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Production of human mast cell chymase in human myometrium and placenta in cases of normal pregnancy and preeclampsia
European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
Production of human mast cell chymase in human myometrium and placenta in cases of normal pregnancy and preeclampsia Ryuji Mitania,*, Kazuhisa Maedaa, Rijin Fukuia, Satoko Endoa, Yoshika Saijoa, Koji Shinoharaa, Masaharu Kamadaa, Minoru Iraharaa, Shuji Yamanoa, Yutaka Nakayab, Toshihiro Aonoa a
Departments of Obstetrics and Gynecology, School of Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan b Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan Received 15 February 2001; accepted 21 September 2001
Abstract Objective: To investigate whether the human mastcell chymase-endothelin-1(1–31) system was present in human myometrium, chorion and umbilical cord in normal pregnancy. Methods: Myometrium, placenta and umbilical cord were obtained from five normal pregnant women and 10 with preeclampsia. Each tissue was stained with antibodies against hMC and ET-1(1–31). Results: Routine cells were located mainly around vessels. The number of hMC-positive cells and production of ET-1(1–31) were significantly higher in myometrium from patients with severe preeclampsia compared to those from normal pregnant women. In contrast, their numbers were significantly lower in placenta and umbilical cord in patients with severe preeclampsia. Conclusions: These results suggest that the hMC-ET-1(1–31) system is active in normal pregnancy. Overproduction of hMC and ET-1(1–31) in the myometrium may be involved in the pathogenesis of severe preeclampsia, and in such cases some defense mechanism may operate in the fetus to cope with the pathological eftect of the hMC-ET-1(1–31) system. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mast cells; Human mast cell chymase; Preeclampsia; Endothelin-1(1–31)
1. Introduction Preeclampsia is a pregnancy-specific syndrome of unknown etiology, characterized by hypertension, proteinuria, edema and activation of the coagulation cascade, which improves delivery, rapidly. Preeclampsia is associated with vasospasm of fetoplacental vessels and endothelial injury, both of which are generally accepted as potential major pathological factors in preeclampsia, although the cause of these changes is unknown. Mast cells release various chemical mediators and proteases, such as chymase, tryptase and carboxypeptidase. Chymase from human mast cell (human mast cell chymase, hMC) converts angiotensin ATI into ATII at renin-angiotensin systems in blood vessels [1–3]. In addition, hMC also cleaves big endothelins (ETs) at the Tyr31–Gly32 bond and produces vasoconstricting 31-amino acid length endothelins ETs(1–31) [4]. An increase in serum levels of endothelin1(ET-1) (1–21), an authentic ET-1 was demonstrated in * Corresponding author. Tel.: þ81-88-631-7177; fax: þ81-88-631-2630. E-mail address: [email protected] (R. Mitani).
patients with severe preeclampsia, eclampsia or HELLP syndrome, and the elevation of ET-1 levels appeared to correlate with the elevation of maternal blood pressure [5]. Those data suggested that ET-1 was implicated in the pathogenesis of preeclampsia, namely hypertension, vasospasm and endothelial injury. In a previous report, we demonstrated that ET-1(1–31), a novel vasoconstrictor, contracted the human umbilical artery, which indicated ET-1(1-31) might play a particulary important role in fetal circulation [6]. Thus ET-1(1–31) and hMC appear to be implicated in the developement of the preeclampsia. The aim of this study was to investigate whether hMC and ET-1(1–31) were producted in human myometrium, placenta and umbilical cord. We also examined their productions in women with a normal pregnancy and in patients with preeclampsia.
2. Materials and methods This study protocol was approved by the Ethics Committee of the University of Tokushima. Ten women with severe
0301-2115/02/$ – see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 1 ) 0 0 5 4 6 - 2
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R. Mitani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
Table 1 Clinical characteristics of study population (mean S:D:) Characteristics
Normal pregnancy (n ¼ 5)
Maternal age (year) Gravity Parity Gestational age (week) Birth weight (g)
30.3 3.2 1.2 37.5 2646
5.4 3.3 0.4 0.9 180
Severe preeclampsia (n ¼ 10)a 35.0 1.5 0.9 34.4 1599
3.5 1.9 1.3 1.9 268
P-value NS NS NS <0.05 <0.05
a Pregnant women manifested hypertension (>160/110 mmHg or a rise of >30 mmHg systolic pressure and of >15 mmHg diastolic pressure from nonpregnant values), proteinuria (>200 mg protein per 24 h), and general edema.
Fig. 1. Human mast cell chymase was producted in mast cells of myometrium (A), placenta (B) and umbilical cord (C) obtained from normal pregnant women. These cells positive for hMC were especially observed around vessels.
R. Mitani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
157
Fig. 1. (Continued ).
preeclampsia and five with a normal pregnant women were enrolled for this study (Table 1). All subjects understood the purpose and protocol of this study and gave their informed consent in writing. The samples were obtained at the time of elective cesarean section. Severe preeclampsia was diagnosed based on the following criteria: (1) blood pressure >160/110 mmHg or a rise of >30 mmHg in systolic pressure and of >15 mmHg in diastolic pressure from non-pregnancy values, (2) proteinuria >200 mg protein per 24 h, and (3) generalized edema. All the patients met above three criteria. After washing with physiological saline, each tissue was fixed in Carnoy’s fluid (60% absolute ethanol, 30% chloroform, and 10% glacial acetate acid). Three consecuive specimens of 3 mm in thickness were obtained from the tissues. Each specimen was then subjected to three sessions of 10 min deparaffinization in xylene (xylol), followed by hydration by immersion twice for 10 min in 100% ethanol, and once for 5 min in 100, 90, 80, 70% and then 50% ethanol and followed by three times in phosphate-buffered saline (PBS,10 mM, pH 7.4; each time). Specimens for hMC and control were blocked with horse plasma and specimens for ET-1(1–31) with goat plasma at room temperature for 1 h. Each specimen was incubated overnight at 48 C with 0.27 mg/ml anti-mast cell monoclonal chymase antibody (Chemicon International, Temecula, CA), same concentration of mouse lgG Kappa antibody as the control, or antihuman ET-1(1–31) monoclonal antibody (diluted 1:100; prepared at the Department of Nutrition, The University of Tokushima). The specimens were then washed with PBS (10 mM, pH 7.4) three times (10 min each time), and incubated for 60 min at room temperature with biotinilated
horse or goat anti-mouse IgG antibody. Color of these specimens developed with avidin-biotin alkaline phosphatase and Vector red was observed under a light microscope (400) [7]. The number of hMC-positive mast cells and production of ET-1(1–31) were estimated by calculating the mean number of the positive cells of five high power fields (hpf) with the aid of an image analyzer (Mac SCOPE, Mitani Corp., Fukui, Japan). Student’s t-test was used to compare continuous variables. Significance was defined as P < 0:05.
3. Results Human mast cell chymase was observed in mast cells localized in myometrium, placenta and umbilical cord obtained from normal pregnant women. The cells positive for hMC were observed mainly around vessels. lmmunoloTable 2 Number of hMC-positive mast cells (mean S:D:) Specimensa
Normal pregnancy (n ¼ 5)
Severe preeclampsia (n ¼ 10)b
P-value
Myometrium Placenta Umbilical cord
8.0 132 10.8 11.6 7.4 1.6
31.5 2.8 3.9 1.4 2.8 1.0
<0.05 <0.05 <0.05
a
All specimens were obtained at elective cesarean section. Pregnant women manifested hypertension (>160/110 mmHg or a rise of >30 mmHg systolic pressure and of >15 mmHg diastolic pressure from non-pregnant values), proteinuria (>200 mg protein per 24 h), and general edema. b
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R. Mitani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
calization of ET-1(1–31) was also observed in mast cells of myometrium, placenta and umbilical cord obtained from normal pregnant women and the cells especially existed around vessels, too. No cells stained for human mast cell chymase in the control without anti-mast cell monoclonal chymase antibody. It was not siginificant that the differrence in the production of hMC and ET-1(1–31) among these three tissues.
In specimens obtained from patients with severe preeclampsia, the production of hMC was significantly (P < 0:05) less in placenta and cord, compared with that observed in normal pregnancy. Whereas, significantly (P < 0:05) stronger staining of hMC was observed in myometrium (Figs. 1 and 2, Table 2). Likewise, the production of ET-1(1–31) was significantly (P < 0:05) weaker in placenta and cord, compared with those in normal pregnancy.
Fig. 2. In specimens obtained from patients with preeclampsia, the expression of hMC was stronger in myometrium (A), compared with those in normal pregnancy, while it was weaker in placenta (B) and umbilical cord (C).
R. Mitani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
159
Fig. 2. (Continued ).
Table 3 Number of ET-1(1-31) positive mast cells (mean S:D:) Specimensa
Normal pregnancy (n ¼ 5)
Severe preeclampsia (n ¼ 10)b
P-value
Myometrium Placenta Umbilical cord
7.8 12.3 11.8 1.6 7.8 1.3
28.5 3.2 2.9 1.2 2.7 1.3
<0.05 <0.05 <0.05
a
AII specimens were obtained at elective cesarean section. Pregnant women manifested hypertension (>160/110 mmHg or a rise of >30 mmHg systolic pressure and of >15 mmHg diastolic pressure from non-pregnant values), proteinuria (>200 mg protein per 24 h), and general edema. b
Whereas, significantly (P < 0:05) stronger staining of ET1(1–31) was observed in myometrium (Table 3). These changes of ET-1(1–31) were in parallel to with the change of hMC in both normal pregnancy and preeclampsia, because ET-1(1–31) was generated by hMC.
4. Discussion In the present study, we clearly demonstrated that cells positive for hMC and ET-1(1–31) were found in myometrium, placenta and umbilical cord, especially around vessels. Furthermore, their presence in each tissue differed greatly between normal pregnant women and patients with severe preeclampsia. Namely, in severe preeclampsia,
productions of hMC and ET-1(1–31) were weaker in placenta and umbilical cord and stronger in myometrium compared to those in normal pregnant women, respectively. Okunishi et al. suggested the presence of an ATII synthesis system that did not depend on the angiotensin converting enzyme in renin-angiotensin system (RAS) in the excised blood vessels of humans, monkeys and dogs [2]. They assumed that the new ATII synthetase was the hMC present in the tunica externa and perivascular connective tissue and demonstrated that the chymase-dependent ATII production was the main constituent in human [4]. Urata et al. cloned the genome of AT II chymase which is present in large quantities in human heart and found that base sequence of the gene was completely identical to that of hMC [8]. Because of its widespread tissue distribution hMC appears to play an important role in other tissues including uterus [9]. Recently, we showed that hMC processed big ET to ET1(1–31), a novel peptide consisting of 31-amino acid residues. Because of its strong vasoconstricting activity, ET-1(1–21), an authentic type of ET-1 consisting of 21amino acid residues, is thought to be involved in the pathogenesis of hypertension and angiospasm and to induce preeclampsia, thereby. As a matter of fact, ET1(1–21) produced in vascular endothelial cells of the umbilical cord contracts the umbilical artery through the ET-1 receptor present in the smooth muscles of the artery. Furthermore, the concentration of ET-1 in maternal blood
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R. Mitani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
of the patients with pregnancy induced hypertension was significantly higherthan in that of the normal pregnancy group [10]. In a previous study, we demonstrated that the contracting activity of ET-1(1–31) in the uterine artery, a systemic vessel, was weaker than that of ET-1(1–21). However, ET-1(1–31) exerted similar or even stronger effect on the umbilical artery compared to ET-1(1–21). Thus, regulation of the contraction of the umbilical artery would differ from that in systemic blood vessels. These results indicated that ET-1(1–31) might play important roles in fetal circulation [6]. In the present study, hMC and ET-1(1–31) were widespread in the patients with severe preeclampsia. Mast cells are closely related to various pathological conditions, such as atherosclerosis, bronchial asthma and hypertrophy of tonsils. Thus, hMC and hMC-induced ET-1(1–31) are also considered to be closely related to these pathological conditions. The present study indicates that overproduction of hMC in the myometrium might cause a vicious circle between vasoconstriction and vascular endothelial injury through an overproduction of ET-1(1–31).In contrast, the number of hMC-positive mast cells and production of ET-1(1–31)in the placental villi and umbilical interstitium of the fetus delivered from patients with severe preeclampsia were less compared to those observed in normal pregnant women. This result is compatible with that of Cervar et al. Cervar et al. reported that the release of ET-1(1–21) by chorionic cells from patients with preeclampsia, was significantly declined compared to that in normal pregnancy [11]. The decreased activity of the hMC system in fetal tissues obtained from patients with severe preeclampsia cannot be explained based on the findings of the present study. Some defense mechanisms might work in the fetus to improve its circulation, though the factor(s) that prevents the production of hMC in fetal tissue is unclear.
Acknowledgements This study was supported in part by Grants-in-Aid for Scientific Research (no. 11770941) from the Ministry of Education, Science and Culture, Japan. References [1] Okunishi H, Miyazaki M, Toda N. Evidence for a putatively new angiotensin(II)-generating enzyme in the vascular wall. J Hypertens 1984;2:277–84. [2] Okunishi H, Miyazaki M, Okamura I, Toda N. Different distribution of the angiotensin(II)-generating enzymes in the aortic wall. Biochem Biophy Res Commun 1987;149:1186–92. [3] Okunishi H, Oka Y, Shiota N, Kawamoto I, Song K, Miyazaki M. Marked species-difference in the vascular angiotensin(II)-forming pathways: humans versus rodents. Jpn J Pharmacol 1993;62:207–10. [4] Nakano A, Kishi F, Minami K, Wakabayashi H, Nakaya Y, Kido H. Selective conversion of big endothelins to tracheal smooth muscleconstricting 31-amino acid length endothelins by chymase from human mast cells. J lmmunol 1997;159:1987–92. [5] Greer IA, Leask R, Hodoson BA, Dawes J, Kilpatrick DC, Liston WA. Endothelin, elastaseand endothelial dysfunction in preeclampsia. Lancet 1991;337:558. [6] Takeji T, Nakaya Y, Kamada M, Maeda K, Saijo Y, Mitani R, et al. Effect of a novel vasoconstrictor endothelin-1(1–31) on human umbilical artery. Biochem Biophy Res Commun 2000;270:622–4. [7] Irani AM, Bradford TR, Kepley CL, Schechter NM, Schwartz LB. Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and antichymase antibodies. J Histochem Cytochem 1989;37:1509–15. [8] Urata H, Kinoshita A, Misono KS, Bumpus FM, Husain A. Identification of a highly specific chymase as the major angiotensin(II)forming enzyme in the human heart. J Biol Chem 1990;265: 22348–57. [9] Urata H, Boehm KD, Philip A, Kinoshita A, Gabrovsek J, Bumpus FM, et al. Cellular localization and regional distribution of an angiotensin(II)-forming chymase in the heart. J Clin Invest 1993;91:1269–81. [10] Taylor RN, Varma M, Teng NNH, Roberts JM. Women with preeclampsia have higher plasma endothelin levels than women with normal pregnancy. J Clin Endocrinol Metab 1990;71:1675–7. [11] Cervar M, Kainer F, Jones CJP, Desoye G. Altered release of endothelin-1,2 and thromboxane B2 from trophoblastic cells in preeclampsia. Eur J Clin Invest 1996;26:30–7.
Production of human mast cell chymase in human myometrium and placenta in cases of normal pregnancy and preeclampsia Ryuji Mitania,*, Kazuhisa Maedaa, Rijin Fukuia, Satoko Endoa, Yoshika Saijoa, Koji Shinoharaa, Masaharu Kamadaa, Minoru Iraharaa, Shuji Yamanoa, Yutaka Nakayab, Toshihiro Aonoa a
Departments of Obstetrics and Gynecology, School of Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan b Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan Received 15 February 2001; accepted 21 September 2001
Abstract Objective: To investigate whether the human mastcell chymase-endothelin-1(1–31) system was present in human myometrium, chorion and umbilical cord in normal pregnancy. Methods: Myometrium, placenta and umbilical cord were obtained from five normal pregnant women and 10 with preeclampsia. Each tissue was stained with antibodies against hMC and ET-1(1–31). Results: Routine cells were located mainly around vessels. The number of hMC-positive cells and production of ET-1(1–31) were significantly higher in myometrium from patients with severe preeclampsia compared to those from normal pregnant women. In contrast, their numbers were significantly lower in placenta and umbilical cord in patients with severe preeclampsia. Conclusions: These results suggest that the hMC-ET-1(1–31) system is active in normal pregnancy. Overproduction of hMC and ET-1(1–31) in the myometrium may be involved in the pathogenesis of severe preeclampsia, and in such cases some defense mechanism may operate in the fetus to cope with the pathological eftect of the hMC-ET-1(1–31) system. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mast cells; Human mast cell chymase; Preeclampsia; Endothelin-1(1–31)
1. Introduction Preeclampsia is a pregnancy-specific syndrome of unknown etiology, characterized by hypertension, proteinuria, edema and activation of the coagulation cascade, which improves delivery, rapidly. Preeclampsia is associated with vasospasm of fetoplacental vessels and endothelial injury, both of which are generally accepted as potential major pathological factors in preeclampsia, although the cause of these changes is unknown. Mast cells release various chemical mediators and proteases, such as chymase, tryptase and carboxypeptidase. Chymase from human mast cell (human mast cell chymase, hMC) converts angiotensin ATI into ATII at renin-angiotensin systems in blood vessels [1–3]. In addition, hMC also cleaves big endothelins (ETs) at the Tyr31–Gly32 bond and produces vasoconstricting 31-amino acid length endothelins ETs(1–31) [4]. An increase in serum levels of endothelin1(ET-1) (1–21), an authentic ET-1 was demonstrated in * Corresponding author. Tel.: þ81-88-631-7177; fax: þ81-88-631-2630. E-mail address: [email protected] (R. Mitani).
patients with severe preeclampsia, eclampsia or HELLP syndrome, and the elevation of ET-1 levels appeared to correlate with the elevation of maternal blood pressure [5]. Those data suggested that ET-1 was implicated in the pathogenesis of preeclampsia, namely hypertension, vasospasm and endothelial injury. In a previous report, we demonstrated that ET-1(1–31), a novel vasoconstrictor, contracted the human umbilical artery, which indicated ET-1(1-31) might play a particulary important role in fetal circulation [6]. Thus ET-1(1–31) and hMC appear to be implicated in the developement of the preeclampsia. The aim of this study was to investigate whether hMC and ET-1(1–31) were producted in human myometrium, placenta and umbilical cord. We also examined their productions in women with a normal pregnancy and in patients with preeclampsia.
2. Materials and methods This study protocol was approved by the Ethics Committee of the University of Tokushima. Ten women with severe
0301-2115/02/$ – see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 1 ) 0 0 5 4 6 - 2
156
R. Mitani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
Table 1 Clinical characteristics of study population (mean S:D:) Characteristics
Normal pregnancy (n ¼ 5)
Maternal age (year) Gravity Parity Gestational age (week) Birth weight (g)
30.3 3.2 1.2 37.5 2646
5.4 3.3 0.4 0.9 180
Severe preeclampsia (n ¼ 10)a 35.0 1.5 0.9 34.4 1599
3.5 1.9 1.3 1.9 268
P-value NS NS NS <0.05 <0.05
a Pregnant women manifested hypertension (>160/110 mmHg or a rise of >30 mmHg systolic pressure and of >15 mmHg diastolic pressure from nonpregnant values), proteinuria (>200 mg protein per 24 h), and general edema.
Fig. 1. Human mast cell chymase was producted in mast cells of myometrium (A), placenta (B) and umbilical cord (C) obtained from normal pregnant women. These cells positive for hMC were especially observed around vessels.
R. Mitani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
157
Fig. 1. (Continued ).
preeclampsia and five with a normal pregnant women were enrolled for this study (Table 1). All subjects understood the purpose and protocol of this study and gave their informed consent in writing. The samples were obtained at the time of elective cesarean section. Severe preeclampsia was diagnosed based on the following criteria: (1) blood pressure >160/110 mmHg or a rise of >30 mmHg in systolic pressure and of >15 mmHg in diastolic pressure from non-pregnancy values, (2) proteinuria >200 mg protein per 24 h, and (3) generalized edema. All the patients met above three criteria. After washing with physiological saline, each tissue was fixed in Carnoy’s fluid (60% absolute ethanol, 30% chloroform, and 10% glacial acetate acid). Three consecuive specimens of 3 mm in thickness were obtained from the tissues. Each specimen was then subjected to three sessions of 10 min deparaffinization in xylene (xylol), followed by hydration by immersion twice for 10 min in 100% ethanol, and once for 5 min in 100, 90, 80, 70% and then 50% ethanol and followed by three times in phosphate-buffered saline (PBS,10 mM, pH 7.4; each time). Specimens for hMC and control were blocked with horse plasma and specimens for ET-1(1–31) with goat plasma at room temperature for 1 h. Each specimen was incubated overnight at 48 C with 0.27 mg/ml anti-mast cell monoclonal chymase antibody (Chemicon International, Temecula, CA), same concentration of mouse lgG Kappa antibody as the control, or antihuman ET-1(1–31) monoclonal antibody (diluted 1:100; prepared at the Department of Nutrition, The University of Tokushima). The specimens were then washed with PBS (10 mM, pH 7.4) three times (10 min each time), and incubated for 60 min at room temperature with biotinilated
horse or goat anti-mouse IgG antibody. Color of these specimens developed with avidin-biotin alkaline phosphatase and Vector red was observed under a light microscope (400) [7]. The number of hMC-positive mast cells and production of ET-1(1–31) were estimated by calculating the mean number of the positive cells of five high power fields (hpf) with the aid of an image analyzer (Mac SCOPE, Mitani Corp., Fukui, Japan). Student’s t-test was used to compare continuous variables. Significance was defined as P < 0:05.
3. Results Human mast cell chymase was observed in mast cells localized in myometrium, placenta and umbilical cord obtained from normal pregnant women. The cells positive for hMC were observed mainly around vessels. lmmunoloTable 2 Number of hMC-positive mast cells (mean S:D:) Specimensa
Normal pregnancy (n ¼ 5)
Severe preeclampsia (n ¼ 10)b
P-value
Myometrium Placenta Umbilical cord
8.0 132 10.8 11.6 7.4 1.6
31.5 2.8 3.9 1.4 2.8 1.0
<0.05 <0.05 <0.05
a
All specimens were obtained at elective cesarean section. Pregnant women manifested hypertension (>160/110 mmHg or a rise of >30 mmHg systolic pressure and of >15 mmHg diastolic pressure from non-pregnant values), proteinuria (>200 mg protein per 24 h), and general edema. b
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R. Mitani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
calization of ET-1(1–31) was also observed in mast cells of myometrium, placenta and umbilical cord obtained from normal pregnant women and the cells especially existed around vessels, too. No cells stained for human mast cell chymase in the control without anti-mast cell monoclonal chymase antibody. It was not siginificant that the differrence in the production of hMC and ET-1(1–31) among these three tissues.
In specimens obtained from patients with severe preeclampsia, the production of hMC was significantly (P < 0:05) less in placenta and cord, compared with that observed in normal pregnancy. Whereas, significantly (P < 0:05) stronger staining of hMC was observed in myometrium (Figs. 1 and 2, Table 2). Likewise, the production of ET-1(1–31) was significantly (P < 0:05) weaker in placenta and cord, compared with those in normal pregnancy.
Fig. 2. In specimens obtained from patients with preeclampsia, the expression of hMC was stronger in myometrium (A), compared with those in normal pregnancy, while it was weaker in placenta (B) and umbilical cord (C).
R. Mitani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
159
Fig. 2. (Continued ).
Table 3 Number of ET-1(1-31) positive mast cells (mean S:D:) Specimensa
Normal pregnancy (n ¼ 5)
Severe preeclampsia (n ¼ 10)b
P-value
Myometrium Placenta Umbilical cord
7.8 12.3 11.8 1.6 7.8 1.3
28.5 3.2 2.9 1.2 2.7 1.3
<0.05 <0.05 <0.05
a
AII specimens were obtained at elective cesarean section. Pregnant women manifested hypertension (>160/110 mmHg or a rise of >30 mmHg systolic pressure and of >15 mmHg diastolic pressure from non-pregnant values), proteinuria (>200 mg protein per 24 h), and general edema. b
Whereas, significantly (P < 0:05) stronger staining of ET1(1–31) was observed in myometrium (Table 3). These changes of ET-1(1–31) were in parallel to with the change of hMC in both normal pregnancy and preeclampsia, because ET-1(1–31) was generated by hMC.
4. Discussion In the present study, we clearly demonstrated that cells positive for hMC and ET-1(1–31) were found in myometrium, placenta and umbilical cord, especially around vessels. Furthermore, their presence in each tissue differed greatly between normal pregnant women and patients with severe preeclampsia. Namely, in severe preeclampsia,
productions of hMC and ET-1(1–31) were weaker in placenta and umbilical cord and stronger in myometrium compared to those in normal pregnant women, respectively. Okunishi et al. suggested the presence of an ATII synthesis system that did not depend on the angiotensin converting enzyme in renin-angiotensin system (RAS) in the excised blood vessels of humans, monkeys and dogs [2]. They assumed that the new ATII synthetase was the hMC present in the tunica externa and perivascular connective tissue and demonstrated that the chymase-dependent ATII production was the main constituent in human [4]. Urata et al. cloned the genome of AT II chymase which is present in large quantities in human heart and found that base sequence of the gene was completely identical to that of hMC [8]. Because of its widespread tissue distribution hMC appears to play an important role in other tissues including uterus [9]. Recently, we showed that hMC processed big ET to ET1(1–31), a novel peptide consisting of 31-amino acid residues. Because of its strong vasoconstricting activity, ET-1(1–21), an authentic type of ET-1 consisting of 21amino acid residues, is thought to be involved in the pathogenesis of hypertension and angiospasm and to induce preeclampsia, thereby. As a matter of fact, ET1(1–21) produced in vascular endothelial cells of the umbilical cord contracts the umbilical artery through the ET-1 receptor present in the smooth muscles of the artery. Furthermore, the concentration of ET-1 in maternal blood
160
R. Mitani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 101 (2002) 155–160
of the patients with pregnancy induced hypertension was significantly higherthan in that of the normal pregnancy group [10]. In a previous study, we demonstrated that the contracting activity of ET-1(1–31) in the uterine artery, a systemic vessel, was weaker than that of ET-1(1–21). However, ET-1(1–31) exerted similar or even stronger effect on the umbilical artery compared to ET-1(1–21). Thus, regulation of the contraction of the umbilical artery would differ from that in systemic blood vessels. These results indicated that ET-1(1–31) might play important roles in fetal circulation [6]. In the present study, hMC and ET-1(1–31) were widespread in the patients with severe preeclampsia. Mast cells are closely related to various pathological conditions, such as atherosclerosis, bronchial asthma and hypertrophy of tonsils. Thus, hMC and hMC-induced ET-1(1–31) are also considered to be closely related to these pathological conditions. The present study indicates that overproduction of hMC in the myometrium might cause a vicious circle between vasoconstriction and vascular endothelial injury through an overproduction of ET-1(1–31).In contrast, the number of hMC-positive mast cells and production of ET-1(1–31)in the placental villi and umbilical interstitium of the fetus delivered from patients with severe preeclampsia were less compared to those observed in normal pregnant women. This result is compatible with that of Cervar et al. Cervar et al. reported that the release of ET-1(1–21) by chorionic cells from patients with preeclampsia, was significantly declined compared to that in normal pregnancy [11]. The decreased activity of the hMC system in fetal tissues obtained from patients with severe preeclampsia cannot be explained based on the findings of the present study. Some defense mechanisms might work in the fetus to improve its circulation, though the factor(s) that prevents the production of hMC in fetal tissue is unclear.
Acknowledgements This study was supported in part by Grants-in-Aid for Scientific Research (no. 11770941) from the Ministry of Education, Science and Culture, Japan. References [1] Okunishi H, Miyazaki M, Toda N. Evidence for a putatively new angiotensin(II)-generating enzyme in the vascular wall. J Hypertens 1984;2:277–84. [2] Okunishi H, Miyazaki M, Okamura I, Toda N. Different distribution of the angiotensin(II)-generating enzymes in the aortic wall. Biochem Biophy Res Commun 1987;149:1186–92. [3] Okunishi H, Oka Y, Shiota N, Kawamoto I, Song K, Miyazaki M. Marked species-difference in the vascular angiotensin(II)-forming pathways: humans versus rodents. Jpn J Pharmacol 1993;62:207–10. [4] Nakano A, Kishi F, Minami K, Wakabayashi H, Nakaya Y, Kido H. Selective conversion of big endothelins to tracheal smooth muscleconstricting 31-amino acid length endothelins by chymase from human mast cells. J lmmunol 1997;159:1987–92. [5] Greer IA, Leask R, Hodoson BA, Dawes J, Kilpatrick DC, Liston WA. Endothelin, elastaseand endothelial dysfunction in preeclampsia. Lancet 1991;337:558. [6] Takeji T, Nakaya Y, Kamada M, Maeda K, Saijo Y, Mitani R, et al. Effect of a novel vasoconstrictor endothelin-1(1–31) on human umbilical artery. Biochem Biophy Res Commun 2000;270:622–4. [7] Irani AM, Bradford TR, Kepley CL, Schechter NM, Schwartz LB. Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and antichymase antibodies. J Histochem Cytochem 1989;37:1509–15. [8] Urata H, Kinoshita A, Misono KS, Bumpus FM, Husain A. Identification of a highly specific chymase as the major angiotensin(II)forming enzyme in the human heart. J Biol Chem 1990;265: 22348–57. [9] Urata H, Boehm KD, Philip A, Kinoshita A, Gabrovsek J, Bumpus FM, et al. Cellular localization and regional distribution of an angiotensin(II)-forming chymase in the heart. J Clin Invest 1993;91:1269–81. [10] Taylor RN, Varma M, Teng NNH, Roberts JM. Women with preeclampsia have higher plasma endothelin levels than women with normal pregnancy. J Clin Endocrinol Metab 1990;71:1675–7. [11] Cervar M, Kainer F, Jones CJP, Desoye G. Altered release of endothelin-1,2 and thromboxane B2 from trophoblastic cells in preeclampsia. Eur J Clin Invest 1996;26:30–7.