The authors have declared that no competing interests exist.
Conceived and designed the experiments: BL JN ZLW. Performed the experiments: BL YX FHQ MZZ YDL. Analyzed the data: BL YX. Wrote the paper: BL CV. Supervised the work and critically revised the manuscript: JN ZLW.
To investigate the placental proteome differences between pregnant women complicated with gestational diabetes mellitus (GDM) and those with normal glucose tolerance (NGT).
We used two-dimensional electrophoresis (2DE) to separate and compare placental protein levels from GDM and NGT groups. Differentially expressed proteins between the two groups were identified by MALDI-TOF/TOF mass spectrometry and further confirmed by Western blotting. The mRNA levels of related proteins were measured by realtime RT-PCR. Immunohistochemistry (IHC) was performed to examine the cellular location of the proteins expressed in placenta villi.
Twenty-one protein spots were differentially expressed between GDM and NGT placenta villi in the tested samples, fifteen of which were successfully identified by mass spectrometry. The molecular functions of these differentially expressed proteins include blood coagulation, signal transduction, anti-apoptosis, ATP binding, phospholipid binding, calcium ion binding, platelet activation, and tryptophan-tRNA ligase activity. Both protein and mRNA levels of Annexin A2, Annexin A5 and 14-3-3 protein ζ/δ were up-regulated, while the expression of the Ras-related protein Rap1A was down-regulated in the GDM placenta group.
Placenta villi derived from GDM pregnant women exhibit significant proteome differences compared to those of NGT mothers. The identified differentially expressed proteins are mainly associated with the development of insulin resistance, transplacental transportation of glucose, hyperglucose-mediated coagulation and fibrinolysis disorders in the GDM placenta villi.
Gestational diabetes mellitus (GDM), defined as glucose intolerance that first occurs or is first identified during pregnancy
Although the etiology of GDM has not been clarified, increasing evidence demonstrates that the placenta, the organ connecting the developing fetus with the maternal body, plays an important role in the development of GDM
Previous studies have identified a host of gene expression changes in the GDM placenta. These altered genes were generally associated with inflammatory responses, endothelial reorganization
Given the clinical significance of GDM, and the power of proteomics approaches, we performed the present study to analyze the proteome changes of GDM placenta villi, to provide evidence for a potential pathway associated with GDM pathogenesis or placental remodeling in GDM.
This study was approved by the research ethical committee of The First Affiliated Hospital of Sun Yat-sen University and written informed consent was provided by all volunteers.
Twenty-five pregnant women complicated with gestational diabetes mellitus were recruited for the GDM group. The diagnosis of GDM was based on American Diabetes Association (ADA) criteria
To improve homogeneity and comparability, placentas selected for the study were from pregnant women that received Cesarean sections. Placenta villi of approximately 3 cm3 were obtained from five different intact cotyledons immediately following delivery. After the maternal decidual layer was removed, the tissue was quickly washed with ice-cold PBS, frozen in liquid nitrogen, and stored at −80°C for further protein or mRNA extraction. Samples for immunohistochemistry were fixed in 4% paraformaldehyde for three days and embedded in paraffin before sectioning.
Eight placenta villi samples from the GDM group and eight from the NGT group were randomly chosen for two-dimensional electrophoresis (2DE). For each sample, a total of 300 mg placental tissue was disrupted in liquid nitrogen and solubilized in 1000 µL lysis buffer consisting of 7 M urea, 2 M thiourea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v IPG buffer (pH 3–10), 1% v/v PMSF and 1% v/v protease inhibitor cocktail. After incubating on ice for 2 hours, the sample was centrifuged at 16000 g for 30 min at 4°C to remove debris. The supernatant was collected and further purified using a ReadyPrep 2D Clean-up kit (Bio Rad, Hercules, CA, USA) to improve 2DE gel quality by removing contaminating substances in the sample. Next, the concentrations of the purified protein samples were determined by Bradford protein assay according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA), using bovine serum albumin (BSA) as standard. The purified protein samples were stored at −80°C until 2DE was performed.
The eight purified protein samples from above were pooled to form a final sample for each group. For 2 DE analysis, 1 mg of sample was diluted in sample buffer consisting of 50 mM Tris-HCl (pH 8.5), 7 M urea, 2 M thiourea, 2% CHAPS, 0.3% dithiothreitol (DTT), 0.5% IPG buffer (pH 3–10 linear), and 10 µL of a protease inhibitor mixture to a final volume of 450 µL. Protein samples were applied on immobilized pH 3–10 linear gradient IPG strips (24 cm) (Bio-Rad, Hercules, CA, USA) and active rehydration was performed at 30 V for 6 h then 60 V for 6 h. Focusing was performed using an Ettan IPGphor III IEF system (GE Healthcare, Waukesha, WI, USA) at 200 V for 2 h, 500 V for 2 h, 1000 V for 2 h, 5000 V for 2 h, after which the voltage was gradually increased to and held at 10000V until a total of 90000Vhs was reached, then kept at 500 V for 5 h.
After focusing, strips were equilibrated for 15 min in 75 mM Tris-HCl (pH 8.8), 6 M urea, 2% w/v SDS, 29.3% v/v glycerol, and 1.0% DTT, followed by a 15 min incubation in the same buffer containing 2.5% iodoacetamide in place of DTT. Second-dimension electrophoresis was performed on a 12.5% polyacrylamide gel, using the Ettan DALT Six system (GE Healthcare, Waukesha, WI, USA). Each gel was run at 2w for 1 h at 15°C, and then increased to 15w until the tracking dye migrated to within 1 cm of the bottom of the gel.
2-D gels were fixed in 50% methanol for 2 h, visualized by Coomassie Blue Colloidal Staining (GE Healthcare, Waukesha, WI, USA), and scanned using the ImageScanner system (GE Healthcare, Waukesha, WI, USA) combined with LabScan software(GE Healthcare, Waukesha, WI, USA). All gel images were analyzed using ImageMaster 2D Platinum software (GE Healthcare, Waukesha, WI, USA). Gel images from each group were edited, and spots were matched manually. A unique identification number was assigned to matching spots on different gels. Normalization of the spot intensities was conducted according to the total optical density of the gel. Spots which had more than 2 fold change in relative spot volume were identified as significantly differential spots between gels.
Spots from 2-DE gels selected for further analysis were excised using a blade and gel pieces were transferred to microfuge tubes. After rinsing with distilled water and destaining with potassium ferricyanide and sodium thiosulfate, the gel pieces were dehydrated in 100% acetonitrile. 2 µL (25 ng/µL) of modified porcine trypsin in 25 mM ammonium bicarbonate, pH 8, was added to each sample then incubated at 37°C overnight. The trypsin-digested solutions were collected and the remaining peptides were extracted from the gel pieces by incubating in 0.1% Trifluoroacetic acid/60% acetonitrile for 15 min prior to drying in a vacuum centrifuge.
MALDI-TOF/TOF mass spectrometry measurements were performed using a Bruker Ultraflex III MALDI-TOF/TOF MS (Bruker Daltonics, Leipzig, Germany) operating in reflectron mode with 20 kV accelerating voltage and 23 kV reflecting voltage. A saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid was used as the matrix. 1 µL of the matrix solution and sample solution at a ratio of 1∶1 was applied onto the Score384 target well. By routine, a standard peptide calibration mix in the mass range 800–3200 Da (Bruker Daltonics, Leipzig, Germany) was analyzed for external calibration of the mass spectrometer. The calibration mix contained: Angiotensin II, Angiotensin I, Substance P, Bombesin, ACTH clip 1–17, ACTH clip 18–39, Somatostatin 28. A series of eight samples were spotted around one external calibration mixture. The SNAP algorithm (S/N threshold: 5; Quality Factor Threshold: 30) in FlexAnalysis 3.0 was used to pick up the 100 most prominent peaks in the mass range m/z 700–4000. The subsequent MS/MS analysis was performed in a data-dependent manner, and the 5 most abundant ions fulfilling certain preset criteria (S/N higher than 3 and Quality Factor higher than 30) were subjected to high energy CID analysis. The collision energy was set to 1 keV, and nitrogen was used as the collision gas.
Peptide mass fingerprints (PMFs) were searched using the program Mascot 2.1 (Matrix Science Ltd) against the database of SwissProt (version 20091028, 510076 sequences). The search parameters were as follows: trypsin digestion with one missed cleavage; carbamidomethyl modification of cysteine as a fixed modification and oxidation of methionine as a variable modification; peptide tolerance maximum, ±0.3 Da; MS/MS tolerance maximum, ±50 ppm; peptide charge, +1; monoisotopi mass. p<0.05 was used for a local PMF search. For unambiguous identification of proteins, more than 5 peptides must be matched for a PMF search.
Western blot on Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ, and Ras related protein Rap1A was performed on 10 cases (other than the cases for 2DE/MS study) of placenta villi from both the GDM and NGT groups. 30 µg of protein from each sample was loaded onto each lane of 12% resolving and 4% stacking polyacrylamide gels (GE Healthcare, Waukesha, WI, USA) and electrophoresed through a Bio-Rad system (Bio-Rad, Hercules, CA, USA) combined with Laemmli SDS buffering system (25 mM Tris-base, 192 mM glycine, 0.1%SDS). Next, proteins were transferred to PVDF membranes at 70V for 50 min in ice cold transferring buffer and blocked in 5% skimmed milk for 1 h at room temperature. The PVDF membranes were then incubated separately with antibody: rabbit anti-Annexin A2 (Abcam, ab41803, Cambridge, MA, USA, 1 µg/ml), rabbit anti-Annexin A4 (Abcam, ab65846, Cambridge, MA, USA, 0.5 µg/ml), rabbit anti-Annexin A5 (Epitomics, 3225-1, Burlingame, CA, USA, 0.5 µg/ml), rabbit anti-14-3-3 ζ/δ (BioLegend, 614802, San Diego, CA, USA, 1 µg/ml), rabbit anti-Rap (Epitomics, 1726-1, Burlingame, CA, USA, 2 µg/ml), and mouse anti-β-actin (Sigma-Aldrich, A1978, Saint Louis, MO, USA, 0.1 µg/ml ) at 4°C overnight. The membranes were incubated with the indicated secondary antibody for 1 h at room temperature. The immunopositive bands were visualized using Western Lightning chemiluminescence reagents (Invitrogen, Carlsbad, CA, USA). All Western blot exposures were in the linear range of detection, and the intensities of the resulting bands were quantified using Image J software.
Total RNA from placenta villi was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions, and the concentration and purity of RNA was assessed by NanoDrop® ND-1000 spectrophotometry (Thermo Scientific, Rockford, IL, USA). 2 µg of total RNA was used in the first strand cDNA synthesis with TaqMan® Reverse Transcription Reagents (Takara Bio Inc., Otsu, Shiga, Japan) according to manufacturer’s instructions.
Real-time PCR were performed on 15 samples from each group with the ABI PRISM 7500 Sequence Detection System (Applied Biosystem, Foster City, CA, USA), using SYBR green real-time PCR mix (Takara Bio Inc., Otsu, Shiga, Japan) according to the manufacturer’s specifications. Primers for real-time PCR are shown in
To study the location of Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ, and Ras related protein Rap1A in human placenta villi, immunohistochemistry (IHC) was performed on samples from each group. The detection of these proteins in the placental tissues was carried out using a two-step IHC procedure. First, paraffin blocks were cut into 5 µm-thick sections. Then, the sections were deparaffinized in xylene and rehydrated in graded alcohol concentrations. Nonspecific binding was blocked by pre-incubation with blocking solution for 5 min, then the sections were incubated for 2 h at room temperature with antibodies against Annexin A2 (10 µg/ml), Annexin A4(5 µg/ml), Annexin A5(5 µg/ml), 14-3-3 ζ/δ (10 µg/ml) and Rap(20 µg/ml). The primary antibodies used for IHC were the same products used in the Western blot analysis. Following incubation with primary antibodies, sections were incubated with appropriate secondary antibody for 1 h. Substrate-chromogen DAB reagent was then added to each section following a rinse, and finally hematoxylin solution was used to stain nuclei.
Data were input and analyzed by SPSS11.0 database. The results were expressed as mean ± SD or mean ± S.E.M, and statistical analysis was carried out using independent student’s t-test.
Twenty-five pregnant women were recruited for each group. Clinical and laboratory data were compared between pregnant women complicated with gestational diabetes mellitus (GDM) and normal glucose tolerance (NGT). As shown in
Gestational diabetes Mellitus (n = 25) | Normal Glucose Tolerance(n = 25) |
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Age (year) | 31.28±3.13 | 30.80±3.44 | 0.608 |
Gravidity | 1.52±0.82 | 1.60±0.82 | 0.732 |
Parity | 1.00 | 1.04±0.20 | 0.327 |
Pre-gravidity BMI |
22.13±2.11 | 21.96±3.55 | 0.838 |
Pre-partum BMI (kg/m2) | 27.83±2.84 | 27.56±3.46 | 0.767 |
OGTT‡-fast (mmol/l) | 4.65±0.45 | 4.21±0.32 | <0.001 |
OGTT-1h (mmol/l) | 11.75±2.61 | 7.97±0.95 | <0.001 |
OGTT-2h (mmol/l) | 9.94±1.63 | 6.33±1.00 | <0.001 |
HbA1c§ (%) | 5.94±0.50 | 4.69±0.44 | <0.001 |
Basal insulin (µU/ml) | 16.29±5.89 | 9.40±2.35 | <0.001 |
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Gestational Age (day) | 271.64±5.45 | 273.84±5.37 | 0.157 |
Gender | 1.000 | ||
Male (n, %) | 12, 48 | 12, 48 | |
Female (n, %) | 13, 52 | 13, 52 | |
Birth Weight (kg) | 3.55±0.33 | 3.28±0.44 | 0.018 |
Birth Length (cm) | 50.12±1.45 | 49.92±1.58 | 0.643 |
Placental weight (kg) | 0.59±0.043 | 0.56±0.049 | 0.026 |
BMI: body mass index; ‡OGTT: oral glucose tolerance test; §HbA1c: Glycated hemoglobin.
To investigate the proteomic alterations in the GDM placenta villi, the protein levels in samples from GDM and NGT pregnant women were compared using 2 DE. In order to account for fetal gender differences, placenta villi from four male and four female pregnancies comprised each group. As shown in
(
Spot No |
%Vol Ratio |
Protein ID | Protein name | Mw(kDa) | pI | Sequence coverage (%) | Mascot MS/MS score | Functional association | Subcellular location |
11822 | 1000000 | P62879 | Guanine nucleotide-binding proteinG(I)/G(S)/G(T) subunit beta-2 | 37.331 | 5.60 | 7 | 142 | Transducer | Cytoplasm |
11827 | 1000000 | O00487 | 26S proteasome non-ATPase regulatory subunit 14 | 34.577 | 6.06 | 7 | 92 | Hydrolase Metalloprotease Protease | Proteasome |
11862 | 1000000 | P08758 | Annexin A5 | 35.937 | 4.94 | 9 | 95 | Blood CoagulationSignal Transduction | Cytoplasm |
11882 | 1000000 | P09525 | Annexin A4 | 35.883 | 5.85 | 20 | 57 | Anti-ApoptosisSignal Transduction | Cytoplasm |
11980 | 1000000 | P63104 | 14-3-3 protein zeta/delta | 27.745 | 4.73 | 9 | 62 | Anti-ApoptosisSignal Transduction | Cytoplasm |
10896 | 2.4378 | P41250 | Glycyl-tRNA synthetase | 83.140 | 6.61 | 3 | 84 | ATP Binding | CytoplasmMitochondrion |
12101 | 2.42479 | P24844 | Myosin regulatory light polypeptide 9 | 19.827 | 4.80 | 12 | 131 | Calcium Ion Binding | Muscle Myosin Complex |
11782 | 2.14439 | P07355 | Annexin A2 | 38.604 | 7.56 | 13 | 276 | Calcium-Dependent Phospholipid Binding | Basement MembraneExtracellular MatrixSecreted |
11519 | 2.08983 | P07954 | Fumarate hydratase, mitochondrial | 54.637 | 6.99 | 1 | 31 | Fumarate Hydratase Activity | Cytoplasm Mitochondrion |
11343 | 2.15177 | P23381 | Tryptophanyl-tRNA synthetase, cytoplasmic | 53.165 | 5.83 | 7 | 146 | Tryptophan-Trna Ligase Activity | Cytoplasm |
11693 | 2.20513 | P02675 | Fibrinogen beta chain | 55.928 | 8.54 | 6 | 136 | Platelet Activation | External Side Of Plasma Membrane |
11671 | 2.73296 | P02675 | Fibrinogen beta chain | 55.928 | 8.54 | 6 | 150 | Platelet Activation | External Side Of Plasma Membrane |
11852 | 2.77444 | P02671 | Fibrinogen alpha chain | 94.973 | 5.7 | 1 | 69 | Platelet Activation | External Side Of Plasma Membrane |
12096 | 5.23071 | P62834 | Ras-related protein Rap-1A | 20.987 | 6.38 | 15 | 160 | Signal Transduction | Cell Membrane |
11195 | 20.7913 | P02675 | Fibrinogen beta chain | 55.928 | 8.54 | 6 | 108 | Platelet Activation | External Side Of Plasma Membrane |
Spot No is the unique protein spot number referring to the labels in
%Vol Ratio is analyzed by ImageMaster software and calculated by the ratio of relative spot volume of the two groups.
The differentially expressed spots were excised from gels, digested with trypsin and identified by MALDI-TOF/TOF mass spectrometry. The peptide mass fingerprinting (PMF) and MS/MS identification of Annexin A2 by MALDI-TOF-MS/MS is presented in
(
As shown in
Western blots were performed to confirm differential protein expression between GDM and NGT groups. Ten placenta villi samples from each group (in addition to the samples for the 2DE/MS study) were obtained for Western blot analysis of five differentially expressed proteins: Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ, and Ras related protein Rap1A. As shown in
(
To compare the mRNA levels of Annexin A2, Annexin A4, Annexin A5, 14-3-3 protein ζ/δ, and Ras related protein Rap1A in GDM and NGT placenta villi, fifteen samples from each group were analyzed by realtime RT-PCR. As shown in
To investigate the localization of differentially expressed proteins in the placenta villi, we performed IHC on Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ, and Ras related protein Rap1A. As shown in
Annexin A2, Annexin A4, Annexin A5, 14-3-3 ζ/δ and Ras related protein Rap1A are mainly expressed in trophoblast cells of placenta villi. Magnification: 200x Arrows: indicate location of each protein. Bar: 50 µm.
The placenta plays an important role in the development of gestational diabetes mellitus (GDM), while hyperglycemia, hyperinsulinemia, and other metabolic dysfunctions in GDM patients in turn induce structural and functional alterations of the placenta. Previous research has identified changes in placental gene expression in women suffering from GDM
Insulin resistance is the pivotal mechanism of both type 2 diabetes mellitus (T2DM) and GDM. Insulin induces cell glucose uptake by sequentially activating the insulin receptor (IR), insulin receptor substrates (IRS), PI3K, AKT and GLUT4
The insulin signal pathway has been previously described by Pessin (Ref. 15). When stimulated by insulin, the insulin receptor activates insulin receptor substrates (IRS), PI3K, AKT and GLUT4 sequentially, leading to the localization of GLUT4 to the cell membrane for transfer of glucose.
IR internalization is the major mechanism by which cells maintain the response to insulin stimulation within a normal range
The 14-3-3 proteins are also involved in the insulin signal pathway. Upon stimulation by insulin, the insulin receptor (IR) phosphorylates tyrosine residues in insulin receptor substrate-1 (IRS-1) and subsequently, phosphorylated IRS-1 associates with phosphoinositol-3-kinase (PI3K). 14-3-3 proteins can bind IRS-1 and thus inhibit insulin signaling in several ways. Firstly, 14-3-3 proteins compete with IR for IRS binding
Combining the alterations in expression of Annexin A2 and 14-3-3 complex, we speculate that GDM placental cells make adjustments in response to chronic insulin stimulation by down-regulating insulin receptors and blocking the insulin signal pathway, thereby reducing the uptake of glucose.
Hyperglycemia and hyperinsulinemia are two major pathophysiological changes in GDM. Maternal hyperglycemia is able to induce fetal hyperglycemia due to an increased transplacental transfer of glucose, while maternal insulin is not able to cross the placenta. Fetal hyperglycemia can stimulate insulin secretion by the fetal pancreas, subsequently leading to normalization of fetal glycemia. However, intrauterine hyperglycemia and hyperinsulinemia are also associated with macrosomia in GDM pregnancy, by a mechanism known as the Pederson hypothesis
Proteins found to have altered expression between GDM and NGT placental groups in the present study are associated with additional cellular functions including coagulation and fibrinolysis balance.
A hypercoagulable state becomes more common during pregnancy, especially in those complicated with GDM. However, we found Annexin A5, a major protein involved in hypocoagulation, was up-regulated in GDM placenta villi. Annexin A5, otherwise known as placenta anticoagulant protein 1, can form a shield around negatively-charged phospholipid molecules and inhibit blood coagulation
(
Besides hypercoagulation, GDM has long been as associated with an attenuated fibrinolysis state
On the contrary, Annexin A2 may contribute to enhanced fibrinolysis in the GDM placenta villi. Previous studies suggest Annexin A2 may increase plasmin generation by binding to tissue plasminogen activator (t-PA) and plasminogen
Interestingly, three protein spots (numbers: 11693, 11671, and 11195) were identified as the same protein (fibrinogen beta chain). It is a common phenomenon in 2DE for the same protein to migrate to multiple locations on the gel
In summary, the present study mainly focuses on placental proteome alterations in pregnant women complicated with GDM, which may either be the cause or result of physiological changes in GDM. Differentially expressed proteins found and identified in the current study include Annexin A2, Annexin A5, 14-3-3 protein ζ/δ, and Ras related protein Rap1A, which are involved in the regulation of the insulin pathway and coagulation/fibrinolysis and may play important roles in insulin resistance and diabetic-related coagulation/fibrinolysis alterations. To the best of our knowledge, the present work is the first proteomic study on GDM placenta villi. Further studies may be performed to uncover additional proteomic changes and to confirm the relationship between alterative proteins and GDM physiology.
(DOC)
We thank Dr. Chang-zhao Li and Li-li Chen for technological support for immunohistochemistry examination.