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Published ahead of print on June 16, 2005, doi:10.1165/rcmb.2005-0144OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 297-302, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0144OC

Cyr61 Protects against Hyperoxia-Induced Cell Death via Akt Pathway in Pulmonary Epithelial Cells

Yang Jin, Hong Pyo Kim, Emeka Ifedigbo, Lester F. Lau and Augustine M. K. Choi

Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; and Department of Biochemistry and Molecular Genetics, University of Illnois at Chicago, Chicago, Illinois

Correspondence and requests for reprints should be addressed to Augustine M. K. Choi, M.D., Division of Pulmonary, Allergy and Critical Care Medicine, 628 NW MUH, University of Pittsburgh Medical Center, 3459 5th Ave., Pittsburgh, PA 15213. E-mail: ChoiAM{at}upmc.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We have used gene expression profiling approaches to identify new molecular targets in various models of lung injury and human lung diseases. Among the many genes that are significantly induced in these studies, cysteine-rich61 (Cyr61) consistently ranks as one of the most significant genes. Here, we use the well-established model of hyperoxia to better understand the function of Cyr61 in acute lung injury. Cyr61, a stress-related immediate-early response gene, has known diverse functions involving angiogenesis, tumorigenesis, and wound repair. It belongs to the newly discovered "CCN" family containing six growth and regulatory factors. We showed that hyperoxia induces Cyr61 expression in a variety of pulmonary cells and in lung tissue in vivo. Loss of function studies, by suppressing Cyr61 expression by siRNA, accelerated lung epithelial cell death after hyperoxia. Gain of function studies, by overexpressing Cyr61, significantly conferred increased resistance to hyperoxia-induced cell death. Moreover, cells overexpressing Cyr61 induce Akt activation. Inhibition of Akt by siRNA abrogated the protective effects of Cyr61-overexpressing cells in response to hyperoxia. Taken together, our data demonstrate that Cyr61 expression provides cytoprotection in hyperoxia-induced pulmonary epithelial cell death and that this effect was in part mediated via the Akt signaling pathway.

Key Words: Akt • cell death • Cyr61 • hyperoxia • lung


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
A newly discovered extracellular matrix–associated protein, cysteine-rich61 (Cyr61), has been shown to have diverse physiologic functions including angiogenesis (1), tumorigenesis (1–5), tumor metastasis (6), tumor suppression (7), wound healing (8, 9), development (10), and cell survival/death/differentiation/migration/adhesion (11, 12). Cyr61 belongs to the Cysteine-rich 61, Connective tissue growth factor and Nephroblastoma-overexpressed (CCN) gene family of growth regulators. Six members have been identified currently, consisting of Cyr61 (CCN1), CTGF (CCN2), Nov (CCN3), Wnt-inducible secretory protein-1 (WISP-1) (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6) (13). This family of proteins is believed to be involved in multifunctional signaling pathways (14) and expressed in wide variety of tissues during normal development (12). Expression of Cyr61 has also been shown to confer resistance to apoptosis in breast cancer MCF-7 cells (15), and to facilitate cell proliferation in prostate cancer, benign prostate hypertrophy (BPH), and gliomas (16–18). It further facilitates pancreatic cancer metastases (6). Furthermore, Cyr61 can suppress the growth of human endometrial cancer cells (19) and non–small cell lung cancer cells (20). In addition, Cyr61 has been shown to mediate Coxsackievirus B3 infection–induced cell death (21). These reports strongly suggest differential physiologic function of Cyr61 in cell survival/death depending on cell types and cellular stimuli.

More recently, we have observed marked induction of Cyr61 in various gene expression profiling analysis in various models of lung injury and human tissues, including chronic obstructive pulmonary diseases (COPD) and ventilator-associated lung injury (22, 23). Perkowski and coworkers further showed that Cyr61 mRNA was upregulated by microarray analysis of murine lung tissue after hyperoxia (24). However, the functional role of Cyr61 in hyperoxia-induced lung injury and pulmonary cell death remains unclear. We hypothesized that Cyr61 may play an important role in the pathogenesis of acute lung injury. The goals of our current study were to explore: (1) the expression of Cyr61 in lung cells (in vitro) and lung tissues (in vivo); (2) initial functional role of Cyr61 in lung cells, especially in hyperoxia-induced pulmonary cell death; and (3) the potential mechanisms/pathways by which Cyr61 indeed has an effect on hyperoxia-induced lung cell death. To our knowledge, this is the first study to investigate the functional role of Cyr61 in hyperoxia-induced lung cell death.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Chemicals and Reagents
Rabbit polyclonal anti-Cyr61 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-Akt and anti–phosphor-Akt from Cell Signaling Technology, Inc. (Beverly, MA); tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin (IL)-1ß, interferon (IFN)-{gamma}, and recombinant human transforming growth factor (TGF)-ß from R&D Systems, Inc. (Minneapolis, MN). Wortmannin was purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). All other chemicals were from Sigma (St. Louis, MO).

Cell Culture
Pulmonary cell lines including H292, A549, Beas2B, and MRC5 cells were purchased from the American Type Culture Collection (Manassas, VA), cultured in RPMI or DMEM medium, supplemented with 10% FBS (Hyclone, Logan, UT) and at 37°C in a humidified atmosphere of 5% CO2–95% air. Stably transfected Beas2B cells were grown in DMEM with 10% FBS and G418 (500 ng/ml; GIBCO BRL, Life Technologies, Grand Island, NY). Pulmonary artery smooth muscle cells (PASMC) were digested from freshly isolated rat pulmonary arteries with a modified technique described by Smirnov and colleagues (25). Briefly, main pulmonary arteries were isolated and opened via a longitudinal incision, and allowed to recover for 30 min in cold HEPES-buffered physiologic saline solution (HPSS) containing 1.5 mM Ca2+. The pulmonary artery was then placed at room temperature in low-Ca2+ HPSS (20 µM Ca2+) for 20 min before enzymatic digestion at 37°C for 20 min in low-Ca2+ HPSS containing 1 mg/ml of collagenase (type I, 1,750 U/mg), 1 mg/ml of papain (9.5 U/mg), 2 mg/ml of bovine serum albumin (BSA), and 1 mM dithiothreitol (Sigma). The artery was then transferred to enzyme-free Ca2+-free HPSS, and PASMC were isolated. Primary cultures of rat main pulmonary artery endothelial cells (PAEC) were generously provided by Dr. Mark N. Gillespie (University of South Alabama, Mobile, AL). The endothelial cells were cultured in one-half DMEM and one-half Ham's F-12 medium (Mediatech, Herndon, VA) supplemented with 10% FBS and gentamicin (50 µg/ml).

Animal Studies
C57BL/6 male mice were obtained at 6–8 wk of age (Jackson Laboratories, Bar Harbor, ME) and housed in a specific pathogen–free (SPF)/barrier animal facility at the University of Pittsburgh. All experimental procedures were performed under the guidelines set by University of Pittsburgh. Mice were exposed to room air or hyperoxia (100% oxygen). After 48–72 h, mice were killed and lung tissues were obtained to perform Western blot analysis or immunofluorescence.

Cell Viability Assays
Cell viability assays were performed using the CellTiter-Glo Luminescent Cell Viability Assay according to the protocol provided by Promega (Madison, WI). Briefly, cells were plated into the 96-well plates. After transfection and exposure to hyperoxia, cells were washed twice with cold PBS. One hundred microliters of PBS was added into each well, followed by 100 µl CellTiter-Glo Substrate. Cells were incubated at room temperature for at least 10 min; luminescent signal was then measured using the Lmax luminometers (Molecular Devices, Sunnyvale, CA).

Transfection of siRNA and Stable Transfection of Cyr61
Cyr61 siRNA was purchased from Santa Cruz Biotechnology (Catalog no. sc 39331) and Akt siRNA was purchased from Cell Signaling Technology, Inc. (Catalog no. 6211). Nonspecific control siRNA was also obtained from Santa Cruz Biotechnology. pCDNA3.1 vector was purchased from Invitrogen (Carlsbad, CA). Cyr61 cDNA was a kind gift from Dr. Lester Lau at University of Illinois. Transfection of siRNA was performed per commercial protocol coming along with each product. Briefly, for the transient transfection of siRNA, Beas2B cells were placed in 24- or 96-well plates, optimal confluent condition for transfection was determined, and 50–80 nM siRNA was used for each transfection. The same amount of nonspecific control siRNA was also transfected. After 30 h, cells were exposed to room air or hyperoxia. For the stable transfection of Cyr61, Beas2B cells were transfected with pcDNA3.1 vector + Cyr61 or vector alone. G418 (500 ng/ml) was used to select the clones with successful transfection. Clones were examined by Western blot analysis for Cyr61 expression.

Western Blot Analysis
For Western blot analysis, cells were harvested and washed with cold PBS twice, then cells were scraped and resuspended in cell lysis buffer with protease inhibitor mixtures (New England Biolabs, Beverly, MA). Protein concentration was measured using the Bradford method. Cell lysate was electrophoresed under denaturing conditions (12% SDS-polyacrylamide gels) and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). A quantity of 30–50 µg protein was loaded in each lane. After being transferred onto nitrocellulose membranes, the blot was blocked with 5% nonfat milk and incubated with primary antibodies from 1–16 h. The membranes were then washed, blotted with the respective secondary HRP-conjugated antibodies, and developed using the ECL assays (Amersham Life Science, Arlington Heights, IL) per manufacturer's instructions.

Statistical Analysis
Data were reported as means ± SD. Differences between two groups were determined by Student's t test. Data shown here were representatives of the identical results obtained from three independent experiments. Statistically significance was set at P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Hyperoxia Induces Cyr61 Expression in Lungs In Vivo and In Vitro
We first examined the expression of Cyr61 protein expression in vivo by Western blot analysis. Lung tissues isolated from C57/Bl6 mice after exposure to room air or hyperoxia (95% oxygen) were subjected to Western blot analysis. After 48 h of hyperoxia, Cyr61 expression was significantly induced after hyperoxia when compared with control normoxia tissues (Figure 1A). To further confirm the expression of Cyr61 in lung tissue after hyperoxia, we performed immunofluoroscence studies in mice lungs after 48 h of hyperoxia. We observed marked increased Cyr61 expression in both alveolar and bronchial cells after hyperoxia (Figure 1B).



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  		Figure 1. Expression of Cyr61 in murine lung tissues. Mice were treated with and without hyperoxia (95% oxygen). After 48 h, murine lung tissues were homogenized for Western blot analysis or fixed in 2% paraformaldehyde, followed by 30% sucrose overnight for Immunofluorescence. (A) Cyr61 expression in murine lung tissues detected by Western blot analysis; 50 µg of homogenized lung tissues were loaded in each lane. Upper row was blotted with anti-Cyr61 antibodies and lower row was blotted with anti–ß-actin antibody. (B) Cyr61 expression in murine lung tissues detected by Immunofluorescence. Red arrows: Cyr61 expression.

 
We then examined Cyr61 protein expression after hyperoxia in vitro using a variety of pulmonary cell lines and primary cells isolated from rat lungs. Cyr61 protein expression was highly induced by hyperoxia in a time-dependent manner in a variety of pulmonary cell types (Figure 2). Hyperoxia-induced Cyr61 expression was observed not only in fibroblasts (MRC5) and bronchial epithelial cells (Beas2B and H292), but also in primary cells isolated from pulmonary artery (PAEC, PASMC) (Figure 2B). Interestingly, A549 cells did not exhibit increased Cyr61 protein expression after hyperoxia. We used TGF-ß1, known inducer of Cyr61, as our positive controls for these studies. Furthermore, we examined whether some proinflammatory cytokines and endotoxin can induce the expression of Cyr61 in lung epithelial cells (Figure 2). In our study, TNF-{alpha} seems the strongest inducer, followed by IL-1ß, cytomix, and IFN-{gamma}. Endotoxin, lipopolysaccharide (LPS) can also induce Cyr61 expression at the dose of 1 µg/ml.



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  		Figure 2. Expression of Cyr61 in pulmonary cells. Cells were treated with or without hyperoxia (95% oxygen). After 48 h (except in A), cells were collected and protein concentration was determined. Equal amount of protein (30 µg/each lane) was loaded. Cyr61 protein was detected by Western blot analysis. (A) Time course of Cyr61 expression after hyperoxia in H292 cells. (B) Primary cells were obtained from rat lung pulmonary artery. Upper rows: endothelial cells from rat pulmonary artery. Lower rows: smooth muscle cells from rat pulmonary artery. (C) Cyr61 expression in Beas2B (upper), A549, (middle), and MRC5 cells (lower). Beas2B, A549, and MRC5 cells were treated with hyperoxia or TGF-ß. Cells treated with TGF-ß (2 ng/ml, 48 h) were used as positive controls. (D) Effects of cytokines and endotoxin on Cyr61 expression in Beas2B cells. Beas2B cells were treated with TNF-{alpha} (1,000 U/ml), IFN-{gamma} (250 U/ml), IL-1ß (100 U/ml), and their mixture or LPS (1µg/ml, or as labeled).

 
Effects on Cell Survival after Hyperoxia by Suppressing Cyr61 Expression
To determine the potential role of Cyr61 in hyperoxia-induced cell death, we performed loss of function studies in Beas2B epithelial cells by transfection of Cyr61 siRNA and nonspecific control siRNA. Control cells (nonspecific siRNA) exhibited increased cell death after hyperoxia as expected (Figure 3). However, inhibition of Cyr61 expression by siRNA significantly decreased cell survival after hyperoxia when compared with control cells (Figure 3). Interestingly, inhibition of Cyr61 by siRNA also conferred increased cell death in normoxic conditions.



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  		Figure 3. Effect of suppressing Cyr61 on cell viability. Beas2B cells were transiently transfected with control siRNA or Cyr61 siRNA. Thirty hours after transfection, cells were exposed to room air or hyperoxia (95% oxygen). After 48 h, cell viability was analyzed as described in MATERIALS AND METHODS. Open bars: Beas2B cells transfected with control siRNA; filled bars: cells transfected with Cyr61 siRNA. *P < 0.05, Cells transfected with control siRNA versus cells transfected with Cyr61 siRNA. +P < 0.05, normoxia versus hyperoxia in cells transfected with control siRNA. {ddagger}P < 0.05, normoxia versus hyperoxia in cells transfected with Cyr61 siRNA. #P < 0.05, after exposure to hyperoxia, cells transfected with Cyr61 siRNA were significantly less viable compared with cells transfected with control siRNA.

 
Effects on Cell Survival after Hyperoxia by Overexpressing Cyr61
After demonstrating that the inhibition of Cyr61 resulted in increased hyperoxia-induced cell death, we then performed gain of function studies by overexpressing Cyr61 in Beas-2B cells. We hypothesized based on the siRNA studies above that overexpression of Cyr61 will render the cells to exhibit increased survival after hyperoxic stress. Cell clones overexpressing Cyr61 protein were assessed by Western blot analysis (Figure 4A). We picked cell clone #1 (vector control) and cell clone #4 (overexpression of Cyr61) for our studies (Figure 4A). After 48 h, we observed that vector control cells exhibited increased cell death after hyperoxia as expected (Figure 4B), whereas Cyr61 overexpressing cells were significantly more resistant to cell death in response to hyperoxia when compared with cells transfected with empty vectors (control cells) (Figure 4B). Interestingly, we also observed that overexpression of Cyr61 conferred cytoprotective effects in response to another cellular stress, nutrient depletion (data not shown).



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  		Figure 4. Effect of overexpressing Cyr61 on cell viability. Beas2B cells were stably transfected with pCDNA3.1 vector alone (without insert) or with Cyr61. (A) Western blot analysis to detect the expression of Cyr61 in each clone. Clone 1 and 2, Beas2B cells were transfected with pCDNA3.1 vector alone (Beas2B/vector). Clones 3–5, cells were transfected with pCNDA3.1 + Cyr61 (Beas2B/Cyr61). An equal amount of protein (30 µg/lane) was loaded in each lane. Blot was then stained by PonceauS to verify equal loading (not shown). (B) Effect of hyperoxia on viability of Cyr61-overexpressing cells. Both Beas2B/vector and Beas2B/Cyr61 cells were exposed to room air and hyperoxia. After 48 h, cell viability assays were performed using the method described in MATERIALS AND METHODS. Open bars: Beas2B/vector cells (clone 1). Filled bars: Beas2B cells/Cyr61 (clone 4). *P < 0.05, Normoxia versus hyperoxia in Beas2B/vector cells. + P < 0.05, after exposure to hyperoxia, viable Beas2B/vector cells compared with viable Beas2B/Cyr61 cells.

 
Akt Cell Signaling Pathways Mediate Cytoprotective Effects of Cyr61 after Hyperoxia
Using cells overexpressing Cyr61, we examined the effect of Cyr61 on cell signaling involved in cell survival/death. We observed that hyperoxia could induce high levels of phospho-Akt activation in Cyr61-overexpressing cells but not in the vector control cells (Figure 5A). The induction of phospho-Akt by hyperoxia in Cyr61-overexpressing cells is time-dependent. There were no significant changes on the total Akt level between vector control cells and Cyr61-overexpressing cells (Figure 5B).



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  		Figure 5. Expression and activation of Akt in Beas2B cells stably overexpressing Cyr61 after hyperoxia. (A) Beas2B cells stably overexpressing Cyr61 (clone 4) and control cells (transfected with vector only, clone 1) were exposed to room air or hyperoxia (95% oxygen). After 48 h, cells were collected and fractioned in 10% SDS gel, then blotted with anti–phosphor-Akt and ß-actin antibodies, respectively. (B) Time course of phosphor-Akt expression after hyperoxia. As above, cells from clones 1 and 4 were exposed to hyperoxia, after each time point, cells were collected, franctioned on 10% SDS gel, and blotted with either anti–phosphor-Akt or total Akt antibodies. Fifty micrograms of protein was loaded in each lane.

 
To further examine the potential role of Akt pathway in Cyr61-mediated cytoprotection after hyperoxia, we transiently transfected the vector control cells and Cyr61 overexpressing cells with Akt siRNA to suppress Akt expression. Transfection of Akt siRNA reversed the cytoprotective effects of overexpressing Cyr61 compared with the transfection of control siRNA (Figure 6).



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  		Figure 6. Effect of suppressing Akt on the survival of Cyr61-overexpressing cells under hyperoxia. Control cells (clone 1) and Cyr61-overexpressing cells (clone 4) were transiently transfected with control siRNA or Akt siRNA. Thirty hours after transfection, cells were exposed to room air or hyperoxia. After another 48 h, cell viability was analyzed as described in MATERIALS AND METHODS. Open bars: Beas2B cells/vector alone (clone 1). Filled bars: Beas2B/Cyr61 (clone 4). *P < 0.05, normoxia versus hyperoxia in Beas2B/vector cells. # P < 0.05, normoxia versus hyperoxia in Beas2B/Cyr61 cells. {ddagger}P < 0.05, after exposure to hyperoxia, Beas2B/vector cells transfected with control siRNA compared with Beas2B/Cyr61 cells transfected with control siRNA.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Our study investigated the potential functional role of Cyr61 in hyperoxia-induced cell death in lung epithelial cells. Although Perkowski and coworkers had shown that hyperoxia induced Cyr61 mRNA in murine lung tissues (24), for the first time, we demonstrated that after hyperoxia, Cyr61 was induced in a variety of pulmonary cells and in murine lung tissues at protein level. The epithelial cells, fibroblasts, pulmonary endothelial cells, and pulmonary artery smooth muscle cells all expressed certain level of Cyr61 constitutively. The basal level expression of Cyr61 was significantly enhanced by hyperoxia in a time-dependent manner. Previous reports demonstrated differential Cyr61 expression in different tumor types (26–28). In our study, one commonly used tumor cell line, A549 cells, failed to yield any constitutive expression of Cyr61 and had no response to hyperoxic induction, suggesting cell specificity of Cyr61 expression.

Given the robust expression of Cyr61 protein induced by hyperoxia in the lungs and its known roles involved in wound healing and repair, one immediate question would be what function Cyr61 plays in hyperoxia-induced cell death. Although there were some studies on the role of Cyr61 in cell survival and death previously, most of them focused on tumor cells (7, 11, 15). Depending on the tumor type and location, both suppression and promotion of cell death by Cyr61 expression were found (1–5, 7, 11, 19), indicating its diverse physiologic function to specific stimuli. In our study, Cyr61 played a protective role in hyperoxia-induced cell death in Beas2B epithelial cells. This protective effect was confirmed by both "loss" and "gain" function assays, using siRNA to inhibit Cyr61 expression or overexpressing this protein by stable transfection, respectively. The recombinant Cyr61 protein is not commercially available at this time. When it is available, we plan to further confirm our observation by directly applying the Cyr61 protein into cell culture media given its presumable "autocrine" and "paracrine" effect (12).

Furthermore, we were interested in the intracellular pathways by which Cyr61 confers cytoprotective function. By examining a few of the known candidate pathways (12, 29–32), we found that after being exposed to hyperoxia, phosphor-Akt was markedly elevated in the Cyr61-overexpressing cells. Akt had been shown to involve in cytoprotective signaling pathways in hyperoxia-induced lung epithelial cell death (33, 34). Lu and colleagues further showed that Akt protected mice from hyperoxic pulmonary damage and delayed cell death. In their study, a constitutively active form of Akt was introduced intratracheally into the lungs of mice by adenovirus gene transfer techniques. The elevated Akt protected hyperoxia-induced lung injury in these mice in vivo (33). Therefore, we hypothesized that Cyr61 protects the hyperoxia-induced cell death via Akt pathway. By suppressing the expression of Akt, Akt siRNA markedly reduced the protective effect of Cyr61 on hyperoxia-induced cell death compared with the nonspecific control siRNA. This observation confirmed our hypothesis that Cyr61 protected against hyperoxia-induced epithelial cell death via Akt signaling pathway.

Kim and coworkers showed previously that Coxsackievirus B3 infection induced Cyr61 activation via JNK to mediate Hela cell death (21). We thus examined the phosphor-JNK level in Cyr61-overexpressing cells with and without exposure to hyperoxia. We failed to find any differences on phosphor-JNK level between Cyr61-overexpressing cells and vector control cells, indicating that in our hyperoxia model, Cyr61 might not function via JNK-related pathways (data not shown). In addition, Tong and colleagues reported that Cyr61 suppressed the growth of non–small cell lung cancer cells via the p53 pathways (20). In our study, there were no changes found on p53 level between Cyr61-overexpressing cells and vector controls treated with or without hyperoxia (data not shown).

Further direction in our group will focus on studying the role of Cyr61 in hyperoxia-induced lung injury in vivo. We would test the function of overexpressing Cyr61 in vivo after hyperoxia exposure. In addition, we would further explore the function of Cyr61 in other pulmonary cells, such as endothelial cells, fibroblasts, and smooth muscle cells under hyperoxic stress. Cyr61 may play an important role in hyperoxia-induced lung injury, repair, and remodeling. However, given the complexity of physiologic functions of this molecule, Cyr61 may possess pleiotropic functions in response to other cellular stresses such as TNF-{alpha} and LPS, both impotant mediators in lung injury and ARDS.

In summary, our data suggested that Cyr61 played a protective role in hyperoxia-induced cell death in Beas-2B lung epithelial cells via Akt-related signaling pathways. Further characterization of this observation in other cell types and in vivo is under investigation.


   Footnotes
 
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form April 18, 2005

Received in final form June 9, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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