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Cardiovascular Research 2005 66(1):179-185; doi:10.1016/j.cardiores.2005.01.001
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Copyright © 2005, European Society of Cardiology

Tumor necrosis factor-{alpha} promotes atherosclerotic lesion progression in APOE*3-leiden transgenic mice

Lianne S.M. Boestena,d,*, A. Susanne M. Zadelaarb,d, Anita van Nieuwkoopa,d, Marion J.J. Gijbelse,f, Menno P.J. de Winthere, Louis M. Havekesb,d and Bart J.M. van Vlijmenc

aDepartment of General Internal Medicine, Leiden University Medical Center, The Netherlands
bDepartment of Cardiology, Leiden University Medical Center, The Netherlands
cDepartment of Hematology, Leiden University Medical Center, The Netherlands
dTNO-PG/Gaubius Laboratory, Leiden, The Netherlands
eDepartment of Molecular Genetics, Cardiovascular Research Institute, Maastricht University, Maastricht, The Netherlands
fDepartment of Pathology, Maastricht University, Maastricht, The Netherlands

* Corresponding author. Department of General Internal Medicine, Leiden University Medical Center c/o TNO Prevention and Health, Gaubius Laboratory, Zernikedreef 9, P.O. Box 2215, 2301 CE Leiden, The Netherlands. Tel.: +31 71 5181421; fax: +31 71 5181904. Email address: lsm.boesten{at}pg.tno.nl

Received 3 November 2004; revised 24 December 2004; accepted 3 January 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Tumor necrosis factor-{alpha} (TNF{alpha}) is a pleiotropic cytokine exerting both inflammatory and cell death modulatory activity, and is thought to play a role in the pathogenesis of atherosclerosis. Studies in mice indicated that TNF{alpha} affects atherosclerosis minimally or not under conditions that allow fatty streak formation. Here, we examined the possible role of TNF{alpha} in advanced and complex atherosclerotic lesions.

Methods and results: To induce atherosclerosis, TNF{alpha}-deficient (Tnf–/–) APOE*3-Leiden and control APOE*3-Leiden only mice were fed a cholesterol-rich diet. Comparable levels of plasma cholesterol and triglycerides and the systemic inflammatory parameters, serum amyloid A and soluble intercellular adhesion molecule-1 were found in APOE*3-LeidenTnf–/– and control mice. Although absence of TNF{alpha} did not affect the quantitative area of atherosclerosis, APOE*3-LeidenTnf–/– mice had a higher relative number of early lesions (46.1% vs. 21.4%) and a lower relative number of advanced lesions (53.9% vs. 78.6%, P=0.04). In addition, the advanced lesions in APOE*3-LeidenTnf–/– mice showed less necrosis (9.9 ± 12.1% vs. 23.4 ± 19.3% of total lesion area, P=0.04) and an increase in apoptosis (1.5 ± 1.5% vs. 0.4 ± 0.6% of total nuclei, P=0.03).

Conclusions: Our data indicate that TNF{alpha} stimulates the formation of lesions towards an advanced phenotype, with more lesion necrosis and a lower incidence of apoptosis.

KEYWORDS Atherosclerosis; Cytokines; Apoptosis; Necrosis; Transgenic animal models


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Inflammatory processes are involved in all stages of atherosclerotic lesion development [1,2]. Cytokine-mediated pro-inflammatory responses are thought to positively contribute to the atherogenic process. Tumor Necrosis Factor-{alpha} (TNF{alpha}) is a central mediator of inflammatory reactions [3]. TNF{alpha} is a member of the TNF ligand family and binding to its receptors TNFR1 (p55) and TNFR2 (p75) leads to activation of downstream targets [4]. Whereas binding of TNF{alpha} to TNFR1 activates responses associated with induction of adhesion molecule expression [5], apoptosis [6], and resistance to bacterial infection [7,8], binding to TNFR2 activates induction of T cell proliferation [9], induction of TNF{alpha}-mediated skin tissue necrosis [10], and modulation of TNF{alpha}-mediated pulmonary inflammation [11].

Although TNF{alpha} and its receptors are thought to be considerably important in a number of biological activities relevant to atherosclerosis, its function in atherogenesis remains unclear. The TNF{alpha} gene has a number of polymorphisms, some of which affect transcription and secretion [12]. These polymorphisms have been associated with a number of infectious, inflammatory and immune diseases including atherosclerosis and coronary artery disease (CHD) [12]. However, human association studies are controversial varying from no [12–14], a weak [15] or a strong [16] association between TNF{alpha} polymorphisms and CHD. Moreover, studies on the role of TNF{alpha} in atherosclerosis using several transgenic or knock out mouse models also yielded controversial results. TNF{alpha} ligand deficiency on a wild type C57BL/6 background did not affect early lesion development [17]. However, on the same background, TNF receptor 1 deficiency did affect atherosclerosis formation, resulting in enhanced (early) lesion formation. This unexpected result was attributed to increased macrophage scavenger receptor activity and consequent increased uptake of atherogenic lipoproteins [18]. Very recently, as a part of mouse studies on a non-cleavable transmembrane form of TNF{alpha}, TNF{alpha}-deficiency on a C57BL/6 background was demonstrated to reduce atherosclerosis [19]. This effect however coincided with a significant decrease in plasma VLDL and increase in plasma HDL levels. Hence, a direct anti-atherosclerotic effect of TNF{alpha} deficiency could not be concluded from this experiment. Overall, the above recombinant mouse studies suggest that TNF{alpha} may have subtle pro-atherogenic properties, possibly related to an unexpected TNF{alpha}-mediated effect on atherogenic lipoproteins, at least in a setting of a C57BL/6 background.

Variable results were also obtained upon (pharmacological) modulation of TNF{alpha} expression. Specific immunization against TNF{alpha} was not effective in preventing the formation of advanced lesions in apoE-deficient mice [20]. Capturing TNF{alpha} using a TNF{alpha} receptor homologue had only a modest and gender-dependent inhibitory effect on the formation of early lesions in the apoE-deficient setting [21]. On the contrary thalidomide, a TNF{alpha} production inhibitor, was capable of inhibiting the early atherogenesis in apoE-deficient mice [22].

The majority of these mouse studies focuses on the role of TNF{alpha} in early lesion development in relatively atherosclerosis-resistant C57BL/6 mice [17–19] and young apoE-deficient mice [20,22] that display only minimal advanced lesion formation. Inflammation and cell death are important processes in the development and transition towards advanced and complex atherosclerotic lesions and may be modulated by TNF{alpha}. Therefore, we examined the role TNF{alpha} under conditions of advanced lesion formation. To this end, we crossbred TNF{alpha} deficient (Tnf–/–) mice with APOE*3-Leiden transgenic mice. APOE*3-Leiden is a mutant form of apoE, characterized by a 7-amino acid tandem repeat of residues 120–126 and yields a mature protein of 306 amino acid residues. Family studies have demonstrated that this mutation is associated with a dominantly inherited form of familial dysbetalipoproteinemia (FD) [23–25]. APOE*3-Leiden transgenic mice have a lipoprotein profile that is very similar to the profile of patients with FD in which the elevated plasma cholesterol and triglyceride levels are mainly confined to the VLDL/LDL-sized lipoprotein fractions. Therefore, APOE*3-Leiden transgenic mice are an established model for hyperlipidemia and atherosclerosis [26]. We investigated the effect of TNF{alpha} on advanced lesion formation and on lesion composition. Our data indicate that TNF{alpha}, independent of the atherogenic lipoprotein profile, stimulates lesional necrosis, decreases the incidence of apoptosis and the progression of lesions towards a more advanced phenotype.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Mice and diet
The experimental animals were obtained by crossbreeding Tnf–/– mice [27] with APOE*3-Leiden transgenic mice [28]. Offspring was analyzed for their TNF{alpha} status using polymerase chain reaction (PCR) analysis and presence of the human APOE*3-Leiden transgene by ELISA [29].

For experiments, 8 weeks old female APOE*3-LeidenTnf–/– and littermate control APOE*3-LeidenTnf+/+ mice were used. Mice (n=31) were fed a semi-synthetic cholesterol-rich diet composed essentially according to Nishina et al. [30]. supplemented with cocoa butter (15%, by weight) and cholesterol (1%, by weight), without dietary cholate. The diet was purchased from Hope Farms, Woerden, the Netherlands. Mice were bred and housed under specific pathogen-free conditions and given food and water ad libitum. All animal work was approved by institutional regulatory authority and carried out in compliance with guidelines issued by the Dutch government. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Blood sampling and analysis
Blood samples were collected in EDTA-coated vials (Sarstedt, Nümbrecht, Germany) by bleeding from the tail vein. Plasma cholesterol and triglyceride levels were measured enzymatically using commercially available kits (Roche Diagnostics GmbH, Mannheim, Germany).

Total blood leukocyte (CD45+), T-cell (CD3+), B-cell (CD19+) and monocyte/granulocyte (CD11b+) numbers were determined by FACS analysis (FACSCalibur, BD Biosciences, California, USA) of whole blood using a PerCP-CY5.5-conjugated rat anti-mouse CD45 monoclonal antibody, a fluorescein isothiocyanate (FITC)-conjugated hamster anti-mouse CD3 monoclonal antibody, a R-phycoerythrin (R-PE) conjugated rat anti-mouse CD19 monoclonal antibody, and an allophycocyanin (APC)-conjugated rat anti-mouse CD11b monoclonal antibody, respectively following standard protocol (TruCOUNT, BD Biosciences, California, USA).

Serum Amyloid A (SAA, acute phase protein) ELISA (BioSource International, Inc., Camarillo, CA) and Mouse soluble Intercellular Adhesion Molecule-1 (sICAM-1, marker for endothelial cell activation) ELISA (Pierce Biotechnology, Rockford, IL) were performed on plasma samples according to standard protocols [31].

2.3. Atherosclerosis analysis
After 20 weeks of a cholesterol-rich diet, mice were sacrificed, heart and aorta were isolated and embedded in tissue tek (Sakura Finetek Europe BV, Zoeterwoude, The Netherlands) and stored at –80 °C. From the entire aortic root area of the heart, four 7 µm cross-sections with an interval of 42 µm were used for quantification of atherosclerotic lesion area [32]. Sections were routinely stained with toluidin blue for morphometric analysis, characterization and categorization of the lesions, and with Sirius red for quantification of collagen area. Lesion area was determined using Leica Qwin image software (EIS, Asbury, NJ). All analyses were performed blindly without prior knowledge of the genotype.

Atherosclerotic lesions were categorized on severity. Two types of categories were discerned: (1) early lesions were fatty streaks containing only foam cells (types I–II lesions) and (2) advanced lesions showed foam cells in the media and presence of fibrosis, cholesterol clefts, mineralization and/or necrosis covered by a collagenous cap (types III–V lesions) [29]. The number observed in each category is expressed as a percentage of the total number of lesions observed within one group of mice (APOE*3-LeidenTnf–/– or APOE*3-LeidenTnf+/+ control group).

Sections were stained for macrophages using rat FA11 antibody to mouse macrophages (a kind gift from S. Gordon, Oxford University, UK), for smooth muscle cells using a monoclonal {alpha}-smooth muscle cell actin antibody (Sigma-Aldrich, St. Louis, USA), for T-cells using KT3 rat antibody (a kind gift from G. Kraal, VUMC, The Netherlands), and areas were quantified as described previously [33,34]. For the detailed phenotypic sub-analysis of the advanced lesions (macrophage area, SMC area, collagen area and the number of T-cells) only those mice were included that displayed advanced atherosclerotic lesions (APOE*3-LeidenTnf+/+ n=13, APOE*3-LeidenTnf–/– n=14).

2.4. Quantification of lesion necrosis and apoptosis
During pathological examination of the lesions, lesional necrosis was defined by the presence of pyknosis, karyorrhexis, or complete absence of nuclei, as described before [33]. Necrosis area was measured using the method for total lesion area measurements, as described above. Apoptosis was quantified using the TUNEL technique [35]. Only those TUNEL-positive nuclei were included that displayed morphological features of apoptosis including cell shrinkage, aggregation of chromatin into dense masses, and nuclear fragmentation. For the detailed phenotypic sub-analysis of the advanced lesions, on lesion necrosis and apoptosis, only those mice were included that displayed advanced atherosclerotic lesions (APOE*3-LeidenTnf+/+ n=13, APOE*3-LeidenTnf–/– n=14).

2.5. Statistical analysis
Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, California, USA). Data were tested for normality using the Kolmogorov–Smirnov test. All data following normality were tested using Welch's corrected t-test. Data are expressed as mean ± SD, unless stated otherwise. P-value <0.05 was regarded as significant. Frequency data for lesion categorization were compared by means of the Fisher's exact test.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. General
Female APOE*3-LeidenTnf–/– (n=18) mice and APOE*3-LeidenTnf+/+(n=13) littermate controls were fed a cholesterol-rich diet. The mice appeared healthy and displayed no signs of abnormalities. After 20 weeks feeding the cholesterol-rich diet, APOE*3-LeidenTnf–/– mice were significantly lower in body weight as compared to APOE*3-LeidenTnf+/+ control mice (Table 1).


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  		Table 1 Characteristics of female APOE*3-LeidenTnf+/+ and APOE*3-LeidenTnf–/– mice after 20 weeks feeding a cholesterol-rich diet

 
Following cholesterol-rich diet feeding, both APOE*3-LeidenTnf–/– and control mice had comparable levels of plasma cholesterol and plasma triglyceride levels (Table 1). As determined by FPLC analysis on pooled plasma, lipoprotein distribution did not differ between APOE*3-LeidenTnf–/– and APOE*3-LeidenTnf+/+ control mice (Fig. 1).


Figure 1
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  		Fig. 1 Plasma lipoprotein profile after size fractionation of pooled plasma samples of APOE*3-LeidenTnf+/+ (closed symbols) and APOE*3-LeidenTnf–/– (open symbols) after 20 weeks of feeding the cholesterol-rich diet.

 
Analysis of CD45+ blood leukocytes showed that absence of TNF{alpha} resulted in an approximate doubling of circulating CD3+ (P=0.01) and CD19+ (P=0.06) cells (T- and B-cells, respectively), leaving the CD11b+ cell population (monocytes/granulocytes) unaffected (Table 1).

Plasma levels of serum amyloid A (SAA) and soluble intercellular adhesion molecule-1 (sICAM-1) were comparable in APOE*3-LeidenTnf–/– and APOE*3-LeidenTnf+/+ control mice indicating no difference in inflammation (SAA) or endothelial cell activation (sICAM) between the two groups (Table 1).

3.2. Atherosclerosis analysis
Mice fed the cholesterol-rich diet for 20 weeks were sacrificed for collection of heart and aorta. Histopathological analysis of atherosclerotic lesions of the aortic valve area in both APOE*3-LeidenTnf–/– and control mice revealed that the majority of the lesions were fibrous plaques with a lipid core and a cap covering necrotic material (pyknosis, karyorrhexis, or complete absence of nuclei), cholesterol clefts and extra-cellular lipids. As determined by computer-assisted morphometry, mean total lesion area did not differ significantly between APOE*3-LeidenTnf+/+ and APOE*3-LeidenTnf–/– mice (5.8 ± 2.8 and 5.1 ± 4.5 x 104 µm2, respectively, P=0.64, Fig. 2). While a difference in total lesion area was absent, lesion categorization (early vs. advanced) revealed a difference between APOE*3-LeidenTnf–/– and control mice (Fig. 3). APOE*3-LeidenTnf–/– mice had a significant higher incidence of early lesions (46.1% vs. 21.4%) and a significant decreased incidence of advanced lesions (53.9% vs. 78.6%) as compared to APOE*3-LeidenTnf+/+ controls (P=0.04).


Figure 2
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  		Fig. 2 After 20 weeks of feeding the cholesterol-rich diet, the extent of atherosclerosis in APOE*3-LeidenTnf+/+ (closed symbols, n=13) and APOE*3-LeidenTnf–/– (open symbols, n=18) mice was quantified at the level of the aortic valves. Each data point represents the lesion area per mouse. Line represents mean area for each group.

 

Figure 3
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  		Fig. 3 Lesion categorization of APOE*3-LeidenTnf+/+ (black bars) and APOE*3-LeidenTnf–/– (white bars) mice. Lesions were categorized according to severity and are shown as percentage of the total number of lesions present. Frequency data were compared by means of the Fisher's exact test, P=0.04.

 
A more detailed phenotypic sub-analysis analysis of the advanced lesions only, did not yield any effect of TNF{alpha} status on lesion macrophage area, lesion smooth muscle cell area or collagen area. In addition, the number of lesional T cells did not differ between the groups (Table 2). However, pathological analysis revealed that advanced lesions in APOE*3-LeidenTnf–/– mice had smaller areas of pyknosis, karyorrhexis, or complete absence of nuclei as compared to advanced lesions in control mice, suggesting that the TNF{alpha}-status affects lesional necrosis. The early atherosclerotic lesions did not contain any necrotic area in both groups. Computer-assisted morphometric analysis demonstrated that advanced lesions in APOE*3-LeidenTnf–/– mice had significant smaller areas covered by necrosis as compared to advanced lesions in controls (9.9 ± 12.1% vs. 23.4 ± 19.3% of total lesion area, P=0.04, Fig. 4a).


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  		Table 2 Characteristics of advanced atherosclerotic lesions in APOE*3-LeidenTnf+/+ and APOE*3-LeidenTnf–/– mice

 

Figure 4
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  		Fig. 4 Necrosis (a) (P=0.04) and apoptosis (b) (P=0.03) in advanced lesions of APOE*3-LeidenTnf+/+ (black bar, n=13) and APOE*3-LeidenTnf–/– (white bar, n=14). Error bars indicate SEM. Inserts: representative pictures of advanced lesions in APOE*3-LeidenTnf+/+ and APOE*3-LeidenTnf–/– mice. (a) The black dotted line indicates the necrotic area (magn. 40 x, scale bar 100 µm). (b) The arrows indicate TUNEL-positive nuclei. L: lumen, P: plaque, M: media, (magn 100 x, scale bar 100 µm).

 
TUNEL-positive nuclei (apoptotic cells) were only found in advanced lesions of both APOE*3-LeidenTnf–/– and control mice. The location of the TUNEL-positive nuclei (i.e. core or cap) did not differ between APOE*3-LeidenTnf–/– and control mice (data not shown). In addition, the majority of the TUNEL-positive nuclei was located in the core of the advanced lesions, in both APOE*3-LeidenTnf–/– and control mice. Remarkably, the incidence of TUNEL-positive nuclei in advanced lesions of APOE*3-LeidenTnf–/– mice was 3.4-fold higher as observed for lesions in control mice (1.5 ± 1.5% vs. 0.4 ± 0.6% of total number of cells, P=0.03, Fig. 4b). Hence, in APOE*3-LeidenTnf–/– mice a decrease in lesional necrosis coincides with increased apoptosis.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
In the present study we examined the role of TNF{alpha} in the formation of advanced lesions. Using atherosclerosis-susceptible APOE*3-Leiden mice crossbred with TNF{alpha}-deficient mice we found that TNF{alpha} modulates lesional cell death by increasing necrosis and decreasing the incidence of apoptosis. Moreover, TNF{alpha} progresses the lesions towards a more advanced phenotype.

Very recently, Branén et al. also showed that TNF{alpha} stimulates atherosclerosis development using apoE/Tnf knock out mice [36]. ApoE deficiency affects local cholesterol homeostasis in macrophages and inflammatory reactions within the atherosclerotic vessel [37]. In the current study the role of TNF{alpha} was assessed specifically in advanced atherosclerotic lesion development on an APOE*3-Leiden background (with endogenous apoE still present) preventing the possible local effects due to the absence of apoE. Importantly, besides the differences in study-design, both studies demonstrate that TNF{alpha} can be considered a pro-atherogenic cytokine in atherosclerosis development.

Inflammation and cell death are important processes in the development and transition towards advanced and complex atherosclerotic lesions [38–40]. Our observation that TNF{alpha} promotes advanced lesion formation, and on top of that increases the extent of necrosis in these lesions, is in line with the role of TNF{alpha} function in modulating inflammatory processes and cell death [3,4,6–8,10,11].

Studies on human endarterectomy specimens showed that unstable atherosclerotic lesions are characterized by a necrotic core consisting of dead macrophages, macrophage debris and extracellular lipid covered by a fibrocellular cap [41]. Therefore, necrosis is often considered to be a negative risk factor for plaque stability. The observation that TNF{alpha} increases the ratio of necrosis versus apoptosis suggests that TNF{alpha} modulates the lesion towards a more instable phenotype. Moreover, in vitro studies have shown that TNF{alpha} stimulates both macrophages and smooth muscle cells to synthesize matrix proteases [42,43] and in such a way can contribute to plaque instability by degrading the fibrotic cap. Hence, both through increased necrosis and increased matrix protease activation TNF{alpha} may contribute to the formation of instable plaques that are prone to rupture.

The reduction in lesional necrosis upon TNF{alpha} deletion coincided with an increase in lesional apoptosis. TNF{alpha} is known to exert its effector actions, partially, through the activation of the pro-inflammatory transcription factor, NF-{kappa}B [4]. Absence of TNF{alpha} may promote apoptotic cell death via a reduction in NF-{kappa}B activation. Reduced NF-{kappa}B activation does not only lead to a beneficial reduced transcription of pro-inflammatory cytokines and chemokines, but also to a reduction in transcription of anti-apoptotic factors (i.e. Bcl-xL, Bcl-2, IAPs) [44]. Apoptosis is often considered to be beneficial for atherosclerosis, since in contrast to necrosis, it may prevent the release of matrix-degrading enzymes and pro-inflammatory substances from the dying foam cells. Our data suggest that apoptosis forms part of a beneficial process since it coincides with a less advanced lesion phenotype.

Uysal et al. demonstrated that the total weight gain in obese Tnf+/+ mice on a high fat diet was larger than that of obese Tnf–/– mice, which was related to a decrease in fat-pad weight [45]. Also our APOE*3-Leiden mice, fed a cholesterol-rich diet, that additionally contained 15% fat, displayed reduced body weight upon TNF{alpha} deletion (Table 1). Hence, our data support a role for TNF{alpha} in body weight development. Although body weight is not shown to be a predictive parameter for atherosclerosis development in mice [46,47], it remains open to question whether TNF{alpha}-mediated reduction in body weight contributes to the observed TNF{alpha}-mediated reduction in advanced lesion formation.

Upon characterization of the TNF{alpha} deficient mice Marino et al. reported that these mice have no significant abnormalities in the distribution of lymphocytes, granulocytes or monocyte population in thymus, spleen or peripheral blood [48]. Remarkably, we found an increase in circulating T-cells in TNF{alpha} deficient mice, that was independent of the diet and of the APOE*3-Leiden transgene (data not shown). This suggests that TNF{alpha} might play a role in determining circulating T-cells in mice already under basal conditions. In this light it is worthwhile to mention that the higher levels of circulating T-cells did not translate to a higher number of T-cells in the advanced atherosclerotic plaque.

In conclusion, we have demonstrated that TNF{alpha} enhances progression of lesions towards a more advanced phenotype. One may speculate that drugs that inhibit TNF{alpha} expression or capture TNF{alpha} biological action may inhibit advanced lesion formation. Future studies are required to demonstrate whether, next to a reduction in early lesions [21,22], TNF{alpha} can be used as a target to prevent the formation of advanced lesions.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the Netherlands Heart Foundation (NHS 2000.051), the Netherlands Organization for Scientific Research (NWO 902-26-242), M.P.J. de Winther is a NWO-fellow (906-02-075), and B.J.M. van Vlijmen is a fellow of the Royal Netherlands Academy of Arts and Sciences. The authors wish to thank Ingeborg van der Made for her technical assistance.


   Notes
 
Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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