Original Article Augmentation of Arginase Ⅱ Expression in ...lib.tmd.ac.jp/jmd/5904/01_Tajima.pdf · pathway, decreasing vascular resistance, and thereby possibly improving blood

J Med Dent Sci 2012； 59： 75－82

L-Arginine is the common substrate for arginase and nitric oxide synthase (NOS). Arginase converts L-arginine to urea and L-ornithine. L-Ornithine is the principal precursor for the production of polyamines and L-proline, which are required for cell proliferation and collagen synthesis. Endothelial NOS is expressed in the human endometrial glandular epithelium, but the expression and physiological roles of arginase in the human endometrium are not clear. The objective of this study was to investigate the expression and distribution patterns of arginases Ⅰ (A-Ⅰ) and Ⅱ (A-Ⅱ) in the human endometrium by us ing immunoh is tochemistry , reverse transcription-polymerase chain reaction (RT-PCR), and western blotting. A-Ⅰ and A-Ⅱ were detected by immunohistochemistry in human endometrial epithelial cells during the proliferative and secretory phases of the menstrual cycle. RT-PCR showed that A-Ⅰ and A-Ⅱ mRNA were expressed in human endometrial tissue. Western blotting analysis results showed the expression of A-Ⅱ protein. Immunohistochemistry and western blotting results showed that expression levels of A-Ⅱ were significantly higher in the secretory phase than in the proliferative phase. Increased A-Ⅱ levels in the secretory phase may be responsible for endometrial growth by increasing polyamines and proline products.

Key words: arginase, endometrium, enzyme, L-arginine, menstrual cycle

Introduction

The human uterine endometrium undergoes cyclic morphological and functional changes. These dynamic structural changes are crucial for normal uterine physiological function and occur under the control of the sexual steroid hormones estrogen and progesterone.1 After fertilization, the endometrium plays a role in the preparat ion for embryo implantation and in the maintenance of pregnancy. In the endometrium, biological communications between maternal cells and the embryo occur, and many local factors, including cytokines and growth factors, are suggested to be involved in this phenomenon.2-7 The mechanisms of acceptance of the embryo are not clear, although many factors have been investigated. Thus, the aim of this study was to elucidate the mechanisms that are important for implantation in women, especially in infertile women. L-Arginine, a semi-essential amino acid, is the common substrate for 2 enzymes, nitric oxide synthase (NOS) and arginase. NOS catalyzes the oxidation of L-arginine to nitric oxide (NO) and citrulline. However, arginase converts L-arginine to L-ornithine and urea. L-Ornithine, an amino acid, is the precursor of polyamines and L-proline, which are essential components for cell proliferation and is involved in collagen synthesis, respectively. 8 These 2 types of enzymes were observed to work competitively in the living body.9 Thus, an alteration in the concentration balance of these enzymes may affect many physiological phenomena. Recent studies showed that L-arginine intake

Corresponding Author: Tatsuya Harada, M.D. Ph.D.Department of Comprehensive Reproductive Medicine, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, JapanTel: +81-3-5803-5322　Fax: +81-3-5803-0295E-mail: [email protected] June 27；Accepted September 14, 2012

Original Article

Augmentation of Arginase Ⅱ Expression in the Human Endometrial Epithelium in the Secretory Phase

Makiko Tajima, Tatsuya Harada, Tomonori Ishikawa, Yuki Iwahara and Toshiro Kubota

Department of Comprehensive Reproductive Medicine, Graduate School, Tokyo Medical and Dental University

76 J Med Dent SciM. Tajima et al.

significantly has improved uterine blood flow and augmented endometrial thickness in inferti le women.10,11 NO dilates the human uterine artery through the cyclic guanosine monophosphate pathway, decreasing vascular resistance, and thereby possibly improving blood flow.10,11 However, an improvement in blood flow is not an adequate explanation of the mechanism underlying the L-arginine-induced increase in endometrial thickness; thus, to further elucidate this mechanism, given that arginase also uses L-arginine as a substrate, we investigated its role in increased endometrial thickness. Arginase is present in 2 isoforms, arginases Ⅰ (A-Ⅰ) and Ⅱ (A-Ⅱ). Because arginase shares L-arginine as a substrate with NOS, it may also play a role in the regulation of NO synthesis. 9 Therefore, there is significant interest in arginase beyond its role in the urea cycle. The expression and regulation of NOS in the human endometrium have been reported previously. Endothelial NOS was found to be expressed in the human endometrium and mainly immunolocalized to the glandular epithelium.12 However, the expression of arginase and the physiological role of the L-arg in ine-orn i th ine pathway in the human endometrium are not clear. In this study, we examined the expression and distribution patterns of the 2 distinct isoforms of arginase, A-Ⅰ and A-Ⅱ, in the human endometrium in the proliferative and secretory phases, using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and western blotting.

Materials and Methods

1. Endometrial tissue collection The study protocol was approved by the ethical committee of Tokyo Medical and Dental University Hospital (Tokyo, Japan). Human endometrial tissue samples were obtained from women who underwent hysterectomies for myoma uteri or adenomyosis without hormone therapy at Tokyo Medical and Dental University Hospital. Written informed consent was obtained from each patient before the operation. The menstrual cycle stage, which was determined based on the date of the last menstrual period, was histologically confirmed according to the standard criteria. 13

Immediately after hysterectomy, the endometrial tissue samples were scraped approximately 0.5 to

1 cm3 from the uterine wall, placed in cold Dulbecco’s phosphate-buffered saline (D-PBS; D8662, Sigma Chemical Co, St. Louis, MO, USA), and rapidly transported to the laboratory. The specimens were washed thoroughly in cold PBS (D8537, Sigma Chemical Co) and cut into small sections. For the immunohistochemistry, the specimens were fixed in 4% paraformaldehyde. For the RT-PCR and western blot analyses, the samples were snap frozen in liquid nitrogen and stored at －80℃ until use.

2. Immunohistochemistry Paraffin-embedded sections (4-μm thick) of uterine samples were mounted onto silane-coated slides and then deparaffinized. Antigen retrieval was performed by autoclaving the sections at 121℃ for 15 min, and the slides were incubated in 3% hydrogen peroxidase in absolute methanol for 10 min to block endogenous peroxidase activity. After washing with PBS (pH 7.2) to prevent nonspecific antibody binding, the sections were incubated with PBS containing 5% goat serum for 30 min at room temperature. Next, the sections were incubated with an anti-A-Ⅰ monoclonal antibody (1:100; 610708, BD Biosciences Pharmingen, San Diego, CA, USA) for 1 h at room temperature or an anti -A-Ⅱ polyclonal antibody (1:200; sc-20151, Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) overnight at 4℃. The slides were washed with PBS 3 times after incubation with the primary antibodies and then finally incubated with the Histofine Simple Stain MAX PO (Nichirei Biosciences Inc, Tokyo, Japan) for 30 min at room temperature. The histochemical reaction method used to detect peroxidase activity was performed as described in our previous report. 14 The immunostained sections were counterstained with Mayer’s hematoxylin. Negative controls were prepared by replacing the primary antibodies with appropriately diluted normal mouse or rabbit antibodies. The absolute intensity of the diaminobenzidine (DAB) was evaluated in at least 10 nonoverlapping and randomly selected epithelial gland areas in each slide of the endometrium, using the National Institutes of Health Image program (National Institutes of Health, Bethesda, MD, USA). The mean DAB intensity in the glandular epithelium was calculated for each slide, using more than 4 independent specimens, to determine the arginase expression changes throughout the menstrual cycle. Statistical analyses of the differences in DAB

77Arginase expression in the human endometrium

intensity were performed using unpaired t tests. P < .05 was considered statistically significant.

3. RT-PCR Total RNA was isolated from frozen tissue with TRIzol (Invitrogen Corporation, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions, and the concentration was determined at 260 and 280 nm by spectrophotometry. Complimentary DNA was prepared from 4 μg of DNase-treated RNA with the SuperScript First-Strand Synthesis System (Invitrogen Corporation), and one-tenth of the mixture treated with reverse transcriptase was used as a template for PCR. The primer sequences for human A-Ⅰ, A-Ⅱ, ornithine decarboxylase (ODC), and ornithine aminotransferase (OAT) were designed according to the published cDNA sequences. 15,16 Sense and antisense primers for A-Ⅰ, 5′-CTTAAAGAACAAGAGTGTGATG-3′ and 5′-TTCTTCCTAGTAGATAGCTGAG-3′; A-Ⅱ, 5′ -GACACTGCCCAGACCTTTGT-3′ and ′-CGTTCCA TGACCTTCTGGAT-3′; ODC, 5′-GAGCACATCCCAAA GCAAAGT-3′ and 5′-TCCAGAGTCTGACGGAAAGT A-3′; and OAT, 5′-ACACATGAAGCTACCTTCTGA-3′ and 5′-ACGTTGTTCTATTATGTATCA-3′, respectively, were used to amplify 550-, 304-, 374-, and 402-bp fragments, respectively. The housekeeping gene β-tubulin was also amplified. The resulting cDNA samples were amplified using the PCR core kit (Roche Diagnostics, Indianapolis, IN, USA). The PCR experiments were carried out in a GeneAmp PCR System 2700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) in a 50-μL solution (10 mM Tris-HCl at pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, and 2.5 IU Taq polymerase). The amplification procedure consisted of 30 cycles of the following sequential steps: denaturation at 94℃ for 1 min, annealing at 55℃ for 2 min, and extension at 72℃ for 3 min. To increase the sensitivity of detecting the A-Ⅰ transcript, the cDNA was assayed by a nested PCR amplification, for which a second PCR amplification was undertaken with the inner primers 5′-CCCTTTGCTGACATCCCTAA-3′ and 5′-GACTCCAAGATCAGGGTGGA-3′ to amplify a 201-bp fragment using one-fifth of the first amplified cDNA as a template. PCR products were loaded onto a 2.0% agarose gel (Takara Bio Inc, Tokyo, Japan), electrophoresed, and visualized by ethidium bromide staining. The samples that were subjected to PCR amplifications without prior RT and without further sample preparation were used as negative controls.

4. Western blotting The endometrial tissues were lysed with lysis buffer (50 mM Tris-HCl at pH 7.5, 0.15 M NaCl, 1% Nodidet P-40, 0.1% deoxycholic acid, 10 μM phenyl methylsulphonyl fluoride, 0.5-mU/mL aprotinin, 1 μM leupeptin, and 1 μM pepstatin) to analyze the arginase protein expression. The protein content of the endometrial tissues was determined using the Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA) according to manufacturer’s instructions. Each sample, which contained 20 μg of protein, was separated by 10% sodium dodecyl sulfate -polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc, Hercules, CA, USA). After blocking with Block Ace (Dainippon Sumitomo Pharma Co, Ltd, Osaka, Japan) containing 10% fetal bovine serum to prevent nonspecific binding of the antibodies, the membranes were incubated with the same antibodies used in the immunohistochemistry as follows: anti-A-Ⅰ monoclonal antibody (1:1,000; BD Biosciences Pharmingen) overnight at 4℃ and anti-A-Ⅱ polyclonal antibody (1:2,000; Santa Cruz Biotechnology, Inc) for 90 min at room temperature. After washing with TBS with 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (1:5,000; GE Healthcare, Little Chalfont, UK). The bound antibodies were detected using enhanced chemiluminescence (GE HealthCare) according to the manufacturer’s instructions. β-Actin was used as an internal standard. The same statistical analyses were performed as those for the immunochemistry data.

Results

1. Localization of arginase by immunohistochemistry To determine the expression and localization of the 2 arginase isoforms in the human endometrium, paraffin-embedded sections were immunostained with anti-A-Ⅰ or anti-A-Ⅱ antibody. We have already demonstrated the specificity of the antibodies in a previous study.17 Representative photomicrographs are shown in Figure 1. Immunostaining for A-Ⅰ and A-Ⅱ was observed in all of the human endometrial tissue specimens examined. Immunostaining for A-Ⅰ was detected in endometrial epithelial cells. No differences in A-Ⅰ expression in the endometrium were found throughout the menstrual cycle.


Immunostaining for A-Ⅱ was also clearly detected in endometrial epithelial cells. In contrast, stronger immunostaining for A-Ⅱ protein was found in the endometrial tissue samples obtained from the secretory phase than those from the proliferative phase. No immunostaining was observed in the negative controls.

The mean DAB intensities of A-Ⅱ immunohistochemistry in endometrial epithelial cells in the proliferative and secretory phases are shown in Figure 2 and Table 1. The A-Ⅱ expression levels in the epithelial gland in the secretory phase were significantly higher than those in the proliferative phase (P < .05).

A-I

A-II

P S

Cont.(A-I)

Cont.(A-II)

30 μm 30 μm

30 μm 30 μm

30 μm 30 μm

30 μm 30 μm

Figure 1. Immunohistochemical localization of arginase Ⅰ (A-Ⅰ) and Ⅱ (A-Ⅱ) proteins in the human endometrium during the proliferative (P) and secretory phases (S). Representative photomicrographs are shown. No staining was observed in the control sections (Cont.). Objective lens: 20 ×. Scale bar = 30 μm.


Table: Patient profile and results of arginase Ⅱ expression levels as determined by immunohistochemistry and western blotting

Patient No. Age Diagnosis Cycle

Date Phase*1

Arginase II expression levels as determined by

immunohistochemistry (DAB intensity)

western blot analysis (A-II/β-actin ratio)

1 45 retroperitoneal neoplasm 16 p 67.8 ND*2

2 37 leiomyoma 10 p 113.1 ND









11 51 leiomyoma 10 p ND 1.3673




15 43 leiomyoma 21 s 69.5 ND




19 42 adenomyosis 29 s 122.7 ND

20 43 leiomyoma+adenomyosis 41 s 156.4 ND





25 43 leiomyoma+adenomyosis 35 s ND 1.2207

26 40 leiomyoma 16 s ND 1.8138



*1) histological endometrial menstrual phase, P: proliferative phase S: secretory phase*2) Not done.

2. Detection of arginase mRNA by RT-PCR Messenger RNAs for A-Ⅱ, ODC, and OAT were detected by RT-PCR, and mRNA for A-Ⅰ was detected by nested RT-PCR in RNA extracted from human endometrial tissue(proliferative phase, n = 4; secretory phase, n = 4). Amplification of human endometrial tissue cDNA with A-Ⅰ-, A-Ⅱ-, ODC-, and OAT-specific primers generated the expected 201-, 304-, 374-, and 402-bp fragments, respectively, for all the samples (Figure 3). The product sequences were identical to those of A-Ⅰ, A-Ⅱ, ODC, and OAT, respectively. The amounts of the 4 PCR products were similar in the proliferative and secretory phases. No mRNAs were detected in the negative controls (samples without prior RT and without further sample preparation).

Figure 2. Result of the diaminobenzidine intensity analysis of the A-Ⅱ expression levels in the epithelial gland area in the human endometrium (proliferative phase, n = 10; secretory phase, n = 10). The expression levels in the secretory endometrium were significantly higher than in the proliferative phase. *P < .05.

A-I

A-II

β-tubulin

P S

201bp

304bp

298bp

Cont.

OAT

ODC 374bp

402bp

Figure 3. Nested reverse transcription-polymerase chain reaction (RT-PCR) for A-Ⅰ and RT-PCR for A-Ⅱ, ODC, and OAT using total RNA extracted from the human endometrium during the proliferative and secretory phases of the menstrual cycle. The amplified cDNA fragments were electrophoresed on 2.0% agarose gels and visualized by ethidium bromide staining. DNA fragment sizes generated by RT-PCR for A-Ⅰ, A-Ⅱ, ODC, OAT, and β -tubulin are indicated at the right of each panel. The data presented are from a single experiment that is representative of 5 separate experiments. The samples that were subjected to PCR amplification without prior RT and without further sample preparation were used as negative controls. No mRNAs were detected by PCR without prior RT (data not shown) and without further sample preparation (Cont.).


3. Detection of arginase isoforms by western blotting To evaluate the expression levels of the 2 types of arginase in endometrial tissues, western blotting analyses of the endometrial tissue lysate was performed. We have already demonstrated the specificity of the antibodies in a previous study. 17 The bands for A-Ⅰ protein were not observed in all the samples. The expression levels of A-Ⅰ in the endometrium were observed to be low. The representative results showing the A-Ⅱ band patterns during the proliferative and secretory phases are shown in Figure 4. Western blotting using an anti-A-Ⅱ antibody probe resulted in bands near

39 kDa. A-Ⅱ proteins were detected in the proliferative and secretory phases, and a higher intensity was clearly observed in the secretory phase. The A-Ⅱ: ß-actin ratios in the endometrium during the proliferative and secretory phases are shown in Figure 5. The A-Ⅱ protein levels were significantly higher in the secretory phase than in the proliferative phase (P < .05).

Discussion

The p resen t s t udy demons t ra tes us i ng immunohistochemistry, RT-PCR, and western blot analyses that the human endometrium expresses 2 distinct arginase isoforms, A-Ⅰ and A-Ⅱ. To the best of our knowledge, this is the first report on the expression of the 2 arginase isoforms in the human endometrium. To the results of the immunohistochemistry showed that immunostaining for the 2 arginase isoforms was apparently localized in endometrial epithelial cells in the proliferative and secretory phases. Furthermore, we observed that the expression A-Ⅱ levels changed throughout the menstrual cycle. Immunostaining for A-Ⅱ in endometrial epithelial cells was stronger in the secretory phase than in the proliferative phase. In addition, we confirmed with western blot analyses that the A-Ⅱ protein levels were higher during the secretory phase of the endometrium. Arginase is present in 2 isoforms, A-Ⅰ and A-Ⅱ. They are encoded by different genes on different chromosomes, 18-20 differ in their cellular location and t i ssue d is t r ibut ion , and are regu lated i ndependen t l y . 8 ,21 -24 A rg inase syn thes i zes L-ornithine, which is catalyzed to polyamines and L-proline, by ODC and OAT, respectively. A-Ⅰ, which is a cytosolic enzyme, catalyzes the final step in the urea cycle in hepatocytes and is expressed to a limited extent in a few other tissues. In this study, a small amount of A-Ⅰ was detected in the human endometrium. Furthermore, we could not detect any differences in A-Ⅰ expression levels during the menstrual cycle. However, our findings show that the expression levels of A-Ⅱ in endometrial epithelial cells changed throughout the menstrual cycle. A-Ⅱ, a mitochondrial enzyme, is expressed mainly in extrahepatic tissues such as the kidney, brain, small intestine, mammary gland, and macrophages. Recent studies have indi-cated that increased levels of arginase activity may be involved in airway remodeling by cell proliferation

A-II

β-actin

39 kD

42 kD

P S

Figure 4. Result of the western blot analysis of A-Ⅱ using an anti-A-Ⅱ polyclonal antibody against protein obtained from human endometrium lysate during the proliferative and secretory phases of the menstrual cycle. The A-Ⅱ antibody reacted with a protein band at 39 kDa, which is the appropriate molecular weight for A-Ⅱ. Representative results are shown.

Figure 5. Results of the densitometric analyses of the A-Ⅱ protein levels in human endometrium lists during the proliferative and secretory phases of the menstrual cycle (proliferative phase, n = 4; secretory phase, n = 4). The bar represents the ratio of A-Ⅱ to β-actin and is shown as the mean ± SD of 2 independent experiments. *P < .05.


and collagen deposition in the airway wall and con-tribute to persistent airway hypertensiveness in asthma.25-27 In the cardiovascular system, arginase activation contributes to age-related vascular changes through a number of mechanisms, including polyamine-dependent vascular smooth muscle prolif-eration and collagen synthesis, which leads to aber-rant vessel wall remodeling and neointima forma-tion. 28-30 In the reproductive system, the relationship between arginase and endometrial receptivity has not been elucidated. In this study, the increased A-Ⅱ expression levels in the secretory phase might have led to the increase in cell proliferation and collagen synthesis in the endometrium, possibly causing the thickening and differentiation of the endometrium to prepare for implantation. However, further study is needed to verify this hypothesis. A thin endometrium is thought to be related to low pregnancy rates in patients treated by in vitro fertilization programs, and it is very difficult to improve endometrial growth in patients with a thin endometrium.31-35 Recent research has shown that L-arginine supplementation may improve endometrial thickness and receptivity. 10,11,36 Although increased uterine blood flow has been suggested as an important factor for endometrial growth, we consider local cell proliferation and collagen synthesis also very important. L-Arginine supplementation may lead to an increase in the levels of L-ornithine, which is metabolized to polyamine and L-proline, augmenting endometrial cell proliferation and collagen synthesis as a result. L-Ornithine is metabolized to polyamines by ODC and to pyrroline-5-carboxylate by OAT, which can subsequently be utilized for the synthesis of L-proline. In addition, we detected the mRNA expressions for OAT and ODC in human endometrium using RT-PCR. These findings suggest that cell proliferation and collagen synthesis might be augmented by increased arginase activity in the endometrium. In the metabolic pathway of L-arginine, a semi-essential amino acid in the human body, 2 enzymes, NOS and arginase, share L-arginine as a substrate. NOS, which catalyzes the oxidation of L-arginine to NO and citrulline, has been well studied. eNOS was detected in the human endometrial epithelial cells with immunohistochemistry. 12 However, the role of eNOS in the endometrium is not clear. A-Ⅰ and A-Ⅱ expressions were also localized in endometrial epithelial cells with the same distribution as eNOS. NOS and arginase may work competitively in the

endometrium, or the consumption of L-arginine may be increased. However, arginase may also regulate a number of cellular functions by NO-independent mechanisms.8,37,38 In the present study, we were able to detect arginase expression but were not able to elucidate the regulation of arginase expression and the possible role of arginase in the human endometrium. Clarification of these problems might be key for improving the outcomes of infertility treatments. In conclusion, the present study shows that 2 functional arginase isoforms were expressed in the human endometrial epithelial cells and that the A-Ⅱ levels were altered during the menstrual cycle. Elevated A-Ⅱ expression levels in the secretory phase may be responsible for endometrial growth by increasing polyamine and proline products. Thus, the L-arginine-ornithine-proline and polyamine pathways may play roles in the periods of receptivity for embryo implantation.

References1. Curry TE, Jr., Osteen KG. The matrix metalloproteinase

system: changes, regulation, and impact throughout the ovarian and uterine reproductive cycle. Endocr Rev. 2003; 24(4): 428-65.

2. King A. Uterine leukocytes and decidualization. Hum Reprod Update. 2000; 6(1): 28-36.

3. Norwitz ER, Schust DJ, Fisher SJ. Implantation and the survival of early pregnancy. N Engl J Med. 2001; 345(19): 1400-8.

4. Dey SK, Lim H, Das SK, et al. Molecular cues to implantation. Endocr Rev. United States 2004; 341-73.

5. Makker A, Singh MM. Endometrial receptivity: clinical assessment in relation to fertil ity, infertil ity, and antifertility. Med Res Rev. 2006; 26(6): 699-746.

6. Pafilis J, Batistatou A, Iliopoulou A, et al. Expression of adhesion molecules during normal pregnancy. Cell Tissue Res. 2007; 329(1): 1-11.

7. Munro SK, Farquhar CM, Mitchell MD, Ponnampalam AP. Epigenetic regulation of endometrium during the menstrual cycle. Mol Hum Reprod. England 2010; 297-310.

8. Wu G, Morris SM, Jr. Arginine metabolism: nitric oxide and beyond. Biochem J. 1998; 336 (Pt 1): 1-17.

9. Boucher JL, Moali C, Tenu JP. Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for L-arginine utilization. Cell Mol Life Sci. 1999; 55(8-9): 1015-28.

10. Battaglia C, Salvatori M, Maxia N, Petraglia F, Facchinetti F, Volpe A. Adjuvant L-arginine treatment for in-vitro fertilization in poor responder patients. Hum Reprod. 1999; 14(7): 1690-7.

11. Takasaki A, Tamura H, Miwa I, Taketani T, Shimamura K,


Sugino N. Endometrial growth and uterine blood flow: a pilot study for improving endometrial thickness in the patients with a thin endometrium. Fertil Steril. United States: 2010 American Society for Reproductive Medicine. Published by Elsevier Inc 2010; 1851-8.

12. Khorram O, Garthwaite M, Magness RR. Endometrial and myometrial expression of nitric oxide synthase isoforms in pre- and postmenopausal women. J Clin Endocrinal Metal. 1999; 84(6): 2226-32.

13. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol. 1975; 122(2): 262-3.

14. Harada T, Kubota T, Aso T. Usefulness of CA19-9 versus CA125 for the diagnosis of endometriosis. Fertility and Sterility. 2002; 78(4): 733-9.

15. Gobert AP, Cheng Y, Wang JY, et al. Helicobacter pylori induces macrophage apoptosis by activation of arginase Ⅱ. J Immunol. 2002; 168(9): 4692-700.

16. Bidwell GL, Rancher D. Application of thermally responsive polypept ides directed against c-My transcriptional function for cancer therapy. Mol Cancer Ther. 2005; 4(7): 1076-85.

17. Ishikawa T, Harada T, Koi H, Kubota T, Azuma H, Aso T. Identification of arginase in human placental villa. Placenta. 2007; 28(2-3): 133-8.

18. Sparkes RS, Dizikes GJ, Lisa I, et al. The gene for human liver arginase (ARG1) is assigned to chromosome band 6q23. Am J Hum Genet. 1986; 39(2): 186-93.

19. Goth T, Araki M, Mori M. Chromosomal localization of the human arginase Ⅱ gene and tissue distribution of its mRNA. Biochem Biopsy’s Res Commun. United States 1997; 487-91.

20. Morris SM, Jr. Recent advances in arginine metabolism: roles and regulation of the artiness. Br J Pharmacology. England 2009; 922-30.

21. Dizikes GJ, Grody WW, Kern RM, Cederbaum SD. Isolation of human liver arginase cDNA and demonstration of nonhomology between the two human arginase genes. Biochem Biophys Res Commun. United States 1986; 53-9.

22. Haraguchi Y, Takiguchi M, Amaya Y, Kawamoto S, Matsuda I, Mori M. Molecular cloning and nucleotide sequence of cDNA for human liver arginase. Proc Natl Acad Sci U S A. 1987; 84(2): 412-5.

23. Gotoh T, Sonoki T, Nagasaki A, Terada K, Takiguchi M, Mori M. Molecular cloning of cDNA for nonhepatic mitochondrial arginase (arginase Ⅱ) and comparison of its induction with nitric oxide synthase in a murine macrophage-like cell line. FEBS Lett. Netherlands 1996; 119-22.

24. Vockley JG, Jenkinson CP, Shukla H, Kern RM, Grody WW, Cederbaum SD. Cloning and characterization of the human type Ⅱ arginase gene. Genomics. United States 1996; 118-23.

25. Meurs H, Maarsingh H, Zaagsma J. Arginase and asthma: novel insights into nitric oxide homeostasis and airway hyperresponsiveness. Trends Pharmacology Sci. England 2003; 450-5.

26. Bouquet J, Jeffery PK, Busse WW, Johnson M, Vignola AM. Asthma. From bronchoconstriction to airways inflammation and remodeling. Am J Respire Crit Care Med. 2000; 161(5): 1720-45.

27. Ricciardolo FL, Zeugma J, Meurs H. The therapeutic potential of drugs targeting the arginase pathway in asthma. Expert Opin Investig Drugs. 2005; 14(10): 1221-31.

28. Durante W, Johnson FK, Johnson RA. Arginase: a critical regulator of nitric oxide synthesis and vascular function. Clin Exp Pharmacol Physiol. Australia 2007; 906-11.

29. Ryoo S, Gupta G, Benjo A, et al. Endothelial arginase Ⅱ: a novel target for the treatment of atherosclerosis. Circ Res. United States 2008; 923-32.

30. Santhanam L, Christianson DW, Nyhan D, Berkowitz DE. Arginase and vascular aging. J Appl Physiol. United States 2008; 1632-42.

31. Alam V, Bernardini L, Gonzales J, Asch RH, Balmaceda JP. A prospective study of echographic endometrial characteristics and pregnancy rates during hormonal replacement cycles. J Assist Reprod Genet. 1993; 10(3): 215-9.

32. Abdalla HI, Brooks AA, Johnson MR, Kirkland A, Thomas A, Studd JW. Endometrial thickness: a predictor of implantation in ovum recipients? Hum Reprod. 1994; 9(2): 363-5.

33. Richter KS, Bugge KR, Bromer JG, Levy MJ. Relationship between endometrial thickness and embryo implantation, based on 1,294 cycles of in vitro fertilization with transfer of two blastocyst-stage embryos. Fertil Sterile. 2007; 87(1): 53-9.

34. El-Toukhy T, Coomarasamy A, Khairy M, et al. The relationship between endometrial thickness and outcome of medicated frozen embryo replacement cycles. Fertil Steril. 2008; 89(4): 832-9.

35. Miwa I, Tamura H, Takasaki A, Yamagata Y, Shimamura K, Sug ino N . Pathophys io log ic features of “ th in ” endometrium. Fertil Steril. 2009; 91(4): 998-1004.

36. Battaglia C, Regnant G, Marsella T, et al. Adjuvant L-arginine treatment in controlled ovarian hyperstimulation: a double-blind, randomized study. Hum Reprod. 2002; 17(3): 659-65.

37. Shearer JD, Richards JR, Mills CD, Caldwell MD. Di f ferent ia l regulat ion of macrophage argin ine metabolism: a proposed role in wound healing. Am J Physiol. 1997; 272(2 Pt 1): E181-90.

38. Satriano J. Arginine pathways and the inflammatory response: interregulation of nitric oxide and polyamines: review article. Amino Acids. 2004; 26(4): 321-9.

Documents

Original Article Augmentation of Arginase Ⅱ Expression in ...lib.tmd.ac.jp/jmd/5904/01_Tajima.pdf · pathway, decreasing vascular resistance, and thereby possibly improving blood