Transforming Growth Factor-β Expression in Human Placenta and Placental Bed in Third Trimester Normal Pregnancy, Preeclampsia, and Fetal Growth Restriction
2001; Elsevier BV; Volume: 159; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63029-5
ISSN1525-2191
AutoresFiona Lyall, H. Blair Simpson, Judith N. Bulmer, Andrew Barber, Stephen C. Robson,
Tópico(s)Prenatal Screening and Diagnostics
ResumoNormal human pregnancy depends on physiological transformation of spiral arteries by invasive trophoblasts. Preeclampsia (PE) and fetal growth restriction (FGR) are associated with impaired trophoblast invasion and spiral artery transformation. Recent studies have suggested that transforming growth factor (TGF)-β3 is overexpressed in the placenta of PE patients and that this may be responsible for failed trophoblast invasion. There are, however, no studies on TGF-βs in the placenta in FGR or in the placental bed in PE or FGR. In this study we have used immunohistochemistry, Western blot analysis, and enzyme-linked immunosorbent assay to examine the expression of TGF-β1, TGF-β2, and TGF-β3 in placenta and placental bed of pregnancies complicated by PE and FGR and matched control pregnancies. The results show that TGF-β1, -β2, and -β3 are not expressed in villous trophoblasts but are present within the placenta. TGF-β1, -β2, and, to a much lesser extent, TGF-β3 were present within the placental bed but only TGF-β2 was present in extravillous trophoblast. No changes in expression of either isoform were found in placenta or placental bed in PE or FGR compared with normal pregnancy. These data are not consistent with overexpression of TGF-β3 being responsible for failed trophoblast invasion in PE. Our findings suggest that the TGF-βs do not have a pathophysiological role in either PE or FGR. Normal human pregnancy depends on physiological transformation of spiral arteries by invasive trophoblasts. Preeclampsia (PE) and fetal growth restriction (FGR) are associated with impaired trophoblast invasion and spiral artery transformation. Recent studies have suggested that transforming growth factor (TGF)-β3 is overexpressed in the placenta of PE patients and that this may be responsible for failed trophoblast invasion. There are, however, no studies on TGF-βs in the placenta in FGR or in the placental bed in PE or FGR. In this study we have used immunohistochemistry, Western blot analysis, and enzyme-linked immunosorbent assay to examine the expression of TGF-β1, TGF-β2, and TGF-β3 in placenta and placental bed of pregnancies complicated by PE and FGR and matched control pregnancies. The results show that TGF-β1, -β2, and -β3 are not expressed in villous trophoblasts but are present within the placenta. TGF-β1, -β2, and, to a much lesser extent, TGF-β3 were present within the placental bed but only TGF-β2 was present in extravillous trophoblast. No changes in expression of either isoform were found in placenta or placental bed in PE or FGR compared with normal pregnancy. These data are not consistent with overexpression of TGF-β3 being responsible for failed trophoblast invasion in PE. Our findings suggest that the TGF-βs do not have a pathophysiological role in either PE or FGR. During early human pregnancy, extravillous cytotrophoblasts from anchoring villi invade the decidualized endometrium and myometrium (interstitial trophoblast) and also migrate in a retrograde direction along the spiral arteries (endovascular trophoblast) transforming them into large diameter conduit vessels of low resistance.1Pijnenborg R Bland JM Robertson WB Brosens I Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy.Placenta. 1983; 4: 397-414Abstract Full Text PDF PubMed Scopus (605) Google Scholar Endovascular trophoblast invasion has been reported to occur in two waves; the first into the decidual segments of spiral arteries at 8 to 10 weeks of gestation and the second into myometrial segments at 16 to 18 weeks of gestation.1Pijnenborg R Bland JM Robertson WB Brosens I Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy.Placenta. 1983; 4: 397-414Abstract Full Text PDF PubMed Scopus (605) Google Scholar This physiological transformation is characterized by a gradual loss of the normal musculoelastic structure of the arterial wall and replacement by amorphous fibrinoid material in which trophoblast cells are embedded.2Brosens I Robertson WB Dixon HG The physiological response of the vessels of the placental bed to normal pregnancy.J Pathol Bacteriol. 1967; 93: 569-579Crossref PubMed Scopus (599) Google Scholar, 3Pijnenborg R Dixon G Robertson WB Brosens I Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy.Placenta. 1980; 1: 3-19Abstract Full Text PDF PubMed Scopus (698) Google Scholar, 4Sheppard BL Bonnar J The ultrastructure of the arterial supply of the human placenta in pregnancy complicated by fetal growth retardation.J Obstet Gynaecol Br Cwlth. 1976; 83: 948-959Crossref Scopus (186) Google Scholar, 5De Wolf F De Wolf-Peeters C Brosens I Ultrastructure of the spiral arteries in human placental bed at the end of normal pregnancy.Am J Obstet Gynecol. 1973; 117: 833-848Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 6Khong TY De Wolf F Robertson WB Brosens I Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants.Br J Obstet Gynaecol. 1986; 93: 1049-1059Crossref PubMed Scopus (1438) Google Scholar, 7Blankenship TN Enders AC King BF Trophoblastic invasion and modification of uterine veins during placental development macaques.Cell Tissue Res. 1993; 274: 135-244Crossref PubMed Scopus (71) Google Scholar These physiological changes are required for a successful pregnancy. Failure of trophoblast invasion and spiral artery transformation has been documented in preeclampsia (PE), one of the leading causes of maternal death.8Roberts JM Redman CW Pre-eclampsia: more than pregnancy-induced hypertension.Lancet. 1993; 341: 1447-1451Abstract PubMed Scopus (1163) Google Scholar In this syndrome reduced uteroplacental perfusion is associated with widespread endothelial dysfunction and fetal growth restriction (FGR) leading to significant maternal and perinatal morbidity. Similar spiral artery abnormalities have been reported in the placental bed of women with FGR in the absence of maternal hypertension as well as in miscarriages.4Sheppard BL Bonnar J The ultrastructure of the arterial supply of the human placenta in pregnancy complicated by fetal growth retardation.J Obstet Gynaecol Br Cwlth. 1976; 83: 948-959Crossref Scopus (186) Google Scholar, 9Michel MZ Khong TY Clark DA Beard RW A morphological and immunological study of human placental bed biopsies in miscarriage.Br J Obstet Gynaecol. 1990; 97: 984-988Crossref PubMed Scopus (62) Google Scholar, 10Jauniaux E Zaidi J Jurkovic D Campbell S Hustin J Comparison of colour Doppler features and pathological findings in complicated early pregnancy.Hum Reprod. 1994; 12: 2432-2437Google Scholar, 11Hustin J Jauniaux E Schaaps JP Histological study of the materno-embryonic interface in spontaneous abortion.Placenta. 1990; 11: 477-486Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 12Khong TY Liddell HS Robertson WB Defective haemochorial placentation as a cause of miscarriage: a preliminary study.Br J Obstet Gynaecol. 1987; 94: 649-655Crossref PubMed Scopus (194) Google Scholar, 13Khong TY Placental changes in fetal growth retardation. Fetus and Neonate.in: Hanson MA Spencer JAD Rodeck CH Physiology and Clinical Applications. vol 3. Cambridge University Press, Cambridge1995Google Scholar, 14McFadyen IR Price AB Geirsson RT The relation of birth-weight to histological appearances in vessels of the placental bed.Br J Obstet Gynaecol. 1986; 93: 476-481Crossref PubMed Scopus (81) Google Scholar, 15Pijnenborg R Anthony J Davey DA Rees A Tiltman A Vercruysse L Van Assche FA Placental bed spiral arteries in the hypertensive disorders of pregnancy.Br J Obstet Gynaecol. 1991; 98: 648-655Crossref PubMed Scopus (594) Google Scholar, 16Sheppard BL Bonnar J An ultrastructural study of utero placental arteries in hypertensive and normotensive pregnancy and fetal growth retardation.Br J Obstet Gynaecol. 1981; 88: 695-705Crossref PubMed Scopus (278) Google Scholar Despite the importance of trophoblast invasion and vascular remodeling these processes are still not well understood. However they are thought to include changes in expression of cell adhesion molecules, matrix metalloproteinases, and their tissue inhibitors and growth factors and their receptors.17Lyall F Robson SC Defective EVT function and pre-eclampsia.in: Kingdom JCP Jauniaux ERM SPM O'Brien The Placenta: Basic Science and Clinical Practice. RCOG Press, London2000: 79-96Google Scholar, 18Lyall F Kaufmann P The uteroplacental circulation: EVT.in: Baker PN Kingdom JCP Intrauterine Growth Restriction. Springer-Verlag, London2000: 85-119Google Scholar Transforming growth factor-βs (TGF-βs) are members of a large superfamily of cytokines including activins, inhibins, and bone morphogenic proteins.19Pepper MS Transforming growth factor-beta: vasculogenesis and vessel wall integrity.Cytokine Growth Factor Rev. 1997; 8: 21-24Abstract Full Text PDF PubMed Scopus (611) Google Scholar The family is composed of three related 25-kd homodimeric proteins TGF-β1, -β2, and -β3. TGF-β exerts its biological effects through binding to cell surface receptors designated types I, II, and III. Studies have suggested that TGF-β, produced primarily by the decidua, may regulate trophoblast invasion.20Lala PK Hamilton GS Growth factors, proteases and protease inhibitors in the maternal-fetal dialogue.Placenta. 1996; 17: 545-555Abstract Full Text PDF PubMed Scopus (138) Google Scholar Recently Caniggia and colleagues21Caniggia I Grisaru-Gravnosky S Kuliszewsky M Post M Lye SJ Inhibition of TGFBβ3 restores the invasive capability of EVTs in preeclamptic pregnancies.J Clin Invest. 1999; 103: 1641-1650Crossref PubMed Scopus (320) Google Scholar reported that TGF-β3 was a major regulator of trophoblast invasion in vivo and in vitro. Expression of TGF-β3 in placental villous tissue peaked at 7 to 8 weeks of gestation and was virtually undetectable by 9 weeks. The same group also reported that TGF-β3 was weakly expressed in third trimester placentas but was dramatically up-regulated in placentas obtained from women with PE. It was suggested that overexpression of TGF-β3 may account for failure of trophoblast invasion in PE. Nothing is known about expression of TGF-βs the placental bed in PE and FGR where there is failure of normal spiral artery transformation. Thus in this article we have used immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA) to examine the expression of TGF-β1, -β2, and -β3 in placentas and placental bed biopsies from normal pregnancies and from pregnancies complicated by PE or FGR. Samples were obtained from pregnant women at the Royal Victoria Infirmary, Newcastle-on-Tyne, UK. The study was approved by the Joint Ethics Committee of Newcastle and North Tyneside. Three groups of women were studied: control pregnancies with no hypertension or FGR, women with pregnancies complicated by PE, and women with pregnancies complicated by FGR in the absence of maternal hypertension. In some of the cases placentas but not placental bed biopsies were collected andvice versa therefore some of the clinical details differed between placental and placental bed experiments. Thus these are presented as two separate tables (Table 1, Table 2). The overall clinical details for the two groups were similar.Table 1Clinical Details for Placenta Immunohistochemistry StudiesControl (n = 18)PE (n = 19)FGR (n = 10)Age (years)30.65 ± 4.9831.21 ± 7.528.8 ± 8.0Gestational age at delivery (weeks)36.28 ± 3.7134.27 ± 3.7233.67 ± 2.84Birth weight (kg)2.93 ± 0.862.16 ± 1.05*P < 0.005 compared with control pregnant group.1.4 ± 0.53*P < 0.005 compared with control pregnant group.Systolic BP (mmHg)112.24 ± 9.27165 ± 13.64*P < 0.005 compared with control pregnant group.124.5 ± 10.66Diastolic BP (mmHg)67.8 ± 7.5109.95 ± 8.34*P < 0.005 compared with control pregnant group.73.5 ± 9.14Maternal plasma urates (mmol/L)—407.26 ± 52.64—Values are shown as mean ± S.D.* P < 0.005 compared with control pregnant group. Open table in a new tab Table 2Clinical Details for Placental Bed Immunohistochemistry StudiesControl (n = 18)PE (n = 23)FGR (n = 9)Age (years)29.4 ± 6.0729.33 ± 7.829.75 ± 7.9Gestational age at delivery (weeks)35.5 ± 3.833.92 ± 4.2734.33 ± 2.39Birth weight (kg)2.67 ± 0.912.09 ± 0.971.38 ± 0.36*P < 0.005 compared with control pregnant group.Systolic BP (mmHg)116.33 ± 8157.14 ± 13.93*P < 0.005 compared with control pregnant group.119.44 ± 8.08Diastolic BP (mmHg)71.83 ± 7.59110.73 ± 7.51*P < 0.005 compared with control pregnant group.73.5 ± 9.14Maternal plasma urates (mmol/L)—419.05 ± 67.15—Values are shown as mean ± S.D.* P < 0.005 compared with control pregnant group. Open table in a new tab Values are shown as mean ± S.D. Values are shown as mean ± S.D. PE was defined as pregnancy-induced hypertension (blood pressure ≥ 140/90 mmHg) and proteinuria (≥300 mg/24 hours) in women who were normotensive before pregnancy and had no other underlying clinical problems such as renal disease. FGR was defined ultrasonically as fetal abdominal circumference 1.5 SD22Robson SC Chang TC Measurement of human fetal growth.in: Hanson MA Spencer JAD Rodeck CH Fetus and Neonate. Cambridge University Press, Cambridge1995: 297-325Google Scholar and umbilical artery pulsatility index ≥95th centile.23Arduini D Rizzo G Normal values of pulsatility index from fetal vessels; a cross-sectional study of 1556 healthy fetuses.J Perinat Med. 1990; 18: 165-172Crossref PubMed Scopus (447) Google Scholar We have previously shown that a fall in abdominal circumference SD score of >1.5 SD is the optimal cut-off to define a group of fetuses with evidence of wasting at birth and morbidity associated with FGR.22Robson SC Chang TC Measurement of human fetal growth.in: Hanson MA Spencer JAD Rodeck CH Fetus and Neonate. Cambridge University Press, Cambridge1995: 297-325Google Scholar Birth weight centiles were obtained from charts of the Northern Region population of England;24Tin W Wariyar UK Hey EN Selection biases invalidate current low birthweight-for-gestation standards.Br J Obstet Gynaecol. 1997; 104: 180-185Crossref PubMed Scopus (54) Google Scholar small for gestational age was defined as a birth weight below the 10th centile. The majority of placental bed biopsies were obtained from women undergoing elective cesarean section as described previously.25Lyall F Barber A Myatt L Bulmer JN Robson SC Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.FASEB J. 2000; 14: 208-219PubMed Google Scholar, 26Lyall F Robson SC Bulmer JN Kelly H Duffie E Human trophoblast invasion and spiral artery transformation: the role of nitric oxide.Am J Pathol. 1999; 154: 1105-1114Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Briefly, after delivery of the infant, the position of the placenta was determined by manual palpation. Six placental bed biopsies were then taken under direct vision using biopsy forceps (Wolf, UK). In three cases placental bed biopsies were collected after vaginal delivery. These biopsies were taken under ultrasound guidance using the same biopsy forceps introduced through the cervix. Placental bed biopsies were included in this study if they contained decidual and/or myometrial spiral arteries with interstitial trophoblasts. Placental samples of ∼1 cm3 were also collected. All samples were collected directly into liquid nitrogen-cooled isopentane and stored sealed at −70°C until required. Samples were used for subsequent immunohistochemical analysis and Western blotting experiments. Cryosections (7 μm) from each specimen were stained with hematoxylin and eosin (H&E) for histological analysis. Desmin (NCL-DES-DERII, 1:100) and cytokeratin (NCL-LP34, 1:800) monoclonal antibodies were obtained from Novocastra, Newcastle-on-Tyne, UK. The Factor VIII monoclonal antibody was obtained from DAKO, Cambridge, UK, and used at 1:800. Rabbit polyclonal antibodies raised against TGF-β1 (SC146), TGF-β2 (SC90), and TGF-β3 (SC820) were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA. Full-length human recombinant TGF-β1 (12.5 kd), TGF-β2 (12.5 kd), and TGF-β3 (12.5 kd) were obtained from Santa Cruz and used as positive controls in Western blots. All other reagents were obtained from Sigma Chemical Co., Poole, UK, unless stated otherwise. After immunostaining with the above antibodies we assessed the integrity of the muscle wall of the spiral artery by the degree of medial smooth muscle remaining around the spiral artery (desmin immunostaining). Morphological assessment was based on the method described by Pijnenborg and colleagues.1Pijnenborg R Bland JM Robertson WB Brosens I Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy.Placenta. 1983; 4: 397-414Abstract Full Text PDF PubMed Scopus (605) Google Scholar, 3Pijnenborg R Dixon G Robertson WB Brosens I Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy.Placenta. 1980; 1: 3-19Abstract Full Text PDF PubMed Scopus (698) Google Scholar The muscle was graded as preserved, separated, disorganized, or grossly disorganized. Absent or incomplete medial changes was deemed when the smooth muscle was preserved or separated and presence of medial changes was deemed when the muscle was disorganized or grossly disorganized/absent. A representative sample of 10 placentas were studied from each group. Before homogenization, a cryosection from each block was cut and stained with H&E to confirm that the specimens were placenta rather than decidua. Each frozen piece of tissue was weighed without allowing the tissue to thaw. Tissue samples were ground to a fine powder in liquid nitrogen with a mortar and pestle and added to 4 volumes of cold lysis buffer [25 mmol/L Tris, 0.25 mol/L sucrose, 1 mmol/L ethylenediaminetetraacetic acid, pH 7.6, and 50 μl/g tissue protease inhibitor cocktail (Sigma)]. Using a Polytron homogenizer at setting 10, the sample containers were surrounded by ice and homogenized for 3 × 10 second intervals. The homogenate was spun at 5000 ×g for 10 minutes at 4°C to remove debris. The supernatant was aliquoted and stored at −70°C until required. Protein concentrations were determined by the method of Bradford27Bradford MM A refined and sensitive method for the quantitation of proteins utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (225102) Google Scholar using bovine serum albumin (BSA) as a standard, and then diluted to the required concentration. Samples were mixed 1:1 with loading buffer (1.2 ml of 1 mol/L Tris, pH 6.8, 2 ml of glycerol, 4 ml of 10% sodium-dodecyl-sulfate, 2 ml of 1 mol/L dithiothreitol, 0.8 ml of distilled water with bromophenol blue added to give a deep blue color) and boiled for 5 minutes before loading. Samples were separated on 15% sodium dodecyl sulfate-polyacrylamide-resolving gels with a 4% stacking gel using Protean II apparatus (BioRad, Hemelhempstead, UK) at a constant current of 30 mA. Each well was loaded with 75 μg of protein. Low molecular weight range markers (20 to 106 kd range; BioRad Laboratories, Richmond, CA) were loaded beside the samples. Protein was transferred overnight in buffer containing 25 mmol/L Tris, 190 mmol/L glycine, 20% methanol at a constant 30 V to BioBlot NC nitrocellulose membranes (Costar; Corning Inc., NY). Filters were blocked for 1 hour at room temperature in phosphate-buffered saline (PBS) containing 5% Marvel and 0.25% Tween-20. The antibodies (diluted 1:1000 in PBS containing 3% Marvel and 0.25% Tween-20) were added for 1 hour at room temperature. The filters were rinsed once, washed twice for 5 minutes in PBS containing 0.25% Tween-20 and then incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Diagnostics Scotland, Carluke, UK) diluted 1:2000 in PBS containing 0.25% Tween-20 for 1 hour at room temperature. Blots were washed once for 5 minutes, followed by two 15-minute washes in PBS containing 0.25% Tween-20, and then one 5-minute wash in distilled water. Proteins were detected using the Amersham ECL detection system and filters were exposed to Hyperfilm ECL (Amersham, Buckinghamshire, UK). Immunohistochemistry was performed using an avidin-biotin peroxidase method (Vectastain Elite rabbit kit; Vector Laboratories, Peterborough, UK). Placenta and placental bed cryosections (7 μm) were mounted on APES-coated slides, air-dried overnight, fixed in acetone for 10 minutes at room temperature, and then wrapped in pairs and frozen at −20°C until required. Each specimen was stained with H&E for histological analysis. In addition, placental bed biopsies were immunostained for cytokeratin (1:800) to detect trophoblasts, desmin (1:100) to detect muscle, and Factor VIII (1:800) to detect endothelium. To determine TGF-β localization, sections were blocked with 1% BSA for 30 minutes followed by the kit blocker for 20 minutes. Sections then underwent a further 45-minute incubation in 0.1% phenylhydrazine to block endogenous peroxidase staining. These and all subsequent steps were performed at room temperature. Sections were then incubated for 1 hour with antibodies at a dilution of 1:200 for TGF-β1 and 1:250 for TGF-β2 and TGF-β3. The remaining steps were performed according to the instructions supplied with the kit. The reaction was developed with Fast diaminobenzidine tablets. Washes between each step were performed in TBS buffer (0.15 mol/L Tris-buffered saline, pH 7.6). Sections were counterstained in Mayer's hematoxylin (BDH, Poole, UK) and mounted in DPX synthetic resin. Omission of primary antibody or substitution with nonimmune serum for the primary antibody were both included as controls. Intensity of immunostaining was scored on an arbitrary scale of 0 to +++ where 0 represents no staining, + represents weak staining, ++ represents moderate staining, and +++ represents dark staining. The scoring of the samples was performed by two separate observers blinded to the tissue identity (FL and HS). Sections were all stained on the same day for each antibody to eliminate day to day variations in immunostaining. Because the antibodies for this study had previously been used on skin tissue,28Frank S Madlener M Werner S Transforming growth factors TGF β1, β2 and β3 and their receptors are differentially regulated during normal and impaired wound healing.J Biol Chem. 1996; 271: 10188-10193Crossref PubMed Scopus (324) Google Scholar normal human skin was used as a positive control tissue and processed as for placental samples. Placental TGF-β2 was measured using the Promega Emax Immunoassay System. Assays were performed on aliquots of the homogenates prepared for Western blot analysis. The assay detects biologically active TGF-β2 in an antibody sandwich format. Flat-bottomed 96-well plates were coated with TGF-β2 monoclonal antibody, which binds soluble TGF-β2 in the test sample. A second antibody to TGF-β2 was added to complete the sandwich. After washing, an antibody-conjugate (horseradish peroxidase-TGF-β2) was added which binds to the sandwich complex. Finally the chromogenic substrate 3,3′,5,5′-tetramethyl benzidine was added. Plates were read in a Labsystems Multiscan Bichromatic plate reader connected to a PC with Genesis software. The samples were quantified against a standard curve generated with known amounts of TGF-β2. The range of the assay is 32 to 1000 pg/ml. The specificity of the assay is <5% cross-reactivity with TGF-β1 and TGF-β3 at 10 ng/ml. Because some of the samples were higher than the highest standard a separate standard curve was made and the samples were reassayed so that they were measured within the linear range of the standards. For assays the samples were diluted 1:100 in the sample buffer supplied with the kit. After assay the final concentration of TGF-β2 in the sample was calculated and expressed as pg TGF-β2 per mg protein. In vivo TGF-β2 is processed from a latent form to a bioactive form. Only the bioactive form is immunoreactive with this kit. In vitro, the total amount of TGF-β2 (bioactive and nonbioactive) can be determined by acid treatment of samples. However because it is the bioactive form that is most likely to influence trophoblast invasion the samples were not acid treated. The assay for TGF-β3 was performed on the same samples as for TGF-β2 using an in-house assay developed from reagents obtained from R&D Systems, Oxon, UK. The assay was modified from a protocol supplied by R&D Systems. All incubations were performed at room temperature. Plates were coated with 100 μl of 4 μg/ml capture antibody (anti-TGF-β3, mAb 643) in PBS overnight. After three washes (0.05% Tween-20 in PBS, pH 7.4), plates were blocked for 1 hour with PBS containing 1% BSA and 5% sucrose. Plates were washed again and then 100 μl of standards (human recombinant TGF-β3 (243-83) or samples were added. Standards ranged from 2000 pg/ml to 4 pg/ml. Samples were diluted 1:2 or 1:5 in TBS, pH 7.3, containing 0.05% Tween-20 and 0.1% BSA. After a 2-hour incubation and three washes, 100 μl of biotinylated anti-TGF-β3 (BAF-243) diluted 1:250 in sample diluent buffer was added for 2 hours. After three further washes, 100 μl of 1:200 streptavidin-HRP (DY998) diluted in PBS containing 0.1% BSA was added for 20 minutes. Three more washes were performed and then 100 μl of TMB substrate (DY999) was added for ∼20 minutes or until a blue color developed. The reaction was stopped with 50 μl of 1 mol/L H2SO4 and the plates were read at 450 nm with a correction wavelength of 540 nm. As for TGF-β2 the final concentration of TGF-β3 in the sample was calculated and expressed as pg TGF-β3 per mg protein. Clinical details were compared using analysis of variance and post hoc testing was performed using the Fisher's protected least significant difference (PLSD) test. For ELISA and immunohistochemical studies, statistical comparisons were also performed using analysis of variance. Statistical differences were considered to be significant at P < 0.05. Western blot analysis was performed on a sample of 10 placental homogenates from each group (Table 1). These were randomly selected from the patient group shown in Table 1 all of which were used for subsequent immunohistochemical studies. Gestational age at delivery was comparable in the three groups. All infants in the control group had a birth weight greater than the 10th centile for gestational age. Mean birth weight was reduced in the PE group although only three infants were small for gestational age. Umbilical artery PI was abnormally elevated in four PE cases and one had reversed end-diastolic frequency. Birth weight was significantly reduced in the FGR and PE groups when compared with the control groups and the FGR group was significantly reduced when compared to the PE group; all infants in the FGR group had a birth weight below the 10th centile with six below the fifth centile. Umbilical artery PI was abnormally elevated in all of the FGR fetuses; four had absent and three had reversed end-diastolic frequencies. Figure 1 shows the results of Western blot analysis of placental samples from each group. The number of samples necessitated running the samples on two gels. Running of gels, blotting, and hybridization were performed during the same two days to eliminate day to day variability for each antibody. The positive controls for TGF-β1, TGF-β2, and TGF-β3 were clearly visible however TGF-β1, TGF-β2, and TGF-β3 were not detected on any of the samples. These data suggest that either there is no TGF-β in the placenta during late pregnancy or the levels are below the sensitivity of Western blot analysis that was ∼0.5 ng for the positive control TGF-β2 and >2 ng for TGF-β1 and TGF-β2. Normal human skin was the positive control for all three anti-TGF-β antibodies (Figure 2). TGF-β1 was present in basal epidermis and pilosebaceous units. TGF-β2 was detected throughout the epidermis, but strongest basally. TGF-β2 reactivity was also seen in pilosebaceous units and, diffusely and weakly, in the dermis. Staining for TGF-β3 was in the epidermis, predominantly in basal layers, and diffusely in the dermis. TGF-β3 immunoreactivity was weaker than that for TGF-β1 and TGF-β2. These reactivity patterns are primarily in agreement with those reported by Frank and colleagues.28Frank S Madlener M Werner S Transforming growth factors TGF β1, β2 and β3 and their receptors are differentially regulated during normal and impaired wound healing.J Biol Chem. 1996; 271: 10188-10193Crossref PubMed Scopus (324) Google Scholar Omission controls were negative. Controls in which the primary antibody was replaced with 1:200 normal rabbit serum showed variable staining of Hofbauer cells in villous stroma in a minority of normal and pathological samples. This reactivity exactly mirrored that of all three anti-TGF-β antibodies, which labeled Hofbauer cells in the same samples. Immunostaining of Hofbauer cells was therefore considered to reflect Fc receptor binding in frozen sections. The clinical details for patients used for the placenta immunohistochemistry studies are also shown in Table 1. Villous syncytiotrophoblast was consistently negative for TGF-β1, TGF-β2, and TGF-β3 (a few samples were +) (Figure 3; a to c and g to j). Villous cytotrophoblast, which were scanty in normal placentas and more prominent in placentas from pregnancies complicated by PE or FGR, were also negative for all three TGF-β isoforms. As discussed above, Hofbauer cells showed nonspecific reactivity for all three TGF-β isoforms in a minori
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