Advanced glycation end products induce secretion of chemokines and apoptosis in human first trimester trophoblasts

H. Konishi, M. Nakatsuka1, C. Chekir, S. Noguchi, Y. Kamada, A. Sasaki and Y. Hiramatsu

Department of Obstetrics and Gynecology, Okayama University Medical School, 2-5-1 Shikata, Okayama City, Okayama, 700-8558, Japan

1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Okayama University Medical School, 2-5-1 Shikata, Okayama City, Okayama, 700-8558 Japan. Tel: 086 223 7151 (ext. 7320); Fax: 086 225 9570; Email: mikiya{at}cc.okayama-u.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: We studied the effects of advanced glycation end products (AGEs), which are known to accumulate in patients with diabetes, autoimmune diseases, or that smoke, on human trophoblasts. METHODS: First trimester human chorionic villi of 6–10 week gestation were obtained. Expression and localization of the receptor for AGEs (RAGE) was examined by western blotting and immunohistochemistry. Macrophage inflammatory protein (MIP)-1{alpha} and MIP-1{beta}, regulated upon activation, normal T-cell expressed and secreted (RANTES), and human chorionic gonadotropin (hCG) in culture medium were measured by ELISA. Trophoblastic apoptosis was evaluated by the Hoechst 33258 staining and the in situ nick end labeling technique. RESULTS: RAGE was localized in trophoblasts. AGEs significantly stimulated secretion of both MIP-1{alpha} and MIP-1{beta} from trophoblasts in a time- and dose-dependent manner. AGEs significantly induced apoptosis and reduced secretion of hCG. Increased secretions of MIP-1{alpha} and MIP-1{beta} by AGEs were significantly suppressed by inhibitors of nitric oxide synthase (NOS) or nafamostat mesilate, a synthetic serine protease inhibitor and a suppressor of transcription factor, NF-{kappa}B activation. These agents also suppressed the effects of AGEs on hCG secretion and trophoblastic apoptosis. CONCLUSIONS: These AGE-mediated changes in trophoblasts may lead to impairment of implantation and placentation. NOS inhibitors or nafamostat mesilate may modify these effects.

Key words: advanced glycation end product/apoptosis/chemokines/protease inhibitor/trophoblasts


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Advanced glycation end products (AGEs) have reactive, cross-linking molecules that are formed from the non-enzymatic reaction (also called the Maillard reaction) of reducing sugars and the amino groups of proteins, lipids and nucleic acids. Plasma or serum AGE levels are high in patients with hyperglycemia and/or oxidative stress, such as diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), renal insufficiency, or cigarette smokers (Cerami et al., 1997Go; Rodriguez-Garcia et al., 1998Go; Nicholl and Bucala, 1998Go; Ligier et al., 1998Go; Mohamed et al., 1999Go; Schmidt et al., 1999Go,2000Go; Singh et al., 2001Go). Animals injected with exogenous AGE–albumin exhibit increased vascular wall permeability and increased mononuclear cell migratory activity in subendothelial and periarterial spaces (Vlassara et al., 1992Go). Chemokines, macrophage inflammatory protein (MIP)-1{alpha}, MIP-1{beta}, and regulated upon activation, normal T-cell expressed and secreted (RANTES) protein are known to play a major part in the trafficking, extravasation, and recruitment of leukocytes to inflammatory sites (Garcia-Velasco and Arici, 1999Go; Reape and Groot, 1999Go). AGEs may modify cell functions by stimulating chemokines secretion in the inflammatory sites.

Human trophoblasts in placenta are known to be targets of oxidative stress when they are treated with lipopolysaccharide (LPS), a bacterial endotoxin (Asagiri et al., 1998Go; Nakatsuka et al., 2000Go). We have previously reported that LPS stimulates inflammatory cytokines and causes apoptosis in human trophoblasts (Asagiri et al., 1998Go; Nakatsuka et al., 2000Go). We observed that AGEs accumulated in first trimester chorionic villi from women with abortion caused by SLE and/or antiphospholipid syndrome (our unpublished data). However, the effects of AGEs on trophoblasts have not been elucidated.

AGEs may cause tissue injury both directly through phenomena such as trapping and cross-linking and indirectly by binding to specific receptors for AGE (RAGE) on the surface of various cells (Schmidt et al., 1999Go,2000Go; Singh et al., 2001Go). The binding of AGEs to RAGE results in generation of intracellular oxidative stress and subsequent activation of the redox-sensitive transcription factors such as NF-{kappa}B (Mohamed et al., 1999Go; Schmidt et al., 1999Go, 2000Go; Singh et al., 2001Go), which regulates expression of inflammatory cytokines or inducible nitric oxide synthase (iNOS) (Mohamed et al., 1999Go). AGEs are also reported to induce apoptosis in cultured human umbilical vein endothelial cells (Min et al., 1999Go).

In the light of these studies, the cytotoxic effects of AGEs can be attenuated by a suppressor of NF-{kappa}B pathway or an inhibitor of NOS. We previously reported that nafamostat mesilate, a clinically-used synthetic serine protease inhibitor, is a potent suppressor of NF-{kappa}B in lipopolysaccharide (LPS)-stimulated macrophages (Noguchi et al., 2003Go). We also reported that nafamostat mesilate or aminoguanidine, an inhibitor of NOS (Loske et al., 2000Go), suppresses LPS-induced secretion of inflammatory cytokines, overproduction of NO, and apoptosis in cultured human trophoblasts (Nakatsuka et al., 2000Go).

In the present study, we examine the effects of AGE on secretion of chemokines, which are chemoattractants at the maternal–fetal interface, and trophoblastic death or secretion of human chorionic gonadotropin (hCG), which indicates placental function in a culture system of human first trimester trophoblasts. We also examine the effects of NOS inhibitors or nafamostat mesilate on AGEs-induced changes in trophoblasts.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Sample collection
Chorionic villi were obtained from healthy nonsmoking pregnant women immediately after the first trimester termination (6–10 week gestation). Informed consent was obtained from each women involved in this study. The protocol of this study was approved by the local institutional review board.

Chorionic villi were washed with ice-cold physiological saline, and cut into 1–2-mm pieces and homogenized in ice-cold homogenization buffer (10 mM Hepes, pH 7.5, containing 320 mM sucrose, 100 µM EDTA, 1.5 mM dithiothreitol, 10 µg/ml trypsin inhibitor, 10 mg/ml leupeptin, 2 µg/ml aprotinin, and 1 mg/ml phenylmethylsulfonyl fluoride; Knowles et al., 1990Go) with the use of a teflon homogenizer. The homogenate was stored at –70°C for use in subsequent western blot analysis. Portions of chorionic villi were also fixed in 10% buffered formalin and then embedded in paraffin for immunohistochemistry. For culture study, chorionic villi were freshly prepared for isolation of trophoblasts.

Western blot analysis
Western blot analysis was performed as described previously (Nakatsuka et al., 1998Go). The homogenate (30 µg of protein) of chorionic villi or isolated trophoblasts was analyzed with the use of SDS–polyacrylamide gel electrophoresis (7% gel). The gels were blotted onto a nitrocellulose membrane, blocked with 0.2 mg/ml thimerosal in blocking buffer and probed with a goat polyclonal antibody against RAGE (N-16, Santa Cruz Biotechnology, Santa Cruz, CA; 1:500). An anti-goat IgG antibody conjugated to peroxidase (Santa Cruz Biotechnology; 1:5000) was used as the secondary antibody. An ECL reagent and Hyperfilm ECL (Amersham Pharmacia Biotech, Buckinghamshire, UK) were used to detect the peroxidase conjugate as described by the manufacturer. Films were scanned using a flatbed scanner and the bands were quantified using Basic Quantifier software (Bio Image, Ann Arbor, MI), an image analysis program, on a Macintosh computer.

Immunohistochemistry
Immunohistochemical detection was performed using 4-µm sections of samples embedded in paraffin as previously described (Kamada et al., 2000Go). We used a DAKO CSA kit (DAKO Corporation, Carpinteria, CA) and antibodies to RAGE (N-16, Santa Cruz Biotechnology; 1:100) as the primary antibodies. As a negative control, preimmune goat sera were used instead of the primary antibodies.

Measurement of protein concentration
Protein concentrations of these samples were determined by the method of Bradford (Bio-Rad Laboratories, Hercules, CA) with the use of bovine serum albumin (BSA) as a standard.

Preparation of BSA–AGE
BSA–AGE was prepared as previously described (Li et al., 1997Go). AGEs were produced by incubation of BSA (Fraction V, fatty acid free, low endotoxin, Sigma Chemical Co., St Louis, MO) at a concentration of 30 mg/ml with 0.5 M glucose in 0.2 M phosphate-buffered saline (PBS) containing 0.5 mM EDTA, pH 7.4, at 37°C for up to 8 weeks. Unbound glucose was removed by extensive dialysis against PBS. AGEs were lyophilized and resuspended in PBS. The density of brown color, which is the typical physical appearance of AGEs, was quantified by measuring the optical density at 405 nm. The optical density at 405 nm of BSA, which was prepared under the same conditions without glucose, was confirmed to be very low (less than 0.1) at a concentration of 3% (w/v). The optical density at 405 nm of each batch of BSA–AGE was between 1.8 and 2.1 at the same concentration. BSA–AGE was resuspended in the culture medium before use.

Isolation and culture of trophoblasts
Trophoblasts were isolated by digestion with trypsin and DNase I, purified by percoll gradient, and further immunopurified with magnetic beads (Dynabeads, Dynal, Oslo, Norway) as described previously (Blaschitz et al., 2000Go). The purity of the trophoblasts was examined by immunocytochemistry using antibodies to cytokeratin-7 (OVTL/12/30, DAKO Corporation) and antibodies to vimentin (clone V9, DAKO Corporation) (Blaschitz et al., 2000Go). The purity of the trophoblasts was found to be >95% while cell viability was >97%. Isolated trophoblasts were suspended to a concentration of 5 x 105 cells/ml in phenol red free Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 25 mM Hepes, 2 mM glutamine, 10% fetal calf serum (FCS), and 50 µg/ml gentamicin, and placed into 6-well Falcon multidishes (Becton Dickinson Labware, Lincoln Park, NJ) or chamber slides (Nunc, Inc. Naperville, IL). Cells were incubated in humidified 5% CO2 and 95% air at 37°C. The culture medium was changed to FCS-free fresh medium with various doses of BSA–AGE on the next day of isolation of trophoblasts. Aminoguanidine (100 µM, Sigma Chemical Co.), an inhibitor of glycation and NOS, NG-monomethyl-L-arginine (L-NMMA) (100 µM, Sigma Chemical Co.), a selective inhibitor of NOS, or nafamostat mesilate (10 µM, 6-amidino-2-naphthyl p-guanidinobenzoate dimethane sulfonate, Torii & Co., Ltd., Tokyo, Japan) was also added to the indicated culture at the same time as BSA–AGE was added. Media and cells were harvested at the indicated time.

Detection of apoptosis
Apoptosis in trophoblasts was identified morphologically under fluorescence microscopy after staining with Hoechst 33258 (Molecular Probes, Eugene, OR) (Nakatsuka et al., 1999Go). Apoptosis was confirmed by detection of DNA degradation using the in situ nick end labeling technique with an In Situ Apoptosis Detection Kit (TaKaRa, Shiga, Japan) following the manufacturer's instructions (Nakatsuka et al., 1999Go; Nakatsuka et al., 2000Go).

Measurement of chemokines and human chorionic gonadotropin in culture medium
Concentrations of MIP-1{alpha}, MIP-1{beta}, RANTES and human chorionic gonadotropin (hCG) in culture medium were measured using the ELISA kits (hMIP-1{alpha} ELISA kit from R&B systems, Minneapolis, MN; hMIP-1{beta} ELISA kit and hRANTES ELISA kit from BioSource International, Camarillo, CA; {beta}-hCG/hCG ELISA kit from Design Resource Group International, Pine Brook, NJ). The concentration was presented as a value in which the concentration in blank medium was subtracted.

Statistical analysis
Statistical significance was determined by Mann–Whitney U test or one-way analysis of variance and Fisher's multiple comparison post test. Data are expressed as mean±SD and a P-value <0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Localization of RAGE in human chorionic villi
Western blotting showed expression of RAGE in chorionic villi from normal pregnant women in the first trimester (Figure 1A). The anti-RAGE antibody detected a single band at ~50 kDa whereas pre-immunized goat serum failed to react by immunoblotting (data not shown). Immunohistochemical detection showed that RAGE was localized both in a syncytiotrophoblast and in cytotrophoblasts of chorionic villi from the first trimester human placenta (Figure 1C). RAGE was also localized in endothelial cells and macrophages in chorionic villi from the first trimester human placenta. We observed no significant differences in intensity of imunoreactivities to RAGE between villous cytotrophoblasts and extravillous cytotrophoblasts.



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Figure 1. Expression and localization of RAGE in human chorionic villi. (A) RAGE protein (arrow) was detected in the first trimester human chorionic villi by western blotting (lane 1, 6 week gestation; lane 2, 8 week gestation; lane 3, 10 week gestation). (B and C) Immunoreactivity to RAGE protein was localized in both syncytiotrophoblast and cytotrophoblasts (B, negative control; C, anti-RAGE antibody). Scale bar (B and C) is 50 µm.

 
Effects of BSA–AGE on secretion of MIP-1{alpha}, MIP-1{beta}, or RANTES from human trophoblasts
MIP-1{alpha}, MIP-1{beta} and RANTES were secreted from isolated human trophoblasts. Treatment with BSA–AGE stimulated secretion of MIP-1{alpha} from trophoblasts in a time-dependent manner (Figure 2A). After 24 h culture, the concentration of MIP-1{alpha} (Figure 2B) in culture medium of trophoblasts treated with BSA–AGE was significantly higher than that in the culture medium of trophoblasts treated with BSA (6, 12, and 24 mg/ml). Treatment with BSA–AGE also stimulated secretion of MIP-1{beta} from trophoblasts in a time-dependent manner (Figure 3A). After 24 h culture, the concentration of MIP-1{beta} (Figure 3B) in culture medium of trophoblasts treated with BSA–AGE was significantly higher than that in the culture medium of trophoblasts treated with BSA (12 and 24 mg/ml). There was no significant difference in the concentrations of RANTES between the culture medium of trophoblasts treated with BSA–AGE and that of the trophoblasts treated with BSA (Figure 4).



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Figure 2. Secretion of MIP-1{alpha} from trophoblasts treated with BSA–AGE. (A) Time course of MIP-1{alpha} secretion. Trophoblasts were treated with 12 mg/ml BSA–AGE. (B) Dose-dependent MIP-1{alpha} secretion. Trophoblasts were treated with BSA–AGE for 24 h. Circles: BSA, squares: BSA–AGE. Data are expressed as mean±SD (n=3–6). Significant difference: *P<0.05, **P<0.01. Each figure is representative of three independent experiments.

 


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Figure 3. Secretion of MIP-1{beta} from trophoblasts treated with BSA–AGE. (A) Time course of MIP-1{beta} secretion. Trophoblasts were treated with 12 mg/ml BSA–AGE. (B) Dose-dependent MIP-1{beta} secretion. Trophoblasts were treated with BSA–AGE for 24 h. Cicles: BSA, squares: BSA–AGE. Data are expressed as mean±SD (n=3–6). Significant difference: *P<0.05, **P<0.01. Each figure is representative of three independent experiments.

 


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Figure 4. Secretion of RANTES from trophoblasts treated with BSA–AGE. (A) Time course of RANTES??secretion. (B) Dose-dependent RANTES secretion. Trophoblasts were treated with BSA–AGE for 24 h. Cirlces: BSA, squares: BSA–AGE. Data are expressed as mean±SD (n=3–6). Each figure is representative of three independent experiments.

 
Effects of BSA–AGE on apoptosis in human trophoblasts
After 24 h culture, BSA–AGE induced apoptosis in human trophoblasts in a dose-dependent manner (Figure 5). BSA–AGE at 6 mg/ml, 12 mg/ml and 24 mg/ml induced apoptosis in trophoblasts up to 10.7%, 27.3% and 38.7%, respectively, which were significantly higher rates compared to apoptosis in trophoblasts treated with BSA.



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Figure 5. Apoptosis in trophoblasts treated by BSA–AGE. Trophoblasts were treated with BSA–AGE for 24 h. Circles: BSA, squares: BSA–AGE. Data are expressed as mean±SD (n=3–6). Significant difference: *P<0.05, **P<0.01. Each figure is representative of three independent experiments.

 
Effects of BSA–AGE on secretion of hCG from human trophoblasts
After 24 h culture, BSA–AGE significantly inhibited the secretion of hCG from human trophoblasts in a dose-dependent manner (Figure 6). BSA–AGE at 12 mg/ml and 24 mg/ml significantly suppressed hCG secretion to 55.0% and 34.3%, respectively, of those observed in BSA-treated trophoblasts.



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Figure 6. Secretion of hCG from trophoblasts treated with BSA–AGE. Trophoblasts were treated with BSA–AGE for 24 h. Circles: BSA, squares: BSA–AGE. Data are expressed as mean±SD (n=3–6). Significant difference: *P<0.05, **P<0.01. Each figure is representative of three independent experiments.

 
Concentration of hCG in the culture medium was lower than that estimated from apoptotic cell death. After normalization by number of viable cells, BSA–AGE at 12 mg/ml and 24 mg/ml significantly suppressed secretion of hCG from viable trophoblasts to 75.1% and 56.7%, respectively.

Effects of aminoguanidine, NG-monomethyl-L-arginine (L-NMMA), or nafamostat mesilate on trophoblasts treated with BSA–AGE
Increased secretion of MIP-1{alpha} (Figure 7A) or MIP-1{beta} (Figure 7B) by treatment wi th BSA–AGE (12 mg/ml, 24 h) was significantly suppressed by co-treatment with aminoguanidine (100 µM), l-NMMA (100 µM), or nafamostat mesilate (10 µM). Increase in trophoblastic apoptosis by treatment with BSA–AGE (12 mg/ml) was significantly suppressed by co-treatment with aminoguanidine (100 µM), L-NMMA (100 µM), or nafamostat mesilate (10 µM) (Figure 7C). Decrease in hCG secretion by treatment with BSA–AGE (12 mg/ml, 24 h) was significantly suppressed by co-treatment with aminoguanidine (100 µM), L-NMMA (100 µM), or nafamostat mesilate (10 µM) (Figure 7D). Treatment with aminoguanidine (100 µM), L-NMMA (100 µM), or nafamostat mesilate (10 µM) without BSA–AGE did not cause any significant effects on secretion of MIP-1{alpha}, MIP-1{beta}, or hCG from trophoblasts, or trophoblastic apoptosis.



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Figure 7. Effects of aminoguanidine (AG), NG-monomethyl-L-arginine (L-NMMA), or nafamostat mesilate (NM) on trophoblasts treated with BSA–AGE. (A) Effects of various agents on BSA–AGE induced MIP-1{alpha} secretion. (B) Effects of various agents on BSA–AGE induced MIP-1{beta} secretion. (C) Effects of various agents on BSA–AGE induced trophoblastic apoptosis. (D) Effects of various agents on hCG secretion inhibited by BSA–AGE. Data are expressed as mean±SD (n=3–6). Each figure is representative of three independent experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We showed expression of RAGE in trophoblasts of the first trimester human chorionic villi from healthy women, and AGEs may affect placental functions via RAGE. Binding of AGEs to RAGE leads to oxidative stress, which could be of importance at sites of inflammation (Bierhaus et al., 1997Go; Loske et al., 1998Go; Mohamed et al., 1999Go; Neumann et al., 1999Go; Schmidt et al., 1999Go,2000Go; Loske et al., 2000Go; Singh et al., 2001Go). RAGE is also known to be expressed during physiological embryonic development (Hori et al., 1995Go). Accumulation of AGEs in placenta may interfere with the possible physiological role of RAGE (Kuniyasu et al., 2003Go).

The effects of AGEs are mediated by growth factors, cytokines and other bioactive molecules, which modulate in a paracrine/autocrine fashion, cell proliferation, extracellular matrix accumulation, hemodynamics, permeability and hemorheological changes (Kirstein et al., 1990Go; Yui et al., 1994Go; Tsuchida et al., 1999Go; Singh et al., 2001Go). We observed that MIP-1{alpha} and MIP-1{beta} were secreted constitutively from the first trimester trophoblasts and that their secretion was upregulated by treatment with AGEs. The purity of the trophoblasts isolated by our methods varies between 95% and 99%. Based on our preliminary study, contaminated cells were syncytial fragments, endothelial cells and macrophages. Therefore, contaminated cells may also secrete these chemokines. However, levels of MIP-1{alpha} or MIP-1{beta} were similar among the preparations. This finding indicates that contaminated cells are not a major source of these chemokines.

Chemokines, although considered to be members of the cytokine superfamily, are rapidly establishing an identity of their own with respect to reproduction (Garcia-Velasco and Arici, 1999Go; Asagiri et al., 2000Go; Ishii et al., 2000Go; Drake et al., 2001Go; Chantakru et al., 2001Go; Moussa et al., 2001Go). They are potent soluble chemoattractants that provide directional cues to summon leukocytes, which are involved in endometrial proliferation, decidualization, blastcyst implantation, immunologic tolerance, regulation of trophoblast invasion and control of infectious agents in a pregnant uterus. Trophoblasts can attract monocytes and specialized maternal natural killer (NK) cells, CD56bright NK cells, by producing MIP-1{alpha} (Drake et al., 2001Go), although this is still controversial (Chantakru et al., 2001Go). Upregulated secretion of MIP-1{alpha} and MIP-1{beta} by treatment with AGEs may alter the immunological environment in the uterus and impair implantation and/or placentation.

Administration of MIP-1{alpha} or RANTES is known to upregulate proliferation and hCG production of choriocarcinoma cell lines (Ishii et al., 2000Go). Although we observed increased secretion of MIP-1{alpha}, the hCG concentration in the culture medium of trophoblasts treated with BSA–AGE was lower than that of BSA-treated trophoblasts. Because the hCG concentration in the culture medium was lower than that estimated from apoptotic cell death, BSA–AGE likely suppressed secretion of hCG from viable cells. It is also possible that AGEs might suppress secretion of hCG from contaminated syncytial fragments. HCG has been known to play a role in endometrial vascularization, placentation and implantation by regulating secretions of prolactin, vascular endothelial growth factor, or other bioactive factors (Licht et al., 2001Go). Therefore, suppression of hCG secretion by AGEs may also be involved in placental dysfunction.

We observed that AGEs induced apoptosis in trophoblasts. AGEs have been also reported to induce apoptosis in mesangial cells (Yamagishi et al., 2002Go) and endothelial cells (Kaji et al., 2003Go). Oxygen free radicals are known to be major participants in AGE-induced cell death (Loske et al., 1998Go; Min et al., 1999Go). AGEs cause generation of NO and superoxide anion in various cells including endothelial cells (Loske et al., 1998Go), macrophages (Neumann et al., 1999Go) and mesangial cells (Sugimoto et al., 1999Go) through binding to RAGE in vitro. AGEs-induced overproduction of NO may cause apoptosis in human trophoblasts as LPS-induced overproduction of NO did (Asagiri et al., 1998Go; Nakatsuka et al., 2000Go).

In the present study, we showed that aminoguanidine, which is an inhibitor of both AGE formation and NO synthesis (Nilsson, 1999Go), suppressed apoptosis in trophoblasts. Because AGE formation is very slow (Singh et al., 2001Go), the suppressive effect of aminoguanidine on AGE formation is negligible in this culture system. Furthermore, L-NMMA, a more specific NOS inhibitor, had similar effects to aminoguanidine. Therefore, NO and/or peroxynitrite is more likely to be involved in trophoblastic apoptosis in the present study. Aminoguanidine is considered to be a candidate agent in retarding the development of atherosclerosis (Baynes and Thorpe, 2001Go), complications of diabetes or Alzheimer's disease (Singh et al., 2001Go; Ulrich and Cerami, 2001Go). However, aminoguanidine has not been a widely used clinical drug.

Nafamostat mesilate has been used clinically without severe adverse effects in patients with disseminated intravascular coagulopathy or pancreatitis, even in some patients during pregnancy (Ohmoto et al., 1999Go). In the present study, nafamostat mesilate had cytoprotective effects on AGE-affected trophoblasts. We previously reported that nafamostat mesilate suppressed LPS-induced secretion of IL-6 and IL-8, NO overproduction, and apoptosis in trophoblasts (Nakatsuka et al., 2000Go). It may be worth studying the efficacy of nafamostat mesilate on AGE-induced pathological changes of various organs in experimental animals and humans.

In the present study, we observed involvements of BSA–AGE in secretion of chemokines, trophoblastic apoptosis, and secretion of hCG. These observations suggest that AGEs may impair placental functions. However, under pathological conditions, proteins other than BSA may also be glycosylated, and it is possible that the effects of AGEs on trophoblasts are different depending on modified proteins. Further investigation is necessary to elucidate the roles of AGEs and the RAGE system in pregnancy.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A part of this work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, from the JAOG Ogyaa Donation Foundation, and from Kanzawa Medical Research Foundation.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on January 12, 2004; accepted on May 27, 2004.





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