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Reproductive Endocrinology |
Department of Obstetrics and Gynecology (K.F., O.K., A.I., S.M., Y.T.), Faculty of Medicine, University of Nagoya, Showa-ku, Nagoya 466; Research Institute of Environmental Medicine (Y.K., Y.M., H.S.), University of Nagoya, Chikusa-ku, Nagoya 464; Division of Biochemistry (T.N.), Biomedical Research Center, Osaka University Medical School, Suita, Osaka 565, Japan
Address all correspondence and requests for reprints to: Osamu Kurauchi M.D., Department of Obstetrics and Gynecology, Faculty of Medicine, University of Nagoya, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan.
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Abstract |
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Introduction |
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The human placenta is one of the original sources from which HGF was purified to homogeneity (10, 11), and the factor is now generally considered to be critical for placental growth and organogenesis. Recently, it has been shown that mice lacking HGF have severely impaired placentas with markedly reduced numbers of labyrinthine trophoblast cells, and fetuses die before birth (12, 13). This result indicates that HGF is essential for maintenance of pregnancy.
Preeclampsia is characterized by maternal hypertension, proteinuria, and generalized edema. In addition to these effects on the mother, there is a profound impact on the fetus, resulting in increased perinatal mortality and frequent intrauterine growth retardation. Despite numerous studies, the pathogenesis of preeclamptic pregnancies remains obscure. The symptoms of preeclampsia disappear soon after termination of pregnancy, and the placenta must be an important factor because the disease can occur even in the absence of a fetus as in molar pregnancy. In light of the results from mice lacking HGF, we studied possible changes in placental HGF and its mRNA in messenger RNA (mRNA) preeclamptic placentas. An analysis of the localization of HGF mRNA and c-met protein, using in situ hybridization and immunohistochemical staining, respectively, was also performed to cast light on its physiological function.
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Materials and Methods |
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Placental tissues of 12–41 weeks’ gestation (n = 68) were obtained by therapeutic abortion (12–21 weeks), elective cesarean (26–40 weeks), or spontaneous vaginal delivery (22–41 weeks) from uncomplicated subjects (n = 54) and from patients with preeclampsia (n = 14). Uncomplicated subjects were normotensive throughout pregnancy and had no proteinuria or other signs of preeclampsia. Although these subjects included women undergoing spontaneous preterm labor, the cases with clinical chorioamnionitis (fever, maternal or fetal tachycardia, uterine tenderness, foul odor, and leukocytosis) were excluded. The gestational weeks of preeclamptic pregnancies ranged from 23–39 weeks. The gestational ages of all cases were determined by ultrasonographic examination in the first trimester.
The mean birth weight in preeclamptic pregnancies was 1316 ± 501 g. The birth weight in all cases of preeclamptic pregnancy was below 1.5 SD from the mean birth weight in appropriate for gestational age (AGA) neonates. The standard birth weight was based on the Japanese fetal body weight standard. The criterion for preeclamptic pregnancy was systolic and diastolic blood pressure above 140 mm Hg and 90 mm Hg, on at least two occasions 6 or more hours apart, together with generalized edema or proteinuria. Proteinuria was defined as 300 mg or more of urinary protein per 24 h or 100 mg/dL or more in at least two random urine specimens collected 6 or more hours apart. All cases of preeclamptic pregnancy did not have any other maternal complications. For Northern blot analysis and measurement of HGF concentration, tissue blocks were taken from the decidual side of the placentas, and villous portions of the placenta were exercised, washed extensively in saline, frozen immediately in liquid nitrogen, and stored at -80 C until use. For in situ hybridization, placental tissues were immediately fixed as will be described below.
Northern blot analysis
Frozen tissues (0.5 g) were pulverized in liquid nitrogen and RNA was isolated by the method of Chomczynski and Sacchi (14). The amount of total RNA was determined by optical density at 260 nm. Northern blot analysis was carried out according to the method described previously (15). Conditions for hybridization were described previously (16). cDNA for human HGF (6) was labeled with [32P] deoxy-CTP (New England Nuclear, Boston, MA) and used as a probe. cDNA for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) house-keeping gene was used for control.
Measurement of HGF levels in placental tissues
Extracts of placental tissue were prepared by modification of the method described previously (17, 18, 19). Briefly, each frozen tissue (0.2g) was quickly thawed at room temperature, minced, and rinsed with ice-cold 0.1M Tris-HCl buffer, pH 7.4. The minced pieces were finally suspended in 2 mL of ice-cold solution containing 50 mM Tris-HCl buffer and 0.25% Triton X-100, followed by homogenization in an ice bath, using a tight fitting pestle. The homogenate was centrifuged at 12,000 g for 20 min at 4 C. The resulting supernatant was appropriately diluted according to protein levels, and then HGF levels were determined by HGF-ELISA (enzyme linked immunoabsorbent assay) as previously described (20). The detection limit of the assay is 0.030 ng/mL; intraassay variation is less than 15% at three different levels of HGF.
Statistics
Statistical significance was determined by the Mann-Whitney U-test.
In situ hybridization
Placentas were manually dissected from other tissues, and samples were cut into 0.5–1.0 cm3 portions, fixed in 4% paraformaldehide dissolved in phosphate buffered saline (PBS, pH7.2), embedded in paraffin, and cut serially at 5 µm thickness. A 2.3 kbp complementary DNA (cDNA) for human HGF cloned into the pBluescript SK(-) (Stratagene, La Jolla, CA) plasmid was used for preparation of digoxygenin-11-UTP-labeled single strand cRNA probes. The plasmid was either linealized with BamH I and transcribed with T7 RNA polymerase to generate an antisense cRNA probe or linealized with Sal I and transcribed with T3 RNA polymerase to generate a sense cRNA probe. The probes were incubated in 40 mmol/L NaHCO3/60 mmol/L Na2CO3 at 60 C for 35 min to hydrolyze them into small fragments. Tissue sections were deparaffinized, and in situ hybridization was performed using protocols modified from Birren et al. (21). Briefly, following pretreatments, hybridization was carried for 16 h using 1.5 µg/mL of probe. The alkaline phosphatase reaction product was developed using the 4-nitroblue tetra-zolium chloride/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) reagents. Controls were hybridized with the sense (cRNA) probe.
Immunohistochemistry
Placental tissue blocks were embedded in paraffin, sections of 5 µm thickness were cut and then fixed on a slide with acetone. The paraffin was removed by xylene and ethanol. The slides were then incubated for 15 min at room temperature with 3% H2O2. After two washes with PBS, the slides were incubated for 10 min with 10% normal goat serum. After removing the serum, the slides were incubated for 2 h at room temperature with anti-HGF receptor (anti c-met proto-oncogene product) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), negative control sections were incubated in the absence of primary antibody. After two washes with PBS, the slides were incubated for 10 min at room temperature with biotin-labeled antirabbit IgG. After two additional washes with PBS, the slides were incubated for 5 min at room temperature with peroxidase-labeled streptoavidin. The immunoreaction was visualized using diaminobenzidine as the chromogenic substrate. The slides were counterstained with hematoxylin.
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Results |
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depicts the Northern blot showing changes
in the expression of HGF mRNA in placental tissues from uncomplicated
subjects at different gestational stages. The mRNA levels were highest
in the placentas obtained from the subjects of second trimester and
decreased in those of third trimester. Figure 2 compares
the expression of HGF mRNA levels in placentas from uncomplicated
subjects with those from patients with preeclampsia. In some of
placentas during third trimester, although there was no significant
difference of the mean gestational weeks in the two groups: 32.13
± 2.70 weeks (preeclamptic pregnancy, ±SD, n = 8,
28–36 weeks gestation) and 33.44 ± 3.88 weeks (uncomplicated
subjects, ±SD, n = 9, 28–40 weeks gestation), the
mean HGF mRNA level of preeclamptic pregnancies corrected by GAPDH mRNA
was significantly lower than that of uncomplicated subjects
(P < 0.05): 0.19 ± 0.07 (preeclamptic
pregnancies, ±SD) and 0.33 ± 0.05
(uncomplicated subjects, ±SD) by densitometric
quantitation.
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, the concentration of HGF in uncomplicated
placental tissue extracts reached a maximum in second trimester,
correlating with the mRNA level. Furthermore, during third trimester,
although there was no significant difference of the mean gestational
weeks between preeclamptic and uncomplicated groups: 33.62 ± 3.04
weeks (preeclamptic pregnancy, ±SD, n = 13, 28–39
weeks gestation) and 35.64 ± 3.84 weeks (uncomplicated subjects,
±SD, n = 33, 28–41 weeks gestation), the mean value
of HGF concentration in the preeclamptic placental tissue was
significantly lower than that in the uncomplicated subjects
(P < 0.05): 4.05 ± 1.44 ng/mg of protein
(preeclamptic pregnancy, ±SD), and 6.16 ± 3.32 ng/mg
of protein (uncomplicated subjects, ±SD) (Fig. 4).
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shows tissue section hybridized with the
antisense HGF cRNA. The message in samples of first trimester placentas
was primarily localized in mesenchymal cells in villous cores.
Hybridization signals of HGF mRNA were not detected in
syncytiotrophoblast and cytotrophoblast cells. Figure 5B shows control
of tissues hybridized with the sense orientation of the HGF cRNA.
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). No staining was shown in
the negative control (Fig. 6B).
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Discussion |
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The fact that mRNA levels and concentrations of immunoreactive HGF were highest in the placentas from uncomplicated subjects in second trimester is of interest, as expression of many placental proteins is maximal either in first trimester with a descending tendency to term (22) or in third trimester with an ascending tendency (23, 24, 25, 26). With gestation advanced, cytotrophoblasts differentiate to syncitiotrophoblasts, and consequently, the number of cytotrophoblasts relative to syncitiotrophoblasts decreases. Decreased expression of mRNA for HGF from second trimester to term may reflect the reduced number of target cells. The uncomplicated placentas from 22–36 weeks were obtained from women undergoing spontaneous preterm labor, which is sometimes associated with chorioamnionitis. Chorioamnionitis is associated with an increase in the production of cytokines, and it is possible that inflamation affects HGF levels. Although we excluded cases with clinical chorioamnionitis, there is still the possibility that cases with histologic chorioamnionitis were included.
In the present study, HGF mRNA was localized to placental mesenchymal cells, whereas c-met protein was demonstrated mainly on cytotrophoblasts. This result is in line with the finding that HGF is expressed in mesenchymes and acts on epithelium in a variety of organs. Placental tissue contains blood vessels as well as trophoblasts and stromal cells. In the placenta, HGF produced in stromal cells could thus act not only on cytotrophoblasts but also on fetal endothelial cells in a paracrine manner, as it is also known to be a potent angiogenic factor (27). However, its receptor, c-met, was detected in cytotrophoblasts, suggesting that HGF mainly exerts its activity on cytotrophoblasts. Actually, we previously reported that HGF promotes the growth of cytotrophoblasts by the paracrine mechanism (28).
A major finding of the present study was that the expression of HGF mRNA and the concentration of immunoreactive HGF were significantly lower in the placentas of some patients with preeclampsia compared with those in the uncomplicated subjects. Until now, abnormal expression of placental proteins involved in the pathogenesis of preeclampsia has been shown only for trophoblasts (29, 30). To our knowledge, this is the first report of abnormal expression in placental mesenchymal cells from preeclamptic placentas.
In normal placental organogenesis, invasion of the uterus and its arterial system by cytotrophoblasts extends through the entire decidua and the adjacent third of the myometrium. However, in preeclamptic placentas, cytotrophoblast invasion is limited to the superficial decidua, and few arterioles are breached, leading to the reduction in uteroplacental blood flow observed in preeclampsia. HGF is known to be a mesenchymal effector for epithelial morphogenesis and invasion in a variety of tissues. Weidner et al. (9) have suggested that HGF induces the progression of epithelial cells to an invasive phenotype. Our results indicate that decreased expression of HGF may affect acquisition of an invasive phenotype by cytotrophoblasts, resulting in the abnormally shallow trophoblast invasion of the uterus that is associated with preeclampsia. Recent observations have suggested that vascular endothelial dysfunction is an important component of preeclampsia (31). It is likely that endothelial dysfunction is a secondary effect of a disturbance in trophoblast migration.
Zhou et al. (29) reported that cytotrophoblast invasion was primarily confined to the superficial portions of the decidua in preeclamptic placenta and showed no evidence of cytotrophoblast invasion of the uterine vessels. This morphological appearance is not directly equivalent to that in mice lacking HGF, where the network of embryonic vessels and maternal sinuses was poorly developed and the size of labyrinth was reduced (13), although it should be remembered that there are fundamental differences in morphological structure between human and mouse. However, malnutrition because of reduced transport of nutrient to the fetus is observed in both cases. We thus speculate that a closely related mechanism is responsible for the pathogenesis of preeclampsia and the fetal loss in mice lacking HGF.
In summary, the expression of HGF in placental mesenchymal cells is most abundant in the second trimester and is significantly low in the placentas of some patients with preeclampsia. Our data may provide a molecular explanation for the abnormally shallow trophoblast invasion of the uterus and point to HGF as a factor in the etiology of preeclampsia.
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Footnotes |
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Received December 2, 1996.
Revised April 28, 1997.
Accepted May 13, 1997.
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uncomplicated subjects. •, patients with preeclampsia.
