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Original Paper

Lipid Metabolism in Vascular Smooth Muscle Cells Infuenced by HCMV Infection

Li L.a · Li Y.b · Dai Z.b · Liu M.b · Wang B.b · Liu S.b · Wang L.b · Chen L.b · Tan Y.b · Wu G.b

Author affiliations

aDept. of Vasculocardiology, Xiangya Hospital, bDept. of Microbiology, School of Basic Medical Sciences, Central South University, Changsha, Hunan, China

Corresponding Author

Wu Guojun and Tan Yurong

Department of Microbiology, School of Basic Medical Sciences, Central South

University, Changsha 410078, Hunan, (China)

Tel. +8673182355003, E-Mail wuguojun@csu.edu.cn; hope7@126.com

Related Articles for ""

Cell Physiol Biochem 2016;39:1804-1812

Abstract

Background: The present study was designed to observe the infection of human cytomegalovirus (HCMV) to human vascular smooth muscle cells (VSMCs), and the effect of viral infection on lipid metabolism in VSMCs. Methods: The cytopathic effects were observed by inverted microscopy and viral infection were examined by electron microscopy and RT-PCR. The lipid metabolism related gene profiling of VSMCs after HCMV infection was assayed by cDNA assay and the abnormal expression of genes were validated by quantitative RT-PCR. The content of cholesterol in VSMCs after HCMV infection was assayed by cholesterol detection kit. Results: VSMCs showed obvious cytopathic effects after HCMV infection. Intact viral particles could be detected in VSMCs using electron microscope. By use of RT-PCR technology, IE gene of HCMV could be amplified from VSMCs. The expression of cell lipid metabolism related gene profiling showed obvious disorders. The expression levels of HMG-CoA synthase and HMG-CoA reductase after infection increased significantly. The cellular cholesterol content (µmol/106 cells) was significantly higher than that of mock infected group at 72h post infection. Conclusion: HCMV can infect VSMCs and the infection can affect cellular lipid metabolism related gene expression, which get involved in the occurrence and development of atherosclerosis (AS).

© 2016 The Author(s) Published by S. Karger AG, Basel


Keywords

Human cytomegalovirus · AS · Lipid metabolism · Vascular smooth muscle cells ·


Introduction

Atherosclerosis is thickening of arterial wall and narrowing of lumen due to accumulation of fatty substances, blood cells (wbc) and proliferation of intimal-smooth muscle cells creating a fibrofatty plaque [1]. It is a major public health problem in developed and developing countries that may lead to various life-threatening complications such as coronary artery disease, stroke, and peripheral artery disease. Lipid accumulation and migration and proliferation of vascular smooth muscle cells (VSMCs) play key roles in the development of AS. Studies have shown that the VSMCs are highly proliferated in a region prone to atherosclerosis whereas VSMCs are found normal in a region not prone to atherosclerosis [2]. Clinical and epidemiological investigations showed that the majority of atherosclerosis patients have abnormal lipid metabolism such as increased level of cholesterol and triglycerides [3]. Pathological observation of atherosclerotic plaque also confirmed the deposition of lipid in these plaques. Although traditional risk factors include hyperlipidemia, hypertension, diabetes mellitus, smoking, and a positive family history, yet these do not fully explain the extent and severity of the conditions. In recent years, numerous studies have implicated that human cytomegalovirus (HCMV) might play an important role in the pathogenesis of AS. HCMV is a virus belong to β- herpes virus family with common infection in population with more than 60 % of infection rate in developed countries and 80% in developing countries [4]. The incidence of infection can vary but in general 50-90% of the population is infected. In the healthy host, the acute phase of infection is self-limiting and the virus establishes a latent infection that can be reactivated in response to a variety of cellular stresses [5]. The long-term effects of viral persistence are the focus of many studies as HCMV infection has been implicated as a co-factor in the development of cardiovascular disease. The association of HCMV infection with atherogenic lesions has been well documented but viral infection has not been established as a proximal cause of disease. HCMV infection can contribute to the development of atherosclerotic plaques at different stages. First endothelial cells are permissive for HCMV infection, which can lead to the activated state that initiates the inflammatory cascade at the site. It is also possible that latently infected monocytes are recruited to primary lesions and subsequently induced to differentiate, thereby triggering lytic replication. The viral progeny produced could then infect endothelial or smooth muscle cells at the site. Infection of smooth muscle cells has been shown to induce their migration in vitro. Fabricant reported that similar human atherosclerotic pathological lesions in chicken could be induced by avian herpes virus infection, suggesting herpes virus was one of important risk factors for AS [6]. Subsequent epidemiological investigations showed that HCMV is also an important risk factor for AS. Abnormal lipid metabolism and HCMV infection in patients with AS indicates that there may be necessary link between viral infection and the body's lipid metabolism.

Taking the results of these studies into consideration, it necessary for us to explore the relationship between HCMV infection and abnormal lipid metabolism for further evaluation of the potential role of HCMV infection in the development of AS.

Material and Methods

Materials

Human VSMCs were supplied by Modern Analysis and Testing Center of Central South University. Human embryonic lung fibroblasts (HLF), human HCMV strain AD169 were preserved by Department of Microbiology, Central South University. DMEM, DMEM/F12 (1:1) medium were purchased from Hyclone, USA. Fetal bovine serum (FBS) was from GIBCO. LB broth was from Tianhe Co., Ltd, Hangzhou, China. Ampicillin was from Amresco, USA. Plasmid extraction kit and DNA gel extraction kit were supplied by OMEGA, USA; pGM-T vector kit and PCR master mix were from Tiangen (Beijing) Co., Ltd. TRIzol and trypsin were from Life Technologies, USA. cDNA first strand synthesis kit was from Toyobo (Shanghai) biological Technology Co., Ltd. Primers for HMG-CoA-synthetase (HMGCS1), HMG-CoA- reductase (HMGCR) were synthesized by Shanghai Sage Biotechnology Co., Ltd. and primers for HCMV IE were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd.

Methods

HCMV preparation. HLF were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. HLF cells (80% confluent) were infected with 100 µL HCMV for 2 h at 37°C, washed two times, and then was added DMEM containing 3% FBS and observed daily for cytopathic effects. The cells were gently blown and subjected to three successive freeze-thaw cycles after obvious cytopathic effects. Supernatants were harvested and tested for TCID50 followed by centrifugation to remove cell debris. Then the virus was aliquoted and stored at -80°C until use.

Cell culture and HCMV infection. Human VSMCs were cultured in a mixture medium of DMEM/F12 (1:1) adding 100 U/mL penicillin, 100 µg/mL streptomycin, and 12% heat inactivated FBS and incubated at 37°C in 5% CO2. Passages were carried out when the cells grow into a monolayer and generation 3-8 were used in the study. 80% confluent monolayer cultures were infected with HCMV (MOI=1). The virus was allowed to incubate for 2 h at 37°C in serum-free DMEM. Thereafter, non-absorbed virus was removed by washing with DMEM twice, then the cells were cultured in fresh medium with 3% FBS and observed and photographed daily until day 8. VSMCs in 3% FBS DMEM/F12 without HCMV acted as mock-infected group. HCMV infection was verified by electron microscopy (School of Basic Medical School, Central South University) and RT-PCR amplification of HCMV IE gene (5'-GCAGCGGCAGAAGAAGAT-3'and 5'-TGGTCACGGGTGTCTCG-3', 414 bp). The mixture was incubated at 95°C for 2 min and then 30 cycles of 94°C, 30 secs; 52°C, 60 secs; 72°C, 60 secs. PCR products were identified by restriction enzyme and sequence analysis.

cDNA expression profiling. Examination of cDNA profiling was assisted by Shanghai Kang Cheng biological technology limited company. 80% confluent monolayer cultures were infected with HCMV (MOI =1). Then the cells were cultured in fresh medium with 3% FBS and harvested at 24h, 48h and 72 post infection. Total RNA was harvested using TRIzol reagent (Invitrogen) and the RNeasy kit (Qiagen) according to manufacturer's instructions, including a DNase digestion step. Then the RNAs were amplified and labeled using the Agilent Quick Amp labeling kit and hybridized with Agilent whole genome oligo microarray which contain 41,000+ unique human genes and transcripts in Agilent's SureHyb Hybridization Chambers. After hybridization and washing, the processed slides were scanned with the Agilent DNA microarray scanner (part number G2505B) using settings recommended by Agilent Technologies. To identify the lipid metabolism genes that are differentially expressed, we performed a Fold-change screening between the two groups obtained from the experiment. The threshold used to screen Up or Down regulated genes is Fold Change>=2.

Real time-PCR to confirm the results of cDNA expression profiling. Total RNA was extracted from the above VSMCs using TRIzol and reverse transcription was performed by reverse transcription kit. 1 µl of the reverse-transcripts was added to a 20 µl PCR mixture for 20 cycles with ABI-7500 detection system (Applied biosystems, USA). The primers were as follows: 5'-GGCCCAAGCCAATGGTATACT-3' and 5'-CTCGGTCACGCTTGCTCTT-3' (HMG-CoA-s, 180bp); 5'-GAGTCACAAGCACGTGGAAGA-3'and 5'-TCTTGTGGCCAGCACCAATA-3' (HMG-CoA-r, 122 bp); 5'-ATCCTACTGGACGGTTCG-3'and 5'-TTGGCTAATGGCACTTGA-3' (β-actin, 139bp). The reaction conditions were as follows: a mixture of 2 µL of 10× PCR buffer, 2 µL of magnesium ion (25 mM), 0.3 µL of dNTPs(25 mmol/L), 0.5 µl of upstream (10 µmol/L) primers , 0.5 µl of downstream (10 µmol/L) primers, Sybr(20×)1 µL, Taq (5 U/µ1) 0.2 µL and cDNA transcripts 1.0 µL . The mixture was incubated at 95°C for 2 min and then 20 cycles of 95°C, 10 secs, 60°C, 30 sec. A melting curve of the reaction system was drawn immediately after the reaction to analyze the specificity of the PCR products. Quantitative analysis of target gene expression data was based on the F=2-ΔΔCt method. ΔΔCt = (average Ct of target gene in experimental groups-average Ct of β-actin in experimental groups)-(average Ct of target gene in control group-average Ct of β-actin in control group).

Determination of cellular total cholesterol

Cells were washed with phosphate buffered saline (PBS) twice, trypsinized, separated sufficiently and were counted after various treatment. Followed the count and centrifugation at 500×g 5 min, 200 µL cell lysate added to lyse the cells. By centrifugation 10000 × g 10 min, supernatant was collected to measure cholesterol content according to previous cholesterol oxidase method [7]. Briefly, cholesterol standard solution provided in the kit was appropriately diluted using cell lysate, and 1000 µL of detection reagent was added to react with 50 µL of cholesterol standard or collected cell lysate supernatant. After 37°C 5min, optical density values at 520 nm were measured. Standard curve was prepared using cholesterol standard solution, and the concentrations of cholesterol in the sample were calculated according to the standard curve. The final result was in terms of 106 cells cholesterol content (µmol / 106 cells).

Statistical analysis

Data are expressed as means ± standard errors of the means. Analysis was performed using SPSS10.0 for windows. Statistical significances were tested using either t test between two samples or variance among multiple samples with q test between groups. A P value of <0.05 was considered statistically significant.

Results

The infection of HCMV to VSMCs

The human VSMCs were cultured in DMEM/F12 and inoculated with HCMV. The results showed that 24 hours after infection, some individual cells appeared swelling. With prolonged incubation time, cytopathic effects became more and more serious and eighth days after infection, almost all the cells showed cytopathic effects (Fig. 1A). On the third day after infection, the cells were harvested and examined by electron microscopy. The results showed the viral particles were visible within the cell (Fig. 1B). Using the technology of RT-PCR, HCMV IE gene could be amplificated from VSMCs (Fig. 1C). The real PCR products were confirmed by direct sequencing. These results showed that HCMV infects human VSMCs, and replicates intact virus particles in the cells.

Fig. 1

The infection of HCMV to VSMCs was assayed by direct inverted microscope (A), electron microscope (B) and RT-PCR (C). A: 1-8: Control group from Day 1 to Day 8; 9-16:HCMV infection from Day 1 to Day 8 (×400); B: On the third day after infection, the viral particles were visible within the cell (Arrow, ×20000). C: HCMV IE gene could be amplificated from VSMCs infected with HCMV. M: 1 Kb DNA marker; 1: IE gene; 2: Mock infection 3: Positive control; 4: Negative control.

http://www.karger.com/WebMaterial/ShowPic/523137

Results of cDNA profiling

After infection VSMCs with HCMV (MOI=1), the cells were harvested at 24, 48, 72 hours and the samples including mocked group were detected by Shanghai Kangchen biological technology limited company. The experimental results showed that there were 27 abnormal expression lipid metabolism genes after HCMV infection, including 12 up-regulated genes and 15 down regulated genes. Among them, low density lipoprotein receptor related protein 10, 11, 12 (LRP 10,11,12), sterol O-acyltransferase 1(SOAT1),fatty acid synthase (FANS), very low density lipoprotein receptor (VLDLR), scavenger receptor B (SCARB), HMG-CoA synthase (HMGCS1), HMG-CoA reductase (HMGCR), acetyl-Coenzyme A acetyltransferase 1 (ACAT1), apolipoprotein E (APOE) and apolipoprotein C1 (APOC1) were significantly up-regulated. However, apolipoprotein (ApoA1, ApoA2, ApoC3, ApoC4, ApoLD1, ApoO, ApoM. ApoH and ApoL1-3), LRP-8,LRP-associated protein gene (LRPAP1), lysophosphatidic acid (LPA) and ACAT2 significantly decreased. The change folds of Hepatic lipase (1.04, -1.17, 1.18), lipoprotein lipase (-1.03, 1.93, 1.99), lecithin cholesterol acyl transferase (-1.28, -1.26, -1.44) and ApoB (-1.03, -1.14, 1.99) at 24h, 48h and 72h did not show any changes. The results showed that HCMV infection influences intercellular lipid metabolism by affecting cell lipid uptake and promoting cellular cholesterol, cholesteryl ester and fatty acid biosynthesis etc, so getting involved in the pathogenesis of AS or arteriosclerosis (Fig. 2).

Fig. 2

Differential expression lipid metabolism genes were screened by cDNA microassay. The experimental results showed that there were 27 abnormal expression lipid metabolism genes after HCMV infection, including 12 up-regulated genes and 15 down-regulated genes. LRP 10,11,12, SOAT1, FANS, VLDLR,SCARB,HMGCS1, HMGCR, ACAT1, APOE and APOC1 were significantly up-regulated. However, ApoA1, ApoA2, ApoC3, ApoC4, ApoLD1, ApoO, ApoM. ApoH and ApoL1-3, LRP-8,L-RPAP1, LPA and ACAT2 significantly decreased.

http://www.karger.com/WebMaterial/ShowPic/523136

Real-time PCR to confirm the results of cDNA microassay

Real-time PCR was used to validate the changes of HMG CoA synthase and HMG CoA reductase expression after HCMV infection. The results showed that, after HCMV infection, the expression level of HMG CoA synthase and HMG CoA reductase in human VSMCs remained unchanged at 24 hours, but at 48 and 72 hours, the expression levels of the two genes increased significantly compared with mock infection, indicating HCMV infection up-regulate HMG CoA synthase and HMG CoA reductase expression (Fig. 3).

Fig. 3

Fig. 3 HMG-CoA synthase and HMG-CoA reductase expression were validated by real-time PCR after HCMV infection (n=4, ** P <0.01). A: HMG-CoA synthase amplification curve. B: HMG-CoA reductase amplification curve. C:β-actin amplification curve. D: HMG-CoA synthase relative expression. E: HMG-CoA reductase relative expression. The results showed that, after HCMV AD169 infection, the expression level of HMG-CoA synthase and HMG-CoA reductase in human VSMCs increased significantly in a time dependent manner.

http://www.karger.com/WebMaterial/ShowPic/523135

Determination of total cholesterol in VSMCs after HCMV infection

VSMCs were harvested after HCMV infection and rinsed twice with PBS (pH7.2, 0.01M). Then cells were lysed by RIPA cell lysis reagent, and the supernatants were collected for total cholesterol content assay. The results showed that at 24 and 48 hours post infection, the contents of cholesterol in the cells had no significant difference between mock and viral infection. However, at 72h after infection, the total cholesterol contents in VSMCs were significantly higher than those of mock infection, indicating that HCMV infection induced the imbalance of cholesterol metabolism in VSMCs and led to intracellular cholesterol accumulation (Fig. 4).

Fig. 4

The total cholesterol content was assayed by cholesterol oxidase reaction (n=6, ** P <0.01). The results showed that at 72h after infection, the total cholesterol contents in VSMCs were significantly higher than those of mock infection.

http://www.karger.com/WebMaterial/ShowPic/523134

Discussion

No matter in developed countries or in developing countries, AS and its cardiac and cerebral complications are the major causes of death and disability. For long time, the studies of the pathogenic factors and intervention of AS related diseases mainly concentrated on hypertension, hyperlipidemia and other traditional risk factors. However, in recent years, although effective interventions were given to obesity, lipid metabolism, diabetes, hypertension, smoking and other traditional risk factors, incidence of AS based cardiovascular diseases is still increasing, indicating that in addition to the traditional risk factors, there are still other pathogenic factors for AS. In the early 20's of last century, some scholars proposed that microbial infection had a certain relationship with AS. It was not until 1970s that Fabricant successfully induced atherosclerotic pathological changes of chicken infected with Marek's disease virus, an avian herpes virus and etiology of AS had gradually attracted the attention of scholars. At present, HCMV, Chlamydia pneumoniae, Helicobacter pylori [8,9,10] are recognized as the important pathogenic factors of AS, especially HCMV, which is recognized as an important pathogenic factor of AS from serum epidemiology, molecular epidemiology and animal model [11,12]. Recent reports showed that HCMV infection induced the increase of arterial blood pressure [13]. However, the mechanism of AS induced by HCMV remains unclear.

There were several reports about the mechanism of AS induced by HCMV infection, including abnormal gene expression and cell proliferation of VSMCs after HCMV infection [14], but these studies did not provide direct evidence for viral infection or replication in human VSMCs. We verified in present study that HCMV directly infected VSMCs by observation of cytopathic effects, immediate early (IE) gene expression, and intact virus particles in cells.

Clinical epidemiological investigation and practice showed that AS patients usually had abnormal lipid metabolism. Lipid accumulation is one of the critical mechanisms contributing to atherogenesis, and the importance of lipids in initiating the atherosclerotic process is exemplified by the characteristic feature of the earliest lesion of atherosclerosis, accumulation of lipid-laden foam cells, which are derived from macrophages and smooth muscle cells (SMCs). Native LDL does not in itself predispose to foam cell development, in part because its receptor, the LDL receptor, down-regulates in response to increasing intracellular levels of LDL. LDL, however, is oxidized in the sub-intimal space to oxidized LDL (Ox-LDL), which is transported into the cell via the scavenger receptor (SR). Most importantly, this receptor, unlike the LDL receptor, does not down-regulate as intracellular cholesterol levels rise, thereby providing a mechanism for large amounts of cholesterol to accumulate. This presumably leads to the development of foam cells and thereby may contribute to the atherosclerosis process. Whether HCMV infection, one of important pathogenic factors for AS is related with abnormal lipid metabolism? We next observed lipid metabolism related gene expression in VSMCs followed by HCMV infection by cDNA microarray and the results of the study showed that many lipid metabolism related genes such as apolipoprotein genes, lipid uptake associated receptor genes and lipid synthase genes were abnormal, indicating that viral infection leads to abnormal expression of lipid metabolism related genes in cells, so promoting the occurrence and development of AS.

The cellular uptake of lipid is mediated by receptors, including very low density lipoprotein receptor, low density lipoprotein receptor related protein, scavenger receptor [15,16] and so on. And the expression of these receptors is not influenced by cholesterol content in cells, resulting in a large number of lipid accumulation in cells without limit and formation of foam cells and AS. The study showed that the expression of LRP 10,11 and 12, VLDLR, SCARB increased significantly after infection, indicating that HCMV infection led to increased uptake and accumulation of lipids in the cells, which was consistent with the reports of Carlquist and Zhou [17,18].

In addition to the increased cellular uptake of lipoprotein, another important reason for accumulation of lipids is increased lipid biosynthesis. It has been reported that many virus promoted cellular cholesterol synthesis, such as HSV-1, HCV and HIV [19,20,21]. On the one hand, the replication of virus promotes the accumulation of cholesterol, on the other hand, the accumulation of cholesterol in cells, participates in or accelerates the formation of AS. A series of catalytic enzymes get involved in De novo synthesis of cholesterol in cells, and the most important enzymes include HMG-CoA synthase and HMG-CoA reductase. HMG-CoA reductase is the rate- limiting enzyme in cholesterol synthesis, which will lead to abnormal cholesterol synthesis if express abnormally. cDNA profiling and real-time PCR confirmed that expression of HMG-CoA synthase and HMG-CoA reductase increased after HCMV infection. In order to confirm increased expression of HMG-CoA synthase and HMG-CoA reductase induced by HCMV infection leads to increased cellular cholesterol synthesis, we observed cholesterol contents in the cells and showed that the contents of cholesterol in cells after infection increased significantly, indicating that HCMV infection increases the cellular cholesterol synthesis and accumulation.

Besides, after HCMV infection, ACAT1 expression in cells also increase obviously, which is the only enzyme in cells to catalyze cholesterol and fatty acyl coenzyme A into cholesterol ester. It has been reported that some pathogens, such as Chlamydia pneumoniae promoted the expression of the enzyme, resulting in synthesis and accumulation of intracellular cholesterol ester and formation of foam cell, thus accelerating the occurrence and development of AS [22]. The vascular lipid deposition is often associated with the pathogenesis of AS. The body fat deposit required mostly from the de novo synthesis of fatty acids, namely fatty acid synthase catalyzes acetyl coenzyme A and malonyl coenzyme A synthesis into fatty acid and triglyceride synthesis [23]. The results of this study also showed that the expression level of fatty acid synthase was significantly higher after HCMV infection, indicating that virus infection promotes fatty acid synthesis.

Although the increase in intracellular cholesterol may also be due to the increase of low density lipoprotein uptake, but in the cell culture system of this study, oxidized low density lipoprotein was not added, and cDNA profiling did not show any changes of hepatic lipase, lipoprotein lipase and lecithin cholesterol acyl transferase that involved in cholesterol uptake and transport, so the increase of intercellular cholesterol after HCMV infection may not be due to an increase in the low density lipoprotein cholesterol uptake and transport, but intracellular cholesterol synthesis .

Apolipoproteins are important components of lipoproteins, which play important physiological roles in lipoprotein metabolism through acting as components and stabilizing the structure of lipoprotein, modifying enzymatic activities, acting as ligands of lipoprotein receptors and participating in the binding of lipoproteins with their receptors etc. Studies showed that some apolipoproteins such as ApoB [24] promote the formation of AS, while other apolipoproteins such as ApoA1, ApoM, ApoH have anti-atherogenic effects [25,26,27]. The results of the study showed that after HCMV infection, ApoA1, ApoM, ApoH was down regulated, indicating that HCMV infection induced abnormal expression of anti-AS apolipoprotein, which is conducive to the formation of intracellular lipid accumulation and the formation of AS.

In conclusion, our results indicate that HCMV infection increases uptake of Ox-LDL by vascular SMCs. The increased uptake appears to be caused by increased class A scavenger receptor gene expression, an effect mediated, at least in part, by the virus's immediate-early gene product, IE72. Thus, HCMV can increase the capacity of SMCs to incorporate Ox-LDL, and can do so in the absence of viral replication. This finding is consistent with the known propensity of herpes viruses to exert cellular effects during abortive, nonpermissive infections. Because HCMV also encodes an IE protein (IE84) that, by interfering with the inhibitory actions of p53, can predispose to SMC accumulation, it appears that HCMV possesses the genetic programs that can alter the function of infected SMCs so that two of the major mechanisms underlying atherogenesis are activated, increased SMC accumulation, and increased uptake of Ox-LDL. The epidemiologic and experimental data and the results of the present investigation provide mechanistic support for the hypothesis that HCMV predisposes to the development of atherosclerosis.

Disclosure Statement

The authors have declared that no competing interests exist.


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Author Contacts

Wu Guojun and Tan Yurong

Department of Microbiology, School of Basic Medical Sciences, Central South

University, Changsha 410078, Hunan, (China)

Tel. +8673182355003, E-Mail wuguojun@csu.edu.cn; hope7@126.com


Article / Publication Details

Accepted: August 12, 2016
Published online: October 17, 2016
Issue release date: October 2016

Number of Print Pages: 9
Number of Figures: 4
Number of Tables: 0

ISSN: 1015-8987 (Print)
eISSN: 1421-9778 (Online)

For additional information: http://www.karger.com/CPB


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References

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