Placental expression of ceruloplasmin in pregnancies complicated by severe preeclampsia

Abstract

There is consensus that ischemia/reperfusion injury associated with preeclampsia (PE) promotes both placental damage and the release of factors leading to maternal endothelium dysfunction, a hallmark of this potentially life-threatening syndrome. These factors include plasminogen activator inhibitor-1 (PAI-1) and soluble fms-like tyrosine kinase-1 (sFlt-1). The goal of this study was to further characterize placental factors involved in the pathophysiology of PE. Thus, DNA microarray gene profiling was utilized to identify mRNA differentially regulated in placentas from women with severe PE compared to both preterm (PC) and term control (TC) groups. Microarray studies detected an upregulation of mRNA for ceruloplasmin, a copper-containing iron transport protein with antioxidant ferroxidase properties, in PE compared to PC and TC placentas, respectively. Quantitative real-time PCR confirmed these results by demonstrating significant increases in ceruloplasmin mRNA in PE vs PC and TC placentas. Supporting previous reports, the expression of sFlt-1 and PAI-1 were also upregulated in PE placentas. Immunohistochemistry localized ceruloplasmin to the intervillous space in PE and PC placentas, whereas stronger syncytial staining was noted in PE. Western blotting confirmed a significant increase in ceruloplasmin levels in placental tissue in PE compared to PC groups. PCR identified the presence of mRNA for ceruloplasmin in primary cultures of syncytiotrophoblasts, but not villous-derived fibroblasts, suggesting that syncytium is the site of ceruloplasmin synthesis in placenta. Hypoxic treatment (1% O(2)) of syncytiotrophoblasts enhanced levels of ceruloplasmin mRNA approximately 25-fold, a significantly greater upregulation than that noted for PAI-1 and sFlt-1, suggesting that enhanced ceruloplasmin expression is a sensitive marker of syncytial hypoxia. We suggest that syncytial ceruloplasmin and its associated ferroxidase activity, induced by the hypoxia accompanying severe PE, is important in an endogenous cellular program to mitigate the damaging effects of subsequent reperfusion injury at this site.

Figure 1

Quantitative real-time PCR (qRTPCR) analysis of placental ceruloplasmin expression. RNA was extracted from whole placental tissue from pregnancies with preeclampsia (PE), preterm control (PC), and term control (TC) groups and converted to cDNA. Levels of ceruloplasmin (CP) expression were then determined by qRTPCR and normalized to levels of 18S RNA (a) or cyclophilin B (b). In (c) the PE group was subdivided into patients without and with IUGR (PE and IUGR + PE, respectively), and the PC group was subdivided into patients without +and with infection (idiopathic preterm delivery (IPTD) and chorioamnionitis (CA), respectively). Placentas from pregnancies with isolated IUGR were also analyzed. Results are expressed as a mean + s.e.m. from the indicated number of different placentas per group, and results were analyzed by ANOVA. *P<0.05 vs PC and TC groups; **P<0.001 vs PC and TC groups; ***P<0.05 vs IPTD and CA groups.

Figure 2

Analysis of placental PAI-1, sFlt-1, and PAI-2 expression in placentas by qRTPCR. Levels of placental PAI-1 (a), sFlt-1(b), and PAI-2 (c) gene expression in PE, PC, and TC groups were determined by qRTPCR and normalized to that of 18S RNA. Nine, sixteen, and seven placentas were analyzed for all three genes in PE, PC, and TC groups, respectively. Results, expressed as a mean + s.e.m., were analyzed by ANOVA. *P<0.01 vs TC and P<0.001 vs PC; **P<0.001 vs PC and TC; ⁺P<0.01 vs PE and PC.

Figure 3

Immunohistochemical detection of ceruloplasmin in placenta. Immunohistochemistry was used to localize ceruloplasmin in villi of a placenta from PE (a, c, and e) and PC (b and d) groups. These representative micrographs were from pregnancies delivered at approximately 31 weeks of gestation. Arrows indicate diaminobenzidine/nickel sulfate staining for ceruloplasmin in the intervillous space for both PE and PC placentas in (a) and (b), respectively. Conversely syncytial staining, denoted by arrowheads, was more intense in the PE placenta (a and c). No staining was observed in the PE placenta when nonspecific goat IgG replaced the anticeruloplasmin antibody (e). (a), (b), and (e), × 200; (c) and (d), × 640.

Figure 4

Detection of placental ceruloplasmin expression by western blotting. Western blot analysis of whole placental tissue extracts was carried out to assess the relative levels of ceruloplasmin in PE and PC groups (n = 6 different placentas per group). Ceruloplasmin (CP) was detected at a molecular weight of approximately 140 kDa (top blot, a), cyclophilin B (Cycl) served as a housekeeping protein control (bottom blot, a). Quantitation of ceruloplasmin protein levels was performed by densitometry following normalization to cyclophilin B expression and is presented in (b). Results are expressed as a mean (cerulolplasmin/cyclophilin ratio × 10) + s.e. The values for the PE and PC groups were 10.9±2.7 and 3.8±3.4, respectively. *P<0.01 vs PC group by Student’s t-test.

Figure 5

PCR analysis of ceruloplasmin expression in tissue and cells isolated from term placentas. RNA was extracted from whole placental tissue, or cultures of syncytiotrophoblasts (SCTs) and fibroblasts (FIBs) obtained from three different placentas. Following conversion to cDNA, endpoint PCR was carried out to detect ceruloplasmin (CP, top panel) and 18S RNA (bottom panel). The size of the amplified product is indicated at the right of the figure.

Figure 6

Induction of ceruloplasmin expression in SCTs by hypoxic treatment. Primary cultures of SCTs were maintained for 24 h under normoxic (N) or hypoxic (H) conditions and levels of PAI-1 (a), sFlt-1 (b), and ceruloplasmin (CP, c) were determined by qRTPCR and normalized to level of 18S RNA. Panels a to c depict results from a single representative experiment. Cumulative results from four independent experiments, expressed as fold induction by hypoxic treatment (mean + s.e.m.), are shown in (d). *P<0.001 vs PAI-1 and sFlt-1 by ANOVA.