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(Circulation. 2004;110:1612-1619.)
© 2004 American Heart Association, Inc.

Hypertension

Antibodies From Preeclamptic Patients Stimulate Increased Intracellular Ca²⁺ Mobilization Through Angiotensin Receptor Activation

Theingi M. Thway, PhD; Sergiy G. Shlykov, PhD; Mary-Clare Day, BSN; Barbara M. Sanborn, PhD; Larry C. Gilstrap, III, MD; Yang Xia, MD, PhD; Rodney E. Kellems, PhD

From the Departments of Biochemistry and Molecular Biology (T.M.T., S.G.S., B.M.S., Y.X., R.E.K.) and Obstetrics, Gynecology, and Reproductive Sciences (M.-C.D., L.C.G.), The University of Texas Health Science Center at Houston.

Correspondence to Dr Rodney E. Kellems, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, 6431 Fannin St, Houston, TX 77030. E-mail rodney.e.kellems{at}uth.tmc.edu

Received October 23, 2003; de novo received March 22, 2004; revision received June 1, 2004; accepted June 23, 2004.

	Abstract

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Background— Preeclampsia is a serious disorder of pregnancy characterized by hypertension, proteinuria, edema, and coagulation and vascular abnormalities. At the cellular level, abnormalities include increased calcium concentration in platelets, lymphocytes, and erythrocytes. Recent studies have shown that antibodies directed against angiotensin II type I (AT₁) receptors are also highly associated with preeclampsia.

Methods and Results— We tested the hypothesis that AT₁ receptor–agonistic antibodies (AT₁-AAs) could activate AT₁ receptors, leading to an increased intracellular concentration of free calcium and to downstream activation of Ca²⁺ signaling pathways. Sera of 30 pregnant patients, 16 diagnosed with severe preeclampsia and 14 normotensive, were examined for the presence of IgG capable of stimulating intracellular Ca²⁺ mobilization. IgG from all preeclamptic patients activated AT₁ receptors and increased intracellular free calcium. In contrast, none of the normotensive individuals had IgG capable of activating AT₁ receptors. The specific mobilization of intracellular Ca²⁺ by AT₁-AAs was blocked by losartan, an AT₁ receptor antagonist, and by a 7-amino-acid peptide that corresponds to a portion of the second extracellular loop of the AT₁ receptor. In addition, we have shown that AT₁-AA–stimulated mobilization of intracellular Ca²⁺ results in the activation of the transcription factor, nuclear factor of activated T cells.

Conclusions— These results suggest that maternal antibodies capable of activating AT₁ receptors are likely to account for increased intracellular free Ca²⁺ concentrations and changes in gene expression associated with preeclampsia.

Key Words: autoimmunity • antibodies • signal transduction • hypertension • pregnancy

	Introduction

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Preeclampsia is a pregnancy-induced hypertensive disorder that affects 3% to 5% of first pregnancies and is a leading cause of maternal and fetal mortality and morbidity. This disorder is characterized by the development of a maternal syndrome that includes hypertension, coagulation abnormalities, edema, proteinuria, and vascular abnormalities. These symptoms normally develop after 20 weeks of gestation and disappear within 7 to 10 days after delivery. However, subtle signs, such as increased protein excretion, may persist for several months. The main target organs affected in preeclampsia are the brain, kidneys, liver, lungs, placenta, and heart. Advanced stages of the disease include cerebral hemorrhage, renal failure, and the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets). Two well-known pathogenic features of preeclampsia are placental and vascular abnormalities. Shallow trophoblast invasion and improper remodeling of spiral arteries are among the best-recognized and most frequently associated features of this disease. Altered levels of circulating vasoactive substances, such as endothelin and prostaglandins, have been reported in preeclampsia.¹ Preeclampsia is also associated with abnormalities in Ca²⁺ metabolism and increased intracellular Ca²⁺ levels in a variety of cell types, such as platelets, erythrocytes, and lymphocytes.^2–4 Haller et al² have shown that basal intracellular free Ca²⁺ in platelets is substantially elevated in preeclamptic patients compared with women with uncomplicated pregnancy. This phenomenon completely disappeared 6 weeks after delivery, which suggests a relevant relationship to preeclampsia. Similar studies were done using lymphocytes and erythrocytes and showed that the intracellular free Ca²⁺ concentration is increased in these cells of preeclamptic patients.^3,4 In addition, a more widespread dysregulation of cellular Ca²⁺ metabolism is also implicated in preeclampsia.⁵ The underlying mechanism dictating changes in intracellular free Ca²⁺ levels and Ca²⁺ metabolism in these patients remains unknown.^6,7 In the present study, we tested the possibility that angiotensin receptor–activating antibodies, known to be associated with preeclampsia,⁸ could account for the increased intracellular Ca²⁺ seen in preeclampsia.

	Methods

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Materials
Tissue culture medium (RPMI 1640), FBS, and antibiotics such as penicillin-streptomycin (100x), and geneticin (G418, 50 mg/mL) were purchased from Invitrogen Life Technologies. Human angiotensin II (Ang II) (catalog No. A9525) was obtained from Sigma. Fura 2-AM cell permeant (catalog No. F-1221) and 35-mm glass-bottom microwell dishes (catalog No. p35G-1.5-10C) were obtained from Molecular Probes Inc and Matek Corp, respectively. Losartan (COZAAR) was a gift from Merck Research Laboratory (Rahway, NJ). The 7-amino-acid peptides P1 (AFHYESQ), an epitope for AT₁-AAs, and P2, another 7-amino-acid peptide (VEGIENE) were synthesized by the Protein Chemistry Core Laboratory, Baylor College of Medicine. These peptides correspond to 2 different portions of the second extracellular loop of the human AT₁ receptor. P1 is the epitope recognized by AT₁-AAs from preeclamptic individuals. GammaBind G Sepharose was purchased from Amersham Pharmacia Biotech. PathDetect nuclear factor of activated T cells (NFAT) cis-reporting system and synthetic Renilla luciferase reporter vector were purchased from Stratagene and Promega Corp, respectively.

Patients
Thirty patients who were admitted to Memorial Hermann Hospital were identified by the obstetrics faculty of the University of Texas Medical School at Houston. Sixteen patients were diagnosed with severe preeclampsia on the basis of the definition set by the National High Blood Pressure Education Program Working Group report. The criteria include the presence of high blood pressure of 160/110 mm Hg and the presence of protein in urine of 0.3 g in a 24-hour period. These women had no previous history of hypertension. Other criteria included the presence of persistent headache, visual disturbances, epigastric pain, or the HELLP syndrome in women with blood pressure of 140/90 mm Hg. For patients with preeclampsia, the gestational age at delivery ranged from 27 to 39 weeks, with an average of 33 weeks. Fourteen normotensive pregnant individuals were characterized by uncomplicated pregnancies with normal-term deliveries. For normotensive patients, the gestational age at delivery ranged from 37 to 41 weeks, with an average of 39 weeks. Blood samples were centrifuged at 18 000g for 10 minutes, and the serum samples were stored at –80°C. Patients were approached for the study during the intrapartum (44%) or early postpartum (56%) period. Patient enrollment occurred between March 2002 and May 2003. The research protocol, including the consent form, was approved by the institutional Committee for the Protection of Human Subjects.

Cell Culture
Chinese hamster ovary cells stably transfected with rat Ang II receptor type 1A (CHO.AT1A) were kindly provided by Dr Terry S. Elton (Brigham Young University, Provo, Utah). Cells were cultured in RPMI 1640 medium containing 5% FBS, 1% antibiotics, 17.5 mg/mL L-proline, and 100 µg/mL gentamicin at 37°C and 5% CO₂.

Preparation of the IgG Fraction
The IgG fraction was prepared from 200-µL serum samples loaded onto 100 µL of GammaBind G Sepharose column. Non-IgG flowthrough was collected. The IgG fraction was washed 8 times with wash buffer (50 mmol/L Tris-HCl, 0.02% NaN₃, pH 7.4) and eluted in 1.6 mL of elution buffer (100 mmol/L glycine-HCl, pH 2.7) according to the manufacturer’s instructions. The eluted IgG fraction was neutralized to pH 7.0 with 1 mol/L Tris-HCl (pH 9.0).

Measurement of Intracellular Ca²⁺ Concentration
A day before the experiment, CHO.AT1A cells were plated at a density of 6.0x10⁴ cells per well in polylysine-coated glass inserts in 35-mm dishes (Matek Corp). After 6 to 8 hours, the cell culture medium was removed and replaced with serum-free medium, and cells were cultured overnight. The next day, cells were loaded with 5 µmol/L fura 2-AM at room temperature for 35 minutes in fluorescence buffer (145 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L Na₂HPO₄, 0.5 mmol/L MgCl₂, 1 mmol/L CaCl₂, 10 mmol/L HEPES, 5 mmol/L glucose, pH 7.4). Excessive fura 2-AM was removed by rinsing twice with fluorescence buffer. Loaded fura 2-AM was allowed to hydrolyze in fluorescence buffer for 30 minutes at room temperature. Experiments were performed at room temperature. Changes in fluorescence in individual cells were monitored at 340- and 380-nm excitation and 510-nm emission wavelengths in an InCyt2 Im2 imaging system (Intracellular Imaging Inc). In each experiment, at least 20 cells were monitored for changes in fluorescence. For detection of intracellular Ca²⁺ changes, cells were treated with different dilutions of serum, immunoglobulin-lacking serum eluant, or immunoglobulin fraction, as indicated. For inhibition of Ca²⁺ response, cells were preincubated with 1 µmol/L losartan for 1.5 to 2 minutes before addition of IgG fraction. For neutralization experiments, synthetic peptide P1 or P2 (50 µmol/L) was incubated with IgG at 37°C for 15 minutes. A mixture of peptide-IgG solution was added to the plate while cells were being monitored.

Transient Transfection Assay
CHO.AT1A cells were plated at a density of 1.2x10⁵ cells in 24-well plates for 2 hours. Cells were transfected using 500 ng of the NFAT-luciferase reporter construct containing 4 copies of the NFAT binding element (PathDetect NFAT cis-Reporting system), 20 ng of phRTK, a synthetic Renilla luciferase reporter construct (for internal control), and 5 µL of Lipofectamine Reagent (Invitrogen Life Technologies) for 5 hours. The cells were serum starved for 24 hours and treated with either Ang II or IgG overnight where indicated. The treated cells were lysed in 100 µL of passive lysis buffer (Promega Inc) at room temperature for 15 minutes. Luciferase activity (measured in relative light units) was measured using 20 µL of lysate with the Dual Luciferase system (Promega Inc).

Statistical Analysis
Mobilization of intracellular free Ca²⁺ by individual IgG samples was performed a minimum of twice. The statistical analysis of changes in free Ca²⁺ concentration in each patient category with or without inhibitors was performed by use of the Mann-Whitney test. For transient transfection assays, either the Mann-Whitney test or a paired t test was used. A probability value of P0.05 was considered significant.

	Results

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Dose-Dependent Stimulation of Intracellular Free Ca²⁺ Is Mediated by Ang II in CHO.AT1A Cells
The primary goal of our study was to examine the ability of AT₁-AAs to stimulate an increase in intracellular Ca²⁺ concentration through AT₁ receptor activation. We chose to use CHO.AT1A because these cells are reliably and widely used to study AT₁ receptor activation by Ang II. These CHO.AT1A cells are responsive to Ang II stimulation^9,10 and retain various signaling components downstream of AT₁ receptor activation. First, we determined the dose-dependent effect of Ang II in stimulating intracellular Ca²⁺ levels in these cells. Fura 2-AM–loaded CHO.AT1A cells were treated with different concentrations of Ang II (0.01, 0.1, and 1 µmol/L), and changes in intracellular Ca²⁺ concentrations were measured. Values represent the means and SEM of fura 2-AM fluorescence obtained by the analysis of at least 20 cells in each experiment. Each experiment was repeated at least 3 times. As shown in Figure 1 (A through C), Ang II stimulated an increase in intracellular free Ca²⁺ in a dose-dependent manner. By 6 to 7 minutes after treatment, intracellular Ca²⁺ concentrations in these cells returned to near basal levels. The increased concentration of Ca²⁺ stimulated by 0.1 µmol/L Ang II was completely inhibited by 1 µmol/L losartan, an AT₁ receptor antagonist (Figure 1D). Losartan itself had no effect on intracellular free Ca²⁺ concentrations (Figure 1E). These data indicate that the CHO.AT1A cells responded to Ang II with an increase in intracellular Ca²⁺ concentration as a result of AT₁ receptor activation.

View larger version (11K): [in this window] [in a new window]   Figure 1. Dose-dependent stimulation of intracellular free Ca2+ is mediated by Ang II in CHO.AT1A cells. Fura 2-AM–loaded CHO.AT1A cells were treated with different concentrations of Ang II [0.01 (A), 0.1 (B), and 1 µmol/L (C)] and change in intracellular free calcium level (Ca2+ [nmol/L]) was monitored for period of time indicated. Calcium response triggered by 0.1 µmol/L Ang II was inhibited by 1 µmol/L losartan (D), whereas losartan itself had no effect in stimulation of intracellular Ca2+ (E). Each graph represents average of changes in intracellular calcium levels in at least 20 different cells in each experiment. Arrows indicate time when activator (solid) or inhibitor (dotted) was added.

IgG Fractions From Preeclamptic Patients Show a Dose-Dependent Stimulation of Intracellular Ca²⁺ Concentrations
The results presented above show that CHO.AT1A cells possess functional AT₁ receptors that are responsive to Ang II stimulation, resulting in the mobilization of intracellular calcium. Next, we examined the ability of IgG fractions from preeclamptic patients to stimulate an increase in intracellular [Ca²⁺]. Values represent the means and SEM of fura 2-AM fluorescence obtained by the analysis of at least 20 cells in each experiment. Fura 2-AM–loaded CHO.AT1A cells were treated with serum from preeclamptic individuals and serum eluant from which the IgG fraction was removed. Initially, serum from preeclamptic patients at 1:200 dilution stimulated a significant Ca²⁺ response (Figure 2A), whereas no stimulation was observed with a serum eluant from which the IgG fraction had been removed (Figure 2B). Next, fura 2-AM–loaded CHO.AT1A cells were treated with different dilutions of the IgG fraction (1:400, 1:40, and 1:10). Similar to those responses seen with Ang II stimulation, AT₁-AAs in the immunoglobulin fraction at 1:400, 1:40, and 1:10 dilutions increased intracellular Ca²⁺ levels in a dose- and time- dependent manner (Figure 2, C–E). The increased [Ca²⁺] stimulated by 1:10 dilution of immunoglobulin was completely inhibited by 1 µmol/L losartan (Figure 2F). In addition, we examined the ability of the immunoglobulin fraction from a normotensive pregnant individual to stimulate an increase in intracellular [Ca²⁺] (Figure 2G). The immunoglobulin fraction from the serum of a normotensive individual had no effect on Ca²⁺ mobilization even at a 1:5 dilution. These data suggest that Ca²⁺ mobilization in CHO.AT1A cells can be used to detect AT₁-AAs.

View larger version (12K): [in this window] [in a new window]   Figure 2. IgG fraction from preeclamptic patients shows a dose-dependent stimulation of intracellular Ca2+ levels. CHO.AT1A cells were treated with 1:200 dilution of preeclamptic sera (A), with 1:200 dilution of IgG-depleted serum eluant (B), and different dilutions of IgG fractions from same preeclamptic patient [1:400 (C), 1:40 (D), and 1:10 (E)]. Losartan (LST) at 1 µmol/L inhibited effect of intracellular calcium mobilization stimulated by immunoglobulin fraction at 1:10 dilution (F). Similarly, CHO.AT1A cells were treated with IgG fractions from a normotensive pregnant individual at 1:5 dilution to stimulate calcium response (G). Arrows indicated time when IgG fraction was added.

Higher Concentrations of Preeclamptic IgG Increased the Number of Responding Cells
In the previous section, we observed that increasing concentrations of IgG increased the average concentration of intracellular calcium. We next determined whether the increased overall intracellular [Ca²⁺] resulted from an increase in the [Ca²⁺] in some or all of the cells in the population. Figure 3 shows the results obtained from 33 cells treated with 2 different concentrations of IgG. Individual cells were monitored for changes in intracellular [Ca²⁺]. Figure 3A represents the overall change in the intracellular [Ca²⁺] of all 33 cells. Figure 3B shows changes in the intracellular [Ca²⁺] for each cell. Figure 3B shows that 33 cells were analyzed for calcium mobilization (ie, increased fluorescence as judged by an increase in the absorbance curve). After the addition of a 1:40 dilution of IgG, 11 of the 33 cells showed increased fluorescence at 100 seconds on the time scale. After the addition of a 1:10 dilution of IgG at 230 seconds, 31 of the 33 cells displayed increased fluorescence (only cells numbered 8 and 24 did not show an increase in fluorescence). In additional experiments, the number of responding cells varied from 50% to 100% among different immunoglobulin preparations from preeclamptic individuals when tested at a 1:10 dilution. These data suggest that the concentration of IgG influenced the percentage of cells in the population that responded and that some cells required higher concentrations of IgG to activate the AT₁R and stimulate an increase in intracellular calcium.

View larger version (37K): [in this window] [in a new window]   Figure 3. Higher concentrations of AT1-AAs increase number of responding cells. A, Graph showing average intracellular Ca2+ concentration in a population of 33 cells. B, Graph shows intracellular Ca2+ concentration in each of 33 cells being monitored in response to IgG treatment with 2 different concentrations. First * indicates time point when a 1:40 dilution of IgG was added. Second * indicates time point when IgG concentration was increased to 1:10 dilution.

Increased Intracellular Ca²⁺ Mobilization Is Stimulated Through AT₁-Receptor Activation by IgG From Preeclamptic Individuals but Not by IgG From Normotensive Pregnant Women
To determine whether our findings can be generalized, we extended our analysis to include 14 normotensive pregnant individuals and 16 individuals diagnosed with severe preeclampsia. On the basis of the dose-dependent activation by IgG shown in Figures 2 and 3 , we chose to use a 1:10 dilution of IgG from both groups of pregnant women. Figure 4A shows that IgG from preeclamptic individuals stimulated a significant increase in intracellular Ca²⁺ concentration, with an average value of 335.4 (± 38.4) nmol/L. The IgG from 14 normotensive pregnant individuals was also analyzed. Among 14 IgG preparations from normotensive individuals, 10 had little or no effect in stimulating Ca²⁺ mobilization (Figure 4). The mean intracellular [Ca²⁺] among these 10 individuals was 62.0±12.3 nmol/L. The 4 normotensive individuals showing a significant Ca²⁺ mobilization did not involve AT₁-receptor activation (see last paragraph of this section). Figure 4B presents the average of changes in intracellular [Ca²⁺] of 16 preeclamptic individuals and the 10 normotensive individuals that showed little to no Ca²⁺ response. These data suggest that IgG from preeclamptic patients significantly stimulates Ca²⁺ mobilization, whereas IgG from normotensive pregnant individuals generally does not (P<0.05). The 4 normotensive pregnant individuals showing a significant Ca²⁺ response will be considered separately below.

View larger version (35K): [in this window] [in a new window]   Figure 4. AT1-AAs stimulate increased intracellular Ca2+ mobilization. A, Calcium response activated by IgG from each of 16 preeclamptic (PE) patients and 14 normotensive (N) pregnant individuals was analyzed. Graph represents changes in Ca2+ concentration by each patient in each patient group. Experiments were repeated a minimum of twice per patient sample. B, Average of changes in intracellular Ca2+ concentration of 16 preeclamptic and 10 normotensive individuals showing little or no Ca2+ response (filled squares). Ca2+ response provoked by IgG from 4 normotensive pregnant individuals (open squares) was not associated with AT1 receptor activation (see Figure 5B). Calcium responses activated by each of 16 preeclamptic patients and 14 normotensive individuals (C) and average values (D) were presented as percent responding cells. *P<0.05.

The data presented above evaluated the Ca²⁺ response in terms of the average Ca²⁺ concentration per cell. To further evaluate the Ca²⁺ mobilization response, we determined the percentage of cells in the population showing a Ca²⁺ mobilization response after treatment with IgG from preeclamptic and normotensive pregnant individuals. Figure 4, C and D, presents the percentage of responding cells in both groups and their average values, respectively. Immunoglobulin from preeclamptic patients increased intracellular free [Ca²⁺] in >50% (average, 79.5±4.8%) of the CHO.AT1A cell population. In contrast, IgG from the majority (10/14) of normotensive individuals was capable of activating calcium mobilization in only a small percentage of cells (average, 8.9±2.3%). IgGs from 4 normotensive pregnant individuals stimulated Ca²⁺ mobilization in a large percentage of cells, but, as we will show below, this was not the result of AT₁-receptor activation. These data suggest that IgG from preeclamptic patients stimulates Ca²⁺ mobilization in a large population of cells, whereas IgG from normotensive individuals does not (Figure 5, A and B).

View larger version (26K): [in this window] [in a new window]   Figure 5. Specificity of increased intracellular Ca2+ concentration by AT1-AAs is determined using AT1 receptor–specific antagonists and an AT1 receptor–specific epitope peptide. A, Determination of specificity of calcium response observed from IgG fractions of preeclamptic patients. Losartan at 1 µmol/L was used to inhibit calcium response stimulated by IgG fraction from all preeclamptic patients. Effects of P1 and P2 were examined using immunoglobulin fraction from 5 patients. Immunoglobulin at 1:10 dilution was preincubated with either P1 or P2 before treatment. P1 is a 7-amino-acid epitope peptide that is target of AT1-AAs associated with preeclampsia. P2 is a 7-amino-acid corresponding to a nonepitope region of AT1 receptors. **P<0.05. B, Determination of specificity of calcium response observed from immunoglobulin fractions of 4 normotensive individuals. Losartan (LST) at 1 µmol/L was used to evaluate involvement of AT1 receptor activation in these calcium responses.

We next determined whether IgG-mediated Ca²⁺ mobilization was acting through AT₁ receptor activation. We initially examined the IgG from the 16 patients with severe preeclampsia. In these experiments, fura 2-AM–loaded CHO.AT1A cells were pretreated with 1 µmol/L losartan, and then IgG was added to the culture at a 1:10 dilution. The results (Figure 5A) show that losartan inhibited the Ca²⁺ response in the preeclamptic samples (335.4±38.4 versus 52.8±18.2 nmol/L, P<0.05). We also tested the specificity of the response by preincubating the IgG (1:10 dilution) from 5 preeclamptic patients with a 7-amino-acid epitope peptide (P1) that corresponds to a portion of that second extracellular loop of the AT₁ receptor (Figure 5A). This P1 peptide inhibited the Ca²⁺ response of the preeclamptic samples tested (251.8±48.1 versus 101.3±23.6 nmol/L, P<0.05). As a nonspecific peptide control, the effect of another peptide, P2, which corresponds to a different region of the AT₁ receptor, was also tested. This peptide had no inhibitory effect on Ca²⁺ mobilization (251.8±48.1 versus 316.7±41.9 nmol/L, P<0.05).

Next, we examined the specificity of the Ca²⁺ response seen in the 4 normotensive individuals who showed a Ca²⁺ response by pretreating cells with losartan (Figure 5B). The average value for increased intracellular [Ca²⁺] for IgG from these 4 normotensive individuals is 391.7±86.1 nmol/L. After pretreatment with 1 µmol/L losartan, the Ca²⁺ peak reached 379.1±69.6 nmol/L. These data suggest that IgG from these 4 normotensive individuals shows a losartan-insensitive Ca²⁺ response, indicating that Ca²⁺ mobilization in these cases was not mediated by AT₁ receptor activation. Overall, the data in Figure 5 suggest that IgG from preeclamptic patients stimulates Ca²⁺ mobilization by AT₁ receptor activation. In contrast, IgG from normotensive individuals does not activate AT₁ receptors leading to Ca²⁺ mobilization.

IgG From Preeclamptic Individuals Activates the Expression of a Reporter Gene–Associated With Calcium-Mediated Intracellular Signaling
To determine whether Ca²⁺ mobilization stimulated by IgG from preeclamptic patients could result in the activation of Ca²⁺-responsive genes, we chose to examine the activation of an NFAT luciferase reporter gene. It has been shown that Ca²⁺, through the action of calcineurin/calmodulin, stimulates the dephosphorylation of NFAT, resulting in its translocation to the nucleus. Dephosphorylated nuclear NFAT participates in the activation of specifically targeted genes. We used a synthetic promoter construct containing 4 copies of NFAT cis-acting elements linked to the firefly luciferase reporter gene to test whether Ang II as well as IgG from preeclamptic and normotensive pregnant individuals could activate the NFAT transcription factor (Figure 6) resulting in an increase in the synthesis of luciferase. First, we examined the ability of Ang II to activate the NFAT/luciferase reporter construct (Figure 6A). The results (Figure 6A) show a dose-dependent activation of the NFAT/luciferase construct that was inhibited by losartan. Next, we determined the ability of IgG from preeclamptic and normotensive pregnant individuals to activate this promoter construct. We observed activation of NFAT-luciferase constructs by 2 different dilutions of preeclamptic IgG (Figure 6B). In addition, we tested the ability of IgG (1:10 dilution) from 2 different preeclamptic and 2 normotensive individuals to activate the reporter construct (Figure 6C). These normotensive individuals were among those 10 normotensive patients with little or no Ca²⁺ response. We found that IgG from the preeclamptic individuals activated the NFAT/luciferase reporter gene, whereas IgG from the normotensive pregnant individuals did not. These data indicate that IgG from preeclamptic individuals activated a calcium-responsive reporter gene, whereas IgG from normotensive pregnant individuals did not.

View larger version (17K): [in this window] [in a new window]   Figure 6. AT1-AAs activate NFAT. A, Ang II induced NFAT-luciferase construct in a dose-dependent manner. Transfected CHO.AT1A cells were treated with different concentrations (0, 0.01, 0.1, and 1.0 µmol/L) of Ang II, and activation of NFAT luciferase reporter gene was measured. B, Stimulation of NFAT-luciferase by different dilutions of AT1-AAs. Transfected cells were treated with 0, 1:10, and 1:5 dilutions of preeclamptic IgG. C, Similarly, 2 different IgGs from preeclamptic (PE) and normotensive (N) IgG at 1:10 dilution were incubated with transfected cells. Ang II at 0.1 µmol/L was added as a positive control. *P<0.05. LST indicates losartan.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Preeclampsia is associated with abnormalities in Ca²⁺ metabolism and increased intracellular Ca²⁺ levels in a variety of cell types, such as platelets, erythrocytes, and lymphocytes.^2–4 In this study, we tested the hypothesis that AT₁ receptor–activating antibodies (AT₁-AAs) are responsible for increased intracellular Ca²⁺ levels in preeclampsia. As a model system for our studies, we used Chinese hamster ovary cells stably transformed with a minigene encoding the rat AT₁A angiotensin receptor. With these cells, we tested the ability of AT₁-AAs to mobilize Ca²⁺ and to activate a calcium-responsive reporter gene. Our findings suggest that AT₁-AAs activate AT₁ receptors in a dose-dependent manner, leading to increased intracellular Ca²⁺ concentrations. Thus, AT₁-AAs have the potential to account for increased basal Ca²⁺ concentrations in a variety of different cell types associated with preeclampsia.

Using Ca²⁺ mobilization as an indicator for AT₁ receptor activation, we determined that AT₁-AAs were highly associated with the severe preeclampsia group. AT₁-AAs were seldom seen in normotensive pregnant individuals. Immunoglobulin from all preeclamptic patients activated AT₁ receptors and increased intracellular free [Ca²⁺] in >50% (average, 79.5±4.8%) of the CHO.AT1A cells. The specificity of the increased intracellular Ca²⁺ response by AT₁-AAs in preeclampsia was confirmed by use of an AT₁ receptor antagonist as well as an AT₁ receptor epitope peptide. Both losartan and P1, an epitope representing a portion of the second extracellular loop, inhibited the increased Ca²⁺ response stimulated by AT₁-AAs. These results suggest that AT₁-AAs are capable of mobilizing calcium specifically by activating AT₁ receptors. These studies are consistent with previous reports showing that AT₁-AAs stimulate AT₁ receptors, resulting in other biological responses, and that AT₁-AAs are highly associated with preeclampsia.^8,11–13

The IgG from most normotensive individuals was capable of activating the AT₁ receptor in no more than 20% of the treated cells. However, increased intracellular Ca²⁺ mobilization was observed in 4 of 14 normotensive pregnant women. This effect was not inhibited by losartan indicating that Ca²⁺ mobilization activated by these IgG fractions was not mediated through AT₁ receptor activation. This suggests that these effects may be mediated by other antibodies circulating in these women and activating different receptors, such as the ₁- or ß₁-adrenergic receptors or the muscarinic receptors or by other immune components.^14,15 If antibodies to these receptors exist in normotensive pregnant individuals, it will be interesting to determine whether they are also found in normotensive nonpregnant women. Additional studies using serum of nonpregnant individuals could provide further insight.

We were able to assess the biological consequences of Ca²⁺ mobilization using a transcriptional activation assay. Activation of Ca²⁺ mobilization should lead to changes in the expression of specific genes. To address this possibility, we used a firefly luciferase reporter construct under the control of the NFAT transcription factor. NFAT is activated by mobilized Ca²⁺ via calcineurin, a phosphatase catalyzing the dephosphorylation of NFAT and allowing its translocation into the nucleus.¹⁶ Nuclear NFAT interacts with cis-acting elements in the NFAT reporter construct, allowing the luciferase reporter to be expressed. We observed a dose-dependent activation of an NFAT luciferase reporter construct that parallels Ca²⁺ mobilization. These results revealed the ability of AT₁-AAs to alter gene expression via increased calcium signaling. Furthermore, activation of NFAT has been shown to regulate the expression of downstream genes encoding plasminogen activator inhibitor-1, interleukin 8, and tumor necrosis factor-, proteins that are elevated in the plasma of preeclamptic patients.^16–20 Thus, increased calcium mobilization resulting from AT₁ receptor activation by AT₁-AAs may underlie many of the cellular changes associated with preeclampsia (Figure 7).

View larger version (12K): [in this window] [in a new window]   Figure 7. Role of AT1-AAs and associated pathophysiologies in preeclampsia. Various cell model systems have been used to determine role of AT1-AAs. AT1-AAs can interact with AT1 receptors on a variety of cell types to provoke a variety of biological responses relevant to preeclampsia. This diagram represents responses that AT1-AA has been shown to have that affect in vitro systems and its possible role in Ca2+-mediated physiological effects.

In view of the available data, we hypothesize that the biological activity of maternal antibodies that activate AT₁ receptors may be responsible for many aspects of preeclampsia. AT₁-AAs are not likely to be secondary to hypertension-related vascular injury, because the antibodies are seldom associated with essential hypertension.²¹ Furthermore, according to Wallukat et al⁸ in their initial study, AT₁-AAs were not associated with 10 pregnant patients with preexisting essential hypertension. Thus, even the combination of essential hypertension and pregnancy does not automatically result in the production of AT₁-AAs. In preeclampsia, AT₁-AAs presumably activate AT₁ receptors present in many cell types found in a number of organs and systems, including the heart, kidneys, placenta, central nervous system, and peripheral vessels. The diverse consequences of widespread AT₁ receptor activation may account for the clinical complexity of preeclampsia. However, many questions regarding the role of AT₁-AAs in preeclampsia remain unanswered. When does AT₁-AA appear during pregnancy? Is there a cause-and-effect relationship between AT₁-AAs and disease development? What is the relative concentration of the antibody in relation to the disease severity? What is the fate of AT₁-AAs after pregnancy? To further understand the role of AT₁-AAs in development of preeclampsia, sensitive, reliable, and convenient high-throughput biological and immunochemical assays that accurately detect AT₁-AAs are needed. There is a potential for the NFAT transfection assay to serve as the basis for development of a high-throughput biological assay to detect the presence of AT₁-AAs.

	Acknowledgments

 
This work was supported by a grant from the National Institutes of Health (HD-34130) to Dr Kellems. We thank Dr Terry S. Elton (Brigham Young University, Provo, UT) for his generosity in providing CHO.AT1A cells and Merck Research Laboratory (Rahway, NJ) for providing losartan.

	Footnotes

 
Guest Editor for this article was Joseph Loscalzo, MD, PhD.

	References

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