Glucose-dependent insulinotropic peptide stimulates thymidine incorporation in endothelial cells: role of endothelin-1

Ke-Hong Ding,1 Qing Zhong,1 and Carlos M. Isales1,2

1Institute of Molecular Medicine and Genetics, Department of Medicine, Medical College of Georgia, and 2Augusta Veterans Affairs Medical Center, Augusta, Georgia 30912

Submitted 21 November 2002 ; accepted in final form 21 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
We have previously characterized the receptor for glucose-dependent insulinotropic polypeptide (GIPR) in vascular endothelial cells (EC). Different EC types were found to contain distinct GIPR splice variants. To determine whether activation of the GIPR splice variants resulted in different cellular responses, we examined GIP effects on human umbilical vein endothelial cells (HUVEC), which contain two GIPR splice variants, and compared them with a spontaneously transformed human umbilical vein EC line, ECV 304, which contains four GIPR splice variants. GIP dose-dependently stimulated HUVEC and ECV 304 proliferation as measured by [3H]thymidine incorporation. GIP increased endothelin-1 (ET-1) secretion from HUVEC but not from ECV 304. Use of the endothelin B receptor blocker BQ-788 resulted in an inhibition of [3H]thymidine incorporation in HUVEC but not in ECV 304. These findings suggest that, although GIP increases [3H]thymidine incorporation in both HUVEC and ECV 304, this proliferative response is mediated by ET-1 only in HUVEC. These differences in cellular response to GIP may be related to differences in activation of GIPR splice variants.

receptor; splice variants


GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE (GIP), also named gastric inhibitory polypeptide, is a 42-residue peptide produced by mucosal K cells of the duodenum and small intestine and secreted into the circulation in response to the ingestion of nutrients (4, 5, 8, 21). GIP is involved in several facets of the anabolic response and is thought to be particularly important in stimulating insulin secretion (9). The GIP receptor is a member of the seven transmembrane domain G protein-coupled receptor superfamily, which includes receptors for glucagon, glucagon-like peptide 1, secretin, vasoactive intestinal polypeptide, calcitonin, corticotropin-releasing factor, pituitary adenylate cyclase-activating polypeptide, and growth hormone-releasing hormone (28). GIP receptors have been detected in many tissues, including endothelium, heart, pancreas, gut, brain, adipose tissue, pituitary, adrenal cortex (28), and bone (3), although no receptors are present on smooth muscle. GIP receptor binding has been shown to stimulate adenylyl cyclase and elevate intracellular cAMP levels in pancreatic islets (23), islet tumor cell lines (1), osteoblast-derived cells (3), and various cell lines transfected with the GIP receptor (10, 17, 30). In addition, GIP has been shown to increase Ca2+ uptake into isolated islets (29) and to increase intracellular Ca2+ levels in HIT-T15 (15), bone (3), and COS cells (30).

Although GIP receptors are widely distributed in the endothelium (28), their role in this tissue is not completely clear. We have recently reported that multiple GIP receptor splice variants are present in different endothelial cell (EC) types (31). Human umbilical vein endothelial cells (HUVEC) were found to contain two GIP receptor splice variants, whereas a spontaneously transformed umbilical vein EC line, ECV 304 cells, had four receptor splice variants. Although GIP increased intracellular calcium in both EC types, GIP activated cAMP-dependent protein kinase in the HUVEC but not in the ECV 304 cells, suggesting that the splice variants could be differentially linked to signaling pathways (31). Interestingly, infusion of GIP has been shown to have different effects on blood flow in vivo; i.e., it increases portal venous flow and decreases hepatic artery blood flow (13), suggesting that GIP has distinct vasoactive properties.

In an attempt to shed more light on GIP's role in the endothelium, GIP effects on vascular EC proliferation (by use of [3H]thymidine incorporation as a measure) and the underlying mechanism of these effects were investigated. Our present results demonstrate that GIP modulates proliferation in HUVEC through stimulation of endothelin (ET)-1 synthesis and release and subsequent activation of the endothelin B receptor (ETBR) subtype. Although GIP also stimulates [3H]thymidine incorporation in ECV 304 cells, this effect does not seem to mediated by ET-1. In view of the differences in cellular responses to GIP in HUVEC vs. ECV 304, linkage of GIP receptor splice variants to different transducers in these two cell types might in fact be responsible for the observed differences.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Reagents

GIP, ET-1, BQ-788, and BQ-610 were from Bachem (Torrance, CA). The 125I-labeled ET-1 radioimmunoassay (RIA) kit was purchased from Peninsula Labs (San Carlos, CA). [Methyl,1',2'-3H]thymidine was from Amersham (Arlington Heights, IL).

Cell Culture

For these studies, we used normal HUVEC (Clonetics, San Diego, CA), as well as the immortalized HUVEC cell line ECV 304 (ATCC, Gaithersburg, MD) (26). Cells were grown to confluence in endothelial growth medium (EGM; Clonetics) or Medium 199 (Whittaker Biologic Products, San Diego, CA), supplemented with 10% FCS (vol/vol; HyClone Laboratories, Logan, UT), penicillin (100 U/ml), streptomycin (100 mg/ml), and amphotericin B (3 mg/ml).

[3H]Thymidine Incorporation Assay

EC were seeded in 6-well plates at 2 x 105/well and allowed to attach and reach 80% confluence. Cells were then placed in a modified Krebs-Ringer bicarbonate (KRB) buffer (containing in mM: 25 sodium bicarbonate, 120 sodium chloride, 4 potassium chloride, 1.2 magnesium sulfate, 1.2 sodium bisphosphate, 5.6 dextrose, 1.25 calcium chloride, and 2 mg BSA/ml) overnight. The KRB buffer was then changed, and the appropriate agonist was added for an additional 24 h. ET receptor antagonists were incubated for 15 min before the addition of agonist. During the last 6 h of incubation, 1 µCi/well of [3H]thymidine was added. Cells were then washed three times with phosphate-buffered saline, treated twice with 5% TCA (ice cold) for 15 min, washed once with water, and solubilized in 0.3 N sodium hydroxide. The cell-associated radioactivity was then determined by liquid scintillation counting.

Microarray Analysis

T7-based RNA amplification. Ten micrograms of total RNA were converted into double-stranded cDNA (ds-cDNA) by use of the SuperScript Choice System (GIBCO-BRL Life Technologies) with an oligo(dT) primer containing a T7 RNA polymerase promoter (Genset). After second-strand synthesis, the reaction mixture was extracted with phenol-chloroform-isoamyl alcohol, and ds-cDNA was recovered by ethanol precipitation.

Labeling, hybridization, and scanning. In vitro transcription was performed on the above ds-cDNA with the Enzo RNA transcript labeling kit. Biotin-labeled cRNA was purified by use of an RNeasy affinity column (Qiagen) and fragmented randomly to sizes ranging from 35 to 200 bases by incubating at 94°C for 35 min. The hybridization solutions contained 100 mM MES, 1 M Na+, 20 mM EDTA, and 0.01% Tween 20. The final concentration of fragmented cRNA was 0.05 µg/µl in the hybridization solution. Target for hybridization was prepared by combining 40 µl of the fragmented transcript with sonicated herring sperm DNA (0.1 mg/ml), BSA, and 5 nM control oligonucleotide in a buffer containing 1.0 M NaCl, 10 mM Tris · HCl (pH 7.6), and 0.005% Triton X-100. Target was hybridized for 16 h at 45°C to a set of oligonucleotide arrays (HG U133A; Affymetrix, Santa Clara, CA). Arrays were washed at 50°C with stringent solution and then at 30°C with nonstringent solutions. Arrays were then stained with streptavidin-phycoerythrin (Molecular Probes). DNA chips were read at a resolution of 3 µm with a Hewlett-Packard GeneArray Scanner and were analyzed with the GENECHIP software (Affymetrix). Detailed protocols for data analysis of Affymetrix microarrays and extensive documentation of the sensitivity and quantitative aspects of the method have been described (14). Briefly, each gene is represented by the use of ~20 perfectly matched (PM) and mismatched (MM) control probes. The MM probes act as specificity controls, which allows the direct subtraction of both background and cross-hybridization signals. The number of instances in which the PM hybridization signal is larger than the MM signal is computed along with the average of the logarithm of the PM-to-MM ratio (after background subtraction) for each probe set. These values are used to make a matrix-based decision concerning the presence or absence of an RNA molecule. Comparison analysis between control and experimental animals was made with Affymetrix software. Further analysis for functional classification was carried out using GeneSpring (Silicon Genetics, Redwood, CA).

ET-1 Measurements

ET-1 measurements were performed as previously described (12). Briefly, EC were grown in 6-well plates, placed in 0.1% FCS overnight before use, and then incubated for an additional 20 h with the appropriate agonist. The cell culture supernatants were removed, and ET-1 contents were measured with a commercially available RIA kit (Peninsula Labs).

Statistics

Results are expressed as means ± SE. Experiments were performed in triplicate except where noted. Data were analyzed with either ANOVA or unpaired t-tests, where appropriate, with a commercial statistical package (Instat; Graph-pad, San Diego, CA).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
GIP Stimulates [3H]Thymidine Incorporation in EC

GIP receptors on ECV 304 cells and HUVEC have previously been identified (31). To determine whether GIP stimulates proliferation, we examined GIP effects on [3H]thymidine incorporation in HUVEC (Fig. 1A) and in ECV 304 (Fig. 1B). GIP concentrations as low as 10-13 M significantly stimulated [3H]thymidine incorporation in HUVEC with an EC50 of 1 pM. Increasing GIP concentrations to 0.01 nM and above had no further stimulatory effect. In fact, the GIP dose-response curve appeared to be bimodal, with supraphysiological doses of GIP (10 nM and above) still significantly elevated over control but below the peak response at 0.1 nM GIP.



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Fig. 1. Glucose-dependent insulinotropic peptide (GIP) stimulates [3H]thymidine incorporation in both human umbilical vein endothelial cells (HUVEC) and spontaneously transformed human umbilical vein (ECV) 304 cells. Primary HUVEC (A) or ECV 304 cells (B) were grown in 6-well plates at a concentration of 200,000 cells/well. Cells were stimulated with increasing concentrations of GIP, and [3H]thymidine incorporation was measured. In HUVEC, GIP at doses of >=10-13 M had a statistically significant stimulatory effect on cell proliferation. In contrast, concentrations of GIP of >=10-8 M were necessary in ECV 304 cells to significantly stimulate proliferation. Results are expressed as percentage increase over control (means ± SE) of 9 different experiments (*P < 0.05; **P < 0.01).

 

GIP also stimulated [3H]thymidine incorporation in ECV 304 cells, but the dose-response curve was shifted to the right with a calculated EC50 of 2.5 nM. A significant increase in [3H]thymidine incorporation was observed at 10 nM GIP, and increasing the agonist concentration provoked a corresponding increase in [3H]thymidine incorporation (Fig. 1B). The response was greatest at 1 µM GIP.

GIP Increases ET-1 Secretion in HUVEC but not in ECV 304

ET-1 is a known mitogen, and stimulated release from EC is regulated by a wide variety of factors, including glucose, cytokines, and mechanical factors (16). To determine whether GIP-induced elevations in [3H]thymidine incorporation were a direct or an indirect effect mediated by ET-1, we first measured GIP's ability to stimulate ET-1 secretion. HUVEC were stimulated with increasing concentrations of GIP (Fig. 2A), and GIP significantly increased ET-1 secretion at a concentration as low as 0.1 nM. Higher concentrations of GIP dose-dependently increased ET-1 secretion with an EC50 of 4.6 nM. In contrast, if ECV 304 cells were used, GIP had no significant effect on ET-1 secretion (Fig. 2B). To assess the underlying mechanism of this effect, we performed microarray analysis of GIP-stimulated HUVEC and ECV 304. HUVEC were stimulated for 6 h with GIP (0.1 nM), and 59 genes were upregulated, including a significant increase in endothelin-converting enzyme (Table 1). When the concentration of GIP was increased to 10 nM, 446 genes were now upregulated at least twofold, although the endothelin-converting enzyme was again upregulated (data not shown). In contrast, when ECV 304 cells were stimulated with 0.1 nM GIP, the expression of 66 genes was significantly changed. However, there was no change in the expression of endothelin-converting enzyme in ECV 304 in response to GIP (Table 2).



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Fig. 2. GIP stimulates endothelin-1 (ET-1) secretion from HUVEC but not ECV 304 cells. HUVEC (A) or ECV 304 cells (B) were grown in 6-well plates and placed in 0.1% FCS overnight before the experiment. Cells were stimulated with increasing concentrations of GIP for 20 h, and ET-1 secretion into the medium was measured using a commercially available RIA kit. GIP at doses of >=0.1 nM had a statistically significant stimulatory effect on ET-1 release in HUVEC (A) but not in ECV 304 cells (B). Results are expressed as pg/well (means ± SE) of 9 different experiments (*P < 0.01; **P < 0.001).

 

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Table 1. Microarray analysis of GIP-stimulated HUVEC

 

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Table 2. Microarray analysis of GIP-stimulated ECV 304

 

BQ-788 Blocks GIP-Stimulated [3H]Thymidine Incorporation in HUVEC but not in ECV 304

To evaluate the role of ET-1 secretion from HUVEC, we utilized the ETBR blocker BQ-788. BQ-788 by itself, at a concentration of 10-6 M, had a small but statistically significant effect on increasing [3H]thymidine incorporation in HUVEC (Fig. 3). However, when increasing concentrations of BQ-788 were added to HUVEC stimulated with GIP (1 nM), [3H]thymidine incorporation was completely inhibited (Fig. 3). In ECV 304 cells, BQ-788 by itself (100 nM) also stimulated [3H]thymidine incorporation; however, when added together with GIP (100 nM)-stimulated cells, there was an additive (rather than inhibitory) effect on [3H]thymidine incorporation (Fig. 4).



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Fig. 3. GIP-stimulated [3H]thymidine incorporation in HUVEC is blocked by the ETB receptor (ETBR) blocker BQ-788 in HUVEC cells. Both HUVEC and ECV 304 cells were grown in 6-well plates and placed in Krebs-Ringer bicarbonate overnight before the experiment. Cells were stimulated with increasing concentrations of GIP in the presence or absence of the ETBR blocker BQ-788 for 24 h, and [3H]thymidine incorporation was measured. BQ-788 at all concentrations tested blocked [3H]thymidine incorporation in HUVEC. Results are expressed as cpm x 10-3 of triplicate samples of 3 different experiments: aP < 0.01 vs. control; bP < 0.001 vs. control; cP < 0.01 vs. control; dP < 0.05 vs. control.

 


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Fig. 4. GIP-stimulated [3H]thymidine incorporation in HUVEC is blocked by BQ-788. However, BQ-788 did not block GIP-stimulated [3H]thymidine incorporation in ECV 304. In fact, a statistically significant potentiation of proliferation was observed when GIP and BQ-788 were added together. Procedure was as described in Fig. 3. Results are expressed as cpm x 10-3 (means ± SE) of triplicate samples of 3 different experiments: *P < 0.01 vs. control; **P < 0.001 vs. control.

 

BQ-610 has no Effect on GIP-Stimulated [3H]Thymidine Incorporation

EC cells contain the ETBR predominantly, whereas smooth muscle cells mainly express the ETA receptor. To evaluate whether block of the ETA receptor had any effect on HUVEC proliferation, we used the selective ETA blocker BQ-610. BQ-610 at concentrations ranging between 10 nM and 1 µM had no effect on GIP-stimulated [3H]thymidine incorporation in HUVEC (Fig. 5), consistent with the low expression of the ETA receptor in EC.



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Fig. 5. BQ-610 does not inhibit GIP-stimulated [3H]thymidine incorporation. HUVEC were grown in 6-well plates and stimulated with GIP (1 nM) in the presence or absence of increasing concentrations of BQ-610 (10-8 to 10-6 M), an ETA receptor blocker, and [3H]thymidine incorporation was measured. There was no significant inhibitory or stimulatory effect of BQ-610 on GIP-stimulated [3H]thymidine incorporation. Results are expressed as cpm x 10-3 (means ± SE) of 9 different experiments (*P < 0.01 vs. control).

 

ET-1 and GIP have Additive Effects on Stimulated [3H]Thymidine Incorporation in ECV 304 but not in HUVEC

To further evaluate the role of ET-1 in GIP-stimulated [3H]thymidine incorporation, we examined the effect of combining ET-1 and GIP (Fig. 6A) in HUVEC. Both GIP (1 nM) and ET-1 (10 nM) alone significantly stimulated [3H]thymidine incorporation. However, when combined, there was no synergistic or additive effect on proliferation. In contrast, if ECV 304 were used (Fig. 6B), not only did GIP and ET-1 by themselves significantly stimulate [3H]thymidine incorporation, there was also an additive effect on proliferation by the combination of these two agonists.



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Fig. 6. ET-1 and GIP effects on [3H]thymidine incorporation in HUVEC vs. ECV 304 cells. A: HUVEC were grown in 6-well plates, stimulated with 10 nM ET-1, 1 nM GIP, or the combination of both agonists, and [3H]thymidine incorporation was measured. Both GIP and ET-1 had a statistically significant effect on [3H]thymidine incorporation. However, in HUVEC, the combination of GIP and ET-1 had no additional effect on proliferation greater than either agonist by itself. B: ECV 304 cells were similarly stimulated with GIP, ET-1, or the combination of both agonists. In contrast to HUVEC, the combination of GIP + ET-1 in ECV 304 had an additive effect on [3H]thymidine incorporation. Values are means ± SE of 9 different experiments (**P < 0.01 vs. control; n.s., not significant).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
GIP has been thought to have a predominant role in synergistically stimulating insulin secretion in the presence of glucose, an "incretin" hormone. However, GIP receptors are widely distributed in the body (28), and GIP more likely plays a role in coordinating and maximizing nutrient absorption and disposition (2). Postprandially, hepatic artery blood flow is known to decrease and portal vein flow to increase. If GIP were mediating this effect, then it is unclear how these opposing effects on blood flow could be mediated through a single GIP receptor. We have previously reported the presence of GIP receptor splice variants in the various endothelial cell types (31). Our present results may account for some of the paradoxical effects of GIP on blood flow in vivo, a decrease in hepatic artery blood flow and a simultaneous increase in portal venous flow (13). We report that two common EC types (HUVEC and EC 304 cells), known to contain a different number of GIP receptor splice variants (2 vs. 4), respond similarly to GIP with an increase in proliferation. However, in HUVEC, this proliferative effect is mediated by a GIP-stimulated increase in ET-1, whereas in ECV 304 it is not. Thus, in HUVEC, one of the GIP receptor splice variants appears to be linked to ET-1 secretion. One caveat, however, is that ECV 304 is a transformed HUVEC line, so some of the observed differences in GIP responses in HUVEC vs. ECV 304 may be unrelated to differences in GIP receptor splice variants. Nevertheless, our previous data (31) and the data presented here strongly suggest that GIP receptor splice variants are linked to different cellular responses.

In a previous publication (31), we reported that the splice variants appeared to be differentially linked to different transduction pathways; the present data suggest, in addition, that the splice variants also have different functional links. As previously mentioned, the GIP receptor belongs to the G protein-coupled seven-transmembrane domain family of receptors, which are dually coupled to two signal transduction mechanisms: phosphoinositol-phospholipase C and adenylate cyclase. Additional diversity in the function of this family of receptors comes from the existence of both different receptor isoforms (e.g., VIP or PACAP) and splice variants [e.g., calcitonin (19), PACAP (24), CRF (6), and GHRH (27)]. Specifically, splice variants of the calcitonin receptor, which differ in the second extracellular domain, lead to differences in binding affinity for the hormone (11), whereas differences in the third intracellular loop of PACAP result in splice variants that display differences in their coupling to the signal transduction pathways (24). Thus there is precedence for the possibility that GIP receptor splice variants may be serving different functions in vascular endothelial cells.

Our data are consistent with the well-known mitogenic effect of ET-1 and the preferential expression of ETB receptors in endothelial cells (16, 18, 22). ET-1 is a potent vasoconstrictor, and if one of the GIP receptor splice variants linked to ET-1 secretion were to be expressed in the hepatic artery, this would account for the observed postprandial vasoconstriction. A difference in sensitivity to GIP was also observed between HUVEC and ECV, with HUVEC responding to concentrations of GIP as low as 10-13 M with an increase in [3H]thymidine incorporation, whereas ECV required a concentration of >=10-8 M. Interestingly, the dose-response curve for [3H]thymidine incorporation in response to GIP was biphasic in HUVEC (Fig. 1A), with concentrations of GIP of 10 nM and above actually leading to a decline in [3H]thymidine incorporation. This biphasic response in [3H]thymidine incorporation was not observed for ECV 304 (Fig. 1B). We have previously demonstrated that GIP at 10 nM increased protein kinase A activity in HUVEC but not in ECV 304 cells (31). cAMP can either inhibit or stimulate cell growth, depending on the cell type studied. cAMP effects on the MAP kinase (or ERK kinase), cell cycle progression, or other ERK-independent pathways have all been implicated in its inhibitory effect on cell proliferation (25). In endothelial cells, in particular, elevations in cAMP have been shown to inhibit the mitogenic effects of vascular endothelial growth factor and bovine fibroblast growth factor (7). Thus it is possible that, in the case of HUVEC, GIP-induced elevations in cAMP lead to an inhibition of proliferation that is not observed in ECV 304 because GIP does not increase cAMP in these endothelial cells.

An unexplained finding is that the ETBR blocker BQ-788 had a small but significant effect on [3H]thymidine incorporation in HUVEC and ECV 304 (Figs. 3 and 4). BQ-788 may result in increased secretion of ET-1 from the endothelium, so this may be partially responsible for the observed small agonist effect of this compound (20). Nevertheless, BQ-788 is still effective in blocking GIP effects on [3H]thymidine incorporation in HUVEC but not in ECV 304.

In summary, GIP stimulates endothelial cell proliferation, and the mechanism for this effect is different in two closely related endothelial cell lines. Differences in GIP receptor splice variants between these two endothelial cell lines may account for the observed differences in response. GIP receptors are widely distributed in the body and can have different and sometimes opposing actions. We speculate that, in vivo, part of the differences in tissue responses to GIP may relate to both differences in GIP receptor splice variants and differences in the GIP concentration required to activate the tissue [related to differences in GIP receptor number (3)]. Further studies are currently in progress, characterizing the individual GIP receptor splice variants.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported in part by grants from the Veterans Administration Merit Review (C. M. Isales) and the National Space Biomedical Research Institute through a National Aeronautics and Space Administration Cooperative Agreement NCC-9–58 (C. M. Isales).


    ACKNOWLEDGMENTS
 
We acknowledge Jianrui Xu for excellent technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. M. Isales, Institute of Molecular Medicine and Genetics, Dept. of Medicine, Medical College of Georgia, 120 15th St., Augusta, GA 30912 (E-mail address: cisales{at}mail.mcg.edu).

Submitted 21 November 2002

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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