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Published online before print October 30, 2008
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From the Department of Immunology,* and the Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas; Biocept, Inc., San Diego, California; and the Departments of Internal Medicine, Microbiology and Immunology, and Pathology, University of Texas Medical Branch, Galveston, Texas
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stable and PCR-amplifiable fetal cell-free DNA (cfDNA) has been detected in maternal plasma and used to predict6,7 and monitor8 the severity of complicated pregnancies. Yet, the low percentage of fetal cfDNA (3% to 6%) in maternal plasma limits its use for routine clinical applications. As a result, many groups, including ours, have focused on methods to enhance recovery and analysis of circulating fetal cfDNA. To achieve this goal, a better understanding of the molecular nature and kinetics of circulating (fetal) DNA is necessary. We hypothesize that circulating DNA could exist as free DNA subject to degradation as well as DNA contained in membrane bound MPs, providing protection from endonucleases. In 2005, we proposed that a significant portion of circulating DNA, including fetal DNA, was membrane bound and suitable for enrichment.9 We reported a 10-fold enrichment of fetal cfDNA from maternal plasma from normal pregnancies using the flow cytometric parameters of forward light scatter and acridine orange (AO) staining of nucleic acids. Although the nature and origin of the AO+ DNA was unknown, we suggested that the mechanism of release of fetal cfDNA might be due to placental apoptosis resulting in release of fetal DNA containing MPs into the circulation. The plausibility of this hypothesis was supported by data concerning turnover of syncytiotrophoblast and release of syncytiotrophoblast MPs (STBMs) into the maternal circulation.10 In addition, Gupta et al detected fetal nucleic acids in the supernatant of STBM samples prepared from placental explants.11 Other studies have also examined STBMs from preeclamptic patients using an enzyme-linked immunosorbent assay based method for quantification.12-14 Whether the STBMs contain nucleic acids similar to the AO+ MPs sorted from maternal plasma has not been studied.
One of the biochemical hallmarks of apoptosis is fragmented DNA. DNA is cleaved between nucleosomes, forming a "ladder" with spacing about 180 bp. Given that the majority of fetal cfDNA is relatively small (<300 bp, as detected by PCR) compared to maternal cfDNA,15 fetal DNA is likely released from apoptotic cells. However, the basis for fetal DNA stability has not been examined. More recently, Tjoa et al demonstrated an association of fetal cfDNA (as detected by β-globin via real-time PCR) and cell death in supernatants from third trimester placental cultures after oxidative stress,16 supporting the hypothesis that fetal cfDNA originates from the placenta via hypoxia-induced cell death, apoptosis and necrosis. However, the DNA detected was not examined as to source or characteristics.
Recovery of trophoblast MPs containing fetal DNA during the first trimester would be of clinical interest to prenatal genetic diagnosis. However, under hypoxic conditions, few syncytiotrophoblasts are detected in first trimester placental explants17 and circulating STBMs are not found until the second and third trimester.13 By contrast, invasive extravillous trophoblasts (EVTs) are most abundant during the first trimester.18 In addition, apoptosis limits invading EVTs during the first trimester,19 suggesting that MPs released by dying EVTs might enter the maternal circulation in the first trimester.
Primary trophoblasts isolated from villous explant cultures contain a heterogeneous population of cytotrophoblasts, syncytiotrophoblasts, fibroblasts, and macrophages.20,21 JEG-3 cells, which model invasive EVTs, most relevant for early genetic testing, have the advantage of being homogeneous with less spontaneous cell death than found with primary trophoblasts.22 Therefore, we used JEG-3 cells to represent EVTs.
Our studies show that cfDNA is contained within membranous MPs derived from dying JEG-3 cells. We also showed for the first time that the DNA within the apoptotic MPs contain fragmented DNA that is protected from plasma DNases. In addition, light scatter and DNA staining properties of in vitro cell derived MPs and maternal plasma MPs are similar, suggesting a common origin or mechanism of release. We also show that in vitro MPs and MPs from normal and preeclamptic pregnancies stain with the DNA dye, PicoGreen. Finally, we show for the first time that significantly higher numbers of DNA+ MPs are found in preeclamptic patients compared to control patients, suggesting that measuring such DNA+ MPs could help predict or monitor preeclampsia in a non-invasive manner. Other clinical and pathological conditions associated with increased levels of cell death (ie, trauma, stroke, organ transplantation, autoimmunity, and cancer) might also benefit from our discovery of circulating nucleic acids containing protected MPs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human trophoblastic cell line, JEG-3 (male), was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in minimum essential medium, supplemented with heat inactivated 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 10 mmol/L HEPES, and 0.1 mmol/L non-essential amino acids (Invitrogen, Carlsbad, CA). The cultures were maintained at 37°C in an atmosphere of 5% CO2.
Induction of Cell Death
Two million cells were cultured in the presence or absence of either 50 µmol/L rotenone, which disrupts the mitochondrial electron transport chain to chemically mimic hypoxia (Sigma-Aldrich, St Louis, MO), or 30 µmol/L etoposide, a DNA topoisomerase II inhibitor (Sigma-Aldrich) that induces apoptosis. For necrosis, cells were heated for 1 hour at 60°C. Twenty-four and 48 hours after induction of cell death, floating cells were collected and adherent cells were detached with trypsin/EDTA (Invitrogen). The cells were pooled, centrifuged at 300 x g at room temperature, rinsed in PBS (pH 7.1; Invitrogen) and centrifuged at 300 x g.
Assessment of Apoptosis by Flow Cytometry
Quantification of Subdiploid Cells
Apoptotic cell death was confirmed by flow cytometric analysis of fixed (permeabilized) cells with a subdiploid DNA content.23 While gently vortexing, 1 x 106 cells (counted with a hemocytometer) were resuspended in 2 ml of 0.9% NaCl, followed by fixation with 5 ml ice cold 70% ethanol added dropwise. Cells were incubated for 1 hour at room temperature, centrifuged, and resuspended in 1 ml PBS containing 50 µg/ml propidium iodide (PI) (Sigma-Aldrich) and 0.1 mg/ml RNase A (Sigma-Aldrich), incubated for 1 hour at 37°C in the dark. Cells were analyzed using a Beckman Coulter EPICS XL2 (Beckman Coulter, Miami Lakes, FL). All data analysis was performed using EXPO32 software (Beckman Coulter).
Cell Viability
PI was used to test for membrane integrity using unfixed cells as previously described.23 One million (nonpermeabilized) cells were resuspended in PBS containing 50 µg/ml PI and analyzed by flow cytometry.
Isolation and Quantification of in Vitro MPs
Isolation and quantification of MPs are performed before all experiments. Double filtered (0.25 µm) PBS (dfPBS) was used in all MP experiments. MPs were centrifuged at room temperature, unless otherwise stated.
Isolation of MPs
MPs from 24 and 48 hours apoptotic and necrotic supernatants were separated from detached cells by two centrifugation steps (300 x g, 5 minutes; 800 x g, 5 minutes) and transferred to a 50 ml conical tube.
Quantification of MPs
MP concentration was determined as previously described.24 Briefly, 100 µl of fluorescent beads (counted at 500/µl with a hemocytometer) were added to 400 µl of each supernatant and 300 µl of dfPBS for a total volume of 800 µl and the number of beads counted was stopped at 10,000. After subtracting the number of background fluorescent beads, the concentration of MPs was determined and the appropriate number of MPs was centrifuged (25,000 x g, 1 hour at 4°C; in a SW40 Ti Swinging-Bucket Rotor in a Beckman L8-M, Class H, ultracentrifuge; Beckman Coulter).
Hoechst 33342 Labeling of MPs
Because lipid membrane MPs protect cell-free RNA (cfRNA) from RNase A activity (Table 1)
, we found it necessary to fix the MPs with 70% ethanol to permeabilize the membranes, before RNase treatment. Two million MPs were fixed with 70% ethanol (as described above) and centrifuged (13,000 rpm, 10 minutes) using an Eppendorf Centrifuge 5415c (Eppendorf, Westbury, NY). The MP pellets were resuspended in 500 µl PBS containing 10 µg/ml of Hoechst 33342 (Invitrogen), with or without 50 µl of 1 mg/ml RNase A. After incubating the samples for 20 minutes at 37°C in a water bath, the samples were placed on ice and analyzed using an LSR II flow cytometer (BD Instruments, San Jose, CA).
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MPs (2 x 106) in 50 µl were placed on a glass slide (Superfrost Plus Micro Slides; VWR Scientific, West Chester, PA) and air-dried at room temperature. The slides were fixed in a Coplin jar containing 25 ml of 100% methanol for 10 minutes and allowed to air-dry. The slides were immersed in a second Coplin jar containing 25 ml of 2x standard saline citrate with 0.2% NP40 for 1 minute and the edges were dried with a tissue. A final concentration of 4,6-diamidino-2-phenylindole (DAPI II, 1 µg/ml, Invitrogen) was placed on the surface of a glass coverslip, inverted onto a slide and analyzed by fluorescence microscopy.
Fluorescence Microscopy
Images of the DAPI-stained MPs were captured through an Olympus BX51 microscope with automated x-, y-, and z-stage movement and a Hamamatsu ORCA-2 Digital Camera using a x100 objective (Hamamatsu Photonics, Hamamatsu, Japan). Data were analyzed using Advanced Digital Imaging Research automated interphase FISH Scanner software (Advanced Digital Imaging Research L.L.C., League City, TX). Each slide was autoscanned for single MPs ranging 1 µm to 6 µm in diameter to avoid clustering of MPs or possible cell contamination. An average of 3000 to 8000 images was taken per slide. Electronic gates (boxes 1 µm to 6 µm in length) were placed around each MP from randomly selected images and the integrated fluorescence intensity was determined. One hundred MPs per slide, per experiment were counted. The amount of DNA per particle was reported as the mean integrated fluorescence intensity (MIFI).
Real-Time PCR
DNA Extraction
DNA was extracted from 1 to 2 million MPs using the QIAamp DNA blood kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. All DNA samples were stored at 4°C before analysis.
PCR Analysis
Real-time PCR was performed as previously described.25 For both the β-globin (102 bp) and sex-determining region Y (SRY, 72 bp), quantitative real-time (RT)-PCR was performed using the Applied Biosystems 7700 sequence detection system (Applied Biosystems, Foster City, CA). Primer and probe sequences were as follows: SRY forward primer: 5':-TGCACAGAGAGAAATACCCGAATTA-3'; SRY reverse primer: 5':-TGCATTCTTCGGCAGCAT-3'; SRY TaqMan probe: 5':-AAGTATCGACCTCGTCGGAAGGCGAA-3'; β-globin forward primer: 5':-GTGCACCTGACTCCTGAGGAGA-3'; β-globin reverse primer: 5':-CCTTGATACCAACCTGCCCAG-3'; β-globin TaqMan probe: 5':-AAGGTGAACGTGGATGAAGTTGGTGG-3'.
Quantification of total and fetal DNA as genome equivalents was based on copies of β-globin and SRY sequences detected. Each reaction contained 5 µl of extracted DNA. Each reaction plate was run simultaneously with a duplicate calibration curve of titrated DNA (standard curve). Each sample was run in triplicate for both loci and the mean of the values was determined using the 7700 software and the standard curve of known DNA concentrations. To determine the relative amount of DNA per MP, the total (β-globin) and fetal DNA (SRY) genome equivalents were divided by the number of MPs.
Apoptotic DNA Ladder Assay
DNA was extracted from fifteen million MPs and analyzed on a 2% agarose gel with a 1-kb DNA ladder (Invitrogen). MPs were resuspended in 300 µl of lysis buffer (10 mmol/L Tris-HCl-EDTA) with 50 µg/ml RNase A for 1 hour at 37°C. MPs were incubated overnight (
12 hours) at 37°C in 0.05% SDS with 2 mg/ml of Proteinase K (Sigma-Aldrich). An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1; Invitrogen) was added and the tubes were gently inverted twenty times. The samples were centrifuged (13,000 rpm, 10 minutes) and the top layer (aqueous) was removed and transferred to second tube. An equal volume of chloroform (99+%; Sigma-Aldrich) was added and the samples were centrifuged (13,000 rpm, 10 minutes). The top layer (aqueous phase) was removed and transferred to a third tube. DNA was precipitated by adding one-third volume of 7.5 mol/L ammonium acetate (Sigma-Aldrich), followed by 2.5 volumes of absolute ethanol, and precipitated at –80°C for 30 minutes. The precipitates were collected by centrifugation (13,000 rpm, 10 minutes) and the pellets were washed with 70% ethanol and air-dried. The proteinase K, phenol/chloroform/isoamyl alcohol (25:24:1), and DNA precipitation steps were repeated a second time. The final DNA pellet was air-dried and resuspended in 60 µl of Tris-HCl-EDTA (10 mmol/L) and DNA concentration determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Three micrograms of DNA were electrophoresed on a 2% agarose gel (stained with 0.2 µg/ml ethidium bromide) and analyzed with Kodak Image Station 4000 mm Pro (Eastman Kodak Company, Molecular Imaging Systems, Rochester, NY).
Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling Assay
DNA nicks were detected by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay as previously described26 with slight modifications. Analysis was by flow cytometry and fluorescence microscopy with the following buffers: 5x TdT reaction buffer (1 mol/L potassium cacodylate [Sigma-Aldrich], 125 mmol/L Tris-Cl, pH 6.6, and 1.25 mg/ml bovine serum albumin [Sigma-Aldrich] stored at –20°C); and fluorescein isothiocyanate-conjugated anti-bromolated deoxyuridine triphosphates (Br-dUTP) monoclonal antibody solution (Anti-Br-dUTP fluorescein isothiocyanate-conjugated monoclonal antibody, 0.5 µg, Phoenix Flow Systems, San Diego, CA; 0.3% (v/v) Triton X-100; 1% (w/v) bovine serum albumin (Sigma-Aldrich); and PBS to 100 µl (stored at 4°C).
Flow Cytometry
Briefly, two million MPs from chemically-induced hypoxic cells and heat stress (HS)-treated cells (at 60°C) were pre-fixed in 1 ml of 2% methanol-free formaldehyde (Electron Microscopy Sciences, Hatfield, PA) diluted in PBS on ice for 1 hour. The MPs were centrifuged (25,000 x g, 1 hour at 4°C) and postfixed in 1 ml of ice-cold 70% ethanol for 5 hours at –20°C. MPs were centrifuged (13,000 RPM, 10 minutes), the ethanol removed, and the MPs washed in 1 ml of PBS. MPs were resuspended in 50 µl of labeling buffer (10 µl 5x TdT reaction buffer, 2 µl Br-dUTP stock solution [2 mmol/L Br-dUTP; Sigma-Aldrich], 0.5 µl [12.5U] TdT enzyme [Invitrogen], 5 µl CoCl2 [10 mmol/L; Sigma-Aldrich] and 33 µl distilled water) and incubated at room temperature overnight. Three hundred microliters of rinsing buffer (0.1% [v/v] Triton X-100, 5 mg/ml BSA, and PBS, pH 7.4) was added and the MPs centrifuged (13,000 RPM, 10 minutes). The supernatant was removed and 100 µl fluorescein isothiocyanate-conjugated anti-Br-dUTP monoclonal antibody was added to the MPs and incubated for 1 hour at room temperature. Four hundred microliters of PBS-based PI staining solution (50 µl of 1 mg/ml PI [Sigma-Aldrich] and 50 µl of 1 mg/ml RNase A [Sigma-Aldrich]) was added and incubated for 30 minutes at room temperature in the dark. The samples were analyzed by a Beckman Coulter EPICS XL2.
Fluorescence Microscopy
After labeling MPs with Br-dUTP incubated at room temperature overnight, a 50 µl aliquot of two million apoptotic or necrotic MPs was air-dried onto slides. MPs were counterstained with DAPI II (Invitrogen, as described above). The double labeled MPs were analyzed with an Olympus BX-51 Microscope and photographed with a Hamamatsu ORCA-2 Digital Camera using an x100 objective (Hamamatsu, Photonics). Scale bars are 10 µm.
PKH26 and Hoechst 33342 Labeling of MPs
Two million MPs from each sample were labeled with PKH26 Red Fluorescent Cell Linker Kit (Sigma) as described by the manufacturer with slight modifications. MPs were washed once with PBS (Invitrogen) and stained with 100 µl of staining solution (5 x 10–5 mol/L PKH26 in diluent C) for 4 minutes at room temperature. The staining reaction was stopped by addition of 100 µl of fetal calf serum. One minute later, 100 µl of complete medium were added and MPs were washed twice in 10% minimum Essential medium, then once in PBS. MPs were resuspended in 500 µl PBS containing 10 µg/ml of Hoechst 33342 (Invitrogen), incubated for 20 minutes in a water bath at 37°C, placed on ice, and analyzed using an LSR II flow cytometer.
Cholera Toxin B and Hoechst 33342 Labeling of MPs
Two million MPs resuspended in 500 µl PBS were stained with 5 µl of cholera toxin B (CTB, 1 mg/ml, Invitrogen) and incubated on ice for 10 minutes. MPs were labeled with 4 µg/ml of Hoechst 33342 (Invitrogen), prepared as described on above, and analyzed using an LSR II flow cytometer.
Plasma DNase Assay
Plasma Samples
After obtaining Institutional Review Board approval from Baylor College of Medicine and written informed consent, 10 ml of peripheral blood was collected from a non-pregnant female in a syringe containing 10 µl/ml of anticoagulant heparin sulfate (5000 U/ml) and allowed to sit at room temperature for 1 hour. Blood was centrifuged for 10 minutes at 800 x g (room temperature), followed by a second centrifugation at 1600 x g. The cell-free plasma was used to incubate the in vitro MPs as described below.
Incubation of in Vitro MPs in Plasma
Five million MPs from each sample were incubated in 800 µl of the non-pregnant female plasma for 24 hours on ice and in a 37°C water bath. DNA was extracted using the MagNA Pure LC System (Roche Diagnostics) according to manufacturer’s instructions. Purified male gDNA (Promega, Madison, WI) and a water blank were used to create a standard curve (1.5 ng to 245 ng DNA) to determine the linearity and range of the PCR products.
PCR Analysis
PCR was performed using primers specific to a 193 bp SRY PCR product, using the following primers: SRY forward primer: 5':-AAAGGCAACGTCCAGGATAGAG-3'; SRY reverse primer: 5':-TGTAATTTCTGTGCCTCCTGGA-3'.
Controls used in all tests included a male positive control for SRY and no DNA (water) control. The PCR reaction (55 µl) included 5 µl 1x PCR buffer, 2 µl 50 mmol/L MgCl, 1.5 µl 100 mmol/L dNTPs, 2.5 µl 100 mmol/L SRY-Forward, 2.5 µl 100 mmol/L SRY-Reverse, 15 µl (30 ng) DNA, 0.3 µl (500 U) Taq polymerase, and 26.2 µl water (Invitrogen). PCR amplification was performed using a hot start method (10 minutes at 95°C.), followed by 35 cycles of denaturation at 94°C for 1 minute, re-annealing at 63.2°C for 1 minute, and extension at 72°C for 1 minute, and one final extension of 10 minutes at 72°C.
DNA Standard Curve
The PCR products were run on a 1% agarose gel, stained with 0.2 µg/ml ethidium bromide, and the band intensities were scanned using the Kodak Digital Science Image Station 440CF (Eastman Kodak Company, Molecular Imaging Systems). Band intensities were determined using the Image J version 1.37v software (developed at the National Institutes of Health). A standard curve was generated by plotting the log of the DNA concentration (x axis) versus ethidium bromide band intensity (y axis). Linear regression analysis was used to determine the slope and intercept for each experiment. The concentration of recovered male DNA was interpolated from a standard curve. The standard curve is representative of three individual experiments (Supplemental Figure 1, see http://ajp.amjpathol.org).
DNase I Sensitivity Assay
To reduce nonspecific binding of PKH26 to the microscope slide, two million MPs from each sample were labeled with 3.6 x 10–5 mol/L PKH26 (Sigma) and washed as previously described above. MPs were resuspended in 24 µl of Promega’s 1x Reaction Buffer and treated with or without 1 U of RNase-free DNase (Promega) for 20 minutes at 37°C. The reaction was stopped by adding 1 µl of Promega’s RQ1 DNase Stop Solution. MPs were fixed as described above, counterstained with DAPI II (Invitrogen), and analyzed with an Olympus BX-51 Microscope as described above.
RNase Protection Assay
Five million MPs were treated with 200 µg/ml or 1000 µg/ml RNase A with and without 0.2% Triton X-100 for 1 hour at 37°C. RNA was isolated using the RNeasy Micro kit (Qiagen Sciences). MPs were lysed using the Qiagen RLT buffer and the lysate homogenized by centrifuging through a QiaShredder (Qiagen Sciences, Valencia, CA). The flow-through was applied to the RNeasy column according to the manufacture’s instructions, treated with DNase I (Promega) and then washed. The RNA was eluted in 14 µl of RNase-free water and subjected to reverse transcription with random primers using Taqman Reverse Transcription Reagents (Applied Biosystems) according to the manufacture’s instructions.
RNA Isolation and Determination
RNA was isolated using the RNeasy Micro kit and treated with DNase I according to the manufacturer’s instructions (Qiagen Sciences, Maryland). RNA was reverse transcribed with random primers using Taqman Reagents (Applied Biosystems), according to the manufacture’s instructions. Reverse Transcription was performed in a PE9700 thermal cycler (Applied Biosystems). After Reverse Transcription, the cDNA products were amplified by real-time PCR using the GeneAmp 7700 sequence detection system and the Taqman Assay (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase mRNA transcript was amplified (Hs00266705-g1 Applied Biosystems) using cDNA. Cycling conditions consisted of a 2-minute incubation at 50°C to activate UNGErase, an initial denaturation step of 95°C for 10 minutes, and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Threshold cycle (Ct) values were used as a measurement of the presence or absence of the transcript. Ct values reflect the cycle number at which the fluorescence generated from accumulating amplicons crosses the background fluorescence. Ct values ranging from 39 to 40 are considered negative for cDNA, suggesting that RNA is not present. At this point during the reaction the number of amplicons is statistically significant above the baseline, indicating the presence of copies of the transcript. Control specimens included reverse transcription blanks and PCR blanks.
Quantification of MPs in Preeclamptic Women
Frozen plasma samples known to come from normal or preeclamptic pregnancies were donated by Dr. Popek (Texas Children’s Hospital, Houston, TX). MP concentrations were determined as previously described in Materials and Methods. Briefly, 20,000 fluorescent beads (counted with a hemocytometer and resuspended in dfPBS) were added to a 1:53.3 dilution of (15 µl) plasma in dfPBS for a total volume of 800 µl and a final concentration of 25 beads/µl. The number of beads counted by flow cytometry (Beckman Coulter EPICS XL2) was stopped at 1000. Next, one million plasma MPs were resuspended in dfPBS for a final volume of 66 µl and labeled with 2 µl of DNA staining dye, PicoGreen (Invitrogen) at room temperature for 10 minutes in the dark. Unlabeled MPs were used as negative controls. Both labeled and unlabeled MPs were resuspended in 350 µl dfPBS and analyzed by flow cytometry (Beckman Coulter EPICS XL2). The number of events was stopped at 10,000 counts.
Statistical Analysis
Comparisons between two groups (Apoptotic MPs vs. Necrotic MPs) and (control pregnancy vs. preeclamptic pregnancy) were performed using the two-sample t-test. Two-sided P-values less than 0.05 were reported to be statistically significant. All statistical analyses were performed using Microsoft Office Excel®.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preeclampsia is associated with hypoxia, which leads to apoptosis of trophoblasts and subsequent elevated levels of fetal cfDNA in maternal plasma.27,28 A trophoblastic cell line, JEG-3, was used to test the effect of chemically-induced hypoxic apoptosis on the release of cfDNA and MPs. Rotenone, which mimics hypoxia by disrupting the mitochondrial electron transport chain, was used.29 Etoposide was used as a positive control for mitochondria-mediated apoptosis,30 whereas 60°C HS for 1 hour was used to model necrosis.
When apoptotic cells are fixed (permeabilized) with ethanol and stained with PI, they appear as a subdiploid peak by flow cytometry.23
Subdiploid DNA is shown in untreated cells (Figure 1A)
and in cells treated with etoposide (Figure 1B)
, rotenone (Figure 1C)
, or HS (Figure 1D)
. Figure 1e
is a composite figure indicating by 24 hours, 30 ± 4% SD of the rotenone-treated cells contained a subdiploid apoptotic region (% Sub-G1), whereas few of the HS-treated cells (9 ± 3%) had a subdiploid region, slightly more than untreated cells (4 ± 2%). These data show that rotenone and etoposide induce apoptosis, whereas HS does not.
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compare light scatter and PI uptake (back-gated on the light scatter and represented in red). Figure 1J
shows that 26 ± 3% of rotenone-treated cells are PI+ after 24 hours, comparable to etoposide treated cells (26 ± 3% PI+). By contrast, nearly all of the HS-treated cells (Figure 1J)
displayed a loss of plasma membrane integrity (99 ± 1% PI+) with light scatter changes and uptake of PI+, typical of necrosis.31
By 48 hours, both etoposide- and rotenone-treated cells were 70 ± 4% and 72 ± 1% PI+, respectively (Supplemental Figure 2, see http://ajp. amjpathol.org), suggesting late apoptosis/secondary necrosis. The vehicle for rotenone and etoposide, dimethyl sulfoxide, had no effect (data not shown). Quantitation of MPs Generated under Chemically Induced Hypoxic Conditions
An increase in circulating DNA as well as endothelial MPs is associated with preeclampsia,32
suggesting that quantitation of MPs could be useful for diagnosing placental pathologies. To test whether the yield of released MPs is indicative of the type of cell death, two million JEG-3 cells were treated with rotenone, etoposide, or HS and cultured up to 48 hours. The numbers of MPs were detected by flow cytometry using a fluorescent bead counting assay.24
By 24 hours, rotenone-induced apoptosis generated 6.7 ± 0.1 x 106 MPs, significantly more MPs than both untreated cells (0.2 ± 0.1 x 106; P = 0.0001, n = 3, Figure 2A
) and 60°C HS-induced necrosis (2.7 ± 0.1 x 106; P = 0.001, n = 3, Figure 2B
). By 48 hours, rotenone-induced cells continued to produce significantly more MPs (13.3 ± 0.2 x 106) than untreated cells (0.2 ± 0.1 x 106; P = 0.001, n = 3, Figure 2A
) or necrotic cells (2.1 ± 0.2 x 106; P = 0.01, n = 3, Figure 2B
). No significant differences were observed between the number of rotenone-induced MPs and etoposide-induced MPs after 24 and 48 hours (Figure 2C)
. From the starting population of two million cells, we detected approximately 3 and 7 rotenone-induced MPs per cell at 24 and 48 hours, respectively (data not shown). By contrast, 60°C HS treatment resulted in only 1 necrotic MP per cell at 24 or 48 hours (data not shown) and untreated cells produced one MP per five cells by 24 and 48 hours (data not shown). The increase of rotenone-induced MPs is concordant with the changes in light scatter and the increase in the number of PI+ cells at 48 hours (Supplemental Figure 2, see http://ajp.amjpathol.org) suggesting that cells undergoing early to late apoptosis/secondary necrosis release continuous levels of MPs.
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To test whether rotenone-induced or 60°C HS MPs contain DNA, MPs were labeled with the DNA dye, Hoechst 33342,33
and DNA content evaluated using the mean fluorescence intensity (MFI). Because we have determined that lipid membranes of rotenone-induced MPs protect cfRNA from RNase A activity (Table 1)
the membranes of the MPs were permeabilzed with 70% ethanol, before RNase treatment. Rotenone-induced (MFI = 91 ± 11) and 60°C HS MPs (MFI = 82 ± 8) have similar amounts of DNA (Figure 3A)
. No significant difference was detected between rotenone-induced MPs treated with or without RNase (P = 0.75, n = 3) or between 60°C HS MPs with or without RNase (P = 0.07, n = 3), suggesting that Hoechst 33342 bound specifically to DNA (Figure 3A)
.
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.
Concentrations of DNA based on amplification of two gene sequences (SRY and β-globin) were determined by real-time PCR (Figure 3C)
. The mean concentration of SRY DNA for rotenone-induced MPs (0.0007 Genome equivalents [Geq] MP ± 0.0003; range 0.0003 to 0.0013) and 60°C HS MPs (0.0007 Geq MP ± 0.0004; range 0.0003 to 0.0017) was similar (n = 9). The mean concentration of β-globin DNA for rotenone-induced MPs (0.0012 Geq MP ± 0.0006; range 0.0004 to 0.0020) and 60°C HS MPs (0.0011 Geq MP ± 0.0005; range 0.0004 to 0.0017) was similar (n = 9). Thus, both apoptosis and necrosis produce MPs with similar concentrations of DNA per particle. However, apoptotic cells generate many more MPs than do necrotic cells (Figure 2)
, so on a per cell basis, there would be 3 to 7 times more DNA from MPs produced after apoptosis induction.
Molecular Form of Micro-Particle DNA (mpDNA)
Apoptotic DNA contains a range of sizes with fragments cut into sizes of 180 bp, which produces a characteristic ladder on gel electrophoresis. Chan et al demonstrated by real-time PCR that the majority of fetal cfDNA detected in maternal plasma is less than 300 bp,15
suggesting that fetal cfDNA is fragmented and may come from apoptotic fetal cells. To determine the molecular form of mpDNA, DNA fragmentation was detected by gel electrophoresis. DNA from 60°C HS MPs has a random and general cleavage pattern (Figure 4A
, Lane 2). By contrast, rotenone-induced mpDNA (Lane 3) is disrupted in a ladder pattern, similar to apoptotic cellular DNA (Lane 1).
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shows a significant difference in the percentage of Br-dUTP+ PI+ MPs between rotenone-induced (20 ± 6%) and 60°C HS MPs (5 ± 2%) (P = 0.02; n = 4). Furthermore, the Br-dUTP MFI (45 ± 3) of rotenone-induced MPs was significantly greater (P = 0.006; n = 4) than the Br-dUTP MFI (27 ± 6) of 60°C HS MPs (Figure 4C)
, suggesting more nicked mpDNA. Fluorescence micrographs also showed that Br-dUTP preferentially bound to apoptotic mpDNA more than necrotic mpDNA (Figure 4D)
. These data suggest that apoptotic mpDNA displays characteristic ladder patterns, indicative of the ordered process of DNA fragmentation during apoptosis. Apoptotic MPs Have More Lipid Staining Membranes than Necrotic MPs
To test whether mpDNA contains lipid membranes, we performed flow cytometry after labeling MPs with membrane (PKH26) and DNA (Hoechst 33342) dyes. There was no significant difference in the percentage of PKH26+ Hoechst 33342+ MPs between apoptotic (92 ± 9%) and necrotic (93 ± 5%) MPs (P = 0.71; n = 3) (Figure 5A)
. However, the PKH26 MFI of apoptotic (66 ± 10) MPs was significantly greater (P = 0.02) than the PKH26 MFI (45 ± 9) of necrotic MPs (Figure 5B)
. Furthermore, apoptotic MPs (35 ± 17; n = 3) and necrotic MPs (35 ± 13; n = 3) bound similar levels of Hoechst 33342 dye (Figure 5C)
.
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. Similar to the PKH26 MFI results, Figure 6b
shows that the CTB MFI was higher in apoptotic MPs (45 ± 6) than in necrotic (33 ± 4) MPs (P = 0.03; n = 3). There was no significant difference detected in Hoechst 33342 MFI between the double stained apoptotic (171 ± 26) and necrotic (169 ± 31) MPs (P = 0.44; n = 3) (Figure 6C)
. These data suggest the DNA+ apoptotic MPs have more material that stains with membrane specific dyes than the necrotic MPs, possibly providing a mechanism for greater DNA stability in maternal plasma.
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To analyze the plasma stability of cfDNA associated with MPs, we incubated apoptotic MPs, necrotic MPs or free male genomic DNA in female plasma for 24 hours at 0°C (endogenous DNase activity is low34
) or 37°C before amplifying the 193 bp SRY fragment. The concentration of recovered male DNA was interpolated from a standard curve as described (see Materials and Methods). More apoptotic mpDNA (28 ± 5%) was detectable by PCR compared to free DNA (6 ± 1%, P = 0.02; n = 3) and necrotic mpDNA (11 ± 1%, P = 0.03; n = 3) after incubation in female plasma for 24 hours at 37°C (Figure 7A)
. These data suggest that apoptotic mpDNA is more resistant to endogenous plasma DNase activity than necrotic mpDNA or free genomic DNA.
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. By contrast, necrotic MPs did not stain with DAPI after DNase I treatment (Figure 7B)
. Thus, DNA packaged in apoptotic MPs is more resistant to both endogenous plasma DNase activity as well as purified DNase I activity than DNA contained within necrotic MPs. Membrane Lipid MPs Protect RNA from RNase Activity
Similar to cfDNA, cell-free RNA (cfRNA) has been suggested as a way of diagnosing fetal pathologies in vivo.35
One study indicated that cfRNA is associated with MPs.36
To determine whether RNA is also found in apoptotic MPs induced in vitro, mRNA was extracted from the MPs and amplified by real-time RT-PCR. To determine whether the RNA was protected by lipid protein complexes, we treated the apoptotic MPs with RNase ± Triton X-100, a nonionic detergent that solubilizes lipids, for 1 hour. Thereafter, the RNA was extracted and RT-PCR was performed using primers that bridge an exon-exon junction to prevent amplification of potential contaminating genomic DNA. Table 1
shows amplification of mRNA from apoptotic MPs treated with either 0.2 mg/ml RNase (Ct value of 21) or 1.0 mg/ml RNase (Ct value of 21). By contrast, the Ct value for apoptotic MPs treated with both Triton X-100 and RNase was 40, which indicates that no RNA could be detected above background. Together, the data suggest that protein/lipid membrane complexes likely protect against RNase activity.
In Vitro MPs Are Similar to Maternal Plasma MPs
To compare similarities between MPs found in maternal plasma and in vitro MPs, samples from each were labeled with or without DNA dye, PicoGreen, and examined by flow cytometry. The dot plots, which display light scatter properties representing size (x axis) and granularity (y axis), are shown in Figures 8A–D
. In vitro MPs (Figure 8, A and B)
had forward light scatter patterns similar to MPs found in normal maternal plasma (Figure 8C)
as well as preeclamptic plasma samples (Figure 8D)
. Although similar in light scatter patterns, dot plots of unlabeled PicoGreen MPs are not shown. These data suggest that the in vitro MPs were similar in size (forward scatter) and structure (side scatter) to the MPs found in maternal plasma. Next, an electronic gate (Gate A) was placed around PicoGreen labeled MPs (Figure 8, C and D)
and unlabeled MPs (data not shown) from control pregnancies and preeclamptic pregnancies and the percentage of PicoGreen+ MPs was determined. Both PicoGreen histograms show three peaks; one negative, one intermediate, and one bright peak (Figure 4, E and F)
. The negative peak is the autofluorescence of unstained MPs as shown in the filled in histogram. The intermediate peak is a heterogeneous population of MPs containing intermediate amounts of DNA, the bright PicoGreen peak has high levels of DNA. To determine MPs with high levels of DNA, the cursor was placed on the PicoGreen bright peak. A threefold increase in the percentage of bright PicoGreen+ MPs from term preeclamptic pregnancies (46.2 ± 22.4%; n = 4) compared to term control pregnancies (14.3.± 7.2%; n = 4; P = 0.03) was detected, showing that the preeclamptic maternal MPs had more DNA per MP.
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Using our fluorescent bead counting assay, we compared the levels of MPs in plasma from control pregnant women and women with preeclampsia. There were higher levels of MPs in maternal plasma from preeclamptic pregnancies (8.8 ± 2.8 x 108; n = 4, Figure 9A
) compared to control pregnancies (1.6 ± 4.6 x 106; P = 0.003, n = 4, Figure 9A
). Furthermore, the number of total PicoGreen+ MPs was significantly higher in preeclamptic women (4.9 ± 3.1 x 108; n = 4, Figure 9B
) compared to control pregnant women (0.6 ± 0.2 x 108; P = 0.031, n = 4, Figure 9B
). These data suggest higher levels of cell death occur in preeclampsia compared to normal control pregnancies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Interestingly, apoptotic MPs containing RNA are also protected from RNase activity (Table 2)
, similar to fetal cfRNA. Our observations further indicate that MPs released from apoptotic cells are similar to plasma MPs based on light scatter and DNA staining properties (Table 2)
, suggesting similar DNA content and cell death mechanism. This report also provides new and convincing evidence that preeclamptic patients have significantly higher amounts of mpDNA than found in normal control pregnancies, suggesting that quantitative analysis of DNA+ MPs could be used to clinically monitor or predict preeclampsia. Indeed, others have shown that levels of STBMs are similar in preeclamptic and control pregnancies at term,13,14
indicating that our methods may be more sensitive and relevant, than methods detecting STBMs.
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During normal pregnancy, fetal cfDNA is released into the maternal circulation from apoptotic fetal cells.37 Therefore, quantification of fetal cfDNA concentrations in maternal plasma has potential for monitoring pregnancy associated-disorders, including preeclampsia. Initial studies reported a five-fold increase in plasma fetal cfDNA concentrations in women with preeclampsia (mean gestational age [MGA] 32 weeks) compared to control pregnant women.38 It was also shown that higher combined maternal and fetal cfDNA concentrations correlated with the severity of preeclampsia (MGA 33 weeks)8 and that higher levels of fetal cfDNA alone were detected in maternal plasma before clinical symptoms (MGA 17 to 20 weeks).6,7 Together, these findings suggest that increased levels of fetal cfDNA in preeclampsia indicate placental damage/cell death and that the pathogenesis occurs before the onset of clinical symptoms.
The cellular mechanism most likely to give rise to circulating MPs is apoptosis. However, it has been suggested that trophoblasts undergo aponecrosis during preeclampsia.17 Aponecrosis is a form of cell death that has overlapping morphological features between apoptosis and necrosis in response to hypoxia39 and chemical-induced hypoxia.40,41 Huppertz et al reported that syncytiotrophoblasts exposed to severe hypoxia displayed both apoptotic and necrotic characteristics, suggesting aponecrosis.17 Consistent with this, our results show that cells treated with rotenone, which blocks the mitochondrial complex I, display fragmented DNA and PI uptake (26%) by 24 hours, whereas by 48 hours, the percentage of PI+ cells increased to 73% (Supplemental Figure 2, see http://ajp.amjpathol.org). Supportive of our results is a study reporting that fibroblastic cells treated with Antimycin A, an inhibitor of mitochondrial respiratory chain complex III, or rotenone, underwent aponecrosis.40 Tjoa et al demonstrated that placental explants challenged with oxidative stress displayed apoptotic as well as necrotic characteristics and released cfDNA into the supernatant, suggesting that aponecrosis may be an alternative mechanism for the release of fetal cfDNA.16 The authors did not address whether cfDNA was associated with MPs, which we conclusively show in this study.
We used JEG-3 cells because they are homogeneous and represent the EVT cells found in early gestation.18 Studies with primary trophoblasts show high background apoptosis,16,22 however background levels of death of JEG-3 cells are low, allowing more interpretable cell death experiments.
Few studies have examined the biochemical properties of cfDNA released from dying cells. Size fractionation of cfDNA extracted from maternal plasma demonstrated that the majority of fetal DNA fragments are less than 300 bp,42 supporting the idea that fetal cfDNA is released from apoptotic cells. Our data show that MPs form a DNA ladder when analyzed by gel electrophoresis. By contrast, necrotic cells released MPs with a DNA smear, consistent with necrotic DNA. Although rotenone-induced MPs are not true hypoxia-derived plasma MPs from preeclamptic women, their characteristics demonstrate that MPs released from apoptotic cells contain small fragments of DNA, similar to fetal DNA in the maternal circulation.42
Our studies show that rotenone-induced MPs contain significantly more membranes than necrotic-released MPs. Most importantly, these lipid membranes protect DNA from DNase activity, which accounts for MP stability in plasma. Others have shown that STBMs prepared by mechanical disruption, thought to represent an aponecrotic form of cell death, also contain high lipid levels,43 consistent with our results.
In addition to fetal DNA, fetal cfRNA is readily found in maternal plasma. Recently, we reported the feasibility of amplifying placental mRNA from dried blood spots,44 suggesting that fetal RNA is stable and easily amplified by reverse transcriptase real-time PCR. Although fetal transcripts have been found in the supernatant of STBMs prepared from placental explants,11 it is unclear whether the MPs contain cfRNA. By contrast, we demonstrate that apoptotic MPs contain cfRNA resistant to RNase activity, protected by lipids.
Syncytiotrophoblasts, which surround the outer layer of the placenta, constantly undergo apoptosis and release STBMs into the maternal blood circulation.12 Because the number of STBMs is increased in preeclampsia, compared to normal pregnancies, STBMs might be used as a predictive biomarker for preeclampsia. However, a limitation is that STBMs are not detected until the second and third trimester of pregnancy.13 Given that preeclampsia is associated with dysfunctional extravillous trophoblasts invasion of both the myometrium and the spiral arteries during the first trimester,18 MPs derived from EVTs might be a more informative biomarker in early gestation than STBMs.
An advantage of measuring total mpDNA from pathological pregnancies is that all sources of MPs would be included. Total cfDNA (maternal and fetal) has been quantitated using β–globin levels, which were higher (MGA 35.6 ± 2.8 weeks) in women with preeclampsia (MGA 32.0 ± 3.8 weeks),45 as well as higher in women (MGA 20.5 ± 3.3 weeks) who subsequently developed preeclampsia (MGA 33.5 ± 3.6 weeks).46 Goswami et al showed that STBMs were elevated in third trimester preeclamptic plasma samples compared to normal pregnancies.13,14 However, a significant difference in the number of STBMs was seen only in women with early-onset preeclampsia (<34 weeks of gestation), whereas no significant difference was seen in women with late-onset preeclampsia (>34 weeks of gestation).
Using our flow cytometry based mpDNA assay, we detected significantly elevated levels of total mpDNA in late-onset preeclampsia compared to control pregnancies, suggesting that our method is better than detecting elevated STBMs by enzyme-linked immunosorbent assay.13 Flow cytometric analysis of total mpDNA is advantageous given that the technique is simple, rapid (no processing of the plasma sample) and inexpensive as compared to real-time PCR. The method is also gender independent. Furthermore, this technique could be applied to other clinical and pathological conditions associated with increased levels of cell death (ie, trauma, stroke, organ transplantations, autoimmunity, and cancer). In its current form, quantifying mpDNA might not be applicable for prenatal diagnosis because it is not tissue specific nor does it discriminate between maternal and fetal DNA. However, such a method could be used to rapidly screen plasma samples before further analysis by more complex assays such as real-time PCR.
Future directions include addressing whether elevated levels of circulating mpDNA can be detected longitudinally in pregnancies that subsequently develop preeclampsia. Furthermore, it will be important to compare mpDNA levels with other known plasma/serum markers for preeclampsia such as Activin A, P-selectin, and vascular endothelial growth factor receptor.47 Finally, addressing whether mpDNA express surface markers from their original cells (ie, maternal endothelial cells or fetal trophoblasts) may help to understand the underlining pathology of preeclampsia or help to enrich fetal DNA from maternal plasma by isolating fetal-specific MPs.
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Acknowledgements |
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Footnotes |
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Supported by grants NIH/NICHD HDO46623 and T32 AI007495. Accepted for publication August 14, 2008.
Supplementary material for this article can be found on http://ajp. amjpathol.org.
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in the Rat Carotid Body Glomus Cells. Histochem Cell Biol 2005, 124:69-76[CrossRef][Medline]
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