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Markers of haemostasis and angiogenesis in placentae from gestational vascular complications: Impairment of mechanisms involved in maintaining intervillous blood flow
Thrombosis Research 125 (2010) 267–271
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Markers of haemostasis and angiogenesis in placentae from gestational vascular complications: Impairment of mechanisms involved in maintaining intervillous blood flow☆ Elena Chinni a, Donatella Colaizzo a, Giovanni L. Tiscia a, Pasquale Martinelli c, Giuseppe M. Maruotti c, Maria Matteo d, Maurizio Margaglione a,b, Elvira Grandone a,⁎ a
Atherosclerosis and Thrombosis Unit, I.R.C.C.S. Casa Sollievo della Sofferenza, S.Giovanni Rotondo, FOGGIA, ITALY Medical Genetics, University of Foggia, FOGGIA, ITALY c Obstetrics and Gynaecology, University Federico II, NAPLES, ITALY d Obstetrics and Gynaecology, University of Foggia, FOGGIA, ITALY b
a r t i c l e
i n f o
Article history: Received 3 June 2009 Received in revised form 24 September 2009 Accepted 25 September 2009 Available online 3 November 2009 Keywords: Haemostasis factors Angiogenesis factors Gestational vascular complications
a b s t r a c t Introduction: Preeclampsia (PE) and fetal growth restriction (FGR) are multifactorial diseases, whose pathogenesis is largely unknown. A significant relationship between haemostasis and angiogenesis in placentae from uneventful pregnancies was previously shown. Materials and Methods: RNA expression of haemostasis (TF, TFPI, TFPI-2, PAI-2, Anx V, TM) and angiogenesis (Ang-1, Ang-2, PlGF, VEGF) markers in placentae from PE (n = 12), PE+FGR (n = 17) and FGR (n = 20) in respect of placentae from uncomplicated pregnancies (n = 21) were investigated. Results: Placentae from complicated pregnancies showed a significant lower expression (p ≤ 0.05 MannWhitney U test) of TF, TFPI, TFPI-2, Anx V, PAI-2 than those from in uncomplicated ones. VEGF and PlGF were not different in the considered groups; Ang-1 and Ang-2 were significantly higher (p ≤ 0.05 Mann-Whitney U test) in the PE group. Correlations between factors involved in haemostasis and those involved in angiogenesis, observed in placentae from uneventful pregnancies are lacking in those from complicated ones. Conclusions: Haemostasis factors are reduced in placentae from complicated pregnancies. The relationship between haemostasis and angiogenesis observed in uncomplicated pregnancies is impaired in PE and FGR. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Preeclampsia (PE) is a multisystem disease of unknown etiology associated with increased perinatal/maternal morbidity and mortality. Although fetal growth restriction (FGR) is often associated with PE, nevertheless it can also be observed in absence of hypertensive disease, and its severity is correlated with the degree of placental infarction and impaired placental nutrient transport. Physiologic changes in the coagulation and fibrinolytic systems are known to result in an increased susceptibility of pregnant women to thrombotic disorders and the formation of fibrin deposits, which can lead to a reduction in nutrient transport across the placenta [1]. Abbreviations: PE, preeclampsia; FGR, fetal growth restriction; TF, tissue factor; TFPI, Tissue Factor Pathway Inhibitor; PAI, Plasminogen Activator Inhibitor; AnxV, Annexin V; TM, Thrombomodulin; Ang-1 and 2, Angiopoietin 1 and 2; VEGF, Vascular Endothelial growth Factor; PlGF, Placental Growth Factor. ☆ From the Atherosclerosis and Thrombosis Unit, I.R.C.C.S. Casa Sollievo della Sofferenza, S.Giovanni Rotondo, FOGGIA, ITALY. ⁎ Corresponding author. Atherosclerosis and Thrombosis Unit, I.R.C.C.S. “Casa Sollievo della Sofferenza”, Poliambulatorio Giovanni Paolo II, S. Giovanni Rotondo (FG), ITALY. Tel./ fax: +39 0 882 416273. 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.09.024
The expression of tissue-factor (TF) renders the trophoblasts procoagulant, thus compromising the risk of bleeding while exposing the placenta to pro-thrombotic risks. Local inhibitory mechanisms, tissuefactor pathway inhibitors (TFPI and TFPI-2), thrombomodulin (TM), Annexin V (Anx V), and the fibrinolytic system, limit coagulation activation and fibrin deposition. Lanir et al. previously showed that gestational vascular complications (GVC) have been associated with abnormalities in the functions of haemostatic and fibrinolytic systems [2]. In a preliminary study, performed on a group of 20 placentae from uncomplicated pregnancies, we found a direct relationship between expression of TFPI or TFPI-2, two inhibitors of TF, and inhibitor of plasminogen activator type 2 (PAI-2) [3]. Furthermore, TFPI-2 expression was directly related also to TF. In addition, a direct relationship between TF and TFPI-2 and vascular endothelial growth factor (VEGF), one of the main regulators of angiogenesis, was found. Thus, in placentae from uncomplicated pregnancies, a relationship between haemostasis and angiogenesis was shown [3]. In PE and in FGR, endovascular invasion of cytotrophoblast remains superficial and uterine blood vessels do not undergo adequate vascular transformation compared with uncomplicated pregnancy [4,5]. Furthermore, in PE invasive trophoblast fails to undergo pseudo-vasculogenesis [6].
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The aberrant vascular transformation is thought to lead to placental insufficiency and consequently placental hypoxia [7]. VEGF, placental growth factor (PlGF), angiopoietins (Ang-1 and Ang-2) are involved not only in the regulation of vascular development and in remodeling during placentation, but also act as growth factors for driving growth and differentiation processes such as invasion [8]. It has been hypothesized that an impairment of trophoblast invasion and a failure of spiral artery remodeling could have a role in the development of PE and FGR [8,9]. In addition, the presence of a haemostatic and angiogenesis balance could be critical for physiological placental function and pregnancy outcome. Aims of this study were to verify whether haemostasis and angiogenesis markers are differently expressed in placentae from PE and FGR and whether the previously described relationships in placentae from uncomplicated pregnancies, are still maintained in placentae from PE and FGR. 2. Methods 2.1. Placental tissue acquisition Placentae were obtained, after an informed consent, from four groups of pregnant women (years age: median [range] = 28 [25-36]) : women with PE (n = 12), PE and FGR (n = 17), FGR “sine causa” (n = 20) and uncomplicated pregnancies (CNT, n = 21) (Table 1). PE was defined as the occurrence of gestational hypertension after 20 weeks’ gestation (≥140/90) and significant proteinuria (≥300 mg in a 24 h urine collection) in a previously normotensive woman on two or more occasions at least 4 hrs apart [10]. FGR fetuses were defined as those whose biometric variables (head circumference, abdominal circumference, femur length) were below the tenth centile, according to fetal size charts for the Italian population [11]. Pericordonal area was sectioned and stored, as previously described [3]. Placentae with marginal or velamentous cord insertion were excluded. Cytotrophoblast was isolated and cultured in a group of PE (n = 8) and in a group of CNT (n = 5) immediately after delivery. 2.2. Cytotrophoblast cell isolation and culture Cells were isolated as previously described, with some modifications [12]. Villous tissue from the decidual side was cut into small pieces. Tissue (~ 50 g) was washed with 0.9% NaCl at room temperature and digested by three steps in warmed Hanks’ solution (Cambrex, Verviers, Belgium) containing 0.125% trypsin (Sigma, St. Louis, USA) and 0.2 mg/ml DNase I (Sigma, St. Louis, USA). Supernatant with 10% FCS (Cambrex, Verviers, Belgium) was layered on Histopaque 1077 (Sigma, St. Louis, USA) and centrifuged (1200 rpm for 20 minutes). Cells in the median layer were separated, washed and plated on culture plates (35 mm) pre-coated with fibronectin (VWR, West Chester USA). Cells were cultured in M199 (Cambrex, Verviers, Belgium) supplemented with 50 μg/ml gentamicin, 1% penicillin-streptomycin (Cambrex, Verviers, Belgium) 20% (vol/vol) FCS (Cambrex, Verviers, Belgium) and incubated in humidified 5% CO2 95% air at 37 °C. After the first 24 hours, apoptotic syncytiotrophoblast was washed out and the remaining cytotrophoblast differentiated and
Table 1 Features of different settings.
PE (n = 10) PE+FGR (n = 17) FGR (n = 20) CNT (n = 21)
Weeks' gestation
Birth wt (g)
Percentile
Placenta weight (g)
33 ± 3,36 33 ± 2,56 33 ± 4,84 39 ± 1,73
2117 ± 893,25 1251 ± 449,87 1373 ± 765,13 3319 ± 445,08
25-50 3-10 ≤3 50-90
600 ± 100 314 ± 149 300 ± 212 506 ± 70,71
matured in culture into new generation of syncytiotrophoblast. Cells were characterized with cytokeratin 7 (DakoCytomation, Denmark). RNA was extracted by means of Invitrogen Trizol, Reagent (Invitrogen Corporation, Carlsbad, California), according to the manufacturer's instructions.
2.3. RNA purification, reverse transcription and quantitative PCR Total RNA was extracted from the frozen samples of placentae by means of Invitrogen Trizol, Reagent (Invitrogen Corporation, Carlsbad, California), according to the manufacturer's instructions. RNA was treated with DNAse (2 U /μg of RNA), incubated in Gene Amp PCR System 2400 Thermal Cycler (Applied Biosystems, Warrington, UK) for 20 minutes at 37 °C, 3 minutes at 85 °C, 10 minutes at 4 °C. Electrophoresis on a 1% agarose gel was performed to confirm the presence and quality of RNA. RNA concentration and purity were determined by UV absorption at 260 nm and 280 nm. Complementary DNA (cDNA) was prepared from the total RNA by means of RT-PCR (Promega Reverse trascription system) (Promega, Madison, Wisconsin, U.S.A.). Quantity mRNA expression of TF, TFPI, TFPI-2, TM, Anx V, PAI-2, VEGF, PlGF, Ang-1, Ang-2 genes was evaluated by ABI 7700TM quantitative real time PCR system (Applied Biosystems, Warrington, UK) and compared to the human housekeeping glyceraldehydes-3-phosphate-dehydrogenase (GAPDH) gene. Primer and probe sequences were either supplied as a ready mix, TaqMan® Gene Expression Assay (VIC-MGB for human GAPDH and FAM-MGB for human TF, human TFPI, human TFPI-2, human TM, human ANX V, human PAI-2, human VEGF, human PlGF, human Ang-1, human Ang-2). Each sample contained: 3 μl (0.04 μg/μl) of cDNA, Taqman Universal PCR Master Mix (12.5 μl), GAPDH primers and probe mix 20x (0.625 μl), target primers and probe mix 20x (1.25 μl) and RNAse free water to a volume of 25 μl. Amplification was performed for 10 min at 95 °C, 45 cycles of 15 seconds at 95 °C, 60 seconds at 60 °C. Gene expression levels were calculated using standard curves generated by serial dilutions of cDNA. A strong correlation between PCR efficiency of the internal control (GAPDH) and the target allowed the use of the ΔCt-method to quantify comparable mRNA levels [13]. Three independent analyses were performed with replicates. Ang-1 and Ang-2 were not determined in cell cultures because of the lack of mRNA.
2.4. Statistical Analysis Statistical analysis was performed by means of SPSS (version 10.0 Macintosh). All the results are presented as median and range values. Mann-Whitney U test was used to compare two independent groups. Correlation between parameters was assessed using the Spearman rank test. Values of p ≤0.05 were considered significant.
3. Results 3.1. Factors involved in the initiation of coagulation and its specific control (TF, TFPI, TFPI-2) TF was significantly less expressed in the FGR group than in the CNT one (Table 2); in PE+FGR group a trend toward significance was observed (p = 0.08). As shown in Table 2, TFPI was significantly less expressed (p ≤ 0.05) in placentae from the PE group than in those from the CNT. TFPI and TFPI-2 were less expressed both in the FGR and in the PE+FGR groups. Placentae from PE did not express significantly different amounts of TF, and TFPI-2 in respect of CNT. Syncytiotrophoblast cells culture showed no significantly different expression of the analyzed haemostasis factors in PE in respect of CNT; a slight, although not significant, decrease was reported for TFPI and TFPI-2 (Table 3).
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Table 2 Expression of haemostasis and angiogenic factors in placentae from uncomplicated pregnancies (CNT) (n = 21), PE (n = 12), PE+FGR (n = 17) and FGR (n = 20).
CNT PE PE+FGR FGR
TF 2^-ΔΔCt Median [Range]
TFPI 2^-ΔΔCt Median [Range]
TFPI-2 2^-ΔΔCt Median [Range]
TM 2^-ΔΔCt Median [Range]
Anx V 2^-ΔΔCt Median [Range]
PAI-2 2^-ΔΔCt Mediane [Range]
VEGF 2^-ΔΔCt Mediane [Range]
PlGF 2^-ΔΔCt Mediane [Range]
Ang-1 2^-ΔΔCt Mediane [Range]
Ang-2 2^-ΔΔCt Mediane [Range]
0,75 [0,01-2,43] 0,35 [0,04-3,29] 0,46 [0,02-2,57] ⁎⁎ 0,22 [0,1-1,17]
0,31 [0,005-4,66] ⁎0,07 [0,01-0,84] ⁎0,10 [0,002-0,68] ⁎0,06 [0,01-0,27]
0,32 [0,001-5,57] 0,05 [0,002-9,34] ⁎⁎0,11 [0,003-5,73] ⁎⁎0,01 [0,002-5,71]
0,69 [0,03-2,72] 0,32 [0,04-2,81] 1,47 [0,06-9,58] 1,08 [0,02-6,37]
0,80 [0,21-3,45] 0,34 [0,003-1,28] 0,99 [0,19-1,71] ⁎0,40 [0,10-1,21]
0,39 [0,01-6,39] 0,23 [0,003- 0,93] ⁎0,09 [0,01-1,36] ⁎⁎ 0,06 [0,003-0,57]
0,22 [0,02-6,60] 0,36 [0,12-3,69] 0,48 [0,04-2,13] 0,16 [0,04-1,43]
0,87 [0,39-2,35] 0,52 [0,21-3,15] 0,81 [0,08-3,64] 0,72 [0,11-5,85]
0,87 [0,06-3,82] ⁎3,33 [0,16-5,19] 0,78 [0,20-2,32] 0,16 [0,06-5,16]
0,77 [0,10-2,66] ⁎2,88 [0,34-6,15] 1,97 [0,74-4,74] 0,23 [0,09-0,79]
In the table the value of median and range of all the groups are reported. ⁎ p ≤ 0.05. ⁎⁎ p ≤ 0.01.
3.2. Factors involved in the coagulation control through the system facilitation and phospholipids modulation ( TM, Anx V) Placentae from women with PE+FGR showed no significant different expression in TM and Anx V (pN 0.05). Anx V was significantly lower in the FGR than in the CNT (pb 0.05) group. No significant difference was reported among the groups in cells culture. 3.3. Fibrinolysis As far as the fibrinolysis is concerned, the PAI-2 was significantly (p b 0.05) less expressed in FGR and in PE+FGR groups (Table 2), while no significant difference was observed in the PE group in the tissues nor in syncytiotrophoblast cells culture (Table 3). 3.4. Angiogenic factors involved in proliferation and differentiation (VEGF, PlGF) and in maturation (Ang- 1 and Ang- 2) The angiogenesis factors VEGF and PlGF were not significantly different in all the considered groups, whereas Ang -1 and Ang -2 were significantly higher in the PE group (Table 2). All the factors involved in the angiogenesis were not significantly (pN 0.05) different in the PE+ FGR or FGR groups. In cell cultures, PlGF was significantly (pb 0.05) higher in the PE group than in the CNT one (Table 3). 4. Correlations 4.1. Correlations between haemostasis factors A significant (Spearman rank test, p≤0,05) direct correlation was found between PAI-2 and both the inhibitors TFPI and TFPI-2. Correlations between TF and its inhibitors and PAI-2, previously observed in placentae from uncomplicated pregnancies, were lacked in the PE, PE+FGR, and FGR groups. 4.2. Correlations between haemostasis and angiogenesis mechanisms An inverse correlation between TF and Ang-1 (Spearman rank test, p≤0.01) was observed in the CNT group. This correlation was lacked in the PE, PE+FGR and FGR groups.
In addition, an inverse correlation between VEGF and Ang-1 was recorded in the CNT group; it was still present in the FGR group, but not in the PE or PE+FGR ones. For each group of cases, the previously observed direct relationship between VEGF and TF and TFPI-2 did not persist. 5. Discussion GVC are multifactorial diseases, whose pathogenesis is not fully understood. It has been postulated that in some of them, like PE with or without FGR , the initiating event could be a reduced utero-placental perfusion as a result of abnormal cytotrophoblast invasion of spiral arterioles [14]. The results obtained in this work show a reduction in the RNA expression of some haemostasis factors in placentae from GVC and the lack of haemostatic balance, critical for normal placental function and pregnancy outcome. Some data are available about the expression of placental TF and its pathway inhibitors. An increase of TF RNA level in placentae and plasma [15,16] from preeclamptic women has been shown by some authors, while others did not confirm these results [17]. Ahron et al. did not find a significant difference in TF RNA in placentae from GVC compared to controls. In agreement with Ahron et al., we did not find any difference in the expression of TF in placentae from PE with/without FGR in respect of controls. We also observed a lower TF expression in placentae from FGR. This could be due to severity of the FGR included, i.e. below the third centile. RNA levels of TFPI are significantly lower in all three groups of GVC in respect of controls, in agreement with data previously reported [17]. The mechanisms which may be associated with the decrease of placental TFPI in women with PE or FGR are currently unknown. It is likely that the observed decrease of TFPI in GVC results from trophoblast which are affected by a hypercoagulability state. This can be potentially due to hypoxia and, in turn, an increased apoptosis of placental trophoblast with secondary reduction of TFPI; alternatively, primary decrease in TFPI results in a local increase of procoagulant activity. This hypothesis seems to be confirmed by the observed imbalance between TF and its inhibitors observed in placentae from GVC.
Table 3 Expression of haemostasis and angiogenic factors in cultures of syncytiotrophoblast from PE (n = 8) and CNT (n = 5).
CNT PE ⁎ p b 0.05.
TF 2^-ΔΔCt Median [Range]
TFPI 2^-ΔΔCt Median [Range]
TFPI-2 2^-ΔΔCt Median [Range]
TM 2^-ΔΔCt Median [Range]
Anx V 2^-ΔΔCt Median [Range]
PAI-2 2^-ΔΔCt Median [Range]
VEGF 2^-ΔΔCt Median [Range]
PlGF 2^-ΔΔCt Median [Range]
0,07 [0,01-3,99] 0,15 [0,01-2,72]
0,01 [0,004-4,90] 0,12 [0,0004-1,28]
0,02 [0,02-4,76] 0,004 [0,0005-1,16]
0,56 [0,04-2,41] 0,18 [0,05-2,96]
0,80 [0,55-2,10] 0,97 [0,42-6,29]
0,50 [0,01-2,99] 0,75 [0,30-12,96]
0,81 [0,27-1,91] 0,22 [0,05-0,82]
0,87 [0,06-2,18] ⁎5,03 [0,22-21,20]
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In more severe forms of GVC, it is possible that both TFPI and TF are less expressed as a consequence of a damage of the syncytiothrophoblast. At variance with our results, Aharon et al. [18] showed that cell damage increases TF expression in cell cultures; discrepancies could be due to different experimental conditions. Our data show that FGR (in presence or absence of PE) is strongly associated with a TFPI-2 reduction. TFPI-2 expression is not significantly lower in culture of syncytiotrophoblasts or in placental sections from PE, whereas it is significantly lower in those from PE+FGR and FGR. Thus, TFPI-2 could be a good marker of FGR. Recently, in a different model Ogawa et al. showed that TFPI-2 is decreased in placentae from FGR, but not from PE [19]. Our data confirm that PE and FGR show distinct distribution of TF and TFPI-2. In agreement with a previous report [20], we did not find significantly different expression of TM in each group in respect of CNT. A significantly lower expression of Anx V in FGR (pb 0.01) was shown. These results suggest a critical role of Anx V in FGR. The apparent discrepancy between data obtained in cultures and in tissues could be, at least in part, explained by the involvement of different types of cells responsible of the production of Anx V [21,22]. In agreement with other authors [1], we report a reduced expression of PAI-2 in sections of placenta from FGR with/without PE, while no difference was observed in syncytiotrophoblast culture. The lack of this difference in cultures could be due to the sample size and to the absence of those cells that contribute to the PAI-2 expression as monocytes/macrophages. In the pathogenesis of PE, VEGF seems to have an important role in dysregulation of endovascular trophoblast invasion as a key step for the development of PE [23]. Although it has been shown in different models that VEGF, PlGF and Ang-2 are decreased in PE [4,5], in placental tissues and in cytotrophoblast cultures we do not show any significant difference in cases’ groups in respect of controls. In agreement with previous data by Tsatsaris, PlGF was not differently expressed in placentae from PE or FGR, whereas it was significantly higher (p=0.03) in cultures [24]. We hypothesise that this increased expression in the cultures could be the result of a compensation of reduced circulating protein levels shown in most studies [25–27]. As reported by other authors, Ang-1 and Ang-2 are significantly more expressed in PE group, confirming that these molecules play an important role in placental biology [28]. We recently showed that TF/TFPI-2 directly correlate with VEGF in term human placentae. Data from present work suggest that in GVC this mechanism of counterbalance is lacking. VEGF and Ang-1 are both involved in vascular development during embryogenesis and certain pathologic conditions and it was shown that Ang-1 may suppress TF expression [29]. In human term placentae an indirect relationship between Ang-1 and TF is present; this relationship is lacked in GVC, due to an increased expression of placental Ang 1. In placentae from uncomplicated pregnancies, the expression of TF and TFPI-2 was significantly associated with VEGF, and a stronger association between haemostasis and fibrinolysis (PAI-2) factors was recorded [3]. In placentae from GVC the relationship between haemostasis and angiogenesis (VEGF) are lacked. These data suggest that the presence of a counterbalance of the angiogenesis by TFPI-2 might have an important role to maintain intervillous blood flow. On the other hand, the relationship between haemostasis (TFPI, TFPI-2) and fibrynolisis is not impaired in the presence of GVC, although they are both reduced. This could be explained by the different cellular sources of the markers [30,31]. There are some limitations to consider in such studies: 1) whether the mRNA levels recorded are primitive or secondary to the development of GVC, 2) the rapid kinetics of the mRNA. In addition, other factors may add variability: the severity of GVC, probably the delivery mode, the duration of the delivery and drugs used by women before and during delivery; we cannot exclude that one or more of these factors have affected our findings.
In conclusion, FGR with or without PE is associated with an impairment of the initiation of coagulation and its specific control (TF, TFPI, TFPI-2), and in the coagulation control phospholipids-modulated (Anx V). Furthermore, in all the GVC a lack of relationship between proand anti-coagulant factors and between haemostasis and angiogenesis factors is observed. Studies aimed at verifying whether an imbalance of these factors is present also in RNA from peripheral blood of pregnant women, could allow to identify new markers of feto-maternal wellbeing. Acknowledgments We wish to thank Dr Giovanna D'Andrea for the significant contribution to cell cultures and all the nurses and midwives of Obstetrics and Gynaecology Department in I.R.C.C.S. “Casa Sollievo della Sofferenza”, S. Giovanni Rotondo for collecting placentae samples. References [1] Estellés A, Gilabert J, Keeton M, Eguchi Y, Aznar J, Grancha S, et al. 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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Markers of haemostasis and angiogenesis in placentae from gestational vascular complications: Impairment of mechanisms involved in maintaining intervillous blood flow☆ Elena Chinni a, Donatella Colaizzo a, Giovanni L. Tiscia a, Pasquale Martinelli c, Giuseppe M. Maruotti c, Maria Matteo d, Maurizio Margaglione a,b, Elvira Grandone a,⁎ a
Atherosclerosis and Thrombosis Unit, I.R.C.C.S. Casa Sollievo della Sofferenza, S.Giovanni Rotondo, FOGGIA, ITALY Medical Genetics, University of Foggia, FOGGIA, ITALY c Obstetrics and Gynaecology, University Federico II, NAPLES, ITALY d Obstetrics and Gynaecology, University of Foggia, FOGGIA, ITALY b
a r t i c l e
i n f o
Article history: Received 3 June 2009 Received in revised form 24 September 2009 Accepted 25 September 2009 Available online 3 November 2009 Keywords: Haemostasis factors Angiogenesis factors Gestational vascular complications
a b s t r a c t Introduction: Preeclampsia (PE) and fetal growth restriction (FGR) are multifactorial diseases, whose pathogenesis is largely unknown. A significant relationship between haemostasis and angiogenesis in placentae from uneventful pregnancies was previously shown. Materials and Methods: RNA expression of haemostasis (TF, TFPI, TFPI-2, PAI-2, Anx V, TM) and angiogenesis (Ang-1, Ang-2, PlGF, VEGF) markers in placentae from PE (n = 12), PE+FGR (n = 17) and FGR (n = 20) in respect of placentae from uncomplicated pregnancies (n = 21) were investigated. Results: Placentae from complicated pregnancies showed a significant lower expression (p ≤ 0.05 MannWhitney U test) of TF, TFPI, TFPI-2, Anx V, PAI-2 than those from in uncomplicated ones. VEGF and PlGF were not different in the considered groups; Ang-1 and Ang-2 were significantly higher (p ≤ 0.05 Mann-Whitney U test) in the PE group. Correlations between factors involved in haemostasis and those involved in angiogenesis, observed in placentae from uneventful pregnancies are lacking in those from complicated ones. Conclusions: Haemostasis factors are reduced in placentae from complicated pregnancies. The relationship between haemostasis and angiogenesis observed in uncomplicated pregnancies is impaired in PE and FGR. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Preeclampsia (PE) is a multisystem disease of unknown etiology associated with increased perinatal/maternal morbidity and mortality. Although fetal growth restriction (FGR) is often associated with PE, nevertheless it can also be observed in absence of hypertensive disease, and its severity is correlated with the degree of placental infarction and impaired placental nutrient transport. Physiologic changes in the coagulation and fibrinolytic systems are known to result in an increased susceptibility of pregnant women to thrombotic disorders and the formation of fibrin deposits, which can lead to a reduction in nutrient transport across the placenta [1]. Abbreviations: PE, preeclampsia; FGR, fetal growth restriction; TF, tissue factor; TFPI, Tissue Factor Pathway Inhibitor; PAI, Plasminogen Activator Inhibitor; AnxV, Annexin V; TM, Thrombomodulin; Ang-1 and 2, Angiopoietin 1 and 2; VEGF, Vascular Endothelial growth Factor; PlGF, Placental Growth Factor. ☆ From the Atherosclerosis and Thrombosis Unit, I.R.C.C.S. Casa Sollievo della Sofferenza, S.Giovanni Rotondo, FOGGIA, ITALY. ⁎ Corresponding author. Atherosclerosis and Thrombosis Unit, I.R.C.C.S. “Casa Sollievo della Sofferenza”, Poliambulatorio Giovanni Paolo II, S. Giovanni Rotondo (FG), ITALY. Tel./ fax: +39 0 882 416273. 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.09.024
The expression of tissue-factor (TF) renders the trophoblasts procoagulant, thus compromising the risk of bleeding while exposing the placenta to pro-thrombotic risks. Local inhibitory mechanisms, tissuefactor pathway inhibitors (TFPI and TFPI-2), thrombomodulin (TM), Annexin V (Anx V), and the fibrinolytic system, limit coagulation activation and fibrin deposition. Lanir et al. previously showed that gestational vascular complications (GVC) have been associated with abnormalities in the functions of haemostatic and fibrinolytic systems [2]. In a preliminary study, performed on a group of 20 placentae from uncomplicated pregnancies, we found a direct relationship between expression of TFPI or TFPI-2, two inhibitors of TF, and inhibitor of plasminogen activator type 2 (PAI-2) [3]. Furthermore, TFPI-2 expression was directly related also to TF. In addition, a direct relationship between TF and TFPI-2 and vascular endothelial growth factor (VEGF), one of the main regulators of angiogenesis, was found. Thus, in placentae from uncomplicated pregnancies, a relationship between haemostasis and angiogenesis was shown [3]. In PE and in FGR, endovascular invasion of cytotrophoblast remains superficial and uterine blood vessels do not undergo adequate vascular transformation compared with uncomplicated pregnancy [4,5]. Furthermore, in PE invasive trophoblast fails to undergo pseudo-vasculogenesis [6].
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The aberrant vascular transformation is thought to lead to placental insufficiency and consequently placental hypoxia [7]. VEGF, placental growth factor (PlGF), angiopoietins (Ang-1 and Ang-2) are involved not only in the regulation of vascular development and in remodeling during placentation, but also act as growth factors for driving growth and differentiation processes such as invasion [8]. It has been hypothesized that an impairment of trophoblast invasion and a failure of spiral artery remodeling could have a role in the development of PE and FGR [8,9]. In addition, the presence of a haemostatic and angiogenesis balance could be critical for physiological placental function and pregnancy outcome. Aims of this study were to verify whether haemostasis and angiogenesis markers are differently expressed in placentae from PE and FGR and whether the previously described relationships in placentae from uncomplicated pregnancies, are still maintained in placentae from PE and FGR. 2. Methods 2.1. Placental tissue acquisition Placentae were obtained, after an informed consent, from four groups of pregnant women (years age: median [range] = 28 [25-36]) : women with PE (n = 12), PE and FGR (n = 17), FGR “sine causa” (n = 20) and uncomplicated pregnancies (CNT, n = 21) (Table 1). PE was defined as the occurrence of gestational hypertension after 20 weeks’ gestation (≥140/90) and significant proteinuria (≥300 mg in a 24 h urine collection) in a previously normotensive woman on two or more occasions at least 4 hrs apart [10]. FGR fetuses were defined as those whose biometric variables (head circumference, abdominal circumference, femur length) were below the tenth centile, according to fetal size charts for the Italian population [11]. Pericordonal area was sectioned and stored, as previously described [3]. Placentae with marginal or velamentous cord insertion were excluded. Cytotrophoblast was isolated and cultured in a group of PE (n = 8) and in a group of CNT (n = 5) immediately after delivery. 2.2. Cytotrophoblast cell isolation and culture Cells were isolated as previously described, with some modifications [12]. Villous tissue from the decidual side was cut into small pieces. Tissue (~ 50 g) was washed with 0.9% NaCl at room temperature and digested by three steps in warmed Hanks’ solution (Cambrex, Verviers, Belgium) containing 0.125% trypsin (Sigma, St. Louis, USA) and 0.2 mg/ml DNase I (Sigma, St. Louis, USA). Supernatant with 10% FCS (Cambrex, Verviers, Belgium) was layered on Histopaque 1077 (Sigma, St. Louis, USA) and centrifuged (1200 rpm for 20 minutes). Cells in the median layer were separated, washed and plated on culture plates (35 mm) pre-coated with fibronectin (VWR, West Chester USA). Cells were cultured in M199 (Cambrex, Verviers, Belgium) supplemented with 50 μg/ml gentamicin, 1% penicillin-streptomycin (Cambrex, Verviers, Belgium) 20% (vol/vol) FCS (Cambrex, Verviers, Belgium) and incubated in humidified 5% CO2 95% air at 37 °C. After the first 24 hours, apoptotic syncytiotrophoblast was washed out and the remaining cytotrophoblast differentiated and
Table 1 Features of different settings.
PE (n = 10) PE+FGR (n = 17) FGR (n = 20) CNT (n = 21)
Weeks' gestation
Birth wt (g)
Percentile
Placenta weight (g)
33 ± 3,36 33 ± 2,56 33 ± 4,84 39 ± 1,73
2117 ± 893,25 1251 ± 449,87 1373 ± 765,13 3319 ± 445,08
25-50 3-10 ≤3 50-90
600 ± 100 314 ± 149 300 ± 212 506 ± 70,71
matured in culture into new generation of syncytiotrophoblast. Cells were characterized with cytokeratin 7 (DakoCytomation, Denmark). RNA was extracted by means of Invitrogen Trizol, Reagent (Invitrogen Corporation, Carlsbad, California), according to the manufacturer's instructions.
2.3. RNA purification, reverse transcription and quantitative PCR Total RNA was extracted from the frozen samples of placentae by means of Invitrogen Trizol, Reagent (Invitrogen Corporation, Carlsbad, California), according to the manufacturer's instructions. RNA was treated with DNAse (2 U /μg of RNA), incubated in Gene Amp PCR System 2400 Thermal Cycler (Applied Biosystems, Warrington, UK) for 20 minutes at 37 °C, 3 minutes at 85 °C, 10 minutes at 4 °C. Electrophoresis on a 1% agarose gel was performed to confirm the presence and quality of RNA. RNA concentration and purity were determined by UV absorption at 260 nm and 280 nm. Complementary DNA (cDNA) was prepared from the total RNA by means of RT-PCR (Promega Reverse trascription system) (Promega, Madison, Wisconsin, U.S.A.). Quantity mRNA expression of TF, TFPI, TFPI-2, TM, Anx V, PAI-2, VEGF, PlGF, Ang-1, Ang-2 genes was evaluated by ABI 7700TM quantitative real time PCR system (Applied Biosystems, Warrington, UK) and compared to the human housekeeping glyceraldehydes-3-phosphate-dehydrogenase (GAPDH) gene. Primer and probe sequences were either supplied as a ready mix, TaqMan® Gene Expression Assay (VIC-MGB for human GAPDH and FAM-MGB for human TF, human TFPI, human TFPI-2, human TM, human ANX V, human PAI-2, human VEGF, human PlGF, human Ang-1, human Ang-2). Each sample contained: 3 μl (0.04 μg/μl) of cDNA, Taqman Universal PCR Master Mix (12.5 μl), GAPDH primers and probe mix 20x (0.625 μl), target primers and probe mix 20x (1.25 μl) and RNAse free water to a volume of 25 μl. Amplification was performed for 10 min at 95 °C, 45 cycles of 15 seconds at 95 °C, 60 seconds at 60 °C. Gene expression levels were calculated using standard curves generated by serial dilutions of cDNA. A strong correlation between PCR efficiency of the internal control (GAPDH) and the target allowed the use of the ΔCt-method to quantify comparable mRNA levels [13]. Three independent analyses were performed with replicates. Ang-1 and Ang-2 were not determined in cell cultures because of the lack of mRNA.
2.4. Statistical Analysis Statistical analysis was performed by means of SPSS (version 10.0 Macintosh). All the results are presented as median and range values. Mann-Whitney U test was used to compare two independent groups. Correlation between parameters was assessed using the Spearman rank test. Values of p ≤0.05 were considered significant.
3. Results 3.1. Factors involved in the initiation of coagulation and its specific control (TF, TFPI, TFPI-2) TF was significantly less expressed in the FGR group than in the CNT one (Table 2); in PE+FGR group a trend toward significance was observed (p = 0.08). As shown in Table 2, TFPI was significantly less expressed (p ≤ 0.05) in placentae from the PE group than in those from the CNT. TFPI and TFPI-2 were less expressed both in the FGR and in the PE+FGR groups. Placentae from PE did not express significantly different amounts of TF, and TFPI-2 in respect of CNT. Syncytiotrophoblast cells culture showed no significantly different expression of the analyzed haemostasis factors in PE in respect of CNT; a slight, although not significant, decrease was reported for TFPI and TFPI-2 (Table 3).
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Table 2 Expression of haemostasis and angiogenic factors in placentae from uncomplicated pregnancies (CNT) (n = 21), PE (n = 12), PE+FGR (n = 17) and FGR (n = 20).
CNT PE PE+FGR FGR
TF 2^-ΔΔCt Median [Range]
TFPI 2^-ΔΔCt Median [Range]
TFPI-2 2^-ΔΔCt Median [Range]
TM 2^-ΔΔCt Median [Range]
Anx V 2^-ΔΔCt Median [Range]
PAI-2 2^-ΔΔCt Mediane [Range]
VEGF 2^-ΔΔCt Mediane [Range]
PlGF 2^-ΔΔCt Mediane [Range]
Ang-1 2^-ΔΔCt Mediane [Range]
Ang-2 2^-ΔΔCt Mediane [Range]
0,75 [0,01-2,43] 0,35 [0,04-3,29] 0,46 [0,02-2,57] ⁎⁎ 0,22 [0,1-1,17]
0,31 [0,005-4,66] ⁎0,07 [0,01-0,84] ⁎0,10 [0,002-0,68] ⁎0,06 [0,01-0,27]
0,32 [0,001-5,57] 0,05 [0,002-9,34] ⁎⁎0,11 [0,003-5,73] ⁎⁎0,01 [0,002-5,71]
0,69 [0,03-2,72] 0,32 [0,04-2,81] 1,47 [0,06-9,58] 1,08 [0,02-6,37]
0,80 [0,21-3,45] 0,34 [0,003-1,28] 0,99 [0,19-1,71] ⁎0,40 [0,10-1,21]
0,39 [0,01-6,39] 0,23 [0,003- 0,93] ⁎0,09 [0,01-1,36] ⁎⁎ 0,06 [0,003-0,57]
0,22 [0,02-6,60] 0,36 [0,12-3,69] 0,48 [0,04-2,13] 0,16 [0,04-1,43]
0,87 [0,39-2,35] 0,52 [0,21-3,15] 0,81 [0,08-3,64] 0,72 [0,11-5,85]
0,87 [0,06-3,82] ⁎3,33 [0,16-5,19] 0,78 [0,20-2,32] 0,16 [0,06-5,16]
0,77 [0,10-2,66] ⁎2,88 [0,34-6,15] 1,97 [0,74-4,74] 0,23 [0,09-0,79]
In the table the value of median and range of all the groups are reported. ⁎ p ≤ 0.05. ⁎⁎ p ≤ 0.01.
3.2. Factors involved in the coagulation control through the system facilitation and phospholipids modulation ( TM, Anx V) Placentae from women with PE+FGR showed no significant different expression in TM and Anx V (pN 0.05). Anx V was significantly lower in the FGR than in the CNT (pb 0.05) group. No significant difference was reported among the groups in cells culture. 3.3. Fibrinolysis As far as the fibrinolysis is concerned, the PAI-2 was significantly (p b 0.05) less expressed in FGR and in PE+FGR groups (Table 2), while no significant difference was observed in the PE group in the tissues nor in syncytiotrophoblast cells culture (Table 3). 3.4. Angiogenic factors involved in proliferation and differentiation (VEGF, PlGF) and in maturation (Ang- 1 and Ang- 2) The angiogenesis factors VEGF and PlGF were not significantly different in all the considered groups, whereas Ang -1 and Ang -2 were significantly higher in the PE group (Table 2). All the factors involved in the angiogenesis were not significantly (pN 0.05) different in the PE+ FGR or FGR groups. In cell cultures, PlGF was significantly (pb 0.05) higher in the PE group than in the CNT one (Table 3). 4. Correlations 4.1. Correlations between haemostasis factors A significant (Spearman rank test, p≤0,05) direct correlation was found between PAI-2 and both the inhibitors TFPI and TFPI-2. Correlations between TF and its inhibitors and PAI-2, previously observed in placentae from uncomplicated pregnancies, were lacked in the PE, PE+FGR, and FGR groups. 4.2. Correlations between haemostasis and angiogenesis mechanisms An inverse correlation between TF and Ang-1 (Spearman rank test, p≤0.01) was observed in the CNT group. This correlation was lacked in the PE, PE+FGR and FGR groups.
In addition, an inverse correlation between VEGF and Ang-1 was recorded in the CNT group; it was still present in the FGR group, but not in the PE or PE+FGR ones. For each group of cases, the previously observed direct relationship between VEGF and TF and TFPI-2 did not persist. 5. Discussion GVC are multifactorial diseases, whose pathogenesis is not fully understood. It has been postulated that in some of them, like PE with or without FGR , the initiating event could be a reduced utero-placental perfusion as a result of abnormal cytotrophoblast invasion of spiral arterioles [14]. The results obtained in this work show a reduction in the RNA expression of some haemostasis factors in placentae from GVC and the lack of haemostatic balance, critical for normal placental function and pregnancy outcome. Some data are available about the expression of placental TF and its pathway inhibitors. An increase of TF RNA level in placentae and plasma [15,16] from preeclamptic women has been shown by some authors, while others did not confirm these results [17]. Ahron et al. did not find a significant difference in TF RNA in placentae from GVC compared to controls. In agreement with Ahron et al., we did not find any difference in the expression of TF in placentae from PE with/without FGR in respect of controls. We also observed a lower TF expression in placentae from FGR. This could be due to severity of the FGR included, i.e. below the third centile. RNA levels of TFPI are significantly lower in all three groups of GVC in respect of controls, in agreement with data previously reported [17]. The mechanisms which may be associated with the decrease of placental TFPI in women with PE or FGR are currently unknown. It is likely that the observed decrease of TFPI in GVC results from trophoblast which are affected by a hypercoagulability state. This can be potentially due to hypoxia and, in turn, an increased apoptosis of placental trophoblast with secondary reduction of TFPI; alternatively, primary decrease in TFPI results in a local increase of procoagulant activity. This hypothesis seems to be confirmed by the observed imbalance between TF and its inhibitors observed in placentae from GVC.
Table 3 Expression of haemostasis and angiogenic factors in cultures of syncytiotrophoblast from PE (n = 8) and CNT (n = 5).
CNT PE ⁎ p b 0.05.
TF 2^-ΔΔCt Median [Range]
TFPI 2^-ΔΔCt Median [Range]
TFPI-2 2^-ΔΔCt Median [Range]
TM 2^-ΔΔCt Median [Range]
Anx V 2^-ΔΔCt Median [Range]
PAI-2 2^-ΔΔCt Median [Range]
VEGF 2^-ΔΔCt Median [Range]
PlGF 2^-ΔΔCt Median [Range]
0,07 [0,01-3,99] 0,15 [0,01-2,72]
0,01 [0,004-4,90] 0,12 [0,0004-1,28]
0,02 [0,02-4,76] 0,004 [0,0005-1,16]
0,56 [0,04-2,41] 0,18 [0,05-2,96]
0,80 [0,55-2,10] 0,97 [0,42-6,29]
0,50 [0,01-2,99] 0,75 [0,30-12,96]
0,81 [0,27-1,91] 0,22 [0,05-0,82]
0,87 [0,06-2,18] ⁎5,03 [0,22-21,20]
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In more severe forms of GVC, it is possible that both TFPI and TF are less expressed as a consequence of a damage of the syncytiothrophoblast. At variance with our results, Aharon et al. [18] showed that cell damage increases TF expression in cell cultures; discrepancies could be due to different experimental conditions. Our data show that FGR (in presence or absence of PE) is strongly associated with a TFPI-2 reduction. TFPI-2 expression is not significantly lower in culture of syncytiotrophoblasts or in placental sections from PE, whereas it is significantly lower in those from PE+FGR and FGR. Thus, TFPI-2 could be a good marker of FGR. Recently, in a different model Ogawa et al. showed that TFPI-2 is decreased in placentae from FGR, but not from PE [19]. Our data confirm that PE and FGR show distinct distribution of TF and TFPI-2. In agreement with a previous report [20], we did not find significantly different expression of TM in each group in respect of CNT. A significantly lower expression of Anx V in FGR (pb 0.01) was shown. These results suggest a critical role of Anx V in FGR. The apparent discrepancy between data obtained in cultures and in tissues could be, at least in part, explained by the involvement of different types of cells responsible of the production of Anx V [21,22]. In agreement with other authors [1], we report a reduced expression of PAI-2 in sections of placenta from FGR with/without PE, while no difference was observed in syncytiotrophoblast culture. The lack of this difference in cultures could be due to the sample size and to the absence of those cells that contribute to the PAI-2 expression as monocytes/macrophages. In the pathogenesis of PE, VEGF seems to have an important role in dysregulation of endovascular trophoblast invasion as a key step for the development of PE [23]. Although it has been shown in different models that VEGF, PlGF and Ang-2 are decreased in PE [4,5], in placental tissues and in cytotrophoblast cultures we do not show any significant difference in cases’ groups in respect of controls. In agreement with previous data by Tsatsaris, PlGF was not differently expressed in placentae from PE or FGR, whereas it was significantly higher (p=0.03) in cultures [24]. We hypothesise that this increased expression in the cultures could be the result of a compensation of reduced circulating protein levels shown in most studies [25–27]. As reported by other authors, Ang-1 and Ang-2 are significantly more expressed in PE group, confirming that these molecules play an important role in placental biology [28]. We recently showed that TF/TFPI-2 directly correlate with VEGF in term human placentae. Data from present work suggest that in GVC this mechanism of counterbalance is lacking. VEGF and Ang-1 are both involved in vascular development during embryogenesis and certain pathologic conditions and it was shown that Ang-1 may suppress TF expression [29]. In human term placentae an indirect relationship between Ang-1 and TF is present; this relationship is lacked in GVC, due to an increased expression of placental Ang 1. In placentae from uncomplicated pregnancies, the expression of TF and TFPI-2 was significantly associated with VEGF, and a stronger association between haemostasis and fibrinolysis (PAI-2) factors was recorded [3]. In placentae from GVC the relationship between haemostasis and angiogenesis (VEGF) are lacked. These data suggest that the presence of a counterbalance of the angiogenesis by TFPI-2 might have an important role to maintain intervillous blood flow. On the other hand, the relationship between haemostasis (TFPI, TFPI-2) and fibrynolisis is not impaired in the presence of GVC, although they are both reduced. This could be explained by the different cellular sources of the markers [30,31]. There are some limitations to consider in such studies: 1) whether the mRNA levels recorded are primitive or secondary to the development of GVC, 2) the rapid kinetics of the mRNA. In addition, other factors may add variability: the severity of GVC, probably the delivery mode, the duration of the delivery and drugs used by women before and during delivery; we cannot exclude that one or more of these factors have affected our findings.
In conclusion, FGR with or without PE is associated with an impairment of the initiation of coagulation and its specific control (TF, TFPI, TFPI-2), and in the coagulation control phospholipids-modulated (Anx V). Furthermore, in all the GVC a lack of relationship between proand anti-coagulant factors and between haemostasis and angiogenesis factors is observed. Studies aimed at verifying whether an imbalance of these factors is present also in RNA from peripheral blood of pregnant women, could allow to identify new markers of feto-maternal wellbeing. Acknowledgments We wish to thank Dr Giovanna D'Andrea for the significant contribution to cell cultures and all the nurses and midwives of Obstetrics and Gynaecology Department in I.R.C.C.S. “Casa Sollievo della Sofferenza”, S. Giovanni Rotondo for collecting placentae samples. References [1] Estellés A, Gilabert J, Keeton M, Eguchi Y, Aznar J, Grancha S, et al. 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