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Mitogen-activated protein kinase (MAPK) p38α was shown to be implicated in the organogenesis of the placenta, and such placental alteration is crucial for the development of hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. We aimed to analyze for the first time human placental expression of MAPK p38α in pregnancies complicated by HELLP. The placental expression of MAPK p38α was investigated by semiquantitative polymerase chain reaction using cDNA extracted from placental tissue of 15 pregnancies with HELLP syndrome and 15 gestational age-matched controls. Seven patients with HELLP also had intrauterine fetal growth restriction (IUGR). In placenta from pregnancy complicated by HELLP, the expression of MAPK p38α is significantly decreased compared to the group with normal pregnancy (p<0.001), while no difference was found between the HELLP and HELLP with IUGR subpopulations. Our study shows for the first time that MAPK p38α is expressed in the human placenta. Pregnancies with placental dysfunction and hypertensive complications are characterized by a significantly decreased expression of MAPK p38α. Our observations suggest that p38 MAPK signaling may be essential in placental angiogenesis and functioning.
Hemolysis, elevated liver enzymes, and low platelets (HELLP), as described by Weinstein (1982), is the syndrome of hepatic dysfunction, thrombocytopenia, and hemolytic anemia and is a severe pregnancy pathology, which occurs in 0.2–0.8% of all pregnancies, and between 4% and 18.9% of patients with preeclampsia–eclampsia develop HELLP syndrome (Barton and Sibai 1991; Sibai 1993).
Patients whose pregnancies are complicated by HELLP syndrome are at a higher risk for renal failure, consumptive coagulopathy, abruptio placentae, pulmonary and cerebral edema, subcapsular liver hematoma, and hypovolemic shock (Vigil-De Gracia et al. 1996; Svenningsen et al. 2006).
The etiology and pathogenesis of HELLP syndrome is not completely understood. Genetic as well as immunologic factors are involved in its pathogenesis (Arngrimsson et al. 1999), and there is an imbalance in the coagulation process in the placenta. Activated leukocytes and macrophages induce cythochine production that may reach the general circulation and cause endothelial dysfunction. In HELLP syndrome, fibrin deposits are also found in the vessels and in liver sinusoids (Haram et al. 2000).
This pregnancy complication results from a pathological trophoblast invasion and development. However, the causative event for the observed shallow trophoblast invasion and disturbed placentation remains unclear.
In most cases, HELLP is initiated by inadequate placental vessel development with subsequent placental ischemia, leading to the release of circulating vasoconstrictors. The ensuing imbalance in vasoactive substances causes intense systemic vasospasm and multiorgan endothelial damage. Multiple genetic, coagulation, and immunologic disorders also appear to contribute to the endothelial damage (Jones 1998).
A recent study investigating the signaling cascades initiated by a host of cellular stimuli, leading to cellular differentiation and the activation of effectors responses shows the importance of mitogen-activated protein kinases (MAPK) (Dong et al. 2002).
MAPKs include ERK-1, ERK-2, JNK-1, JNK-2, JNK-3, and the p38 kinases (α, β, γ, and δ; Johnson and Lapadat 2002). It has been proposed that p38α MAPK regulates some cellular processes unrelated to the stress response, for example, the differentiation and/or apoptosis of several cell types including neurons, myoblasts (Morooka and Nishida 1998; Cuenda and Cohen 1999; Davidson and Morange 2000), and cardiomyocytes (Kolodziejczyk et al. 1999; Mackay and Mochly-Rosen 1999; Clerk et al. 1998; Wang et al. 1998; Zechner et al. 1997) and is involved in signaling from growth factor and G protein-coupled receptors that promote a wide range of biologic processes (Zetser et al. 1999; Nebreda and Porras 2000; Puri et al. 2000; Wu et al. 2000; Lopez-Ilasaca 1998). Several investigators have reported that virus-induced p38 MAPK activation results in cytokine upregulation (Griego et al. 2000; Iordanov et al. 2000; Shapiro et al. 1998). The p38 MAPK pathway plays an essential role in the biosynthesis of proinflammatory cytokines, including TNF-α, IL6, IL-1, and IL-1β, in many different cell types through the regulation of transcriptional and translational events (Han et al. 1994; Raingeaud et al. 1995; Lee et al. 1994, 2000). Issbrücker et al. (2003) provided evidence that p38 MAPK signaling can act as a “molecular switch” between angiogenesis and hyperpermeability. Increasing p38 MAPK activity could, therefore, enhance vascular permeability but concomitantly reduce angiogenesis in tumors in which the vascular endothelium growth factor (VEGF) is upregulated. Adams et al. (2000) showed that loss of p38α causes embryonic death and that p38α appears to be critical only for placenta organogenesis, given that its deficiency leads to placental defects and embryonic lethality (Adams et al. 2000; Allen et al. 2000; Mudgett et al. 2000; Tamura et al. 2000; Barak et al. 1999).
Mudgett et al. (2000) showed through gene targeting that homozygosity for a null mutation in p38α results in embryonic lethality at midgestation stages, most likely as a consequence of defective placental development. In particular, they found that there are two distinct defects in the placenta, corresponding to a severe reduction in the spongiotrophoblast layer, as well as a near absence of the labyrinth layer because of the failure of vascularization by endothelial cells from the underlying chorionic plate (Mudgett et al. 2000).
These works show that placenta as well as embryonic and yolk sac angiogenesis require the activity of p38α MAPK.
In this study, we aimed to elucidate the expression of p38α MAPK in human placental tissue from pregnancies complicated by HELLP syndrome.
We preferred to specifically focus our research on the HELLP syndrome, rather than preeclampsia, given that MAPK had been recently and extensively investigated in preeclamptic patients under different conditions both in vivo (Webster et al. 2006) and in vitro (Cindrova-Davies et al. 2007).
Placental tissues were obtained from 30 placentas of women who gave birth in the Department of Clinical Science, Section of Women's Health of the Polytechnic University of Marche (Ancona, Italy). Fifteen placentas with HELLP syndrome and 15 normal placentas were used for the study; the two groups studied were comparable for age, parity, and body mass index. As expected, the two groups are different in gestational age at delivery, in systolic or in diastolic blood pressure, and in birth weight (Table 1). The study was approved by the local ethics committee, and informed consent was obtained from all patients.
HELLP syndrome was defined as new onset of severe hypertension (systolic blood pressure ≥160 mmHg and/or diastolic blood pressure ≥110 mmHg) with platelet count <100,000 cells/mm3, aspartate aminotransferase activity >70 U/L, and lactate dehydrogenase activity >600 U/L (Sibai et al. 1986), in the absence of other pathologic conditions. The control group included women with normal pregnancy and normal fetal growth. Women with history of hemolysis, liver disorders, thrombocytopenia, hypertension, renal disease, cardiac disease, diabetes mellitus, or evidence of chromosomal and other fetal anomalies were excluded from the study. All controls delivered at term (>37 weeks). In the HELLP group, seven out of 15 cases had fetal growth restriction (defined as abdominal circumference at ultrasound and birth weight <5° centile for the Caucasian population).
All placentas were taken via cesarean section, in nonlaboring women, since contractions during labor may alter the signaling pathway which is the object of our study.
In the HELLP group, a cesarean section was promptly performed because of deterioration in either maternal or fetal status; in the control group, the indications were either abnormal presentation of the fetus or previous cesarean section.
Placental tissue was taken immediately after delivery. Two blocks of whole placenta (approximately 3 cm3) were cut from the lower third of the cotyledons of each placenta and immediately frozen in liquid nitrogen. Once frozen, the placental pieces were wrapped in aluminum to prevent dehydration and stored at −80°C until analysis.
Total RNA was isolated from 30 mg of placenta using SV Total RNA Isolation Systems (Promega) according to the manufacturer's instructions. The integrity of the isolated RNA was evaluated by visualizing 28S and 18S ribosomal RNA on a 1% agarose gel.
cDNA synthesis was performed using 1.2 µg of total RNA in a total volume of 15 µL with 15 µg of oligo d(T)16 using 1 µL of Improm-II Reverse Transcriptase (Promega). The reaction mix was incubated for 5 min at 25°C and then for 60 min at 42°C.
For polymerase chain reaction (PCR) amplifications, 0.5 µL of cDNA template was added to the reaction mixture containing 1.5 mM MgCl, 200 µM dNTPs, 0.2 µM of each primer, and 1.5 U EroTaq DNA Polymerase and PCR Buffer EuroClone. Intron-spanning primers (Invitrogen) were designed to avoid genomic DNA contamination.
Primers for genotyping were: for β-actin, 5′-ACC TTC TAC AAT GAG CTG CGT-3′ (forward primer) and 5′-ATG AGG TAG TCA GTC AGG TCC-3′ (reverse primer); for MAPKp38α, 5′-TTT GAC ACA AAA ACG GGG TTA CGT-3′ (forward primer) and 5′-ACA GAT GAT GAA ATG ACA GGC-3′ (reverse primer; Table 2).
PCR amplifications were performed in a DNA thermal cycler starting with an initial denaturation step at 94°C for 4 min followed by cycles consisting of a denaturation step at 94°C for 15 s, an annealing step for 30 s at predetermined temperatures for each gene (54°C for β-actin and 50°C for MAPK p38α), and an extension step at 72°C for 20 s. To prevent the amplification reaction from reaching the plateau phase, the number of PCR cycles was adjusted for each gene, 22 cycles for β-actin and 28 cycles for MAPK p38α. Ethidium bromide-stained 2% agarose gel was analyzed by densitometry and compared in a semiquantitative mode within a single experiment by using the KODAK EDAS 290 densitometer and KODAK 1D Image Analysis Software. The relative ratio (R) of the net intensities of the MAPK and β-actin bands (used as an internal control) from the same subject was determined to show the relative amount of MAPK mRNA expression.
The Sigmat stat software package, version 3.0, was used for collection, processing, and statistical analysis of all data. Statistical analysis was performed using the t test between the two groups and significance was assessed at p<0.05.
Then, we used a multiple comparison procedure, one-way analysis of variance on ranks followed by Dunn's post hoc test, to test the differences between the control group and the HELLP group with IUGR and without IUGR.
Placental p38α MAPK is significantly lower in HELLP syndrome, compared with the control group (mean ± SEM; 0.0166±0.0012 vs 0.0921±0.0091, p<0.001; Fig. 1).
The amplification of the β-actin gene was similar for all patients, so that the different concentration of p38α MAPK reflects a truly different production between the two groups rather than a generic gene variation.
All pairwise multiple comparison procedures (Dunn's method) showed that the difference between the control group and the HELLP group (17.786±Q 4.414, p<0.05) and HELLP group complicated with IUGR (12.563±Q 3.260, p<0.05) is highly significant, while there is no difference between the HELLP group and HELLP group with IUGR (5.223±Q 1.146; Fig. 2).
This is the first study to show that p38α MAPK is expressed in human placenta and is activated by a variety of extracellular stimuli, including inflammatory cytokines such as IL-1 and TNF, growth factors such as fibroblast growth factor and colony-stimulating factor, osmolarity changes, UV light, and chemical agents that promote a stress response (New and Han 1998).
Many studies have suggested essential requirements for p38 MAPK in inflammatory and environmental stress responses, although the roles of the p38 MAPK pathway in normal development are unclear. The diverse nature of these activators, however, suggests that p38 serves as a point of convergence for a variety of extracellular effectors.
Interestingly, our findings in HELLP syndrome show a significantly reduced expression of placental p38α MAPK. This is consistent with recent reports that showed the correlation between loss of the p38α MAPK gene and the development of placental insufficiency as occurs in HELLP syndrome. Interestingly, we did not observe differences between the group with HELLP and that with HELLP and IUGR. In this case, we may speculate that placental impairment is so high that IUGR may be a result of this impairment and not an independent entity.
Furthermore, our results show that such impairment in HELLP syndrome is greater than what can be extrapolated from the literature in preeclampsia. Compared with the results by Webster et al. (2006), who observed a roughly 50% reduction of phospho-p38 MAPK, our findings show about 80% reduction compared to controls, this means a more severe impairment in HELLP syndrome than preeclampsia.
In addition, a central role for p38α MAPK function in embryogenesis has been demonstrated through analysis of the Drosophila p38α MAPK pathway (Suzanne et al. 1999), as well as its implication in the hypoxia response pathway.
p38 MAPK is a modulator of events in signaling by the VEGF pathway and/or other angiogenic factors and their receptors, negatively regulates angiogenesis by reducing phosphorylation of Erk1/2 MAPK, and increases permeability by yet unidentified mechanisms.
Lamalice et al. (2006) reported that following VEGF treatment of endothelial cells, VEGFR-2 is phosphorylated on Tyr1214 upstream of the Cdc42-SAPK2/p38-MAPKAP K2 pathway. The p38 pathway conveys the VEGF signal to microfilaments inducing rearrangements of the actin cytoskeleton that regulate cell migration. By modulating cell migration, p38 may thus be an important regulator of angiogenesis (Rousseau et al. 1997). Alternatively, hypoxia might activate p38 MAPK, which might indirectly facilitate the expression of VEGF or other angiogenic factors; consistent with this view, the p38α and p38γ isoforms are specifically activated in PC12 cells in response to hypoxic conditions (Conrad et al. 1999).
However, the potential regulatory relationships, if any, between p38α and the other genes in the pathways for placental angiogenesis remain to be examined. Our present analysis of p38α MAPK indicates that it could have a previously unsuspected role in HELLP and IUGR development and raise the possibility that p38α MAPK activity is required for the angiogenic response to the hypoxic environment found during HELLP syndrome or in IUGR.