Study design and population
This cross-sectional study included pregnant women in the following groups: 1) severe PE (n=10); 2) severe PE with SGA (n=10); 3) SGA without PE (n=10); and 4) pregnant women with preterm and term labor (control group, n=10). Women with severe PE or SGA were matched for gestational age at delivery within two weeks of gestation to women in the control group. Patients with multiple pregnancies, preterm prelabor rupture of membranes, histologic chorioamnionitis, stillbirth or fetal congenital or chromosomal abnormalities were excluded. Samples and data were retrieved from the bank of biological samples and clinical databases of the Perinatology Research Branch. All patients were enrolled at Hutzel Women's Hospital, Detroit, MI, USA, and provided written informed consent prior to the collection of samples. The utilization of samples for research purposes was approved by the Institutional Review Boards of both Wayne State University and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD/NIH/DHHS). Many of these samples have been employed to study the biology of inflammation in normal pregnant women and those with pregnancy complications.
PE was defined as hypertension (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg on at least two occasions, 4 hours to 1 week apart) associated with proteinuria (≥300 mg in a 24 hour urine collection, or two dipstick measurements of ≥1+ [89
], or one dipstick measurement of ≥2+)[90
]. Severe PE was defined as systolic blood pressure ≥160 mmHg or diastolic blood pressure ≥110 mmHg and/or proteinuria greater than 5 g in a 24 hour collection or >3+ on dipstick[1
] and in the presence of multi-organ involvement[1
]. SGA was defined as neonatal birthweight below the 10th
percentile for gestational age at birth according to the national birthweight distribution[91
]. Labor was defined by the presence of regular uterine contractions at a frequency of at least 2 contractions every 10 minutes with cervical changes resulting in delivery <37 (preterm) or ≥37 (term) completed weeks of gestation. Control women with preterm or term labor delivered neonates with a birthweight appropriate-for-gestational age (≥10th
mRNA in situ hybridization
The 123bp fragment of human galectin-1 cDNA generated by PCR (forward primer: CATCTCTCTCgggTggAgTC, reverse primer: gAAggCACTCTCCAggTTTg) was subcloned into pGEM-T Easy vector (Promega Corp., Madison, WI, USA) containing SP6 and T7 polymerase promoters. Digoxigenin-labeled anti-sense and sense riboprobes were generated with SP6 and T7 polymerases after linearization of the plasmid with Bam HI and Hind III, respectively. 5 μm sections of paraffin-embedded villous tissues were deparaffinized, hydrated in xylene and graded ethanol and then treated with proteinase K (15 μg/ml) in 0.1 M Tris buffer (pH 8.0) and 50 mM EDTA for 10 minutes at 37°C. Slides were fixed with 4% paraformaldehyde for 20 minutes and acetic anhydride for 10 minutes. Sections were incubated in a hybridization buffer containing digoxigenin-tagged galectin-1 riboprobe (2 μg/ml). Hybridization was carried out in a humidity chamber overnight at 55°C. After repeated post-hybridization washes, sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Diagnostics, Indianapolis, IN) for 1 hour at room temperature. Nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt were used for detection of the hybridization signal.
Total RNA extraction
Total RNA was isolated from snap-frozen placental villous tissues using TRIzol reagent (Invitrogen Carlsbad, CA, USA) and then Qiagen RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturers' recommendations. The 28S/18S ratio and the RNA integrity number (RIN) were assessed using a Bioanalyzer 2100 (Agilent Technologies, Wilmington, DE, USA). An A260nm/A280nm ratio of 1.8, a 28S/18S ratio of 1.3, and a RIN of 6 were minimum requirements for inclusion in expression analysis.
Quantitative real-time reverse transcription–polymerase chain reaction (qRT–PCR)
Total RNA was reverse transcribed with a TaqMan Reverse Transcription Reagent kit using random hexamers (Applied Biosystems, Foster City, CA, USA). The standard curve was run with the LGALS1 TaqMan Gene Expression Assay (Hs00169327_m1; Applied Biosystems, Foster City, CA, USA) to determine the quantity of cDNA needed for an approximate cycle threshold (Ct) of 25. The human RPLPO (large ribosomal protein) TaqMan Endogenous Control (part number: 4326314E) was used as the housekeeping gene for relative quantitation. The LGALS1 and RPLPO genes were then run in triplicates for each case to allow for the assessment of technical variability.
Immunofluorescence confocal microscopy
Five μm sections of snap-frozen villous tissues were fixed with 4% paraformaldehyde for 1 hour at room temperature and acetone for 1 minute at −20°C. Slides were preincubated with Image-it FX signal enhancer (Molecular Probes, Carlsbad, CA, USA) for 30min and CAS blocking solution (Zymed, San Francisco, CA, USA) for 10 minutes. Sections were incubated with goat anti-human galectin-1 IgG (R&D Systems, Minneapolis, MN, USA) and goat isotype control primary IgG at 1:50 dilutions for 1 hour, and with Alexa Fluor 568 conjugated donkey anti-goat IgG (Invitrogen Co., Carlsbad, CA, USA) at 1:1000 dilution for 1 hour. Sytox Green nuclear counter stain (Cambrex, North Brunswick, NJ, USA) was applied at a 1:100,000 dilution for 3 minutes. Stainings were performed on an autostainer (Dako, Carpinteria, CA, USA). Fluorescent and differential interference contrast (DIC) images were captured with a Zeiss Axiovert 200 Ultra-View ERS Rapid Confocal Imager equipped with an argon laser and a Zeiss Fluar 40x / 1.3 oil objective. Images were evaluated by Perkin Elmer ImageSuite 3.0 version 14 (Perkin Elmer Inc., Waltham, MA, USA).
Western blot analysis
Placental villous tissues were lysed with RIPA buffer (Sigma, St Louis, MO, USA) containing protease inhibitor (Roche Diagnostics, Mannheim, Germany). Thirty μg of the protein lysates and 15 ng of recombinant human galectin-1 (R&D Systems, Minneapolis, MN, USA) were electrophoresed on 15% (w/v) SDS-polyacrylamide gels and electroblotted onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were probed with a goat anti-human galectin-1 IgG (R&D Systems, Minneapolis, MN, USA) or with a murine monoclonal anti-β-actin antibody (Sigma, St Louis, MO, USA) at 1:2,000 dilutions for 1 hour; then incubated with horse-radish peroxidase conjugated donkey anti-goat IgG (R&D Systems, Minneapolis, MN, USA) or goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) at 1:4,000 dilutions for 1 hour. Protein bands were detected by ECL chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). The specificity of the anti-human galectin-1 IgG was validated by testing with human recombinant galectins-2,-3,-4,-7, and -8 in the same experimental conditions as galectin-1.
Comparisons among groups were performed using Fisher's exact test for proportions and one-way ANOVA test for normally distributed continuous variables, as well as Kruskal-Wallis test and Mann-Whitney U test for non-normally distributed continuous variables. For the analysis of qRT-PCR data, pair-wise group comparisons were performed using “Generalized estimating equations”[92
]. In parallel, the t-test was also applied by averaging over the three technical replicates (Ct values) of each subject. To determine the influence of the gestational age on LGALS1
gene expression within the groups, a linear model was fitted in which the gestational age was used as a predictor for the Ct values. An adjustment of p-values to account for the six different comparisons among the four groups was performed using the Bonferroni method[93
]. An adjusted p-value of <0.05 was considered to be statistically significant. The R statistical software (www.r-project.org
) including required libraries and SPSS version 12.0 (SPSS Inc., Chicago, IL) were used for the analyses.