Materials
The RXR-selective retinoid used in this study, LGD1069, (Targretin®), was purchased from Sigma Chemicals (St Louis, MO). A stock solution (1 mM) was prepared by dissolving LGD1069 in DMSO and stored at 4°C for <1 month before use. The vehicle (DMSO) was used as a control in all experiments at a final concentration of 0.1%.
Cell culture
HUVECs (Human umbilical vein endothelial cells) were isolated as previously described by Jaffe et al [13], from umbilical cords obtained from a parturient at Beijing Obstetric Hospital who gave written informed consent. The study protocol was approved by the IRB (Institutional Review Board) of Beijing Obstetric Hospital. HUVECs were routinely grown in M199 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Gibco) and endothelial cell growth supplement (ECGS) (BD Biosciences, Bedford, MA) at 37°C and 5% CO2. HUVEC between P3 and P4 were used for all experiments.
MTT assay
Cell viability was assayed using a MTT assay. Briefly, log phase cells were plated in 96-well plates at a density of 3 × 104 cells per well in the presence of 2 ng/ml VEGF (physiological concentration) in 10% FBS media by for 24 h incubation, and then sufficient volumes of stock solution of LGD1069 and DMSO were added to the culture medium, in order to obtain different treatment doses. Then the vehicle and a range of LGD1069 were co-incubated with cells for different time. Three duplicate wells were set up in each sample. At least three independent experiments were carried out. After treatment, cells were incubated with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma Chemical Company) (final concentration 0.5 mg/ml) for 4 h at 37°C. The media was carefully removed from each well and 200 μl of DMSO was added. The plates were gently agitated until the color reaction was uniform and the OD570nm and OD450nm were determined using a microplate reader (WellScan MK3, Labsystems Dragon). The data were analyzed using a SlideWrite program to determine the IC50 value of LGD1069. Media only treated cells served as the indicator of 100% cell viability.
Sulforhodamine B assay
The growth inhibition effect of LGD1069 on endothelial cells was also examined with the sulforhodamine B (SRB) assay. Briefly, HUVEC cells were seeded in 96-well plates (2,000 cells per well) and co-incubated with 2 ng/ml VEGF overnight. Then, triplicate wells were treated with various concentrations of LGD1069. Three duplicate wells were set up in each sample. At least three independent experiments were carried out. After 96 h, the culture medium was discarded, 10% (w/v) pre-cooled (4°C) trichloroacetic acid (100 μl) was added to each well, and the cells were fixed at 4°C in a refrigerator for 1 h. Each well was then stained with 100 μl 0.4% SRB (w/v) in 1% acetic acid (v/v) for 15 min. The plates were washed with 1% acetic acid (v/v) for removal of unbound dye, air dried, and then treated with 150 μl per well 10 mM Tris base (pH 10.5) to dissolve the bound dye. The absorbance in each well was read with a microplate reader (WellScan MK3, Labsystems Dragon) at 515 nm. The data were analyzed using a SlideWrite program to determine the IC50 value of LGD1069. Media only treated cells served as the indicator of 100% cell viability.
Tube Formation Assay
The tube formation assay was used to investigate the effect of LGD1069 on angiogenesis in vitro. The methods were described previously [5]. In briefly, a 96-well plate was coated with 80 μl liquid Matrigel per well, which was allowed to solidify at 37°C for 45 min. HUVEC cells were seeded at a density of 3 × 104 cells per well in 100 μl complete culture medium containing different concentrations of LGD1069 or vehicle (control), then 100 μl serum-free M199 medium contained VEGF (final concentration is 2 ng/ml) was added. Plates were incubated for 24 h at 37°C and 5% CO2 (sufficient for formation of an intact network in the control group). Images were recorded by an inverted microscope (Olympus, IX70, Japan), and total vessel lengths were counted with ImagePro plus 5.0 image analysis software (Media Cybernetics, Inc, Silver Spring, MD).
In vitroassays of HUVECs adhesion
96-well microtiter plates were pre-coated with 10 μl Matrigel (0.5 μg/ml) and incubated at 4°C overnight. Wells were blocked with 20 μl M199 containing 2% BSA for 1h at 37°C. The exponential phase HUVE cells were harvested, and re-suspended in serum-free M199 medium supplemented with 0.1% BSA. The cells (5 × 104/well) were seeded in wells and co-incubated with 2 ng/ml VEGF at 37°C in a 0.5% CO2 atmosphere for 2h with or without LGD1069. The wells were washed thrice with PBS to remove unattached cells, then the attached cells were incubated with MTT and the absorbance was measured at 570 and 450 nm. Each assay was performed in triplicate. Three independent experiments were repeated [14].
Matrigel Invasion Assay
In vitro invasion of HUVEC cells was measured by the invasion of cells through 48-well microchemotaxis plates (AP 48, Neuro Probe, Gaithersburg, MD) with wells separated by a Polyvinylpyrrolidone-free polycarbonate filter with an 8 μm pore size [15]. Briefly, the polycarbonate membrane was pre-coated with 5 μg of fibronectin in a volume of 50 μl on the rough (lower) surface. The Matrigel was diluted to 100 μg/ml with cold PBS and applied to the smooth (upper) surface of the filters (5 μg/filter), and dried at room temperature. The lower compartments of the plates were filled with 30 μl M199 containing 0.1% BSA. Log-phase cells were harvested and washed thrice with serum-free M199, and re-suspended to a final concentration of 2 × 106/ml in M199 with 0.1% BSA. Cell suspensions (100 μl) with or without LGD1069 were added to the upper compartment and with 2 ng/ml VEGF(final concentration) for 24 h at 37°C in a 5% CO2 atmosphere. The filters were fixed with methanol and stained with Haematoxylin for 10 min, washing with distilled water, and then stained with Eosin Y for 30s. The cells on the upper surface of the filters were removed by wiping with cotton swabs. The cells invading the lower surface of the filter through Matrigel and filter were manually counted under a microscope at a magnification of × 200, and each assay was performed in triplicate.
Matrigel Migration Assay
In vitro migration of HUVECs was measured by AP 48 chamber (Neuro Probe, Gaithersburg, MD), similar to in vitro invasion assay. Briefly, the underside of polycarbonate membrane was coated with 20 μg/ml fibronectin overnight at 4°C. 30 μl of M199 (with 10% FBS and 10 μg/ml Collagen I) was added to the lower chamber, and the chamber was covered by filter. HUVECs were trypsinized and washed by FBS-free M199, then 100 μl of cell suspensions (in FBS-free M199, containing 2 × 105 cells) with or without LGD1069 were added to the upper chambers and co-incubated with 2 ng/ml VEGF overnight at 37°C. Determination of migrated cells is the same as what was described in Matrigel Migration Assay.
RT-PCR assay
Untreated and treated cells with LGD1069 were washed twice with cold PBS. Total RNA was isolated by Trizol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water (0.1% DEPC was added to water overnight and then autoclaved 20 min to destroy DEPC). According to the manufacturer's instructions (TaKaRa, Dalian, China), reverse transcription was performed at 42°C in the presence of 5 units of AMV reverse transcriptase and 1 μg of RNA for 60 minutes. AMV RT inactivation and RNA/cDNA/primer denaturation were performed at 95°C for 5 minutes. cDNA was stored at -20°C. The PCR profile was as follows, 10 min at 95°C, followed by several cycles of 30s at 95°C and 1 min at 60°C. The PCR product was separated by 2% agarose gel electrophoresis, and the gels were viewed by UV transillumination, photographed by Kodak 120 gel imaging system. Primers, Tm, and cycles were listed on Table 1.
MMPs activities assay
MMPs activities were measured by SensoLyte® 520 MMPs Assay Kit according to the manufacturer's protocol (AnaSpec, Fremont, CA). Briefly, Untreated and treated cells with LGD1069 were collected and homogenized by assay buffer containing 0.1% Triton-X 100, and centrifuged for 15 min at 10000X g at 4°C. Collect the supernatants and store at -70°C until use. To activate MMPs before the measurement, the supernatants were incubated with 4-aminophenylmercuric acetate (APMA) for 1 h at 37°C. Then, 50 μl MMPs-containing samples were added to 96-well plate per well. Add 50 μl per well of MMPs substrate solution to the sample and control wells. Mix the reagents by shaking the plate gently for 30 sec. Incubate the reaction at 37°C for 50 min. 50 μl stop solution was added per well, and mix the reagents and measure fluorescence intensity at Ex/Em = 490/520 nm(Victor3, Perkin Elmer, Waltham, MA).
Western blot analysis
HUVECs were washed once with PBS and then lysed by the addition of 1 ml lysis buffer (10 mmol/l Tris, pH 7.6, 150 mmol/l NaCl, 5 mmol/l EDTA, pH 8.0, 10 ml/l Triton X-100, 1 mmol/l DTT) containing 0.1 mmol/l PMSF. After 30 min on ice, lysates were collected and clarified by centrifugation at 15,000 g for 10 min at 4°C. Aliquots of whole cell lysates were subjected to 10% SDS-PAGE and then transferred to Hybond nitro blotting membranes. The membranes were blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.5 ml/l Tween-20 (TTBS) and then probed with either anti-Smad2/3(sc-6032), p-Smad2/3(sc-11769), Runx2(sc-8566), and anti-β-actin (sc-1616) antibodies in 30 g/l bovine serum albumin in TTBS. Positive antibody reactions were visualized using UVP EC3-410 luminescence system.
Statistical analysis
The mean values were obtained from at least three independent tests. The data are presented as mean ± S.D., and analyzed with the SPSS software program (version 10.0 for Windows; SPSS INC., Chicago, IL). Comparison among different groups was carried out by analysis of variance (the one-way ANOVA). Differences between means were considered statistically significant at p < 0.05.
