessel segments were gradually stretched over 60 min to a resting tension of 10 mN, which was maintained throughout the experiment, and were allowed to equilibrate for another 30 min. Drugs were added in increasing concentrations to obtain cumulative concentrationeresponse curves: KCl, 20 and 40 mmol/L; phenylephrine, 1 nmol to 10 mmol/L; carbachol, 10 nmol to 100 mmol/L; and nitroglycerin 1 nmol/L to 10 mmol/L. The drug concentration was increased when vasoconstriction or relaxation was completed. Drugs were washed out before the next substance was added, as described previously [18].
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1.9. Analysis of atherosclerotic lesions Hearts of ApoE/ mice with the ascending aorta were embedded in Tissue Tek O.C.T. (Miles), snap frozen, and stored at 80 C. Samples were sectioned on a Leica cryostat (10 mm), starting at the apex and progressing through the aortic valve into the ascending aorta, and were placed on slides. At least 25 consecutive sections per animal were used for analysis. For the detection of atherosclerotic lesions, the sections were fixed with 3.7% formaldehyde for 1 h, rinsed with deionized water, stained with oil red O working solution (0.5%) for 30 min, and then rinsed again. For morphometric analysis, additional hematoxylin staining was performed according to standard protocols. Picrosirius red solution was used to stain collagen fibers. All sections were examined under a Nikon E 600 microscope. Lucia Measurement Version 4.6 software was used to measure the area of histological sections. Two observers independently and blindly performed all measurements and graded standard and immunostaining as described [19]. 1.10. Statistics Data are reported as the mean ± SEM. Differences between 2 experimental groups were tested by 2-tailed Student t tests or by analysis of variance, followed by the application of Bonferroni test when means from 3 or more groups were compared. For quantification of correlations Pearson's correlations coefficient was used. Pvalues less than 0.05 were considered statistically significant. All statistical tests were performed using SPSS software. 2. Results 2.1. Effects on plasma concentrations of cholesterol, plant sterols, and their respective oxides Five groups of apoE/mice were each fed western-type diets for 4 weeks and then cyclodextrin (control) or the respective sterols were delivered by i.p. application. The average cholesterol level in control animals was 417.2 ± 122.1 mg/dL and did not significantly change after the i.p. application of cholesterol (366.3 ± 60.4 mg/dL). Similarly, the i.p.-application of sitosterol (294.0 ± 85.3 mg/dL), 7ßeOHecholesterol (357.7 ± 93.8 mg/dL) or 7-ßeOHesitosterol (450.7 ± 119.1 mg/dL) did not significantly alter plasma cholesterol levels (Fig. 1A). The i.p.-application of sitosterol (8.16 ± 4.11 vs. 3.39 ± 0.96 mg/dL) (Fig. 1B), 7-ßeOHecholesterol (574.57.2 ± 244.92 vs. 33.7 ± 31.5 ng/ml) (Fig. 1C), and 7ßeOHesitosterol (7497 ± 3223 vs. 405.1 ± 151.8 ng/ml) (Fig. 1D) increased the respective plasma levels compared with controls. Importantly, the i.p-application of the non-oxidized cholesterol or sitosterol did not induce an increase in plasma oxysterol (Fig. 1C) or oxyphytosterol (Fig. 1D) concentrations, respectively, indicative for absence of endogenous substrate oxidation. 2.2. Effects on arterial tissue wall concentrations of sterols, phytosterols, and their respective oxides The average cholesterol concentration in the arterial wall in control animals was 5.86 ± 1.93 mg/mg and did not change after the i.p. application of cholesterol (4.14 ± 0.78 mg/mg, Fig. 2A). The concentrations of sitosterol (0.16 ± 0.07 vs. 0.08 ± 0.04 mg/mg) (Fig. 2B) and 7-ßeOHesitosterol (16.82 ± 11.58 vs. 0.24 ± 0.13 ng/ mg) (Fig. 2C) in the arterial wall increased after i.p. application. However, despite the observed increase in plasma concentrations, i.p. application of 7-ßeOHecholesterol had no effect on the respective arterial wall concentration (59.3 ± 18.2 vs.
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Fig. 1. Effects of i.p. application of cholesterol, sitosterol, 7-betaeOHecholesterol, and 7-betaeOHesitosterol on serum levels of cholesterol (A), sitosterol (B), 7betaeOHecholesterol (C), and 7-betaeOHesitosterol (D). *P < 0.05 for sitosterol vs. control; #P, 0.05 for 7-betaeOHecholesterol vs. control; &P, 0.05 for 7-betaeOHesitosterol vs. control.
60.1 ± 60.1 ng/mg). 2.3. Effects on reactive oxygen species in the aortic wall Electron-spin resonance (ESR) spectroscopy demonstrated that oxidative stress (ROS production) in the aorta was increased only in 7-ßeOHesitosterol treated mice (157.7 ± 48.9% compared to control), but not in mice treated with cholesterol (91.9 ± 67.5%), sitosterol (106.4 ± 12.5%), or 7-ßeOHecholesterol (109.0 ± 52.1%) (Fig. 3). 2.4. Effects on endothelial progenitor cell quantification and migration The EPC number and function were determined by DiLDL-lectin staining and a modified Boyden chamber assay. The EPC number did not show a significant difference compared to control animals in any of the four experimental groups (control: 58 ± 22; sitosterol: 52 ± 13; 7-ßeOHecholesterol: 52 ± 28; 7-ßeOHesitosterol: 54 ± 16 cells/microscopic field). There was no difference in the migratory activity of EPCs compared to control animals (control: 41 ± 19; cholesterol: 62 ± 58; sitosterol: 35 ± 26; 7ßeOHecholesterol: 34 ± 16; 7-ßeOHesitosterol: 50 ± 30 migrated EPCs per 105). 2.5. Effects on endothelial dysfunction Despite the changes in plasma and aortic tissue sterol concentrations, the functional analysis of aortic rings showed no difference in endothelial-dependent vasorelaxation after i.p. application of
any of the four experimental sterols compared to the control group. Moreover, endothelial-independent vasorelaxation in response to nitroglycerin was equal in all groups (Fig. 4A and B). 2.6. Effects on atherosclerotic lesion development After 4 weeks of i.p. application of specific sterols, the mice were sacrificed, the aorta was excised, and the aortic lesion area was quantified by histomorphometric analysis. Early atherosclerotic lesion formation was similar between controls (17.2 ± 8.5) and sitosterol treated mice (17.0 ± 9.5%) and showed no difference after i.p. application of cholesterol (14.5 ± 9.1%), 7-ßeOHecholesterol (7.9 ± 4.5%) and 7-ßeOHesitosterol (10.1 ± 6.4%) (Fig. 5A and B). 3. Discussion In this study, we investigated the effects of oxidized cholesterol and oxidized sitosterol in mice. The major novel finding in this study is that the i.p. application of plant sterols or their oxidation products result in elevated plasma levels and increases sterol concentrations in the arterial wall. Interestingly, application of nonoxidized cholesterol or sitosterol increases their respective plasma concentrations but does not give rise to increased oxysterol or oxyphytosterol concentrations suggesting that endogenous oxidation does not play a major role in this context. Finally, only the elevation of 7-betaeOHesitosterol resulted in increased ROS production in the arterial wall, but this did not affect endothelial cell quantification, endothelial cell migration, endothelial function, and early atherosclerotic lesion development. Our group previously observed that a diet supplemented with
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Fig. 2. Effects of i.p.-application of cholesterol (A), sitosterol (B), 7-betaeOHecholesterol (C), and 7-betaeOHesitosterol (D) on arterial wall concentrations. *P < 0.05 for sitosterol vs. control; #P, 0.05 for 7-betaeOHesitosterol vs. control.
Fig. 3. Effects of i.p.-application of cholesterol, sitosterol, 7-betaeOHecholesterol, and 7-betaeOHesitosterol on oxidative stress (ROS). *P < 0.05 for 7-betaeOHesitosterol vs. control.
plant sterols (equivalent to a commercially available spread) induced endothelial dysfunction and led to an increase in ischemic stroke size in wild-type-mice [19,20]. Moreover, we observed that inhibition of cholesterol absorption by diet supplementation with plant sterols was associated with twice the amount of atherosclerotic lesion formation compared with ezetimibe treatment (a drug that reduces both plasma cholesterol and plant sterol levels), despite similar plasma cholesterol levels in apoE/mice [19]. Thus, our previous studies identified a positive correlation between plasma plant sterol concentrations, impaired endothelial function, and the extent of atherosclerotic lesions. In this study, however, there was no negative effect on endothelial function or early atherosclerotic lesion
development. It can be speculated that bypassing the intestine through i.p. application might exert different effects without a negative impact on the vasculature. However, sitosterol concentrations in the plasma in the current model were increased 2-fold, which is comparable to our previous studies [19,21]. Theoretically it is possible that the lipoprotein fraction responsible for transport of plant sterols through the circulation is different after feeding and i.p-application. Moreover, it is important to take into account that lipoprotein transport and plant sterol transport in apoE/ mice differs from the human situation. In the experimental model plant sterols are injected i.p. and the details of their route of entering the circulation are not fully known compared to diet-derived plant sterols that enter the circulation via chylomicrons. Lipoprotein transport and plant sterol transport in apoE/mice differs from the human situation. However, the potential impact related to the route of entrance is independent from the mouse model used but might cause differences in outcome in humans after dietary consumption. However, since this was not evaluated in our experiments, this needs to be studied in more detail in future studies. Another difference to the first study is the shorter time period of sterol exposure. In our previous studies, we investigated dietary plant sterol exposure up to six months [19]. Over this extended period of time, control mice developed atherosclerotic lesions that comprised more than 50% of the lumen in the aortic sinus in apoE/mice. In the current study, a short term 4-week sterol exposure in apoE/mice resulted in only a 17% atherosclerotic lesion area; thus, only the development of early atherosclerotic lesions could be investigated. Data from clinical studies in which a possible pro-atherogenic effect of phytosterols was assessed underscore the hypothesis that time is a crucial factor in the
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Fig. 4. Effects of i.p. application of cholesterol, sitosterol, 7-betaeOHecholesterol, and 7-betaeOHesitosterol on endothelial function. The functional performance of isolated aortic segments was assessed in organ chamber experiments. (A) Endothelium-dependent vasorelaxation induced by carbachol and (B) endothelial-independent vasorelaxation induced by nitroglycerin (NTG), expressed as a percent of the maximal phenylephrine-induced vasoconstriction. Values are mean ± SEM (n ¼ 10 per group).
Fig. 5. Atherosclerotic lesion formation in the aortic sinus of apoE/mice after i.p. application of cholesterol, sitosterol, 7-betaeOHecholesterol, and 7-betaeOHesitosterol for 4 weeks. (A) Representative examples (oil-red-O). (B) Values are mean ± SEM.
assessment. In a young, healthy cohort with low cardiovascular risk and an average age of 40 years, there was no relation between cardiovascular risk and plant sterol plasma levels [22]; however, there appears to be a positive relation between plant sterol plasma levels and an increased cardiovascular risk in older populations with a higher cardiovascular risk and a longer lifetime exposure to circulating plant sterols [19,23,24]. Finally, in the current study, the effects specifically for this study synthesized highly purified cholesterol, sitosterol, and their respective oxides were in focus. In the previous experimental studies, the dietary supplement included a variety of phytosterols and oxyphytosterols that resembled what is currently available for human consumption. Therefore, it might be speculated that other plant sterols and their oxides not tested in the current experiment exert vascular effects. It seems appropriate to further investigate also other plant sterols and their respective oxides over a longer period of time in this model. Our group previously showed that diet supplementation with plant sterols in humans leads to increased plant sterol
concentrations in plasma and aortic valve cusps [20]. Moreover, it has been demonstrated that phytosterols cross the bloodebrain barrier and are incorporated in the central nervous system and various other organ systems such as the liver in both wild-type and apoE/mice [20,25]. Therefore, another aim of this study was to determine whether a short time period of plant sterol exposure results in an increase of plant sterol concentrations in the arterial wall. Results of this study demonstrate for the first time that plant sterols are quickly incorporated in the arterial wall after only a 4week time period in apoE/mice. Recently we investigated in a clinical study in patients with severe aortic stenosis the relationships of phytosterols and oxyphytosterols in plasma and in aortic valve cusps [26]. Interestingly, in this clinical study the plant sterol sitosterol and its oxide 7-ßeOHesitosterol showed only a weak correlation between plasma and aortic valve cusps. The results of the current experimental findings, however, demonstrate a strong correlation for 7-b-OH sitosterol between plasma and the arterial wall. In our opinion these divergent results are due to different pathophysiological processes in atherosclerotic vascular disease and the
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evolution of aortic valve stenosis. Supposedly, the major calcification of cardiovascular tissue in aortic valve stenosis leads to differences in sterol composition compared to atherosclerotic lesion formation. These data are not in line with a previously published study, in which the authors did not find increased phytosterol concentrations in the arterial wall after phytosterol enriched diet supplementation in apoE/mice [27].This study has been criticized [28] and is not in line with our previous findings in clinical studies [19]. Interestingly, only the i.p. application of 7-ßeOHestitosterol resulted in increased plasma levels, a 70-fold increase of vascular sterol deposition, and also increased oxidative stress in the vessel wall. Sitosterol and 7-ßeOHecholesterol did not affect arterial wall ROS production after a 4-week period. A possible explanation is that 7-ßeOHesitosterol cannot be broken down in the vessel wall and leads to a more pronounced sterol deposition. However the increased oxidative stress did not translate into functional consequences like endothelial function and aortic lesion development. Future studies will have to confirm these findings. We also could not detect any influence of the i.p. application of sterols on endothelial progenitor cell quantification and migration. As argued above, this could be due to the sterols investigated or the short time period of the experiment. Future studies will have to elucidate this finding in more detail. In summary, the i.p. application of cholesterol, sitosterol, and their oxides has no effect on plasma cholesterol concentrations in apoE/mice. After i.p. application of sitosterol and 7ßeOHecholesterol, mice show increased plasma levels of the injected sterols. Application of sitosterol and 7-ßeOHesitosterol increased arterial wall deposition and leads to a 70-fold increase of 7-ßeOHesitosterol in the arterial wall, which increased oxidative stress in the arterial wall, however without functional consequences on endothelial function and aortic lesion development. Finally, and probably most importantly, the i.p-application of the non-oxidized sitosterol did not induce an increase in plasma oxyphytosterol concentrations indicative for absence of endogenous substrate oxidation. Conflicts of interest Dieter Lütjohann and Oliver Weing€ artner received funding for analytical contract work by Unilever, Vlaardingen, The Netherlands. €rtner and Ulrich Laufs received lecture fees and Oliver Weinga consulting fees from Merck. Acknowledgments We thank Ellen Becker, Jennifer Kieffer, Silvia Friedrichs and Simone J€ ager for excellent technical assistance. This study was funded by the Universit€ at des Saarlandes and in part by the Dutch Organization for Scientific Research (NOW TOP grant No. 91208006). Sterol and oxysterol analytical work was supported from the EUFP7 project Lipididiet (FP7/2007-2013) under grant agreement n 211696. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2015.02.032. References [1] N.J. Wald, M.R. Law, Serum cholesterol and ischaemic heart disease, Atherosclerosis 118 (Suppl. S1eS5) (1995). [2] A.H. Lichtenstein, L.J. Appel, M. Brands, et al., Summary of American heart association diet and lifestyle recommendations revision 2006, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 2186e2191.
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