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Cardiovascular Research 2004 62(3):481-488; doi:10.1016/j.cardiores.2004.01.024
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Copyright © 2004, European Society of Cardiology

Growth hormone releasing peptide (ghrelin) is synthesized and secreted by cardiomyocytes

María J Iglesiasa, Roberto Piñeiroa, Montserrat Blancob, Rosalía Gallegob, Carlos Diéguezc, Oreste Gualillod, José R González-Juanateya and Francisca Lago*,a

aUnidad de Investigación del Servicio de Cardiología, Laboratorio de Investigación 1, Planta Baja, Area de Investigación y Docencia, Hospital Clínico Universitario de Santiago de Compostela, Travesía Choupana s/n, 15706, Santiago de Compostela, Spain
bDepartamento de Ciencias Morfológicas, Universidad de Santiago de Compostela, Spain
cDepartamento de Fisiología, Universidad de Santiago de Compostela, Spain
dUnidad de Investigación del Servicio de Reumatología, Laboratorio de Investigación 4, Hospital Clínico Universitario, Santiago de Compostela, Spain

* Corresponding author. Tel.: +34-981-950902; fax: +34-981-951068. Email address: frlago{at}usc.es

Received 12 September 2003; revised 12 January 2004; accepted 20 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Ghrelin, the endogenous ligand of growth hormone secretagogue receptor (GHS-R), acts on the pituitary and the hypothalamus to stimulate the release of growth hormone (GH) and promotes appetite and adiposity. It has also been reported to increase myocardial contractility, induce vasodilation, and protect against myocardial-infarction-induced heart failure. Though principally gastric in origin, it is also produced by other tissues. This work investigated whether cardiomyocytes synthesize and secrete ghrelin, and how its production in these cells responds to stress and exogenous apoptotic agents. Methods: Ghrelin and its receptor expression was studied by RT-PCR, immunohistochemistry, and competitive binding studies in mouse adult cardiomyocyte cell line HL-1, and primary cultured human cardiomyocytes. Ghrelin accumulation in cardiomyocyte culture medium was measured by radioimmunoassay. Viability and apoptosis assays were carried on by MTT and Hoechst dye vital staining, respectively. Results: RT-PCR showed that HL-1 cells produce mRNAs for both ghrelin and GHS-R, and that GHS-R1a is expressed in human cardiomyocytes; and competitive binding studies using 125I-labelled ghrelin showed efficient constitutive expression of GHS-R at the surface of HL-1 cells. Immunohistochemistry confirmed the presence of ghrelin in the cytoplasm of HL-1 cells and of isolated human cardiomyocytes in primary culture. Radioimmunoassay showed that ghrelin was secreted by HL-1 cells and human cardiomyocytes into the culture medium. Ghrelin did not modify the viability of HL-1 cells subjected to 12-h starvation, but did protect against the apoptosis inducer cytosine arabinoside (AraC). Finally, production of ghrelin mRNA in HL-1 cardiomyocytes was reduced by AraC but increased if exposure to AraC was preceded by GH treatment. Conclusions: Ghrelin is synthesized and secreted by isolated murine and human cardiomyocytes, probably with paracrine/autocrine effects, and may be involved in protecting these cells from apoptosis.

KEYWORDS Apoptosis; Cell culture/isolation; Growth factors; Hormones; Cardiomyocytes; Receptors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Ghrelin is a 28-amino-acid acylated peptide that was first isolated from rat stomach endocrine cells. It is the endogenous ligand for growth hormone secretagogues receptor (GHS-R) [1], a G-protein-coupled receptor that is found mainly in the pituitary and hypothalamus [2] but also in numerous other tissues [3]. Major interest in this peptide derives from the fact that, in addition to other effects, it is involved in the regulation of energy balance and body weight homeostasis [4]. In rodents, ghrelin stimulates appetite, adiposity and the release of growth hormone (GH) [1,5–8], and regulates the gonadal axis [9] and carbohydrate metabolism [10]. In humans, ghrelin enhances appetite [11], and plasma ghrelin levels correlate negatively with body mass index and are subnormal in the obese [12,13].

Although ghrelin is found mainly in stomach and hypothalamus [1], ghrelin and/or its mRNA have also been detected, at lower levels, in kidney, placenta, pancreas, testis, ovary, adrenal cortex and myocardium (in this last without identification of what type of cell was responsible), and in many of these tissues, including human myocardium, GHS-R mRNA has also been found (see Ref. [14] and references therein). This suggests that ghrelin can act as a paracrine/autocrine hormone in these tissues.

With regard to the cardiovascular system, GHS-R mRNA is present not only in human myocardium but also in the aorta, left ventricle and left atrium of rat [15]. Furthermore, in both humans and experimental animals, ghrelin can have beneficial cardiovascular effects that seem not to be mediated by GH [15–22]. This suggests that one of the multiple mechanisms by which obesity favours cardiac pathology may consist in its being associated, as noted above, with low ghrelin levels, which may reduce cardioprotection.

In the work described here, we investigated whether cardiomyocytes synthesize and secrete ghrelin, and how its production in these cells responds to exogenous apoptotic and anti-apoptotic agents.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
All sera and media were from Life Technologies (Poole, UK), and all other products from Sigma Chemicals Co. (St. Louis, MO, USA), unless otherwise stated.

2.1. Cells
HL-1 cells (a line of adult mouse atrial cardiomyocytes) were a generous gift of Dr. W.C. Claycomb of Louisiana State University Medical Center (New Orleans, LA, USA) and were cultured on fibronectin-covered plates containing ExCell 320 medium (from JRH Biosciences, Andover, UK) supplemented with foetal bovine serum (FBS), insulin, norepinephrine, endothelial cell growth supplement (from Upstate Biotechnology, Lake Placid, NY, USA), and retinoic acid [23]. In experiments on apoptosis induction, HL-1 cells were pretreated for 12 h with 1 µg/ml growth hormone (GH) or 0.1–5.0 µM ghrelin and were then treated for 12 or 24 h with 100 µM AraC as proapoptotic agent.

Primary cultures of human cardiomyocytes were obtained from pieces of right atrial appendage excised to allow catheterization of the right atrium during surgery requiring cardiopulmonary bypass (this excised tissue is normally discarded). The atrial tissue was minced in small pieces and digested at 37 °C in three 5-min cycles with PBS (NaCl, 0.15 M in 0.01 M phosphate buffer of pH 7.4) containing 0.25% trypsin, 0.15% collagenase and 0.02% glucose, and cells were then extracted from the pooled supernatants by centrifuging for 5 min at 580 x g and were cultured in Iscove's modified Dulbecco's medium (Life Technologies) supplemented with 10% FBS, 0.1 mM β-mercaptoethanol, and antibiotics.

2.2. Immunocytochemistry
Tissues: Stomachs and hearts were obtained from eight mice. Samples of normal human stomach and heart from surgical specimens or recent autopsies (n=7) were provided by Prof. J. Forteza of the University Clinical Hospital's Department of Pathology. Samples were fixed in 10% buffered formalin for 24 h, dehydrated, and embedded in paraffin by standard procedures. Sections 5 µm thick were mounted on Histobond adhesive microslides (Marienfeld, Lauda-Königshofen, Germany), dewaxed and rehydrated. Antigens were exposed by microwaving in 0.01 M sodium citrate buffer (pH 6.0) (three 5-min cycles at 750 W). Sections were successively incubated in (1) goat polyclonal anti-ghrelin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:500 (1 h); (2) 3% hydrogen peroxide (Merck, Darmstadt, Germany) to block endogenous peroxidase (10 min); (3) biotinylated donkey anti-goat Ig (Santa Cruz) diluted to 1:100 (30 min); (4) streptavidin–biotin–peroxidase complex (Duet kit, Dakopatts, Glostrup, Denmark), prepared 30 min before use following the manufacturer's protocol (30 min); and (5) 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution, prepared by dissolving a DAB-buffer tablet (Merck) in 10 ml of distilled water (10 min). Between steps, sections were washed twice for 5 min with TBS (0.05 M Tris buffer of pH 7.6 containing 0.3 M NaCl), and after step 5 with distilled water. All dilutions were performed with TBS except that of the primary antibody (step 1), for which a Dakopatts antibody diluent was used. Negative controls were processed using anti-ghrelin antibody exposed overnight to 10 µM ghrelin (Santa Cruz) at 4 °C, or using TBS alone (without antibody, etc.) in some other incubation step.

Cells: HL-1 cells and primary-cultured human cardiomyocytes were fixed by immersion for 15 min in 10% buffered formalin, 5 min in PBS, 4 min in methanol at –20 °C and 2 min in acetone at –20 °C, and were then washed twice for 5 min in PBS. Antigens were exposed by microwaving for 10 min at 750 W in 0.01 M sodium citrate buffer. Anti-ghrelin antibody was used at a dilution of 1:50. Human cells were identified as cardiomyocytes by staining with a 1:50 dilution of an affinity-purified goat polyclonal antibody raised against a peptide sequence near the carboxy terminus of human myosin heavy chain (anti-MHC antibody, from Santa Cruz). Negative controls were processed using anti-ghrelin antibody exposed for 24 h to 10 µM ghrelin at 4 °C, or by omitting some essential step of the reaction.

Microscopy: Sections were observed and photographed using a Provis AX70 microscope (Olympus, Tokyo, Japan).

2.3. Ghrelin binding by HL-1 cells
HL-1 cells (5 x 105) were deprived of serum for 4 h and then left overnight at 4 °C in PBS containing 0.1% of bovine serum albumin (BSA), 105 cpm/ml [125I]hGhrelin (Amersham Biosciences, Freiburg, Germany) and various concentrations of unlabelled ghrelin. Cells were then washed and lysed in 0.1 N NaOH, and total associated radioactivity was measured in a {gamma}-counter. Scatchard analysis was performed using the program Ligand [24].

2.4. Hoechst dye vital staining
HL-1 cells (104) were seeded in 24-well plates and incubated for 45 min at 37 °C in Hoechst 33258 dye. Then HEPES (pH 7.8) was added to a final concentration of 5 mM, and the cells were fixed with 0.4% paraformaldehyde for 30 min and examined by fluorescence microscopy.

2.5. MTT viability assay
HL-1 cardiomyocytes (104 per treatment) were deprived of serum for 12 h and then treated for 72 h with ghrelin (10–12–10–6 M). Four hours before the expiry of this period, 0.5 g/l MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) was added. After overnight incubation at 37 °C, absorbance at 550–600 nm was measured.

2.6. RT-PCR
RT-PCR for ghrelin and GHS-R was performed on RNA from HL-1 cells and from tissues from the same sources as used for immunocytochemistry (vide supra). Total RNA was prepared using TRIzol reagent (from Life Technologies), and 2 µg was back-transcribed into cDNA by incubation for 50 min at 37 °C, 10 min at 42 °C, and 5 min at 95 °C with 200 U of murine leukemia virus reverse transcriptase (from Life Technologies) in 20 µl of a reaction mixture containing 50 mM KCl, 20 mM Tris–HCl (pH 8.4), 2.5 mM MgCl2, 0.1 g/l BSA, deoxy-NTPs (each 1 mM) and 20 U of the ribonuclease inhibitor RNAsin (from Promega, Madison, WI, USA). The resulting cDNA was used as a PCR template in a reaction mixture containing 5 U of Taq DNA polymerase (from Life Technologies), 50 mM KCl, 20 mM Tris–HCl (pH 8.4), 2.5 mM MgCl2, 0.1 g/l BSA, deoxy-NTPs (each 0.2 mM) and the specific primers for mouse preproghrelin (5'-AGCATGCTCTGGATGGACATG-3' (sense) and 5'-AGGCCTGTCCGTGGTTACTTGT-3' (antisense) [25], for mouse GHS-R (5'-CTATCCAGCATGGCCTTCTC-3' (sense) and 5'-GGAAGCAGATGGCGAAGTAG-3' (antisense); from Genebank, accession number AF332997 [GenBank] ), or for human ghrelin (5'-TGAGCCCTGAACACCAGAGAG-3' (sense) and 5'-AAAGCCAGATGAGCGCTTCTA-3' (antisense) [26] (each 0.2 mM). GAPDH was amplified as a control. Thirty-six PCR cycles were performed, each consisting of denaturation at 98 °C for 20 s, annealing for 1 min at 63 °C (58 °C for human ghrelin), and extension at 72 °C for 1 min. The PCR products were electrophoresed on 1% agarose gel, stained with ethidium bromide, and examined under UV light. RT-PCR for human GHS-R1a (the active form [3] of the receptor) was performed as previously described [27].

2.7. Sequence analysis
GHS-R PCR product was sequenced using a BIGDyeTM Terminator kit (Amersham Biosciences) and an ABI Prism automated DNA sequencer (Applied Biosystems, Foster City, CA, USA).

2.8. Southern blots
To confirm their identity, electrophoresed amplimers were transferred from the agarose gels to nylon membranes, hybridized for 18 h at 44 °C with a 32P-labelled antisense cDNA probe for rat ghrelin, washed to remove excess probe, and autoradiographed. Amplimers appearing in the autoradiographs were sized by comparison with calibration lanes in the ethidium–bromide-stained agarose gels.

2.9. Ghrelin radioimmunoassays
Ghrelin levels in HL-1 cells and primary cultured human cardiomyocytes culture media were determined using specific kits from Phoenix Pharmaceuticals (Belmont, CA, USA).

2.10. Statistical analysis
Results shown are the means±S.E.M.s of at least three independent experiments. The significance of differences was estimated by ANOVA followed by Student–Newmann–Keuls multiple comparison tests; p<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Preproghrelin gene expression in cardiomyocytes
RT-PCR for preproghrelin in HL-1 cells afforded a 329-bp cDNA sequence, the identity of which was confirmed by Southern blotting with a probe for rat ghrelin (Fig. 1A). That reverse transcription had proceeded properly was confirmed by amplification of GAPDH. PCR also amplified human ghrelin cDNA obtained from RNA from human myocardium and endocardium (Fig. 1B).


Figure 1
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  		Fig. 1 RT-PCR results showing expression of preproghrelin gene in HL-1 cardiomyocytes (Panel A, lanes 3–6) and of ghrelin gene in human endocardium and myocardium (Panel B, lanes 3 and 4). Positive controls: expression of the target gene in mouse or human stomach (Panel A lane 1 and Panel B lane 2, respectively), and of GAPDH (Panel A lanes 7–11, and Panel B lanes 5–7). Negative controls: Panel A lanes 12 and 13, and Panel B lanes 8 and 9. Molecular weight markers: Panel A lane 2 and Panel B lane 1. (A) Bottom panel: autoradiographs of Southern blots of lanes 1–6 of the upper panel.

 
3.2. Synthesis of ghrelin by cardiomyocytes
The cytoplasm of HL-1 cells and of primary cultures of human cardiomyocytes showed intense immunoreactivity with anti-ghrelin antibody (Fig. 2E and 2G). The possibility that the human cells were fibroblasts rather than cardiac muscle fibres was ruled out by their immunoreactivity with anti-MHC antibody (Fig. 2H). Similarly, the cytoplasm of virtually all muscle fibres in sections of murine and human heart was immunoreactive with anti-ghrelin antibody (Fig. 2J and 2L) (the nuclei of these cells were not stained).


Figure 2
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  		Fig. 2 Ghrelin immunoreactivity in human (g) and HL-1 (e) cardiomyocytes, in human (l) and mouse (j) heart tissue, and, as positive controls, in human (c) and mouse (a) stomach (where immunoreactivity was found in neuroendocrine cells of gastric glands). For murine and human stomach, murine and human heart and HL-1 cells, negative controls were run with pre-saturated primary antibody (b, d, k, m and f, respectively), and for human cardiomyocyte primary cultures by replacing primary antibody with TBS (i). Human cardiomyocyte primary cultures were also stained by anti-MHC antibody (h). (b, d, i, k, m) Nomarsky differential interference contrast. Objective magnifications: C and K, x 20; a, b, d, j, l and m, x 40; e, f, g, h, i, x 60.

 
In the positive controls (mouse and human stomach), immunoreactivity with anti-ghrelin was found in the neuroendocrine cells of the gastric glands (Fig. 2A and 2C). Anti-ghrelin antibody pre-saturated with ghrelin did not stain either these stomach tissues (Fig. 2B and 2D) or heart tissues (Fig. 2K and 2M), and neither was there any staining of the negative controls for the cultured cells (see Methods) (Fig. 2F and 2I).

3.3. Expression of the GHS-R gene and ghrelin binding by cardiomyocytes
RT-PCR showed that, in HL-1 cells, GHS-R mRNA is expressed at levels similar to those observed in mouse heart (Fig. 3A.1). The identity of the PCR product was confirmed by sequencing (data not shown), with mouse pituitary as positive control. Statistical analysis of three independent Scatchard analyses of ghrelin binding by HL-1 cells showed efficient constitutive expression of the receptor gene, yielding values of 3.03±0.8 nM for Kd and 0.14±0.06 nM for Bmax (Fig. 3B), the latter figure being equivalent to about 170,000 binding sites per cell. The presence of GHS-R1a in human cardiomyocytes was confirmed by RT-PCR (Fig. 3A.2).


Figure 3
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  		Fig. 3 (Panel A) Panel A.1: RT-PCR results showing expression of mouse GH secretagogue receptor (GHS-R) gene in HL-1 cardiomyocytes (lanes 4 and 5) and mouse heart (lane 6). Positive controls: expression of the target gene in mouse pituitary (lane 1), and of GAPDH in the aforementioned tissues and cells (lanes 2, 7, 8 and 9, respectively). Negative controls: lanes 10 and 11. Molecular weight marker: lane 3. Panel A.2: RT-PCR results showing expression of human GH secretagogue receptor 1a (GHS-R1a) (lanes 2 and 3) and of GAPDH (lanes 5 and 6) in human cardiomyocytes. Negative controls: lanes 4 and 7. Molecular weight marker: lane 1. (Panel B) A representative experiment (n=3) showing binding of 125I-labelled human ghrelin by HL-1 cells in the presence of various concentrations of unlabelled ghrelin (competitor) (Panel B.1), and Scatchard analysis of binding data from the competition study (Panel B.2).

 
3.4. Secretion of ghrelin by cultured HL-1 cells and human cultured cardiomyocytes
The RIA-measured concentration of ghrelin following 24-h starvation in HL-1 cell culture medium was 16.4±0.3 pg/ml (n=3); and in primary cultured human cardiomyocytes, culture medium was 12.8±1.1 pg/ml (n=4).

3.5. Ghrelin and HL-1 cardiomyocyte viability
Ghrelin did not modify the MTT-assessed proliferation or viability of HL-1 cardiomyocytes (Fig. 4A). However, pre-treatment with 0.1 µM ghrelin for 12 h prevented the apoptosis induced by treatment with 100 µM AraC for 12 h in these cells. Hoechst dye vital staining showed that apoptosis affected 7.6±1.0% of cells with ghrelin pre-treatment, against 14.9±1.8% without (p<0.05, n=3; Fig. 4B). Similar protection was afforded when AraC treatment was preceded by 12-h pre-treatment with GH instead of ghrelin (results not shown). RT-PCR showed that ghrelin mRNA levels were decreased by 33.0±7.1% by 12-h treatment with 100 µM AraC (p<0.05, n=3), but were increased by 48.8±13.9% with respect to control cells if 12-h pre-treatment with GH preceded the AraC treatment (p<0.05, n=3).


Figure 4
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  		Fig. 4 (Panel A) MTT assay results showing that various concentrations of ghrelin did not affect the viability of HL-1 cells (n=3). (Panel B) Results of Hoechst dye vital staining assays of apoptosis in HL-1 cells treated with 100 µM cytosine arabinoside (AraC) for 12 h, with or without pre-treatment with 0.1 µM ghrelin for 12 h. (Panel C) Changes in ghrelin mRNA levels in HL-1 cardiomyocytes following 12-h treatment with AraC, with or without pre-treatment with the antiapoptotic growth hormone (GH). *p<0.05 (n=3).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The above results show in the first place and for the first time that ghrelin can be synthesized by cardiomyocytes of both human and murine origin, and that it is secreted by HL-1 cells (a cultured line derived from murine atrial cardiomyocytes that maintains a heart-specific phenotype [23] and is accordingly used as an in vitro model in studies of cardiomyocyte biology [28]) and also by human cardiomyocytes in primary culture. We also found that HL-1 cells produce GHS-R that efficiently binds ghrelin at the cell surface and that human myocardium expresses GHS-R1a mRNA. Our results are in keeping with the observation of others that GHS-R mRNAs are present in human myocardium [3] and of GHS-R mRNA in rat left ventricle and left atrium [15], and strongly suggest that ghrelin has paracrine/autocrine activity in cardiac muscle.

The discovery that ghrelin is the endogenous GH secretagogue immediately prompted research on its haemodynamic effects, GH being known to play a role in the maintenance of cardiovascular health [29]. Administration of ghrelin has been found to reduce cardiac afterload and increase cardiac output without increasing heart rate in healthy volunteers [15]; to induce vasodilation [19,20]; and to improve the haemodynamics of patients with chronic heart failure (CHF) [16]. CHF-associated cachexia is attenuated by ghrelin in rats [18], and in humans is accompanied by above-normal ghrelin levels, possibly as a compensatory mechanism in response to catabolic–anabolic imbalance [17]. Ghrelin also regulates cardiovascular function in rats suffering septic shock [22]. Similar beneficial cardiovascular effects have been observed in rabbits [21].

That most of the haemodynamic and cardioprotective effects of ghrelin may be direct, i.e. not mediated by GH, is suggested not only by the above-noted evidence of a paracrine/autocrine mode of action, but also, in some cases, by more direct evidence: its vasodilatory effects are not affected by GH release inhibitors [19], and the synthetic GHS-R ligand hexarelin prevents cardiac damage after ischaemia–reperfusion even in hypophysectomized rats [30]. Direct action in vivo is also suggested by the facts that, in vitro, ghrelin stimulates H9c2 cardiomyocyte cell proliferation [31], and reduces the doxorubicin-induced mortality of H9c2 cardiomyocytes and endothelial cells [32] and the AraC-induced mortality of HL-1 cells (this work).

Ghrelin inhibits the in vitro proliferation of cells of breast carcinoma [33], human lung carcinoma [34], and thyroid carcinoma [35], and promotes that of hepatoma cells [36], prostate cancer cells [27], adrenal cells [37] and, as noted above, H9c2 cardiomyocytes [31]. In the absence of AraC, ghrelin administration did not affect the proliferation or viability of HL-1 cardiomyocytes in this study. This difference between HL-1 and H9c2 cells may be due to the former being of adult and the latter of embryonic origin.

Ghrelin reduced the AraC-induced mortality of HL-1 cells, and ghrelin mRNA levels, which were decreased by AraC, were increased by pre-treatment with GH, which protects against AraC-induced apoptosis in these cells [38]. Exactly how GH and ghrelin interact in cardiomyocytes remains to be elucidated. Obesity is an increasingly prevalent condition that increases cardiovascular risk, including risk of heart failure [39]. The fact that ghrelin has beneficial cardiovascular effects, and the anti-apoptotic effects observed in this study, suggests that part of this increased risk may be due to obesity-related reduction of plasma ghrelin levels [12,13], which may reduce protection against the cardiomyocyte apoptosis that is known to contribute to progressive cardiomyocyte loss in heart failure [40]. Weight reduction, which is known to be essential for reducing cardiovascular risk in the obese [41], may therefore owe this effect in part to its restoring normal ghrelin levels.

In conclusion, ghrelin appears to have not only GH-mediated systemic effects, but also paracrine/autocrine actions on the metabolism and viability of a variety of types of cell and tissue. The findings of this study suggest that the cardioprotective effects of ghrelin are due to local mechanisms of this latter kind. It may be hypothesized that failure of these mechanisms may be determinant in the development of several cardiac pathologies.


   Acknowledgements
 
This research was supported by the Xunta de Galicia under projects PGIDIT02SAN91807, PGIDT01PXI90203PR, and PGIDIT02PXIB2080PR. Dr. Francisca Lago and Dr. Oreste Gualillo are recipients of a contract of Research from Spanish Ministry of Health, funded by Instituto de Salud Carlos III and Complexo Hospitalario Universitario de Santiago. (FL:99/3040 and OG: 00/3051).


   Notes
 
Time for primary review 15 days


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

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Y. Mizukami, K. Ono, C.-K. Du, T. Aki, N. Hatano, Y. Okamoto, Y. Ikeda, H. Ito, K. Hamano, and S. Morimoto
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