Ontogeny of Hepatocyte Growth Factor (HGF) and Its Receptor (c-met) in Human Placenta
1998; Elsevier BV; Volume: 153; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)65658-1
ISSN1525-2191
AutoresDavid Somerset, Xiaofeng Li, Simon C. Afford, Alastair J. Strain, Asif Ahmed, Rajabant K. Sangha, Martin Whittle, Mark D. Kilby,
Tópico(s)Renal and related cancers
ResumoSevere intrauterine growth restriction (IUGR) is characterized by abnormal placentation. Mouse gene knockout studies show that an absence of either hepatocyte growth factor (HGF) or its receptor, c-met, leads to intrauterine death secondary to severe IUGR with deficient placentation. In this study, immunocytochemistry localized HGF protein throughout placental villi across gestation, whereas c-met protein was localized only to the perivillous trophoblast and vascular endothelium. Within the IUGR placentae, a reduction in HGF immunostaining within the villous stroma was observed. HGF mRNA was strongly expressed in the perivascular tissue around the stem villous arteries throughout gestation, with weaker expression within the villous stroma and the terminal villi. c-met mRNA expression was limited to the perivillous trophoblast, particularly in the first trimester, with only a faint hybridization signal from the villous stroma. Placental mRNA expression was examined quantitatively using a ribonuclease protection assay: HGF and c-met mRNA expression increased from the first to the second trimester, reaching a zenith before decreasing again through the third trimester to term. HGF mRNA levels were significantly reduced in the IUGR placentae (P = 0.036), whereas c-met mRNA expression was within the normal range for gestation. These findings suggest that HGF derived from the perivascular tissue of stem villous arteries may play an important role in controlling normal villous development. Whereas reduced expression of HGF within IUGR placentae does not prove a causative link with abnormal villous development, the association lends support to this possibility. Severe intrauterine growth restriction (IUGR) is characterized by abnormal placentation. Mouse gene knockout studies show that an absence of either hepatocyte growth factor (HGF) or its receptor, c-met, leads to intrauterine death secondary to severe IUGR with deficient placentation. In this study, immunocytochemistry localized HGF protein throughout placental villi across gestation, whereas c-met protein was localized only to the perivillous trophoblast and vascular endothelium. Within the IUGR placentae, a reduction in HGF immunostaining within the villous stroma was observed. HGF mRNA was strongly expressed in the perivascular tissue around the stem villous arteries throughout gestation, with weaker expression within the villous stroma and the terminal villi. c-met mRNA expression was limited to the perivillous trophoblast, particularly in the first trimester, with only a faint hybridization signal from the villous stroma. Placental mRNA expression was examined quantitatively using a ribonuclease protection assay: HGF and c-met mRNA expression increased from the first to the second trimester, reaching a zenith before decreasing again through the third trimester to term. HGF mRNA levels were significantly reduced in the IUGR placentae (P = 0.036), whereas c-met mRNA expression was within the normal range for gestation. These findings suggest that HGF derived from the perivascular tissue of stem villous arteries may play an important role in controlling normal villous development. Whereas reduced expression of HGF within IUGR placentae does not prove a causative link with abnormal villous development, the association lends support to this possibility. Intrauterine growth restriction (IUGR) remains a major cause of perinatal morbidity and mortality. A specific cohort of pregnancies with IUGR secondary to uteroplacental insufficiency is particularly at risk because of poor placental function.1Hackett GA Campbell S Gamsu H Cohen-Overbeek T Pearce JM Doppler studies in the growth retarded fetus and prediction of neonatal necrotising enterocolitis, haemorrhage, and neonatal morbidity.Br Med J. 1987; 294: 13-16Crossref PubMed Scopus (204) Google Scholar These pregnancies are characterized by abnormal placentation, abnormal umbilical artery diastolic velocities, and reduced liquor.2Macara L Kingdom JC Kaufmann P Kohnen G Hair JMI Lyall F Greer IA Structural analysis of placental terminal villi from growth-restricted pregnancies with abnormal umbilical artery Doppler waveforms.Placenta. 1996; 17: 37-48Abstract Full Text PDF PubMed Scopus (279) Google Scholar The placentae from these pregnancies are significantly smaller than those of controls, with a diminished mean placental surface area and specific morphological abnormalities, including reduced cytotrophoblast proliferation, smaller terminal villi, and abnormal villous vasculature.3Jackson MR Walsh AJ Morrow RJ Mullen JB Lye SJ Ritchie JW Reduced placental villous tree elaboration in small-for-gestational-age pregnancies: relationship with umbilical artery Doppler waveforms.Am J Obstet Gynecol. 1995; 172: 518-525Abstract Full Text PDF PubMed Scopus (179) Google ScholarHepatocyte growth factor (HGF) is a secreted heparin-binding glycoprotein that was originally identified from rat platelets on the basis of its ability to stimulate mitogenesis in hepatocytes.4Strain AJ McGowan JA Bucher NLR Stimulation of DNA synthesis in primary cultures of adult rat hepatocytes by rat platelet-associated substance(s).In Vitro. 1982; 18: 108-116Crossref PubMed Scopus (87) Google Scholar It is now known to be a multifunctional cytokine with angiogenic properties, which variously regulates growth, motility, and morphogenesis in receptive cells.5Boros P Miller MM Hepatocyte growth factor: a multifunctional cytokine.Lancet. 1995; 345: 293-295Abstract PubMed Google Scholar, 6Matsumoto K Nakamura T Emerging multipotent aspects of hepatocyte growth factor.J Biochem. 1996; 119: 591-600Crossref PubMed Scopus (609) Google Scholar, 7Zarnegar R Michalopoulos GK The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis.J Cell Biol. 1995; 129: 1177-1180Crossref PubMed Scopus (552) Google Scholar All these functions are potentially important in placentation, in which proliferation and cellular orientation of trophoblast, mesenchyme, and vascular endothelia are required.HGF is secreted as an inactive, single-chain 92-kd glycoprotein by cells of embryological mesenchymal origin. The active form, a heterodimer consisting of a heavy α chain (55–65 kd) and light β chain (32–36 kd), results from proteolytic cleavage of the precursor, which is thrombin dependent.8Miyazawa K Shimomura T Kitamura N Activation of hepatocyte growth factor in the injured tissues is mediated by hepatocyte growth factor activator.J Biol Chem. 1996; 271: 3615-3618Crossref PubMed Scopus (199) Google Scholar, 9Shimomura T Kondo J Ochiai M Naka D Miyazawa K Morimoto Y Kitamura N Activation of the zymogen of hepatocyte growth factor activator by thrombin.J Biol Chem. 1993; 268: 22927-22932Abstract Full Text PDF PubMed Google ScholarThe biological effects of HGF are mediated by its high-affinity receptor, c-met, a trans-membranous protein encoded by the MET proto-oncogene.10Bottaro DP Rubin JS Faletto DL Chan AML Kmiecik TE Vande Woude GF Aaronson SA Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.Science. 1991; 251: 802-804Crossref PubMed Scopus (2066) Google Scholar c-metundergoes proteolytic cleavage before expression on the cell surface as a heterodimeric protein. It consists of an extracellular 50-kd α chain and a 145-kd β chain that has extracellular,trans-membranous, and intracellular regions. The intracellular domain of the β subunit contains a tyrosine kinase domain and sites for autophosphorylation through which the cellular effects of c-met are mediated.10Bottaro DP Rubin JS Faletto DL Chan AML Kmiecik TE Vande Woude GF Aaronson SA Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.Science. 1991; 251: 802-804Crossref PubMed Scopus (2066) Google Scholar, 11Comoglio PM: Structure, biosynthesis, and biochemical properties of the HGF receptor in normal and malignant cells. Hepatocyte Growth Factor-Scatter Factor (HGF-SF) and the c-Met Receptor. Edited by ID Goldberg, EM Rosen. Birkhauser Verlag, Basel, pp 131–165Google Scholar c-met is expressed by many epithelial cell types, as well as vascular endothelium.12Sonnenberg E, Weidner KM, Birchmeier C: Expression of the metreceptor and its ligand, HGF-SF, during mouse embryogenesis. Hepatocyte Growth Factor-Scatter Factor (HGF-SF) and the c-Met Receptor. Edited by ID Goldberg, EM Rosen. Birkhauser Verlag, Basel, pp 381–394Google ScholarThe biological activities and expression of HGF and its receptor in embryonic life suggest an important role for them in mesenchymal-epithelial interactions during organogenesis.12Sonnenberg E, Weidner KM, Birchmeier C: Expression of the metreceptor and its ligand, HGF-SF, during mouse embryogenesis. Hepatocyte Growth Factor-Scatter Factor (HGF-SF) and the c-Met Receptor. Edited by ID Goldberg, EM Rosen. Birkhauser Verlag, Basel, pp 381–394Google Scholar, 13Birchmeier C Sonnenberg E Weidner KM Walter B Tyrosine kinase receptors in the control of epithelial growth and morphogenesis during development.BioEssays. 1993; 15: 185-190Crossref PubMed Scopus (34) Google Scholar, 14Kolatsi-Joannou M Moore R Winyard PJ Woolf AS Expression of hepatocyte growth factor/scatter factor and its receptor, MET, suggests roles in human embryonic organogenesis.Pediatr Res. 1997; 41: 657-665Crossref PubMed Scopus (59) Google Scholar Direct evidence on the essential role of HGF/c-met in embryogenesis comes from recent experiments in mice using gene knockout techniques. Mice heterozygous for the disrupted gene (HGF or c-met) were phenotypically normal, whereas those homozygous for the defect became severely growth restricted in the second trimester, resulting in universal lethality by the early third trimester.15Bladt F Riethmacher D Isenmann S Aguzzi A Birchmeier C Essential role for the c-met receptor in the migration of myogeneic precursor cells into the limb bud.Nature. 1995; 376: 768-771Crossref PubMed Scopus (1087) Google Scholar, 16Schmidt C Bladt F Goedecke S Brinkmann V Zschieshe W Sharpe M Gherardi E Birchmeier C Scatter factor/hepatocyte growth factor is essential for liver development.Nature. 1995; 373: 699-702Crossref PubMed Scopus (1222) Google Scholar, 17Uehara Y Minowa O Mori C Shiota K Kuno J Noda T Kitamura N Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor.Nature. 1995; 373: 702-705Crossref PubMed Scopus (914) Google Scholar The placentae of mutant embryos were much smaller than normal and had a poorly developed labyrinthine region. The key abnormality was severe depletion of the labyrinthine trophoblast cells, homologous to the perivillous trophoblast in human placentae, combined with poor vascularization of this region.HGF and c-met mRNA and protein have previously been localized in both first trimester and term human placentae.18Kilby MD Afford S Li XF Strain AJ Ahmed A Whittle MJ Localisation of hepatocyte growth factor and its receptor (c-met) protein and mRNA in human term placenta.Growth Factors. 1996; 13: 1-7Crossref PubMed Scopus (29) Google Scholar, 19Clark DE Smith SK Sharkey AM Sowter HM Charnock-Jones DS Hepatocyte growth factor/scatter factor and its receptor c-met: localisation and expression in the human placenta throughout pregnancy.J Endocrinol. 1997; 151: 459-467Crossref Scopus (43) Google Scholar, 20Kauma S Hayes N Weatherford S The differential expression of hepatocyte growth factor and met in human placenta.J Clin Endocrinol Metab. 1997; 82: 949-954Crossref PubMed Scopus (49) Google Scholar In this study, we report for the first time their cellular localization and quantitative expression in the placentae of human pregnancies throughout gestation. We also note changes in cellular distribution and expression of HGF and c-met in pregnancies complicated by severe IUGR.Materials and MethodsTissue CollectionPlacentae were obtained from normal first, second, and third trimester pregnancies and also pregnancies complicated by severe IUGR (see Table 1). First and second trimester placentae were obtained after surgical termination of pregnancy; other placentas were obtained after nonlaboring cesarean section. Growth-restricted fetuses were identified prospectively using ultrasound, and the diagnosis used in our study was based on the following criteria: 1) either an estimated fetal weight of below the third centile for gestational age or serial growth measurements indicating decreased growth velocity >1.5 standard deviations, 2) absent or significantly reduced umbilical artery end diastolic blood flow, and 3) reduced liquor volume.21Chang TC Robson SC Spencer JA Gallivan S Identification of fetal growth retardation: comparison of Doppler waveform indices and serial ultrasound measurements of abdominal circumference and fetal weight.Obstet Gynecol. 1993; 82: 230-236PubMed Google ScholarTable 1Clinical Details of Pregnancies from Which Placenta Were ExaminedTrimesterFirstSecondThirdIUGRGestation matchedNumber (n)851569Gestation (weeks)11 (9–13)16 (14–17)37 (27–41)31 (26–38)32 (27–38)Birth weight (g)NANA2905 (1100–3860)1083 (595–2060)1700 (1084–3360)AEDFNANA050Oligohydramnious00060Median (range) gestational age and birth weight are displayed. AEDF, number with absent end diastolic flow (in the umbilical artery); oligohydramnios, number with maximum depth of liquor <2 cm; NA, not applicable. Open table in a new tab Full-thickness placental biopsies were taken from a central location lying between the basal and chorionic plates. The samples of fresh placental tissue were divided into small pieces and thoroughly washed in phosphate-buffered 0.9% saline (PBS). Tissues were variously preserved: for immunocytochemistry, biopsies were fixed in Formalin before embedding in paraffin wax; for in situ hybridization, they were fixed in 4% paraformaldehyde for 24 hours before being washed, snap frozen in Cryo-Embed (Bright Instrument Co., Huntingdon, UK) and stored at −70°C; for protein or RNA extraction, they were snap frozen in liquid nitrogen immediately after collection and stored at −70°C. Ethical approval for tissue collection was obtained from the South Birmingham Ethical Committee.Western ImmunoblottingTotal protein was extracted from snap-frozen placental tissue using a lysis buffer (50 mmol/L Tris-HCl (pH 8.0), 1% Triton X-100, 0.1% sodium dodecyl sulfate, 250 mmol/L NaCl, and 5 mmol/L ethylenediaminetetraacetic acid) containing 40 mg/ml phenylmethysulfonyl fluoride, 5 mg/ml pepstatin, and 5 mg/ml leupeptin. Protein concentration was calculated spectrophotometrically at 595 nm using a Coomassie blue reagent and human albumin standards. Analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis on discontinuous acrylamide gels according to the method of Laemmli,22Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (206057) Google Scholar using the Mini Protean II System (Bio-Rad, Herts, UK). One hundred micrograms of protein was applied per lane under reducing conditions, and biotinylated molecular weight markers (Bio-Rad) were run in parallel lanes.After electrophoresis, proteins were transferred to nitrocellulose membranes, which were then blocked for nonspecific binding with a solution containing 5% nonfat milk, 0.1% bovine serum albumin, 0.1% Tween 20, and PBS. Membranes were incubated with a primary antibody (anti-HGF (1 in 2000): rabbit polyclonal antibody raised against human HGF, supplied by Dr T. Ishii (Mitsubushi Kasei Corp., Yokohamu, Japan); anti-c-met (1 in 500): affinity-purified rabbit polyclonal antibody raised against a peptide that corresponds to the C-12 terminal amino acids of the human c-met p140 (anti h-met) (Santa Cruz Biotechnology Inc., Santa Cruz, CA)). Antibody binding was visualized using a secondary antibody (peroxide-conjugated goat anti-rabbit (1 in 2000): Dako Ltd., Bucks, UK) with an enhanced chemiluminescence system (Amersham International, Bucks, UK) and recorded by exposure to preflashed film (Hyperfilm-ECL, Amersham International). The specificity of secondary antibody binding was tested by performing no-primary controls, which were negative (data not shown). The biotinylated molecular weight markers were visualized separately by incubating with an avidin-horseradish peroxidase complex (Bio-Rad, Richmond, CA) followed by enhanced chemiluminescence as above.ImmunocytochemistryImmunocytochemistry was performed as described previously.18Kilby MD Afford S Li XF Strain AJ Ahmed A Whittle MJ Localisation of hepatocyte growth factor and its receptor (c-met) protein and mRNA in human term placenta.Growth Factors. 1996; 13: 1-7Crossref PubMed Scopus (29) Google Scholar Serial 3-μm sections of Formalin-fixed, paraffin-embedded tissue were deparaffinized with xylene and then rehydrated, and endogenous peroxidase activity was quenched by the addition of 0.3% (v/v) hydrogen peroxide. Primary antibodies were as above, anti-HGF (1 in 25) and anti c-met (1 in 5). Amplification of the primary antibody reaction was achieved by incubation with an appropriate secondary antibody, followed by a complex of streptavidin and biotinylated peroxidase. The binding was visualized by incubating the sections with diaminobenzidine and hydrogen peroxide in PBS. Sections were counterstained with Mayer's hematoxylin, dehydrated, and mounted. Negative control reactions were performed by omitting the primary antibodies and also, for anti-HGF, by preabsorbing the primary antibody with an excess of recombinant HGF.Ribonuclease Protection AssayTotal RNA was extracted from snap-frozen placental tissue by the guanidinium thiocyanate/acid phenol extraction method and quantified spectrophotometrically at 260 nm.23Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62993) Google Scholar[32P]CTP-labeled anti-sense riboprobes (specific activity ∼4 × 108 cpm/μl) for HGF (677 bp) and c-met (500 bp) were constructed from plasmid-derived cDNAs that we have described previously.18Kilby MD Afford S Li XF Strain AJ Ahmed A Whittle MJ Localisation of hepatocyte growth factor and its receptor (c-met) protein and mRNA in human term placenta.Growth Factors. 1996; 13: 1-7Crossref PubMed Scopus (29) Google Scholar The pTRI-β-actin control template (Ambion, Whitney, UK) was used to construct a 298-bp [32P]CTP-labeled anti-sense riboprobe (specific activity ∼5 × 107 cpm/μl) corresponding to a fragment of human cytoplasmic actin. This was used as a constitutively expressed gene product to control for differences in RNA loading, allowing comparison of HGF and c-met mRNA expression between RNA samples run on the same gel. A total of 34 placental samples were analyzed on five gels; to allow comparison of results between gels, a minimum of 4 identical placental control samples were run on each gel. The assay was performed as described previously.24Sangha RK Li XF Shams M Ahmed A Fibroblast growth factor receptor-1 is a critical component for endometrial remodeling: localization and expression of basic fibroblast growth factor and FGF-R1 in human endometrium during the menstrual cycle and decreased FGF-R1 expression in menorrhagia.Lab Invest. 1997; 77: 389-402PubMed Google ScholarSample RNA (20 μg) was mixed with the three riboprobes (HGF and c-met, ∼1 × 105 cpm; β-actin, ∼2 × 104 cpm), precipitated with ethanol and redissolved in hybridization buffer. Samples were then denatured at 90°C and incubated overnight at 45°C. Nonhybridized RNA was removed by incubating with an RNase digestion buffer, which was subsequently inactivated by the addition of proteinase K. After phenol-chloroform extraction and ethanol precipitation, the RNA was resuspended in loading buffer, denatured at 85°C, and electrophoresed on a polyacrylamide/urea gel. The gels were then washed, transferred to chromatography paper, and dried under vacuum. Autoradiography was performed using preflashed X-ray film (Hyperfilm MP, Amersham International) with intensifying screens at −70°C for 1 to 6 days. Quantification of autoradiographic signals was performed by densitometric analysis (area under the curve), and transcripts detected were assigned relative densitometric units. The ratio of HGF/actin and c-met/actin was then calculated for each sample. A positive and negative control reaction was carried out for each experiment using yeast tRNA in the presence and absence of the RNase digestion buffer.In Situ HybridizationIn situ hybridization for both HGF and c-metwas performed on 6-μm sections of paraformaldehyde-fixed, frozen tissues as previously described.18Kilby MD Afford S Li XF Strain AJ Ahmed A Whittle MJ Localisation of hepatocyte growth factor and its receptor (c-met) protein and mRNA in human term placenta.Growth Factors. 1996; 13: 1-7Crossref PubMed Scopus (29) Google Scholar[35S]UTP-labeled riboprobes (both sense and anti-sense) were synthesized from a 677-bp cDNA for HGF and a 500-bp cDNA for c-met. Sense and anti-sense hybridizations were performed on serial sections, with the sense riboprobe acting as a negative control to demonstrate any nonspecific hybridization. The hybridized sections were dipped into radiographic emulsion (Ilford K5, Mobberley, UK) and stored at 4°C for 14 to 21 days, before developing and counterstaining with hematoxylin and eosin.StatisticsNormal distribution of results was not assumed. The Kruskal-Wallis test was used to compare groups of medians, Dunn's multiple comparison test for posthoc analysis, and the Mann-Whitney test to compare unpaired groups. All calculations were performed using a computer analysis package (Prism 2.0, GraphPad Software, San Diego, CA).ResultsTable 1 summarizes the clinical data from early gestational, uncomplicated third trimester, and IUGR groups.Cellular Localization of HGF and c-metWestern immunoblotting using protein lysates from placental tissue under reducing conditions revealed bands of appropriate sizes for HGF and c-met throughout gestation and in the presence of IUGR, confirming specificity of the antibodies (Figure 1).In first trimester placental tissue, the perivillous trophoblast was immunopositive to HGF, with the syncytiotrophoblast demonstrating slightly stronger staining than the cytotrophoblast (Figure 2A). The mesenchymal core was relatively loosely structured but also showed immunoreactive staining to HGF along with the Hofbauer cells. The corresponding section immunostained with anti c-met antibody demonstrated similarly intense immunolocalization in the trophoblast layer, but with little apparent difference between cytotrophoblast and syncytiotrophoblast (Figure 2B). Little stromal immunostaining to c-met was noted other than in the vascular endothelium. Both HGF and c-met were also immunolocalized within the decidua (Figure 2, C and D). In control sections no positive staining was found when the primary antibody was omitted (Figure 2E). Similarly, no immunostaining was observed when the antiserum was preabsorbed by excess of HGF (data not shown).Figure 2In the first trimester villus, the syncitiotrophoblast (Sy) and cytotrophoblast (Cy) layers of the villus were stained immunopositive to HGF (A) and c-met(B). The mesenchymal stromal core (str) was relatively loosely structured and stained positively for HGF along with the Hofbauer cells (H). The vascular endothelium (endo) stained weakly positive for both HGF (C) and c-met (D). Control sections (see text) were negative (E).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In normal third trimester placentae, HGF immunostaining was most intense in the fused syncytioblast/cytotrophoblast layer surrounding the villi (Figure 3A). There was also positive immunoreactivity to HGF in the mesenchymal villous core and endothelial cells lining the vasculature. c-metimmunoreactivity was strongest in the fused syncytioblast/cytotrophoblast but was also evident around vascular endothelium (Figure 3B).Figure 3In placentae from uncomplicated third trimester pregnancies, positive cytoplasmic staining was identified using anti-HGF antibody (A), most intensely in the syncytium (Sy). There was also positive immunoreactivity to HGF in the endothelial cells (endo) lining the vasculature of the villi and the cells within the mesenchymal stromal core (str). B:c-met immunolocalization is primarily to the vascular endothelium (endo) within villi and to the syncytium (Sy) around the villi. In third trimester placentae complicated by IUGR, HGF immunoreactivity was localized in the same tissue types as in placentae of "uncomplicated" pregnancies (C). However, the HGF immunostaining intensity was less marked in the mesenchymal stroma. In serial sections immunostained for c-met, there was no discernible alteration in either localization or intensity (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In IUGR placentae HGF immunolocalization was similar to that in normal placentae; however, the immunostaining intensity appeared reduced in the mesenchymal stroma (Figure 3C). A blinded operator reviewing the tissue sections qualitatively scored this (Table 2) and showed that HGF immunostaining within the stroma appeared reduced in the IUGR placentae compared with normal controls. In serial sections immunostained for c-met, no discernible alteration in localization or intensity was noted (Figure 3D).Table 2Immunostaining to HGF and c-met in Normal and IUGR PlacentaeImmunostaining intensityNormal (n = 6)IUGR (n = 6)TissueHGFc-metHGFc-metSyncytium++++++++++++Villous stroma+++−+−Villous endothelium++++++++Decidual cells++++++++Hofbauer cells+++−+++−Amnion/chorion+++++++++++One of the authors (X-FL), blinded to the origin of the sections, graded the intensity of immunostaining in each of the placental cell types. Staining was graded as intense (++++), strong (+++), moderate (++), weak (+) and absent (−) compared with the negative control section. Open table in a new tab Expression of mRNA for HGF and c-met in Placental TissuesFigure 4 shows the quantification of mRNA levels in gestational placental tissues using the ribonuclease protection assay. Both HGF and c-met mRNA expression increased from the first to the second trimester, when they were maximally expressed, before falling to their lowest levels at term (Figure 5, Figure 6). Within IUGR placentae, HGF mRNA was significantly reduced when compared with gestationally matched normal controls (Figure 5B), whereas c-met mRNA levels were within normal limits for gestation (Figure 6B).Figure 4Example of an autoradiograph of the ribonuclease protection assay. The signals of the protected fragments were quantified using computer densitometry in placentae from first, second, and third trimesters and IUGR. The actin RNA probe has been used as an internal control to correct for variations in RNA loading between lanes. The results are expressed as arbitrary optical units for HGF/actin and c-met/actin. A total of 34 placental samples were analyzed on five gels using common control samples to allow direct comparison.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Quantification of HGF mRNA using the ribonuclease protection assay.A: Normal placenta from first (T1), second (T2), and third (T3) trimester placenta.P = 0.0008 (Kruskal-Wallis test, statistic = 14.28). **P < 0.001 (Dunn's multiple comparison test, Δ rank sum = 15.40). B: IUGR and gestationally matched third trimester placenta. *P = 0.036 (Mann-Whitney test, two-tailed Pvalue, U = 9.0). Box and whisker plots are shown, indicating medians, interquartile ranges, and total ranges.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Quantification of c-met mRNA using the ribonuclease protection assay. A: Normal placenta from first (T1), second (T2), and third (T3) trimester placenta. *P = 0.006 (Kruskal-Wallis test, statistic = 10.22). **P < 0.005 (Dunn's multiple comparison test, Δ rank sum = 11.40). B: IUGR and gestationally matched third trimester placenta. #P = 0.53 (Mann-Whitney test, two-tailedP value, U = 21.0). Box and whisker plots are shown, indicating medians, interquartile ranges, and total ranges.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In situ hybridization studies from the first trimester reveal HGF mRNA to be localized to the villous stroma, and in particular to the perivascular tissue (Figure 7, A and B), with no signal from either cytotrophoblast or syncytiotrophoblast. Contrasting this, c-met mRNA is clearly localized to the perivillous trophoblast with little signal from the stroma (Figure 7, C and D). In the third trimester, the localization of HGF to the perivascular tissue around the vasculature of the stem villi is clearly demonstrated, with a much weaker signal from the stroma of the terminal villi (Figure 7, E and F). The
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