The detrimental role of angiotensin receptor agonistic autoantibodies in intrauterine growth restriction seen in preeclampsia.

Growth-restricted fetuses are at risk for a variety of lifelong medical conditions. Preeclampsia, a life-threatening hypertensive disorder of pregnancy, is associated with fetuses who suffer from intrauterine growth restriction (IUGR). Recently, emerging evidence indicates that preeclamptic women harbor AT(1) receptor agonistic autoantibodies (AT(1)-AAs) that contribute to the disease features. However, the exact role of AT(1)-AAs in IUGR and the underlying mechanisms have not been identified. We report that these autoantibodies are present in the cord blood of women with preeclampsia and retain the ability to activate AT(1) receptors. Using an autoantibody-induced animal model of preeclampsia, we show that AT(1)-AAs cross the mouse placenta, enter fetal circulation, and lead to small fetuses with organ growth retardation. AT(1)-AAs also induce apoptosis in the placentas of pregnant mice, human villous explants, and human trophoblast cells. Finally, autoantibody-induced IUGR and placental apoptosis are diminished by either losartan or an autoantibody-neutralizing peptide. Thus, these studies identify AT(1)-AA as a novel causative factor of preeclampsia-associated IUGR and offer two possible underlying mechanisms: a direct detrimental effect on fetal development by crossing the placenta and entering fetal circulation, and indirectly through AT(1)-AA-induced placental damage. Our findings highlight AT(1)-AAs as important therapeutic targets.

Involvement of heparin-like glycosaminoglycans and expression of a novel heparan sulfate binding protein (p24) during human placentation and in a model for human implantation

Previous studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. To investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were selected to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay showed that initial binding between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAG) with heparin-like properties, i.e., heparan sulfate and dermatan sulfate. Furthermore, a single class of highly specific, protease-sensitive heparin/heparan sulfate binding sites exist on the surface of RL95 cells. Three heparin binding, tryptic peptide fragments were isolated from RL95 cell surfaces and their amino termini partially sequenced. Reverse transcription-polymerase chain reaction (RT-PCR) generated 1 to 4 PCR products per tryptic peptide. Northern blot analysis of RNA from RL95 cells using one of these RT-PCR products identified a 1.2 Kb mRNA species (p24). The amino acid sequence predicted from the cDNA sequence contains a putative heparin-binding domain. A synthetic peptide representing this putative heparin binding domain was used to generate a rabbit polyclonal antibody (anti-p24). Indirect immunofluorescence studies on RL95 and JAR cells as well as binding studies of anti-p24 to intact RL95 cells demonstrate that p24 is distributed on the cell surface. Western blots of RL95 membrane preparations identify a 24 kDa protein (p24) highly enriched in the 100,000 g pellet plasma membrane-enriched fraction. p24 eluted from membranes with 0.8 M NaCl, but not 0.6 M NaCl, suggesting that it is a peripheral membrane component. Solubilized p24 binds heparin by heparin affinity chromatography and $\sp{125}$I-heparin binding assays. Furthermore, indirect immunofluorescence studies indicate that cytotrophoblast of floating and attached villi of the human fetal-maternal interface are recognized by anti-p24. The study also indicates that the HSPG, perlecan, accumulates where chorionic villi are attached to uterine stroma and where p24-expressing cytotrophoblast penetrate the stroma. Collectively, these data indicate that p24 is a cell surface membrane-associated heparin/heparan sulfate binding protein found in cytotrophoblast, but not many other cell types of the fetal-maternal interface. Furthermore, p24 colocalizes with HSPGs in regions of cytotrophoblast invasion. These observations are consistent with a role for HSPGs and HSPG binding proteins in human trophoblast-uterine cell interactions. ^

Gene regulation during placenta development: Murine adenosine deaminase as a model to study placenta specific expression

The formation of the placenta is one of the first and most important developmental events that occur in early mammalian embryogenesis. Even given this importance of the placenta, the academic community has largely ignored studying gene regulation during the development and maturation of the placenta. For this reason, an in-depth study of gene regulation in the trophoblast layer of the placenta using murine Adenosine Deaminase (Ada) as a model system has been undertaken. It has been determined that Ada is highly expressed in the placenta and is critical for embryo development. Dr. Kellems' laboratory has previously described a 1.8 kb fragment of the Ada 5 ′ flanking region that is capable of directing trophoblast specific expression in a transgenic model system. Preliminary studies have demonstrated several critical portions of this fragment that are necessary for the correct tissue specific expression in the placenta. My first specific aim was to elucidate the trans factor binding to one of these sequences, the FP3. Through electromobility shift assays (EMSA), the 30 bp FP3 was narrowed to a 5 bp sequence which computer databases predicted bound to Acute Myeloid Leukemia 1 (AML-1). This was confirmed by supershift analysis. The functional importance of this binding was demonstrated by a transgenic approach. A significant difference in expression of the reporter in the placenta was seen when the 5 bp sequence was mutated. This finding is a novel use for the AML-1 transcription factor which is the DNA binding portion of the heterodimer Core Binding Protein (CBP). The 5′ 240 bp region has also been demonstrated to contain functionally significant sequence. Through EMSA assays and computer predictions, the area has been narrowed to two pertinent regions that are predicted to contain GATA binding motifs. ^

The role of AT1 receptor autoantibodies in the pathophysiology of preeclampsia

Preeclampsia is a disease that affects 3–5% of all pregnancies. The cause is unknown and there is currently no treatment. The disease poses significant health risks to both the mother and the fetus. To date, research on the topic has not produced a convincing cause for the development of the hallmark symptoms of preeclampsia. The hypothesis of an agonistic autoimmune response to the AT1 receptor is presented. Immunoglobulin fractions from normotensive and preeclampsia patients were prepared for experimental tests. Model systems were tested in three categories to determine if AT 1 receptor specific activation and receptor-ligand interaction was caused by a suspected autoantibody. Activation was found in rat neonatal cardiornyocytes that caused an increased contraction rate. This activity was found in preeclampsia patients, absent in normotensive patients. The activation was antagonized by losartan, an AT1 receptor antagonist, and by epitope peptide competition of the receptor-ligand type interaction. This epitope was the 7 amino acid peptide fragment, AFHYESQ, a sequence present in the second extracellular loop of the AT1 receptor. The patterns of AT1 receptor activation were also found in a human trophoblast cell line, HTR8, with an effect on Pai-1 secretion, a factor that plays a role in preventing hypercoagulation. In human mesangial cells, the AT1 receptor autoantibody present in the immunoglobulin fraction from preeclampsia patients was found to stimulate the secretion of Pai-1, and IL-6, a factor that plays a role in the activation of an inflammatory response. This activity was found in samples from preeclampsia patients, but absent in normotensive patients. Tests including losartan, AFHYESQ, and a non-competitive peptide demonstrated that the secretion of Pai-1 and IL-6 met the criteria for AT1 receptor activation by the suspected agonistic autoantibody. These three model systems address relevant pathophysiology for preeclampsia patients, including increased cardiac output, abnormal placentation, and renal damage. The AT1 receptor agonistic autoantibody is potentially a key player in the development of the pathology and symptoms of preeclampsia. ^

Expression regulation of AP -2 gamma gene

AP-2γ is a member of the AP-2 transcription factor family, is highly enriched in the trophoblast cell lineage, and is essential for placenta development. In an effort to identify factors regulating AP-2γ gene expression we isolated and characterized the promoter and 5′ flanking region of the mouse and human AP-2γ genes. The transcription start site of the mouse AP-2γ gene was mapped by primer extension and 5′ RACE. Transient gene transfer studies showed that basal promoter activity resides within a highly conserved ∼200 by DNA sequence located immediately upstream of the transcription start site. The conserved region is highly GC-rich and lacks typical TATA or CCAAT boxes. Multiple potential Sp and AP-2 binding sites are clustered within this region. Electrophoretic mobility shift assays demonstrated that Sp1 and Sp3 bind to three sites in the promoter region of the mouse AP-2γ gene. Combined mutation of the three putative Sp sites reduced promoter activity by 80% in trophoblast and non-trophoblast cells, demonstrating the functional importance of these sites in AP-2γ gene expression. ^ Mutational analysis of the 5′-flanking region revealed a 117-bp positive regulatory region of the mouse AP-2γ gene located between −5700 and −5583 upstream of the transcription start site. This 117-bp positive regulatory element provided approximately 7-fold enhancement of reporter gene expression in cultured trophoblast cells. A C/EBP-Sp1 transcription factor-binding module is located in this DNA sequence. Electrophoretic mobility shift assays demonstrated that transcription factors Sp1, Sp3 and C/EBP bind to the enhancer element. Mutation of each protein-binding site reduced the enhanced expression significantly. Mutagenesis assays showed that two other protein-binding sites also contribute to the enhancer activity. In summary, we have shown that Sp1 and Sp3 bind to cis-regulatory elements located in the promoter region and contribute to basal promoter activity. We have identified a 117-bp positive regulatory element of AP-2γ gene, and we have shown that Sp and C/EBP proteins bind to the cis -regulatory elements and contribute to the enhanced gene expression. ^