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
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Abstract |
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Key words: advanced glycation end product/apoptosis/chemokines/protease inhibitor/trophoblasts
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Introduction |
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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., 1998; Nakatsuka et al., 2000
). We have previously reported that LPS stimulates inflammatory cytokines and causes apoptosis in human trophoblasts (Asagiri et al., 1998
; Nakatsuka et al., 2000
). 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in first trimester chorionic villi from women with abortion caused by SLE and/or antiphospholipid syndrome (our unpublished data).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., 1999,2000
; Singh et al., 2001
). 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-
B (Mohamed et al., 1999
; Schmidt et al., 1999
, 2000
; Singh et al., 2001
), which regulates expression of inflammatory cytokines or inducible nitric oxide synthase (iNOS) (Mohamed et al., 1999
). AGEs are also reported to induce apoptosis in cultured human umbilical vein endothelial cells (Min et al., 1999
).
In the light of these studies, the cytotoxic effects of AGEs can be attenuated by a suppressor of NF-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-
B in lipopolysaccharide (LPS)-stimulated macrophages (Noguchi et al., 2003
). We also reported that nafamostat mesilate or aminoguanidine, an inhibitor of NOS (Loske et al., 2000
), suppresses LPS-induced secretion of inflammatory cytokines, overproduction of NO, and apoptosis in cultured human trophoblasts (Nakatsuka et al., 2000
).
In the present study, we examine the effects of AGE on secretion of chemokines, which are chemoattractants at the maternalfetal 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.
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Materials and methods |
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Chorionic villi were washed with ice-cold physiological saline, and cut into 12-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., 1990) 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., 1998). The homogenate (30 µg of protein) of chorionic villi or isolated trophoblasts was analyzed with the use of SDSpolyacrylamide 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., 2000). 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 BSAAGE
BSAAGE was prepared as previously described (Li et al., 1997). 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 BSAAGE was between 1.8 and 2.1 at the same concentration. BSAAGE 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., 2000). 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., 2000
). 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 BSAAGE 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 BSAAGE 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., 1999). 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., 1999
; Nakatsuka et al., 2000
).
Measurement of chemokines and human chorionic gonadotropin in culture medium
Concentrations of MIP-1, MIP-1
, RANTES and human chorionic gonadotropin (hCG) in culture medium were measured using the ELISA kits (hMIP-1
ELISA kit from R&B systems, Minneapolis, MN; hMIP-1
ELISA kit and hRANTES ELISA kit from BioSource International, Camarillo, CA;
-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 MannWhitney 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.
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Results |
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Effects of aminoguanidine, NG-monomethyl-L-arginine (L-NMMA), or nafamostat mesilate on trophoblasts treated with BSAAGE
Increased secretion of MIP-1 (Figure 7A) or MIP-1
(Figure 7B) by treatment wi th BSAAGE (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 BSAAGE (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 BSAAGE (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 BSAAGE did not cause any significant effects on secretion of MIP-1
, MIP-1
, or hCG from trophoblasts, or trophoblastic apoptosis.
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Discussion |
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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., 1990; Yui et al., 1994
; Tsuchida et al., 1999
; Singh et al., 2001
). We observed that MIP-1
and MIP-1
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
or MIP-1
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, 1999; Asagiri et al., 2000
; Ishii et al., 2000
; Drake et al., 2001
; Chantakru et al., 2001
; Moussa et al., 2001
). 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
(Drake et al., 2001
), although this is still controversial (Chantakru et al., 2001
). Upregulated secretion of MIP-1
and MIP-1
by treatment with AGEs may alter the immunological environment in the uterus and impair implantation and/or placentation.
Administration of MIP-1 or RANTES is known to upregulate proliferation and hCG production of choriocarcinoma cell lines (Ishii et al., 2000
). Although we observed increased secretion of MIP-1
, the hCG concentration in the culture medium of trophoblasts treated with BSAAGE 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, BSAAGE 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., 2001
). 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., 2002) and endothelial cells (Kaji et al., 2003
). Oxygen free radicals are known to be major participants in AGE-induced cell death (Loske et al., 1998
; Min et al., 1999
). AGEs cause generation of NO and superoxide anion in various cells including endothelial cells (Loske et al., 1998
), macrophages (Neumann et al., 1999
) and mesangial cells (Sugimoto et al., 1999
) 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., 1998
; Nakatsuka et al., 2000
).
In the present study, we showed that aminoguanidine, which is an inhibitor of both AGE formation and NO synthesis (Nilsson, 1999), suppressed apoptosis in trophoblasts. Because AGE formation is very slow (Singh et al., 2001
), 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, 2001
), complications of diabetes or Alzheimer's disease (Singh et al., 2001
; Ulrich and Cerami, 2001
). 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., 1999). 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., 2000
). 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 BSAAGE 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.
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Acknowledgements |
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Submitted on January 12, 2004; accepted on May 27, 2004.
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