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Placental Biglycan Expression is Decreased in Human Idiopathic Fetal Growth Restriction
Placenta 31 (2010) 712e717
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Placental Biglycan Expression is Decreased in Human Idiopathic Fetal Growth Restriction P. Murthi a, b, *,1, F.A. Faisal a, b,1, G. Rajaraman a, b, J. Stevenson a, b, V. Ignjatovic c, d, e, P.T. Monagle c, d, e, S.P. Brennecke a, b, J.M. Said a, b a
Department of Perinatal Medicine, Pregnancy Research Centre, The Royal Women’s Hospital, Parkville 3052, Australia Department of Obstetrics and Gynaecology, The University of Melbourne, Parkville 3052, Australia Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville 3052, Victoria, Australia d Department of Clinical Haematology, The Royal Children’s Hospital, Parkville 3052, Victoria, Australia e Department of Paediatrics, The Royal Children’s Hospital and The University of Melbourne, Parkville 3052, Victoria, Australia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 24 May 2010
Fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. The majority of FGR cases are idiopathic and are associated with placental insufficiency, which can result from placental thrombosis. Evidence suggests that Dermatan Sulfate (DS) is an important anti-coagulant in placentae of uncomplicated pregnancies. This study hypothesised that the expression of biglycan proteoglycan, a source of DS, is decreased in idiopathic FGR placentae compared with placentae from uncomplicated pregnancies. This study aimed to determine biglycan mRNA, protein expression and spatial distribution in idiopathic FGR placentae compared with the placentae from gestation-matched controls. Biglycan mRNA expression, protein expression and spatial distribution was determined in 26 idiopathic FGRaffected placentae and 27 placentae from gestation-matched controls (27e40 weeks gestation) using real-time PCR, immunoblotting and immunohistochemistry, respectively. Mean biglycan mRNA expression was significantly decreased in FGR placentae compared with control placentae (2.87 0.55, (n ¼ 26) vs. 4.48 0.85, (n ¼ 27); t-test p ¼ 0.01). FGR placentae demonstrated a trend towards decrease in mean biglycan protein expression compared with control placentae (0.86 0.22 (n ¼ 9, FGR) vs, 1.9 0.56 (n ¼ 7, control) p ¼ 0.07). Biglycan immunoreactivity was detected in endothelial cells and sub-endothelial cells of the perivascular region of fetal capillaries. Semi-quantitative analyses demonstrated a significant decrease in immunoreactive biglycan in FGR placentae compared with control placentae (51.1 19.3 vs, 500.7 223, n ¼ 6, p < 0.001). This is the first study to demonstrate decreased biglycan expression in idiopathic FGR placentae compared to gestation-matched controls. Reduced biglycan expression may contribute to placental thrombosis within the fetal vasculature, and may contribute to the pathogenesis of idiopathic FGR. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Placenta Fetal growth restriction Gene expression Proteoglycans Dermatan sulphate
1. Introduction Fetal growth restriction (FGR, also known as intrauterine growth restriction, IUGR) is a significant pregnancy disorder. A common definition of FGR is a birth-weight at or below the 10th percentile for gestational age and gender, failure of the fetus to grow to its genetically determined potential size and the likely presence of an underlying pathologic process that inhibits the
* Corresponding author at: Department of Obstetrics and Gynaecology, The University of Melbourne, Parkville 3052, Australia. Tel.: þ61 3 8345 3747; fax: þ61 3 8345 3746. E-mail address: [email protected] (P. Murthi). 1 Equal contributions. 0143-4004/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.05.009
expression of the normal intrinsic growth potential. Not only are there various serious perinatal complications frequently associated with FGR, including stillbirth and prematurity [1], but also increasing numbers of epidemiological and animal studies provide evidence that the long-term consequences of FGR reach into adulthood [2]. Such consequences include an increased risk of chronic somatic disorders such as cardiovascular disease and diabetes [2] as well as asthma [3] and intellectual impairments such as schizophrenia [4], depression [5] and decreased intelligence quotient [6]. Therefore, it is becoming increasingly important to understand the molecular mechanism of human FGR. Only a third of FGR cases can be accounted for by obvious maternal, fetal and placental causes [7], the remainder being idiopathic. FGR pregnancies are distinguished from healthy small for gestational age
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(SGA) pregnancies by abnormal umbilical artery diastolic velocities, asymmetric growth of the fetus and reduced liquor volume [8]. Idiopathic FGR is frequently associated with placental insufficiency [9]. Placental insufficiency may result from various factors including: constriction of the placental blood vessels due to reduction in vasodilator activity [8], incomplete cytotrophoblastic invasion of the maternal spiral arteries [9] or maldevelopment of the placental villous structures [10]. These factors result in increased resistance to blood flow within the placenta in both the maternal and fetal circulations, ultimately resulting in fetal hypoxia and acidosis. In addition, placental histopathology from FGRaffected pregnancies has demonstrated thrombotic lesions within fetal and chorionic vessels [11], whether the presence of these lesions is the cause of FGR remains uncertain [11,12]. However, mouse models of FGR caused by disruption of key components of coagulation such as syndecan demonstrate that FGR-like effects accompanied by marked thrombosis suggesting a possible causative role [13]. These observations suggest that enhanced thrombosis in the placenta may contribute to FGR, although the underlying mechanism remains uncertain. Normal pregnancy is characterized by a dominant pro-coagulant effect and diminishing anti-coagulant and fibrinolytic activities [14e16]. These changes ultimately result in elevated thrombin and fibrin formation. In addition, Derlome et al. demonstrated that plasma is capable of a more rapid and increased thrombin generation during pregnancy [17]. Despite an increase in thrombin generation, thrombotic events (both systemic and within the placental circulation) are rare in normal pregnancies [18], suggesting that thrombin generation is well regulated in uncomplicated pregnancies. Thrombin generation is regulated by glycosaminoglycans including dermatan sulfate (DS). DS binds and activates heparin cofactor II (HCII), which subsequently inhibits the formation of thrombin [19]. DS was shown to be abundant in the placenta [20,21]. In the placenta, DS containing proteoglycans include the small leucine-rich proteoglycans (SLRP), decorin and biglycan [20]. Decorin usually has one chondroitin sulphate (CS) or DS glycosaminoglycan attached to its core protein, while biglycan normally has two DS and/or CS glycosaminoglycans. In contrast to uncomplicated pregnancies, placentae from diabetic and preeclamptic pregnancies display extensive intervillous fibrin and thrombus deposits. These lesions were associated with a reduction in placental DS [22]. We have recently shown that decorin expression is decreased in human idiopathic FGR [23], however the level of biglycan in FGR placentae has not been evaluated to date. The present work investigated whether changes in mRNA expression and protein expression of biglycan in the placenta is associated with idiopathic FGR. 2. Materials and methods 2.1. Patient details and tissue sampling Placentae from pregnancies complicated by idiopathic FGR (n ¼ 26) and gestation-matched control pregnancies (n ¼ 27) were collected with informed patient consent and approval from the Human Research and Ethics Committees of The Royal Women’s Hospital, Melbourne. Ultrasound data were used to prospectively identify pregnancies complicated by FGR. The inclusion criteria for FGR cases are listed in Table 1 and encompass a birth weight less than the 10th centile for gestation age using Australian growth charts [24] and any two of the following criteria diagnosed on antenatal ultrasound; abnormal umbilical artery Doppler flow velocimetry, oligohydramnios as determined by amniotic fluid index (AFI) < 7 or asymmetric growth of the fetus as quantified from the HC (head circumference) to AC (abdominal circumference) ratio greater than the 95th centile for gestation. Gestation for both FGR and control patients was determined based on last menstrual period (LMP) dates and confirmed by first or second trimester ultrasound. Control patients were selected to match FGR cases according to gestation. For both control and FGR-affected pregnancies used in this study, the exclusion criteria were multiple pregnancies, chemical
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Table 1 Clinical criteria for FGR-affected pregnancies used in this study. Characteristics
Number and % of FGR (n ¼ 26)
Birth-weight percentile <3rd percentile 3rd to < 5th percentile 5th to < 10th percentile 10th percentile
11 (42%) 3 (12%) 12 (46%) 0 (0%)
Doppler velocimetry of umbilical artery Elevated Absent Reversed Normal
8 (31%) 10 (38%) 8 (31%) 0 (0%)
Amniotic fluid index Oligohydramnios (AFI<7) Normal (AFI7) Not recorded
8 (31%) 17 (65%) 1 (4%)
Head circumference : abdominal circumference Asymmetry (HC:AC > 95th centile for gestation) Normal (HC:AC 95th centile for gestation) Not recorded
23 (88%) 0 (0%) 3 (12%)
dependency, maternal smoking, pre-eclampsia, prolonged rupture of the membranes, placental abruption, intrauterine viral infection, and fetal congenital anomalies. Only normotensive patients with idiopathic FGR were included. Control patients were included if they required elective delivery by induction of labour/ cesarean section or presented with spontaneous labour. Preterm control patients presented with spontaneous idiopathic preterm labour or underwent elective delivery for conditions not associated with placental dysfunction (for example, one patient in this study delivered preterm electively by cesarean section because of maternal breast cancer). In other words, no control patient recruited to this study had clinical evidence of pre-eclampsia, fetal growth restriction, placental abruption, ascending infection of the genital tract or prolonged rupture of the membranes. All control patients gave birth to normally formed babies with birth weights appropriate for gestational age and the placentae from these patients were grossly normal. All samples were processed within 10 min of placental delivery. Placental tissue samples were excised from randomly selected areas of central placental cotyledons with any attached decidua carefully removed by dissection. Tissues were divided into small pieces and thoroughly washed in phosphate buffered 0.9% saline (PBS) to minimise blood contamination, and then snap frozen and stored at 80 C for RNA analysis. 2.2. Real-time PCR Quantitation of biglycan mRNA expression in placental samples was performed in an ABI Prism 7700 (Perkin-Elmer-Applied Biosystems) using pre-validated Assays on Demand that consisted of a mix of unlabelled biglycan PCR primers and a TaqMan FAM labeled MGB probe (Biglycan Assays on Demand Cat No. Hs00156076_m1, Applied Biosystems). Gene expression quantitation was performed as the second step in a two-step reverse transcription-polymerase chain reaction (RT-PCR) protocol as described previously [24]. Levels of biglycan expression relative to GAPDH was calculated according to the 2-DDCT method [25,26] with a term-control sample used as a calibrator. 2.3. Western immunoblotting Total protein was extracted from FGR-affected placentae (n ¼ 9) and gestationmatched controls (n ¼ 7) and immunoblotting for biglycan was performed as previously described with modifications [24]. Briefly, 25 mg of total protein per lane was loaded and fractionated using 4e20% gradient gel (Bio-Rad). Proteins were transferred to polyvinylidene difluoride (PVDF) membrane, and blocked with 5% nonfat milk in Tris-buffered saline, pH 7.4 (TBS). An affinity purified mouse monoclonal antibody raised against biglycan of human origin (Abcam) of 5 ng/ml or mouse monoclonal antibody raised against human GAPDH (Abcam) of 0.5 ng/ml was used as the primary antibody. Antibody binding was visualised using peroxidase-conjugated rabbit anti-mouse IgG-HRP secondary antibody (Dako). Biglycan immunoreactivity was detected using ECL-Lumilyte autoradiography kit according to the manufacturer’s instructions (PerkinElmer). Immunoreactive biglycan protein relative to GAPDH was determined semi-quantitatively using scanning densitometry (Image Quant). The specificity of the primary antibody was verified using pre-absorbed primary antibody in the presence of recombinant biglycan. 2.4. Immunohistochemistry Randomly selected FGR-affected placentae (n ¼ 6 of 26) and gestation-matched term control placentae (n ¼ 6 of 27) fixed in 4% paraformaldehyde in phosphate
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buffered saline, pH 7.4 (PBS), paraffin embedded and sections cut to 5 mm. Immunohistochemistry was performed using the Histostain-Plus Broad spectrum kit (Zymed, Australia). Briefly, the sections were deparaffinised in Xylene for 10 min after which the paraffin sections were rehydrated in graded ethanol. The slides were rinsed in PBS and blocked with the blocking agent provided in the kit. Mouse monoclonal antibody to biglycan (Abcam) of 5 ng/ml was applied to all sections. Adjacent sections of representative term FGR and term control placentae were incubated with endothelial specific marker CD34 (QBEND-10, Neomarkers, 2.5 ng// ml), incubated overnight at 4 C. The incubations of the biotinylated secondary antibody and streptavidin-conjugated enzyme from the Histostain-Plus Broad Spectrum kit were carried out according to the manufacturer’s instructions. Chromogen detection was performed using AEC (3-Amino-9-Ethylcarbazole, Zymed) and sections were mounted with 80% glycerol. Negative control slides included omission of primary antibody and substitution of antibody diluents. Antibody specificity control was also used as negative control where the primary antibody was preabsorbed in the presence of recombinant human biglycan. Semi-quantitation was performed at 200 magnification as described previously [23]. Four non-overlapping fields from each placental section were examined. The Axiovision (version 4.3) is a computer based software program that semiquantitatively measures staining intensity by pixel volume density. A numeric value for the pixel volume density of each field examined was obtained as the ‘field densitometric sum’ (FDS). The average FDS of the four non-overlapping fields were taken for each placental section. 2.5. Data analysis All parameters of the FGR-affected and control pregnancies were described as mean SEM. Either the Chi-squared test or Student’s t-test was used where appropriate to analyse the significance of any differences between clinical characteristics of the FGR-affected and the control pregnancies. The relationship between gestation and biglycan mRNA expression in both FGR-affected placentae and in gestation-matched control placentae was examined using linear regression analysis with the statistical package STATA 7Ô. The difference in biglycan mRNA and protein expression between FGR-affected and control groups was assessed by the Student’s t-test and a probability value of <0.05 was considered significant.
3. Results Table 2 describes the clinical features of the FGR-affected and the control pregnancies included in this study. Gestational age, maternal age, parity and mode of delivery were not significantly different between the two groups. However, as expected, mean placental weight and mean birth-weight were significantly lower in FGR-affected patients compared to the controls (p < 0.025). Linear regression demonstrated no significant change in relationship between gestation and biglycan mRNA expression for either FGR-affected placentae (co-efficient 0.02e0.25e0.22, p ¼ 0.88) or gestation-matched controls (co-efficient 0.13e0.12e0.37, p ¼ 0.30). As shown in Fig. 1 the mean biglycan mRNA expression was significantly decreased in FGR-affected placentae compared with gestation-matched control placentae (2.87 0.55, (n ¼ 26) vs. 4.48 0.85, (n ¼ 27); t-test p ¼ 0.01). Biglycan protein expression was determined in control (n ¼ 7) and FGR-affected placentae (n ¼ 9). Fig. 2A shows a representative immunoblot for biglycan protein in FGR-affected placentae (n ¼ 3) and in gestation-matched controls (n ¼ 3). Variable levels of
Fig. 1. Semi-quantitative biglycan mRNA expression relative to GAPDH was determined using real-time PCR as described in the Methods section. Biglycan expression relative to GAPDH was calculated according to the 2DDCT method [25] with a termcontrol sample used as a calibrator. Gene expression values expressed as Mean SEM.
immunoreactive biglycan protein (42 kDa) was observed in all samples verified. GAPDH (36 kDa) was used a loading control. As shown in Fig. 2B, semi-quantitative analyses using scanning densitometry of immunoreactive biglycan protein relative to GAPDH protein indicated a trend towards a decrease in the level of immunoreactive biglycan protein in FGR-affected placentae compared to the controls (p ¼ 0.07).
Table 2 Clinical characteristics of samples from FGR-affected and gestation matched control pregnancies (p < 0.05 is denoted by *). Characteristic
Control (n ¼ 27)
FGR (n ¼ 26)
Significance
Gestation age (weeks) Maternal age (years)
34.4 4.1 32.4 5.6
35.7 3.5 31.0 5.7
p ¼ 0.24 p ¼ 0.36
Mode of delivery Vaginal delivery Cesarean in labour Cesarean not in labour
7 (26%) 3 (11%) 17 (63%)
11 (42%) 3 (12%) 12 (46%)
p ¼ 0.42
Sex of newborn Male Female Newborn birth-weight (grams) Placental weight (grams)
10 (37%) 17 (63%) 2488 927 494 146
12 (46%) 14 (54%) 2028 665 413 119
p ¼ 0.50 p ¼ 0.04* p ¼ 0.03*
Fig. 2. A. Representative immunoblot for biglycan protein (42 kDa) and GAPDH (36 kDa) in idiopathic FGR-affected placentae and gestation-matched control placentae. Bands indicate immunoreactivity to mouse monoclonal antibody raised against human biglycan and GAPDH. B Semi-quantitative analysis of immunoreactive biglycan protein normalised to GAPDH in FGR-affected placentae compared with gestation-matched controls. Results are expressed as mean SEM.
P. Murthi et al. / Placenta 31 (2010) 712e717
Immunohistochemical localisation for biglycan protein provided evidence that biglycan was present in both FGR-affected term placentae and in the term controls. Adjacent sections of representative term FGR (39 weeks) and term matching control (39 weeks) were stained for endothelial specific marker CD34 (QBEND-10, Neomarkers). As shown biglycan protein expression was evident in
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the endothelial and sub-endothelial cells of the fetal capillaries in both FGR-affected placentae and in control (Fig. 3A). As shown in Fig. 3B, semi-quantitative analysis of immunoreactivity showed a significant decrease in levels of immunoreactive biglycan protein in FGR-affected placentae compared with gestation-matched controls (51.1 19.3 vs. 500.7 223.0, n ¼ 6, t-test, p < 0.001).
Fig. 3. A. Immunohistochemical localisation of biglycan and CD34 protein in FGR-affected placentae and in gestation-matched controls. Representative images of term Control (39 weeks), term FGR (39 weeks) stained for biglycan and CD34; and a negative control image displaying primary antibody pre-absorbed with recombinant biglycan protein are shown. Images are of 200 magnification and scale bar represents 50 mm. Staining in the endothelium and in the perivascular region is shown by arrows. E refers to endothelium; PV refers to perivascular region and ST refers to syncytiotrophoblast cells. B. Semi-quantitative analysis of biglycan immunoreactive protein in FGR-affected placentae and in gestationmatched controls. Results are expressed as mean SEM and * denotes a significant difference (p < 0.01).
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4. Discussion
Acknowledgements
Biglycan is a major glycoprotein of the extracellular matrix. This study has demonstrated for the first time that a significant decrease in both biglycan mRNA and protein expression levels is associated with idiopathic FGR. While altered placental proteoglycan expression has been previously linked to pre-eclampsia [27e29], this is the first report of its association with FGR. The mechanism by which altered biglycan expression contributes to FGR is uncertain, but may include a functional role in matrix remodelling or vascular thrombosis. Biglycan has been shown to interact with type I and type VI collagens in vitro, suggesting a role in the control of collagen fibrillogenesis [30,31]. Indeed, this is further confirmed by targeted disruption of the biglycan gene in mice with a phenotype characterized by abnormal collagen fibrils in tendons, together with abnormal bone structure and reduced bone mass [32]. Similar to decorin, biglycan has been shown to bind TGF-b in vitro and participate in collagen aggregation, cell adhesion and matrix remodelling [33]. The decreased levels of biglycan expression observed in FGR-affected placentae may be associated with the structural changes in the placenta and may play an important role in biological functions, including the regulation of the assembly of fibrillar collagens and modulation of cell adhesion. In addition to matrix remodelling, recent reports by Little et al. [34] suggest a significant role for biglycan in atherosclerotic lesions in both animals and humans. Gogiel et al. [35] demonstrated increased concentration of biglycan in the umbilical cord vein wall in pre-eclampsia and suggested that this alteration may affect the mechanical properties of this vessel and may contribute to disturbances in fetal blood circulation. This contrasts with our findings in the placenta which demonstrated reduced biglycan expression in FGR. Finally, the potential role for biglycan in contributing to thrombosis must be considered. Recent advances in thrombosis research have provided further insights into the functional role of many glycosaminoglycans in thrombin generation. He et al. [36] have reported that heparin cofactor II (HCII) interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombin formation in vivo. Decreased biglycan expression (both at mRNA and protein levels) in FGRaffected placentae may contribute to placental thrombosis. The endothelial presence of biglycan ensures that DS attached to its core protein is always in close proximity to fetal blood and can easily come into contact with HCII to catalyse the inhibition of thrombin and thus, prevent clot formation. Therefore, biglycan could play a crucial anti-coagulant role within the fetal circulation. Decreased biglycan expression in the endothelial cells of FGR-affected placentae may reduce DS available to catalyse thrombin inhibition in the fetal circulation. This can contribute to thrombosis within the fetal vasculature and subsequently impair fetal growth, as demonstrated in a proportion of FGR cases [37,38]. To further support this, Tovar et al. [39] described a possible balance between two glycosaminoglycan-dependent anti-coagulant pathways present in the vascular wall. One is based on antithrombin activation by heparan sulphate expressed by the endothelial cells; the other, HCII activation by the DS proteoglycans synthesized by cells from the sub-endothelial layer. This study mainly focused on the level of proteoglycans in idiopathic FGR, many archival control and FGR samples were used to match for gestation, this posed a major limitation to analyse functional activity for thrombosis. In summary, this study demonstrates an association between reduced biglycan expression and idiopathic FGR, but causal relationship between biglycan expression and FGR remains to be determined.
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Placental Biglycan Expression is Decreased in Human Idiopathic Fetal Growth Restriction P. Murthi a, b, *,1, F.A. Faisal a, b,1, G. Rajaraman a, b, J. Stevenson a, b, V. Ignjatovic c, d, e, P.T. Monagle c, d, e, S.P. Brennecke a, b, J.M. Said a, b a
Department of Perinatal Medicine, Pregnancy Research Centre, The Royal Women’s Hospital, Parkville 3052, Australia Department of Obstetrics and Gynaecology, The University of Melbourne, Parkville 3052, Australia Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville 3052, Victoria, Australia d Department of Clinical Haematology, The Royal Children’s Hospital, Parkville 3052, Victoria, Australia e Department of Paediatrics, The Royal Children’s Hospital and The University of Melbourne, Parkville 3052, Victoria, Australia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 24 May 2010
Fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. The majority of FGR cases are idiopathic and are associated with placental insufficiency, which can result from placental thrombosis. Evidence suggests that Dermatan Sulfate (DS) is an important anti-coagulant in placentae of uncomplicated pregnancies. This study hypothesised that the expression of biglycan proteoglycan, a source of DS, is decreased in idiopathic FGR placentae compared with placentae from uncomplicated pregnancies. This study aimed to determine biglycan mRNA, protein expression and spatial distribution in idiopathic FGR placentae compared with the placentae from gestation-matched controls. Biglycan mRNA expression, protein expression and spatial distribution was determined in 26 idiopathic FGRaffected placentae and 27 placentae from gestation-matched controls (27e40 weeks gestation) using real-time PCR, immunoblotting and immunohistochemistry, respectively. Mean biglycan mRNA expression was significantly decreased in FGR placentae compared with control placentae (2.87 0.55, (n ¼ 26) vs. 4.48 0.85, (n ¼ 27); t-test p ¼ 0.01). FGR placentae demonstrated a trend towards decrease in mean biglycan protein expression compared with control placentae (0.86 0.22 (n ¼ 9, FGR) vs, 1.9 0.56 (n ¼ 7, control) p ¼ 0.07). Biglycan immunoreactivity was detected in endothelial cells and sub-endothelial cells of the perivascular region of fetal capillaries. Semi-quantitative analyses demonstrated a significant decrease in immunoreactive biglycan in FGR placentae compared with control placentae (51.1 19.3 vs, 500.7 223, n ¼ 6, p < 0.001). This is the first study to demonstrate decreased biglycan expression in idiopathic FGR placentae compared to gestation-matched controls. Reduced biglycan expression may contribute to placental thrombosis within the fetal vasculature, and may contribute to the pathogenesis of idiopathic FGR. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Placenta Fetal growth restriction Gene expression Proteoglycans Dermatan sulphate
1. Introduction Fetal growth restriction (FGR, also known as intrauterine growth restriction, IUGR) is a significant pregnancy disorder. A common definition of FGR is a birth-weight at or below the 10th percentile for gestational age and gender, failure of the fetus to grow to its genetically determined potential size and the likely presence of an underlying pathologic process that inhibits the
* Corresponding author at: Department of Obstetrics and Gynaecology, The University of Melbourne, Parkville 3052, Australia. Tel.: þ61 3 8345 3747; fax: þ61 3 8345 3746. E-mail address: [email protected] (P. Murthi). 1 Equal contributions. 0143-4004/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.05.009
expression of the normal intrinsic growth potential. Not only are there various serious perinatal complications frequently associated with FGR, including stillbirth and prematurity [1], but also increasing numbers of epidemiological and animal studies provide evidence that the long-term consequences of FGR reach into adulthood [2]. Such consequences include an increased risk of chronic somatic disorders such as cardiovascular disease and diabetes [2] as well as asthma [3] and intellectual impairments such as schizophrenia [4], depression [5] and decreased intelligence quotient [6]. Therefore, it is becoming increasingly important to understand the molecular mechanism of human FGR. Only a third of FGR cases can be accounted for by obvious maternal, fetal and placental causes [7], the remainder being idiopathic. FGR pregnancies are distinguished from healthy small for gestational age
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(SGA) pregnancies by abnormal umbilical artery diastolic velocities, asymmetric growth of the fetus and reduced liquor volume [8]. Idiopathic FGR is frequently associated with placental insufficiency [9]. Placental insufficiency may result from various factors including: constriction of the placental blood vessels due to reduction in vasodilator activity [8], incomplete cytotrophoblastic invasion of the maternal spiral arteries [9] or maldevelopment of the placental villous structures [10]. These factors result in increased resistance to blood flow within the placenta in both the maternal and fetal circulations, ultimately resulting in fetal hypoxia and acidosis. In addition, placental histopathology from FGRaffected pregnancies has demonstrated thrombotic lesions within fetal and chorionic vessels [11], whether the presence of these lesions is the cause of FGR remains uncertain [11,12]. However, mouse models of FGR caused by disruption of key components of coagulation such as syndecan demonstrate that FGR-like effects accompanied by marked thrombosis suggesting a possible causative role [13]. These observations suggest that enhanced thrombosis in the placenta may contribute to FGR, although the underlying mechanism remains uncertain. Normal pregnancy is characterized by a dominant pro-coagulant effect and diminishing anti-coagulant and fibrinolytic activities [14e16]. These changes ultimately result in elevated thrombin and fibrin formation. In addition, Derlome et al. demonstrated that plasma is capable of a more rapid and increased thrombin generation during pregnancy [17]. Despite an increase in thrombin generation, thrombotic events (both systemic and within the placental circulation) are rare in normal pregnancies [18], suggesting that thrombin generation is well regulated in uncomplicated pregnancies. Thrombin generation is regulated by glycosaminoglycans including dermatan sulfate (DS). DS binds and activates heparin cofactor II (HCII), which subsequently inhibits the formation of thrombin [19]. DS was shown to be abundant in the placenta [20,21]. In the placenta, DS containing proteoglycans include the small leucine-rich proteoglycans (SLRP), decorin and biglycan [20]. Decorin usually has one chondroitin sulphate (CS) or DS glycosaminoglycan attached to its core protein, while biglycan normally has two DS and/or CS glycosaminoglycans. In contrast to uncomplicated pregnancies, placentae from diabetic and preeclamptic pregnancies display extensive intervillous fibrin and thrombus deposits. These lesions were associated with a reduction in placental DS [22]. We have recently shown that decorin expression is decreased in human idiopathic FGR [23], however the level of biglycan in FGR placentae has not been evaluated to date. The present work investigated whether changes in mRNA expression and protein expression of biglycan in the placenta is associated with idiopathic FGR. 2. Materials and methods 2.1. Patient details and tissue sampling Placentae from pregnancies complicated by idiopathic FGR (n ¼ 26) and gestation-matched control pregnancies (n ¼ 27) were collected with informed patient consent and approval from the Human Research and Ethics Committees of The Royal Women’s Hospital, Melbourne. Ultrasound data were used to prospectively identify pregnancies complicated by FGR. The inclusion criteria for FGR cases are listed in Table 1 and encompass a birth weight less than the 10th centile for gestation age using Australian growth charts [24] and any two of the following criteria diagnosed on antenatal ultrasound; abnormal umbilical artery Doppler flow velocimetry, oligohydramnios as determined by amniotic fluid index (AFI) < 7 or asymmetric growth of the fetus as quantified from the HC (head circumference) to AC (abdominal circumference) ratio greater than the 95th centile for gestation. Gestation for both FGR and control patients was determined based on last menstrual period (LMP) dates and confirmed by first or second trimester ultrasound. Control patients were selected to match FGR cases according to gestation. For both control and FGR-affected pregnancies used in this study, the exclusion criteria were multiple pregnancies, chemical
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Table 1 Clinical criteria for FGR-affected pregnancies used in this study. Characteristics
Number and % of FGR (n ¼ 26)
Birth-weight percentile <3rd percentile 3rd to < 5th percentile 5th to < 10th percentile 10th percentile
11 (42%) 3 (12%) 12 (46%) 0 (0%)
Doppler velocimetry of umbilical artery Elevated Absent Reversed Normal
8 (31%) 10 (38%) 8 (31%) 0 (0%)
Amniotic fluid index Oligohydramnios (AFI<7) Normal (AFI7) Not recorded
8 (31%) 17 (65%) 1 (4%)
Head circumference : abdominal circumference Asymmetry (HC:AC > 95th centile for gestation) Normal (HC:AC 95th centile for gestation) Not recorded
23 (88%) 0 (0%) 3 (12%)
dependency, maternal smoking, pre-eclampsia, prolonged rupture of the membranes, placental abruption, intrauterine viral infection, and fetal congenital anomalies. Only normotensive patients with idiopathic FGR were included. Control patients were included if they required elective delivery by induction of labour/ cesarean section or presented with spontaneous labour. Preterm control patients presented with spontaneous idiopathic preterm labour or underwent elective delivery for conditions not associated with placental dysfunction (for example, one patient in this study delivered preterm electively by cesarean section because of maternal breast cancer). In other words, no control patient recruited to this study had clinical evidence of pre-eclampsia, fetal growth restriction, placental abruption, ascending infection of the genital tract or prolonged rupture of the membranes. All control patients gave birth to normally formed babies with birth weights appropriate for gestational age and the placentae from these patients were grossly normal. All samples were processed within 10 min of placental delivery. Placental tissue samples were excised from randomly selected areas of central placental cotyledons with any attached decidua carefully removed by dissection. Tissues were divided into small pieces and thoroughly washed in phosphate buffered 0.9% saline (PBS) to minimise blood contamination, and then snap frozen and stored at 80 C for RNA analysis. 2.2. Real-time PCR Quantitation of biglycan mRNA expression in placental samples was performed in an ABI Prism 7700 (Perkin-Elmer-Applied Biosystems) using pre-validated Assays on Demand that consisted of a mix of unlabelled biglycan PCR primers and a TaqMan FAM labeled MGB probe (Biglycan Assays on Demand Cat No. Hs00156076_m1, Applied Biosystems). Gene expression quantitation was performed as the second step in a two-step reverse transcription-polymerase chain reaction (RT-PCR) protocol as described previously [24]. Levels of biglycan expression relative to GAPDH was calculated according to the 2-DDCT method [25,26] with a term-control sample used as a calibrator. 2.3. Western immunoblotting Total protein was extracted from FGR-affected placentae (n ¼ 9) and gestationmatched controls (n ¼ 7) and immunoblotting for biglycan was performed as previously described with modifications [24]. Briefly, 25 mg of total protein per lane was loaded and fractionated using 4e20% gradient gel (Bio-Rad). Proteins were transferred to polyvinylidene difluoride (PVDF) membrane, and blocked with 5% nonfat milk in Tris-buffered saline, pH 7.4 (TBS). An affinity purified mouse monoclonal antibody raised against biglycan of human origin (Abcam) of 5 ng/ml or mouse monoclonal antibody raised against human GAPDH (Abcam) of 0.5 ng/ml was used as the primary antibody. Antibody binding was visualised using peroxidase-conjugated rabbit anti-mouse IgG-HRP secondary antibody (Dako). Biglycan immunoreactivity was detected using ECL-Lumilyte autoradiography kit according to the manufacturer’s instructions (PerkinElmer). Immunoreactive biglycan protein relative to GAPDH was determined semi-quantitatively using scanning densitometry (Image Quant). The specificity of the primary antibody was verified using pre-absorbed primary antibody in the presence of recombinant biglycan. 2.4. Immunohistochemistry Randomly selected FGR-affected placentae (n ¼ 6 of 26) and gestation-matched term control placentae (n ¼ 6 of 27) fixed in 4% paraformaldehyde in phosphate
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buffered saline, pH 7.4 (PBS), paraffin embedded and sections cut to 5 mm. Immunohistochemistry was performed using the Histostain-Plus Broad spectrum kit (Zymed, Australia). Briefly, the sections were deparaffinised in Xylene for 10 min after which the paraffin sections were rehydrated in graded ethanol. The slides were rinsed in PBS and blocked with the blocking agent provided in the kit. Mouse monoclonal antibody to biglycan (Abcam) of 5 ng/ml was applied to all sections. Adjacent sections of representative term FGR and term control placentae were incubated with endothelial specific marker CD34 (QBEND-10, Neomarkers, 2.5 ng// ml), incubated overnight at 4 C. The incubations of the biotinylated secondary antibody and streptavidin-conjugated enzyme from the Histostain-Plus Broad Spectrum kit were carried out according to the manufacturer’s instructions. Chromogen detection was performed using AEC (3-Amino-9-Ethylcarbazole, Zymed) and sections were mounted with 80% glycerol. Negative control slides included omission of primary antibody and substitution of antibody diluents. Antibody specificity control was also used as negative control where the primary antibody was preabsorbed in the presence of recombinant human biglycan. Semi-quantitation was performed at 200 magnification as described previously [23]. Four non-overlapping fields from each placental section were examined. The Axiovision (version 4.3) is a computer based software program that semiquantitatively measures staining intensity by pixel volume density. A numeric value for the pixel volume density of each field examined was obtained as the ‘field densitometric sum’ (FDS). The average FDS of the four non-overlapping fields were taken for each placental section. 2.5. Data analysis All parameters of the FGR-affected and control pregnancies were described as mean SEM. Either the Chi-squared test or Student’s t-test was used where appropriate to analyse the significance of any differences between clinical characteristics of the FGR-affected and the control pregnancies. The relationship between gestation and biglycan mRNA expression in both FGR-affected placentae and in gestation-matched control placentae was examined using linear regression analysis with the statistical package STATA 7Ô. The difference in biglycan mRNA and protein expression between FGR-affected and control groups was assessed by the Student’s t-test and a probability value of <0.05 was considered significant.
3. Results Table 2 describes the clinical features of the FGR-affected and the control pregnancies included in this study. Gestational age, maternal age, parity and mode of delivery were not significantly different between the two groups. However, as expected, mean placental weight and mean birth-weight were significantly lower in FGR-affected patients compared to the controls (p < 0.025). Linear regression demonstrated no significant change in relationship between gestation and biglycan mRNA expression for either FGR-affected placentae (co-efficient 0.02e0.25e0.22, p ¼ 0.88) or gestation-matched controls (co-efficient 0.13e0.12e0.37, p ¼ 0.30). As shown in Fig. 1 the mean biglycan mRNA expression was significantly decreased in FGR-affected placentae compared with gestation-matched control placentae (2.87 0.55, (n ¼ 26) vs. 4.48 0.85, (n ¼ 27); t-test p ¼ 0.01). Biglycan protein expression was determined in control (n ¼ 7) and FGR-affected placentae (n ¼ 9). Fig. 2A shows a representative immunoblot for biglycan protein in FGR-affected placentae (n ¼ 3) and in gestation-matched controls (n ¼ 3). Variable levels of
Fig. 1. Semi-quantitative biglycan mRNA expression relative to GAPDH was determined using real-time PCR as described in the Methods section. Biglycan expression relative to GAPDH was calculated according to the 2DDCT method [25] with a termcontrol sample used as a calibrator. Gene expression values expressed as Mean SEM.
immunoreactive biglycan protein (42 kDa) was observed in all samples verified. GAPDH (36 kDa) was used a loading control. As shown in Fig. 2B, semi-quantitative analyses using scanning densitometry of immunoreactive biglycan protein relative to GAPDH protein indicated a trend towards a decrease in the level of immunoreactive biglycan protein in FGR-affected placentae compared to the controls (p ¼ 0.07).
Table 2 Clinical characteristics of samples from FGR-affected and gestation matched control pregnancies (p < 0.05 is denoted by *). Characteristic
Control (n ¼ 27)
FGR (n ¼ 26)
Significance
Gestation age (weeks) Maternal age (years)
34.4 4.1 32.4 5.6
35.7 3.5 31.0 5.7
p ¼ 0.24 p ¼ 0.36
Mode of delivery Vaginal delivery Cesarean in labour Cesarean not in labour
7 (26%) 3 (11%) 17 (63%)
11 (42%) 3 (12%) 12 (46%)
p ¼ 0.42
Sex of newborn Male Female Newborn birth-weight (grams) Placental weight (grams)
10 (37%) 17 (63%) 2488 927 494 146
12 (46%) 14 (54%) 2028 665 413 119
p ¼ 0.50 p ¼ 0.04* p ¼ 0.03*
Fig. 2. A. Representative immunoblot for biglycan protein (42 kDa) and GAPDH (36 kDa) in idiopathic FGR-affected placentae and gestation-matched control placentae. Bands indicate immunoreactivity to mouse monoclonal antibody raised against human biglycan and GAPDH. B Semi-quantitative analysis of immunoreactive biglycan protein normalised to GAPDH in FGR-affected placentae compared with gestation-matched controls. Results are expressed as mean SEM.
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Immunohistochemical localisation for biglycan protein provided evidence that biglycan was present in both FGR-affected term placentae and in the term controls. Adjacent sections of representative term FGR (39 weeks) and term matching control (39 weeks) were stained for endothelial specific marker CD34 (QBEND-10, Neomarkers). As shown biglycan protein expression was evident in
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the endothelial and sub-endothelial cells of the fetal capillaries in both FGR-affected placentae and in control (Fig. 3A). As shown in Fig. 3B, semi-quantitative analysis of immunoreactivity showed a significant decrease in levels of immunoreactive biglycan protein in FGR-affected placentae compared with gestation-matched controls (51.1 19.3 vs. 500.7 223.0, n ¼ 6, t-test, p < 0.001).
Fig. 3. A. Immunohistochemical localisation of biglycan and CD34 protein in FGR-affected placentae and in gestation-matched controls. Representative images of term Control (39 weeks), term FGR (39 weeks) stained for biglycan and CD34; and a negative control image displaying primary antibody pre-absorbed with recombinant biglycan protein are shown. Images are of 200 magnification and scale bar represents 50 mm. Staining in the endothelium and in the perivascular region is shown by arrows. E refers to endothelium; PV refers to perivascular region and ST refers to syncytiotrophoblast cells. B. Semi-quantitative analysis of biglycan immunoreactive protein in FGR-affected placentae and in gestationmatched controls. Results are expressed as mean SEM and * denotes a significant difference (p < 0.01).
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4. Discussion
Acknowledgements
Biglycan is a major glycoprotein of the extracellular matrix. This study has demonstrated for the first time that a significant decrease in both biglycan mRNA and protein expression levels is associated with idiopathic FGR. While altered placental proteoglycan expression has been previously linked to pre-eclampsia [27e29], this is the first report of its association with FGR. The mechanism by which altered biglycan expression contributes to FGR is uncertain, but may include a functional role in matrix remodelling or vascular thrombosis. Biglycan has been shown to interact with type I and type VI collagens in vitro, suggesting a role in the control of collagen fibrillogenesis [30,31]. Indeed, this is further confirmed by targeted disruption of the biglycan gene in mice with a phenotype characterized by abnormal collagen fibrils in tendons, together with abnormal bone structure and reduced bone mass [32]. Similar to decorin, biglycan has been shown to bind TGF-b in vitro and participate in collagen aggregation, cell adhesion and matrix remodelling [33]. The decreased levels of biglycan expression observed in FGR-affected placentae may be associated with the structural changes in the placenta and may play an important role in biological functions, including the regulation of the assembly of fibrillar collagens and modulation of cell adhesion. In addition to matrix remodelling, recent reports by Little et al. [34] suggest a significant role for biglycan in atherosclerotic lesions in both animals and humans. Gogiel et al. [35] demonstrated increased concentration of biglycan in the umbilical cord vein wall in pre-eclampsia and suggested that this alteration may affect the mechanical properties of this vessel and may contribute to disturbances in fetal blood circulation. This contrasts with our findings in the placenta which demonstrated reduced biglycan expression in FGR. Finally, the potential role for biglycan in contributing to thrombosis must be considered. Recent advances in thrombosis research have provided further insights into the functional role of many glycosaminoglycans in thrombin generation. He et al. [36] have reported that heparin cofactor II (HCII) interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombin formation in vivo. Decreased biglycan expression (both at mRNA and protein levels) in FGRaffected placentae may contribute to placental thrombosis. The endothelial presence of biglycan ensures that DS attached to its core protein is always in close proximity to fetal blood and can easily come into contact with HCII to catalyse the inhibition of thrombin and thus, prevent clot formation. Therefore, biglycan could play a crucial anti-coagulant role within the fetal circulation. Decreased biglycan expression in the endothelial cells of FGR-affected placentae may reduce DS available to catalyse thrombin inhibition in the fetal circulation. This can contribute to thrombosis within the fetal vasculature and subsequently impair fetal growth, as demonstrated in a proportion of FGR cases [37,38]. To further support this, Tovar et al. [39] described a possible balance between two glycosaminoglycan-dependent anti-coagulant pathways present in the vascular wall. One is based on antithrombin activation by heparan sulphate expressed by the endothelial cells; the other, HCII activation by the DS proteoglycans synthesized by cells from the sub-endothelial layer. This study mainly focused on the level of proteoglycans in idiopathic FGR, many archival control and FGR samples were used to match for gestation, this posed a major limitation to analyse functional activity for thrombosis. In summary, this study demonstrates an association between reduced biglycan expression and idiopathic FGR, but causal relationship between biglycan expression and FGR remains to be determined.
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