Tissue collection
Serial paraffin sections of human placental UC specimens were obtained from the University of South Florida under the protocol approved by the Ethics and Human Investigation Committees of the University of South Florida (approvel number: 00015578). Written and verbal informed consents were obtained from each patient. All of the samples were grouped according to clinical diagnosis: UC control (n=12) or PE (n=7). For in vitro studies, previously frozen HEECs (n=2) and HUVEC (n=1) from normal women undergoing hysterectomy (laparoscopy or laparotomy) or normal delivery were thawed and grown to confluence, as previously described(17).
Immunohistochemistry
Collected PE and normal patient UC paraffin blocks were cut into 5 µm sections that were then put into a heater to incubate overnight at 56 °C. The slides were deparaffinized in xylene (x3) for 20 min., followed by 100, 90, 80, and 70% alcohol x1 for 10 min. per gradient. Following deparaffinization, the slides were heated in 10 mM citrate buffer (pH 6.0) for 3x5 min in a microwave oven for antigen retrieval. The slides were then immersed in 3% hydrogen peroxide (in 1:1 v/v methanol/distilled water) for 12 min. to quench endogenous peroxidase activity. After washing with tris-buffered saline (TBS); (pH: 7.4) (x3) for 5 min., the slides were incubated in a humidified chamber with 5% blocking normal goat serum (Vector Labs, Burlingame, CA) for 30 min at room temperature (RT) in TBS. Excess serum was emptied, and then the slides were incubated overnight with a primary rabbit polyclonal anti-LEPR antibody (1:60; Santa Cruz Biotechnology, Dallas, TX) in 1% normal goat serum at 4 °C. Normal rabbit immunoglobulin G (IgG) (Vector Labs) isotypes were used for negative controls at the equal primary antibody concentrations. The slides were rinsed (x3) for 5 min. with TBS, and then biotinylated anti-rabbit IgG (Vector Labs) was used at a 1:400 dilution for 30 min. at RT. The antigen-antibody complex was identified using an avidin-biotin-peroxidase kit (Vector Labs) for 30 min. at RT. 3.3´-Diaminobenzidine tetrahydrochloride dihydrate (Vector Labs) was added as the chromogen to visualize immunoreactivity for 90 seconds. The slides were then counterstained with hematoxylin and mounted. Immunoreactive LEPR levels were semi-quantitatively assessed using the subsequent intensity categories: 0, no staining; 1+, weak but visible staining; 2+, moderate or distinct staining; and 3+, strong staining. As described previously(18), for each tissue, a histologic score (HSCORE) was derived by adding the percentages of cells that were stained at each intensity category and then multiplying that value by the weighted intensity of the staining using the formula HSCORE=Σ Pi (i + l), where i represents the intensity scores, and Pi is the corresponding percentage of the cells. In each slide, three randomly selected areas were assessed under a light microscope (x40 magnification), and the percentage of the cells at each intensity within these regions was evaluated at different times by two blinded researchers. The average HSCORE of the two examiners was used. Human endometrial endothelial cell and human umbilical vein endothelial cell isolation and experimental treatment with leptin. Frozen primary HEECs (n=2) and HUVECs (n=1) were derived from banked samples. The samples had been isolated and categorized as previously described(17) from endometrial specimens obtained from reproductive-age women undergoing hysterectomy (laparoscopy or laparotomy) and UC vein specimens obtained from the delivery of a normal pregnant woman. Written informed consent for sample retrieval was obtained from Yale University Faculty of Medicine, Human Investigation Committee and approved by the University of South Florida. Aliquots of frozen primary HEECs and HUVECs were thawed and grown to confluence in basal medium (BM), and a phenol red-free 1:1 v/v Dulbecco’s Modified Eagle Medium/Ham’s F-12 (Gibco, Grand Island, NY) mixture, containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL Fungizone complex (Gibco) supplemented with 10% charcoal-stripped calf serum (Gibco). At ~80% confluence, HEECs and HUVECs were transferred to 6-well plates at a density of 150x103 cells/well, for the corresponding treatments, as designated by each experimental condition. Confluent HEECs and HUVECs were incubated in parallel in BM with 0.1% ethanol (vehicle control) or leptin (0.1, 1.0, 10, 100 and 1000 ng/mL leptin, respectively). After incubation for 24 h, HEECs and HUVECs were rinsed with ice-cold 1x phosphate buffered saline and stored at -80 °C until used for immunoblotting analysis to measure IL-8 total protein levels.
Immunoblot analysis
Total protein was extracted using a cell extraction buffer (BioSource International, Camarillo, CA) containing 3 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The protein level was determined using a detergent-compatible protein assay (Bio-Rad, Hercules, CA). Samples (40 µg) were loaded on 10% Tris-hydrochloric acid-ready gels (Bio-Rad), electrophoretically separated, and electroblotted onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat milk powder in TBS containing 0.1% Tween 20 (TBS-T) for 1 h to reduce any non-specific antibody binding. Subsequently, the membrane was incubated overnight with a monoclonal mouse IgG1 clone primary antibody against IL-8 (1:800 R&D Systems, Inc., Minneapolis, MN) in 5% non-fat milk powder in TBS-T. The membrane was then rinsed several times with 1x TBS-T for 1 h and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Vector Labs) in TBS-T. Following several washes, IL-8 was visualized through light emission from the film (Denville Scientific, Holliston, MA) with enhanced chemiluminescence substrate (Thermo Scientific, Rockford, IL). Band intensities were quantified using computer densitometry analysis (Image J, National Institutes of Health, Bethesda, MD).
Statistical Analysis
Data from immunohistochemistry and Western blot analysis that were normally distributed, according to the Kolmogorov-Smirnov test, were compared using Student’s t-test or one-way analysis of variance, followed by the post hoc Holm-Sidak test. Immunohistochemistry and Western blot analysis data that were not normally distributed were analyzed using the Kruskal-Wallis nonparametric ANOVA-by-Ranks test, followed by the post hoc Student-Newman-Keuls test. Statistical calculations were performed using Sigmaplot 13 for Windows (Jandel Scientific Corp., San Rafael, CA). Statistical significance was considered as p<0.05.
