Objective
The purpose of this study was to determine whether fetuin-A affects trophoblast viability and invasion, whether growth factors that bind receptors that activate tyrosine kinase are impaired by fetuin-A, and whether elevated maternal serum fetuin-A levels are associated with adverse pregnancy outcomes.
Study Design
We studied viability and invasion in first-trimester extravillous trophoblast cells that were exposed to fetuin-A, insulin-like growth factor, and placental growth factor. Insulin receptor substrates expression was assessed. We compared serum fetuin-A levels in 111 preeclampsia cases and 95 controls.
Results
Fetuin-A reduced extravillous trophoblast cell viability and invasion in the presence or absence of growth factors. Fetuin-A reduced insulin receptor substrate–1 and tyrosine phosphorylated insulin receptor substrate–1 expression in extravillous trophoblast cells that had been treated with insulin-like growth factor. Elevated serum fetuin-A levels were more prevalent in preeclampsia cases than control subjects, even after we controlled for diabetes mellitus, hypertension, and obesity.
Conclusion
Fetuin-A may decrease trophoblast viability and invasion caused by the inhibition of receptor tyrosine kinase activity. Elevated serum levels of fetuin-A may be associated with preeclampsia.
Human fetuin-A is a glycoprotein secreted by the liver that modulates insulin action in adipocytes and skeletal myocytes. Elevated serum fetuin-A levels are associated with insulin resistance and metabolic syndrome. When the insulin receptor is activated, a conformational change occurs in the tyrosine kinase domain that leads to autophosphorylation and to the release of different second messengers. Fetuin-A inhibits the tyrosine kinase activity of the insulin receptor that results in decreased rates of autophosphorylation and downstream signaling intermediates such as insulin receptor substrates (IRS).
Several growth factors that promote trophoblast invasion (insulin-like growth factor-1 [IGF-1], epidermal growth factor [EGF], and placental growth factor [PlGF]) bind to receptors that activate tyrosine kinase. We hypothesized that, analogous to its action on the insulin receptor, fetuin-A inhibits the receptor tyrosine kinase activity of trophoblast growth factors and, as a consequence affects trophoblast growth and viability, results in impaired invasion into the uterine compartment. Consequently, we sought to determine whether treating trophoblast cells with fetuin-A reduces cell viability and invasion and whether elevated fetuin-A levels in maternal serum are associated with adverse obstetric outcomes as the result of failed trophoblast invasion.
Materials and Methods
In vitro experiments with extravillous trophoblast (EVT) cells
We conducted in vitro experiments utilizing primary human EVT cells that display invasive properties through extracellular matrices (ECM). EVT cells were obtained from first-trimester placental tissues (8-13 weeks gestation). Briefly, finely minced chorionic villi were cultured at 37°C with Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY). All cell media contained gentamicin (100 μg/mL) and amphotericin B (2.5 μg/mL). Cells that outgrew from attached villous fragments were separated on days 10-12 of culture. The isolated cells were seeded and propagated in the same culture medium. The EVT cells that were used in these experiments were characterized by immunostaining of the trophoblast cell markers human leukocyte antigen–G and cytokeratin filaments 8 and 18 ( Figure 1 , A-C ). In previous cell preparations in our laboratory, >95% of isolated trophoblast cells stained positive for cytokeratin-18 and human leukocyte antigen–G. In this study, further characterization of the purity of the EVT cells that were used in our assays was accomplished by cytokeratin-7 and integrin alpha-I staining as shown by other investigators ( Figure 1 , D and E).
EVT cells do not express fetuin-A. We exposed EVT cells to commercially available human fetuin-A (Sigma-Aldrich, St. Louis, MO) by adding fetuin-A to culture media at different physiologically relevant concentrations that ranged from 150-900 μg/mL.
We used 2 negative controls: EVT cells that were exposed (1) to culture media without fetuin-A and (2) to media that contained only the blank buffer that was used (vehicle control) to reconstitute fetuin-A. Because fetal bovine serum, a usual component of culture medium, contains fetuin-A of bovine origin, we abstained from using fetal bovine serum in our cell media to avoid a cross reaction that could have altered our results.
Functional assays
Primary EVT cells and culture medium were collected to perform functional assays (cell viability and invasion assays). For these experiments, we used 1 × 10 4 cells per well in 96-well plates (1 × 10 6 cells/mL).
We assessed cell viability using Cyto Tox 96 Non-Radioactive Cytotoxicity Assay kit (Promega Corp, Madison, WI). After 48-hour exposure to fetuin-A, cell medium was collected from exposed and nonexposed cells and transferred to 96-well plates to assess lactate dehydrogenase release. After the addition of a reconstituted substrate mix followed by a stop solution provided with the kit, absorbance that was based on the reaction between lactate dehydrogenase and substrate was recorded at 490 nm, and the percentage of cell cytotoxicity was determined.
Invasion of EVT cells through the ECM Matrigel (BD Biosciences, San Jose, CA) was determined 48 hours after fetuin-A exposure with Cell Invasion Assay Kits (Chemicon International, Temecula, CA). After 48 hours, cells that had been placed in invasion chambers that invaded through the ECM Matrigel and across the semipermeable membrane were stained and treated with 10% acetic acid. A volume of 150 μL of the dye/solute mixture was transferred to 96-well plates, and invasion was measured by colorimetric absorbance at an optical density of 560 nm. Levels of invasion were determined by comparison of average optical density values of fetuin-A–exposed cells with those of nonexposed cells. Invasion rates were normalized to 1.0 in cells not exposed to fetuin-A.
Invasion assays with trophoblast growth factors
To investigate whether fetuin-A interferes with the action of trophoblast growth factors for which receptors exhibit tyrosine kinase activity, we performed separate invasion assays using the same methods described earlier. We used fetuin-A concentrations of 600 μg/mL in cell media because our initial results demonstrated significantly decreased viability and invasion only when EVT cells were exposed to fetuin-A concentrations of ≥600 μg/mL.
For these experiments, first-trimester EVT cells were cultured in media that contained (1) standard Dulbecco’s modified Eagle’s medium without fetuin-A (baseline control); (2) fetuin-A 600 μg/mL; (3) individual trophoblast growth factors: IGF-1 10 ng/mL, EGF 50 ng/mL, or PlGF 10 ng/mL; or (4) these same growth factors at the aforementioned concentrations in the presence of fetuin-A 600 μg/mL that was added 2 hours later.
Assessment of cell invasion was performed at 48 hours as explained earlier.
Western blotting
To determine whether fetuin-A is associated with diminished downstream signaling because of receptor tyrosine kinase activity inhibition, the intracellular expressions of IRS-1 and tyrosine phosphorylated IRS-1 (tp-IRS-1) were assessed in cell lysates from EVT cells that had been exposed to trophoblast growth factors and fetuin-A under the same 4 conditions described earlier. Cells were lysed in a lysis buffer at 0°C. The lysate was centrifuged at 16,000 g for 15 minutes; the supernatant was recovered and assayed for protein concentration measurement, and a standard reference curve was obtained with the use of bovine albumin. Equal amounts of protein extracts (40 μg) were used for standard Western blot as described elsewhere. Primary anti–IRS-1 and anti–tp-IRS-1 antibodies (Upstate Biotechnology, Inc, Lake Placid, NY) and secondary anti-rabbit horseradish peroxidase–linked whole antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA) were used for these experiments. The differences in trophoblast lysate IRS-1 and tp-IRS-1 expression between (1) cells that were exposed to 600 μg/mL fetuin-A after being treated with trophoblast growth factors and (2) cells that were treated with growth factors, but not exposed to fetuin-A, were analyzed by scanning densitometry. Band intensity was expressed in arbitrary optic densitometry units. Beta-actin antibody (Upstate Biotechnology, Inc) was used as a loading control; normalization to beta-actin was performed.
All the aforementioned in vitro experiments were conducted in triplicate with different sets of EVT cells and repeated at least twice. Mean values were subjected to statistical analyses.
Case-control study design
To correlate our in vitro findings with pregnancy outcomes, we conducted a secondary analysis of an existing case-control study. In this study, serum samples were collected prospectively after informed consent had been obtained during the third trimester from women who had been admitted in labor to the Hospital of the University of Pennsylvania. Cases consisted of women whose condition had been diagnosed as preeclampsia that was defined according to the American College of Obstetricians and Gynecologists criteria. Control subjects included women who delivered at term with no obstetric complications. A detailed description of the Preeclampsia: Mechanisms and Consequences study is provided elsewhere.
Enzyme-linked immunosorbent assay (ELISA) experiments to detect fetuin-A in maternal serum
Blood was obtained from subjects in the third trimester within 24 hours of enrollment. Samples were centrifuged for 10 minutes at 10,000 g , and the supernatant (serum) was aspirated, distributed in aliquots into separate tubes, and stored at –80°C. Serum samples were available for 206 women (111 cases and 95 control subjects) who were enrolled in the Preeclampsia: Mechanisms and Consequences study. Commercially available ELISA kits (BioVendor LLC, Candler, NC) that contained fetuin-A (positive control) and dilution buffer (negative control) were used in 96-well plates (Nunc, Roskilde, Denmark). We followed the manufacturer’s protocol: 10 μL of serum samples were diluted 1/10,000 with various dilution buffers. Duplicates of 100 μL of the diluted serum, standard positive control samples, quality control samples, and negative control samples were incubated for 1 hour at 300 rpm on an orbital microplate shaker. After the plates were washed, the wells were incubated with conjugate solution for 1 hour, washed again, and incubated with substrate solution with no exposure to sunlight. The reaction was stopped at 10 minutes, and the absorbance (optical density) was read at 450 nm with an ELISA photometer (VWR International, Bridgeport, NJ). Each assay was performed in duplicate and the average optical density was calculated.
Statistical analysis
SPSS statistical software (SPSS Inc, Chicago, IL) was used for all analyses. Mean optical density values and standard errors were calculated and compared between exposed and control cells with Kruskal-Wallis analysis. Mean and median values and standard deviations were used for comparison of demographics and outcomes data between cases and control subjects. χ 2 or Fisher exact tests for dichotomous variables, t tests for continuous variables, and logistic regression analyses were used where applicable. A probability value of .05 was indicative of statistical significance.
Results
Functional assays
EVT cells that were exposed for 48 hours to increasing concentrations of fetuin-A displayed a dose-response reduction in their viability and invasive properties compared with nonexposed control cells. At 48 hours, the viability of EVT cells was significantly reduced when exposed only to 600 and 900 μg/mL (but not to lower concentrations) of fetuin-A compared with nonexposed cells and cells that had been treated with vehicle control: 66.9% (range, 63.9–71.2%) viable, 63.5% (range, 60.8–67.7%), 89.9% (range, 86.2–95.5%), and 86.5% (range, 84.6–92.0%), respectively ( P < .01; Figure 2 ). Similarly, trophoblast cells that were exposed to 600 and 900 μg/mL of fetuin-A showed a 28% (range, 26.1–32.0%) and 30% (range, 29.2–31.7%) invasion decrease through the ECM, respectively ( P < .01). At lower concentrations of fetuin-A, EVT cells exhibited a nonsignificant decrease in invasion through the ECM compared with control cells ( Figure 3 ).
Invasion assays with trophoblast growth factors
Trophoblast growth factors increased the invasion of EVT cells through the ECM at 48 hours: 26.7% increase with IGF-1 ( P = .004), 12.3% increase with EGF ( P = .057), and 44.9% increase with PlGF ( P = .02) compared with baseline control cells ( Figure 4 ). The addition of 600 μg/mL fetuin-A to cell media after EVT cells were treated with growth factors resulted in decreased cell invasion compared with baseline control cells: 22.1% decrease for IGF-1 + fetuin-A ( P = .01), 10.1% decrease for EGF + fetuin-A ( P = .16), and 30.3% decrease for PlGF + fetuin-A ( P = .007; Figure 4 ).
Western blotting
Intracellular expression of tyrosine kinase activity that signaled intermediates (IRS-1 and tp-IRS-1) was reduced uniformly in EVT cells that were treated with growth factors after being exposed to 600 μg/mL fetuin-A compared with cells not exposed to fetuin-A. This difference was significant for those cells that were treated with IGF-1 + fetuin-A compared with those cells that were treated with IGF-1 only ( Figure 5 ; IRS-1 content 117 ± 13 vs 50 ± 11 optic densitometry units, respectively [ P = .02; Figure 5 , A] and tp-IRS-1 content 193 ± 11 vs 85 ± 14 optic densitometry units, respectively [ P = .03; Figure 5 , B]). A nonsignificant trend for lower expression of IRS-1 and tp-IRS-1 was observed when cells that had been treated with EGF and PlGF were exposed to fetuin-A compared with nonexposed EVT cells ( P = NS; data not shown).
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