Molecular and Cellular Endocrinology 184 (2001) 95 102 www.elsevier.

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The upregulation of angiotensin II receptor AT1 in human preeclamptic placenta

Po Sing Leung a,*, Shaw Jenq Tsai b, Gerd Wallukat c, Tse Ngong Leung d, Tze Kin Lau d
a

Department of Physiology, Faculty of Medicine, The Chinese Uni6ersity of Hong Kong, Shatin N.T., Hong Kong b Department of Physiology, National Cheng Kung Uni6ersity Medical College, Tainan, Taiwan, ROC c Max Delbruck Center for Molecular Medicine, Berlin, Germany d Department of Obstetrics and Gynecology, Prince of Wales Hospital, Hong Kong Received 2 February 2001; accepted 30 July 2001

Abstract The human placenta has been considered to possess a locally generated renin angiotensin system (RAS), which may play a physiological role in the regulation of uteroplacental blood circulation. The changes in the expression of such a placental RAS during pregnancy could be important for the physiological and pathophysiological aspects of some clinical disorders, such as pregnancy-induced hypertension, preeclampsia. In the present study, the alterations of expression and localization of placental angiotensin II receptor subtypes, namely AT1 in patients with preeclampsia (elective caesarean delivery) were investigated and compared with controls (vaginal delivery and elective caesarian delivery) using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry respectively. Results from RT-PCR analysis revealed an upregulated expression of placental mRNA for AT1 receptor subtype in patients with preeclampsia when compared with those in controls. In addition, there was also a signicant activation of placental expression of angiotensinogen mRNA in patients with preeclampsia. Results from Western blot showed that the expression of AT1 receptor was also upregulated. Immunohistochemical results further demonstrated that increased immunoreactivity for placental AT1 receptor was predominantly localized to the thin layers of syncytiotrophoblasts and, to a less extent, the capillaries of the term placental villi. These data indicate that upregulation of placental RAS components, notably AT1 receptor in the syncytiotrophoblasts, could play a pathophysiological role in patients with preeclampsia. 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: RAS; Angiotensinogen; Angiotensin II receptor; Placenta; Preeclampsia

1. Introduction The renin angiotensin system (RAS) has long been believed to be a blood-borne biochemical cascade whose nal and bioactive product, angiotensin II, is a potent vasoconstrictor and a primary stimulus for aldosterone secretion. The so-called circulating RAS plays a crucial endocrine role in the regulation of blood pressure and electrolyte as well as uid homeostasis (Page and Bumpus, 1974; Peach, 1977). Recently, an intrinsic angiotensin-generating system with a
* Corresponding author. Tel.: + 852-2609-6879; fax: +852-26035022. E-mail address: psleung@cuhk.edu.hk (P.S. Leung).

paracrine/autocrine role has been demonstrated in a stable of tissues (Campbell, 1987; Campbell and Habener, 1986; Dzau et al., 1987). Such a tissue RAS has recently been reviewed in the pancreas, which has a potential role in regulating the exocrine and endocrine functions of pancreas (Leung and Carlsson, 2001). In the placenta, an intrinsic angiotensin-generating system has been well documented based on the presence of major RAS components including renin, angiotensinogen, angiotensin-converting enzymes, and angiotensin II as well as its receptor subtypes (Hagemann et al. 1994; Poisner and Downing, 1995; Poisner, 1998). As angiotensin II is well known to be a potent vasoconstrictor peptide, the placental RAS is, therefore, important for the control of the uteroplacental vasculature

0303-7207/01/$ - see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 1 ) 0 0 6 3 7 - 2

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and thus the regulation of uteroplacental blood circulation (Poston, 1997; Poisner, 1998). In addition, angiotensin II can modulate the production of other vasoactive substances from the uteroplacental vasculature such as eicosanoids (Haugen et al. 1996) and other endothelial factors (Yunohara et al. 1994). All these data indicate that RAS in the human placenta should play a crucial role in the physiology and possible pathophysiology during pregnancy. In fact, previous studies have been targeting for elucidating the changes of placental RAS in particular reference to renin activity during pregnancy-induced hypertension such as preeclampsia. Nevertheless, there was discrepancy of results whether placental RAS components are activated or depressed in the peripheral circulation and in the preeclamptic placenta (Brar et al. 1987; Kalenga et al. 1996; Poranen et al. 1996). Accordingly, the present study is to elaborate the changes of expression and precise localization of placental RAS components, particularly angiotensin II receptor AT1 and its precursor angiotensinogen in preeclamptic placenta using molecular biological and immunohistochemical approaches.

They were immediately frozen at 70 C for subsequent immunohistochemical and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses.

2.2. RT-PCR analysis of mRNA expression for AT1 receptor and angiotensinogen
Total RNA was isolated from normal and preeclamptic placenta according to the acid guanidinium thiocyanatephenolchloroform protocol as described previously (Chomczynski and Sacchi, 1987). Briey, placental tissues were homogenized in 4 M guanidinium thiocyanate solution and repeatedly extracted with water-saturated phenol. After extraction with chloroform, RNA was precipitated by isopropanol. The resultant pellet was nally resuspended in water treated with diethylpyrocarbonate. The total RNA was studied by gel electrophoresis and quantied by spectrophotometry. Total RNA (10 mg) was subjected to rst strand cDNA synthesis using random hexamer and Superscript II reverse transcriptase (GIBCO-BRL, USA) in a nal volume of 20 ml. After incubation at 42 C for 1 h, the reaction mixture was treated with RNase H before proceeding to PCR analysis. All resultant RNA was tested to be free of DNA contamination by RT-PCR without addition of reverse transcriptase (Leung et al., 1999, 2000a). Semi-quantitative RT-PCR of mRNA expression for AT1 receptors and angiotensinogen was employed and reported previously by our laboratory (Chan et al., 2000; Leung et al., 2000b). After appropriate validation of the PCR, all samples were analyzed for both human RAS component genes and human b-actin gene in the logarithmic phase of the amplication reactions. Fixed amount of cDNA and optimal PCR cycles were directly employed for individual PCR amplication of RAS component genes. Specic human primers of angiotensinogen, AT1 receptor subtype and b-actin were designed for the present RT-PCR. The oligonucleotide sequences of the genes and brief details are summarized in Table 1. PCR reactions were carried out in a volume of 50 ml system containing the corresponding sense and antisense primers using the PCR Reagent System (GIBCO-BRL, USA). The PCR conditions were as follows: For AT1

2. Materials and methods

2.1. Preparation of human placental tissues

Twenty-ve pregnant women were recruited from the Department of Obstetrics and Gynecology at the Chinese University of Hong Kong and informed consent was obtained from each woman. Human ethics approval was obtained from the Clinical Research Ethics Committee at the Chinese University of Hong Kong. There was one group of pregnant women with preeclampsia using elective caesarean delivery at 36 weeks (n=5). Patients with preeclampsia were dened by hypertension and proteinuria after week 20 of pregnancy. Another two groups of pregnant women with different mode of delivery at term pregnancy (37 41 weeks) were employed as controls. They are normal women with vaginal delivery (n = 10) and normal women with elective caesarean delivery (n = 10). The placental tissues were obtained either during the procedure of termination of pregnancy or after delivery.

Table 1 Human oligonucleotide primers and expected product size designed for the RT-PCR used in the present study Gene A0 AT1 b-actin Sense primer 5%-CATACACCCCTTCCACCTC-3% 5%-TTTGTGGTGGGAATATTTGG-3% 5%-GGCTACAGCTTCACCACCA-3% Antisense primer 5%-GGATTGCCTGTAGCCTGTC%-3% 5%-TGATGATGCAGGTGACTTTG-3% 5%-ACGGATGTCCACGTCACAC-3% Band size 374 bp 340 bp 282 bp

A0, Human angiotensinogen (Hunapuli and Kumar, 1987); AT1, Human AT1 receptor subtype (Bergsma et al., 1992); b-actin, Human b-actin (Nakajuma-Iijima et al., 1985).

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subtype, 26 cycles of denaturing, 94 C, 30 s; annealing, 59 C, 30 s and elongating, 72 C, 30 s. For angiotensinogen, 26 cycles of denaturing, 94 C, 30 s; annealing, 58 C, 30 s and elongating, 72 C, 30 s. For b-actin, 2628 cycles of: denaturing, 94 C, 30 s; annealing, 58 C, 30 s and elongating, 72 C, 30 s. All PCR products were separated on 2% agarose gel electrophoresis. The amplied DNA bands were detected using ethidium bromide staining and quantied with image analyzer (Molecular Dynamics Image Quant, Sunnyvale, CA, USA).

0.2 mg/ml. The primary antibody was detected using fragment anti-rabbit IgG-uorescein F(ab)2 (Boehringer Mannheim, working dilution: 40 mg/ml) for 60 min at 37 C. Sections were again washed briey several times with PBS and embedded in mounting medium (Vectashield, Vector). Sections were then examined by confocal laser scanning microscopy (BioRad MRC-1000). The following controls were used, (1) incubation with rabbit non-immune serum; (2) preadsorption with excess of AT1 receptor peptide antigen (Santa Cruz Biotech Inc, Santa Cruz, CA, USA).

2.3. Western blot analysis of AT1 receptor

The procedures for extraction and immunoblotting of AT1 receptor were reported previously (Leung et al., 2000a). Briey, placenta tissues from normal and preeclampsia were homogenized at 4 C in water, 1:9 (wt./vol.) containing 10 mM EDTA and 1 mM PMSF. The protein from resultant supernatant was determined (Bio-Rad Protein Assay) for sodium dodecy sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). Proteins (10 mg per lane) were subjected to electrophoresis on a 12% (vol./vol.) polyacrylamide gel in SDS and the gel was subsequently processed for electroblotting to polyvinylidene diuoride (PVDF) membrane. The blotted PVDF membrane was saturated with 5% (wt./vol.) of skimmed milk in phosphatebuffered saline (PBS), pH 7.4 and 0.1% (vol./vol.) of Tween 20 for 1 h at room temperature. The membrane was then sequentially incubated in AT1 receptor antibody (Santa Cruz Biotechnology Inc, 1/1000) overnight at 4 C and a peroxidase-labelled IgG serum (1/500) for 1 h at room temperature. After thorough washing, the positive band was revealed using ECL western blotting detection reagents and autoradiography lm (Amersham, England). The band was quantied with image analyzer (Molecular Dynamics Image Quant, Sunnyvale, CA, USA).

2.5. Data analysis

All data from RT-PCR were expressed as the mean9 S.E.M. for each of the normal and the preeclamptic groups. The expression was normalized in % of control of the b-actin expression. Data from Western blot analysis were normalized as a percentage of control. Relative expression in preeclampsia was compared with that of each group of control placenta. Differences between individual mean values were made using Students t-test. Values of *PB0.05 were considered statistically signicant versus respective control groups.

3. Results

3.1. Changes of mRNA expression for AT1 receptor in preeclampsia

The expression changes of mRNA for AT1 receptor was investigated using semi-quantitative RT-PCR coupled with specic oligonucleotide primers corresponding to the sequence of human AT1 receptor subtype. An upregulation of AT1 receptor was consistently detected in preeclamptic placenta with elective caesarean delivery when compared with that in normal placenta with vaginal delivery. The expression of AT1 receptor in preeclampsia was upregulated when compared with that in controls (Fig. 1A) and the relative level of changes was about 2-fold, as demonstrated by image analysis (Fig. 1B). Similar expression changes were also observed in preeclamptic placenta with elective caesarean delivery when compared with those in normal placenta with elective caesarean delivery (Fig. 2).

2.4. Immunohistochemical localization of AT1 receptor

The tissues from the normal (n =4) and preeclamptic (n = 4) placenta were processed for indirect immunouorescent staining as described previously (Leung et al. 1997; Leung et al., 2000a). Consecutive cryosections (8 mm) were cut on Cryotome (Shandon AS 620 Cryotome). After sections were xed with cold and freshly prepared paraformaldehye (4%, wt./vol.) for 30 min, they were processed for indirect immunouorescent staining. Unspecic binding was blocked by 20% (w/v) normal sheep serum in PBS for 60 min at 37 C, followed with several PBS washes. Sections were then incubated overnight at 4 C with anti-human AT1 serum (Santa Cruz Biotech Inc, Santa Cruz, CA, USA), as described previously (Leung et al., 2000b), diluted to

3.2. Changes of protein expression for AT1 receptor in preeclampsia

An upregulation of AT1 receptor was signicantly detected in preeclamptic placenta when compared with that in controls. A marked increase of its protein expression was observed in preeclamptic placenta when compared with that in normal placenta with vaginal

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3.4. Cellular localization of AT1 receptor in preeclampsia

In order to further demonstrate the precise localization of increased expression in preeclamptic placenta, AT1 receptor was investigated by immunohistochemistry using specic antibody against the human AT1 receptor subtype. Immunohistochemical results revealed that enhanced immunoreactivity for AT1 receptor in human preeclamptic placenta appeared to be localized mainly in the trophoblastic cells of the term placenta (Fig. 7). More intense immunoreactivity was predominantly localized to the thin layers of syncytiotrophoblastic cells of the placental villi (Fig. 7C) when compared with those in normal placenta either with vaginal delivery (Fig. 7A) or elective caesarean delivery (Fig. 7B). Specicity of the immunostaining was validated by the negative control experiments when specic antibody was preabsorbed in excess with its peptide antigen either in preeclampsia (Fig. 7D) or in

Fig. 1. (A). RT-PCR analysis of the mRNA expression of AT1 receptor in preeclamptic placenta as compared with that in normal placenta with vaginal delivery. Lane M, DNA marker; Lane 1, AT1 receptor expression in normal placenta; Lane 2, AT1 receptor expression in preeclamptic placenta; Lane 3, b-actin expression in the normal placenta; Lane 4, b-actin expression in preeclamptic placenta. The arrows indicate the expected size of amplied products from AT1 receptor (340 bp) and b-actin (282 bp). (B) The relative expression of AT1 receptor/b-actin mRNA (% of control) in normal placenta (C1) and in preeclamptic placenta (PET). The data are expressed as mean9 S.E.M. (n =6 per group).

delivery (Fig. 3). The relative level of changes was about 1.5-fold (Fig. 3B). There was also a similar upregulation of AT1 receptor protein in preeclamptic placenta when compared with that in normal placenta with elective caesarean delivery (Fig. 4).

3.3. Changes of mRNA expression for angiotensinogen in preeclampsia

In parallel with the gene level of AT1 receptor, the expression of angiotensinogen mRNA in preeclampsia was also markedly enhanced in preeclampsia when compared with controls of either normal placenta with vaginal delivery (Fig. 5) or normal placenta with elective caesarean delivery (Fig. 6). There were about more than 2-fold increase in preeclampsia when compared with the respective controls (Figs. 5B and 6B).

Fig. 2. (A) RT-PCR analysis of the mRNA expression of AT1 receptor in preeclamptic placenta as compared with that in normal placenta with elective caesarean delivery. Lane M, DNA marker; Lane 1, AT1 receptor expression in normal placenta; Lane 2, AT1 receptor expression in preeclamptic placenta; Lane 3, b-actin expression in normal; Lane 4, b-actin expression in preeclampsia. The arrows indicate the expected size of amplied products from AT1 receptor (340 bp) and b-actin (282 bp). (B) The relative expression of AT1 receptor/b-actin mRNA (% of control) in normal placenta (C2) and in preeclamptic placenta (PET). The data are expressed as mean 9S.E.M. (n =6 per group).

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upregulation of RAS components, particularly AT1 receptor subtype, in the placenta could play a pathophysiological role in patients with preeclampsia. Preeclampsia is a pregnancy-induced abnormal condition, which is characterized by the onset of acute hypertension after the 24th week of gestation. The conspicuous and easiest signs that can be diagnosed for the disease are the classical triad of preeclampsia, namely hypertension, proteinuria and edema (Roberts and Redman, 1993; Lindheimer, 1993). Although the etiology of the disease remains largely unknown, the most compelling studies indicate that the placenta is the culprit. This is based on the observation that the signs and symptoms disappear after birth or after pregnancy termination when the placenta is no longer present (Palm Gamiz, 1998). It has long been believed that the preeclampsia may be due to the poorly perfused placenta (Page, 1939), which in turn leads to defective trophoblast invasion of the myometrial portion of the spiral arteries (Pijnenborg et al., 1980, 1981). In such condition, the spiral arteries retain their musculoelas-

Fig. 3. (A). Western blot analysis of AT1 receptor protein expression in preeclamptic placenta as compared with that in normal placenta with vaginal delivery. Kidney was employed as a positive control for AT1 receptor expression. Two protein bands of about 50 70 kDa were detected; C1 denotes AT1 receptor expression in normal placenta; PET denotes AT1 receptor expression in preeclamptic placenta. (B) The relative expression of AT1 receptor protein (% of control) in preeclampsia (PET) compared with that of normal placenta (C1). The data are expressed as mean 9 S.E.M. (n= 4 per group).

normal placenta with elective caesarean delivery (Fig. 7E).

4. Discussion The present study has unequivocally demonstrated the upregulation of mRNA expression for AT1 receptor and angiotensinogen in preeclamptic placenta when compared with their respective controls no matter of caesarean or vaginal delivery, as evidenced by semiquantitative RT-PCR. In consistent with its gene level, results from Western blot analysis showed that AT1 receptor protein was also increased in preeclamptic placenta. Two protein bands of about 50 70 kDa were detected in the kidney while one band of about 50 kDa was observed in the placenta. These values may represent one for an unmodied AT1 receptor and one for mature glycosylated receptor, as reported previously (Leung et al., 1998). Moreover, immunohistochemical localization further demonstrated that enhanced immunoreactivity for AT1 receptor was predominantly localized to the thin layers of syncytiotrophoblastic cells from the term placental villi. These data indicate that

Fig. 4. (A) Western blot analysis of AT1 receptor protein expression in preeclamptic placenta as compared with that in normal placenta with elective caesarean delivery. Kidney was employed as a positive control for AT1 receptor expression. C2 denotes AT1 receptor expression in normal placenta; PET denotes AT1 receptor expression in preeclamptic placenta. (B) The relative expression of AT1 receptor protein (% of control) in preeclampsia (PET) compared with that of normal placenta (C2). The data are expressed as mean 9 S.E.M. (n = 4 per group).

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al., 1991a,b). Instead, patients with preeclampsia have been demonstrated to develop agonistic autoantibodies against the angiotensin AT1 receptor (Wallukat et al., 1999). Recently, an intrinsic RAS has been well documented in the placenta (see review Poisner, 1998). Many studies are, therefore, aimed at elaborating a possible association and role of circulating RAS and placental RAS in preeclampsia. However, some reports indicated increased RAS components (Brar et al., 1987) while other indicated unchanged or decreased levels of renin in the circulating RAS as well as in the placental RAS (Brown et al., 1997; Kalenga et al., 1996; Poranen et al., 1996). The present study is the rst to demonstrate unequivocally an upregulation of mRNA for angiotensinogen and AT1 receptor, and precise localization of enhanced AT1 receptor in patients with preeclampsia when compared with those in controls of vaginal delivery or caesarean delivery. Interestingly, preeclampsia may be associated with a common molecular variant of angiotensinogen and its expression is elevated in decidual

Fig. 5. (A). RT-PCR analysis of the mRNA expression of angiotensinogen in preeclamptic placenta as compared with that in normal placenta with vaginal delivery. Lane M, DNA marker; Lane 1, angiotensinogen expression in normal placenta; Lane 2, angiotensinogen expression in preeclamptic placenta; Lane 3, b-actin expression in normal; Lane 4, b-actin expression in preeclampsia. The arrows indicate the expected size of amplied products from angiotensinogen (374 bp) and b-actin (282 bp). (B) The relative expression of angiotensinogen/b-actin mRNA (% of control) in normal placenta (C1) and in preeclamptic placenta (PET). The data are expressed as mean9 S.E.M. (n = 6 per group).

tic properties and responsiveness to vasoactive peptide substances (Lim et al., 1997). This may trigger placental ischemia, leading eventually to the clinical syndrome of preeclampsia (Roberts et al., 1989). It has also been shown that preeclampsia may result from an imbalance between vasodilators such as nitric oxide (Morris et al., 1996) and vasoconstrictors such as angiotensin II (Pipkin, 1988). In fact, the increased vascular tone in preeclampsia may be mediated by the increased sensitivity to angiotensin II because angiotensin II coupled to its AT1 receptor is a potent vasoconstrictor (Doering et al., 1998), which in turn enhances the stimulation for the generation of other vasoactive substances (Yunohara et al., 1994; Haugen et al., 1996). For example, some of the vasoconstrictive effects of angiotensin II appear to be mediated by lipoxygenase (Kisch et al., 1997). Nevertheless, there was discrepancy of available data whether RAS is upregulated in preeclampsia (Pipkin, 1988). In the circulating levels, angiotensin II appears not to be increased in preeclampsia (Hanssens et

Fig. 6. (A) RT-PCR analysis of the mRNA expression of angiotensinogen in preeclamptic placenta as compared with that in normal placenta with elective caesarean delivery. Lane M, DNA marker; Lane 1, angiotensinogen expression in normal placenta; Lane 2, angiotensinogen expression in preeclamptic placenta; Lane 3, b-actin expression in normal; Lane 4, b-actin expression in preeclampsia. The arrows indicate the expected size of amplied products from angiotensinogen (374 bp) and b-actin (282 bp). (B) The relative expression of angiotensinogen/b-actin mRNA (% of control) in normal placenta (C2) and in preeclamptic placenta (PET). The data are expressed as mean 9 S.E.M. (n =6 per group).

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an activation of expression for placental AT1 receptor subtype both at the gene and protein levels in patients with preeclampsia, as demonstrated by semi-quantitative RT-PCR and Western blot respectively. The upregulation of AT1 receptor subtype in the preeclamptic placenta seems to be plausible for the pathogenesis of preeclampsia because of its potent vasoconstrictive effects on the uteroplacental blood circulation. More importantly, the increased expression of AT1 receptor protein was predominantly localized to the syncytiotrophoblasts, albeit to a less extent the capillaries, of the term placental villi in patients with preeclampsia, as evidenced by the present immunohistochemistry. Interestingly, the syncytiotrophoblasts are responsible for the secretion of numerous pregnancy-associated polypeptides into maternal circulation. One of this peptide, neurokinin B has been recently reported to be in excessive secretion during the third trimester and it may contribute to the pathogenesis of preeclampsia (Page et al., 2000; Page and Lowry, 2000). In addition to the potent vasoconstrictive effects of RAS, the actions of upregulation of RAS components in preeclamptic placenta, notably the upregulation of its AT1 receptor subtype localized to the syncytiotrophoblasts that may stimulate the excessive secretion of peptides such as neurokinin B into the maternal circulation, needs further investigation.

Acknowledgements The authors wish to thank the support from the Research Grants Council of Hong Kong (Project No: CUHK 4075/00M & CUHK 4116/01M), Germany/ Hong Kong Joint Research Scheme (Project No: GHK/ 00/05 & GHK/016/00E) and by the Direct Grant from the Chinese University of Hong Kong, Hong Kong.

Fig. 7. Immunohistochemical localization of AT1 receptor in normal and in preeclamptic placenta. Distinct immunoreactivity for AT1 receptor was observed in trophoblastic cells of the normal placenta either with vaginal delivery (A) or with caesarean delivery (B). More intense immunoreactivity for AT1 receptor was predominantly localized to the syncytiotrophoblastic cells of the placental villi (V) from preeclamptic placenta with caesarean delivery (C). No immunostaining was observed in preadsorption of primary antibody with excess of AT1 receptor antigen either in preeclamptic placenta (D) or in normal placenta with caesarean delivery (E). Note, huge numbers of villi can be seen cut in various planes of section and varying diameter from large villi to small branch villi. The villi are in close proximity to the lacuna (L), which are lled with maternal blood. The bar = 100 mm.

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