Reagents. Endothelial growth medium (EGM) culture medium was from Cambrex Bio Science Walkersville (Walkersville, MD). SB-505124 (ALK 5 inhibitor) was from Glaxo SmithKline (King of Prussia, PA, courtesy of N. Laping) and SB-203580 was from Biomol (Plymouth Meeting, PA). Type B Gelatin, EGTA, NAC, and DPI were purchased from Sigma (St. Louis, MO). SlowFade light antifade kit, Hoechst 33342, CM-H2DCFDA, rhodamine-phalloidin, and FITC-DNaseI were from Molecular Probes (Eugene, OR). TGF-β1 was from R&D Systems (Minneapolis, MN). Formaldehyde 37%, Triton X-100, and No. 1 coverslips were purchased from Fisher Scientific (Pittsburgh, PA). Black walled 96-well plates were purchased from Corning (Corning, NY).
HUVEC were isolated from human umbilical cord veins as described previously (23
) and cultured on 0.2% gelatin-coated dishes in EGM culture medium containing 10% FBS. The cells were routinely passaged with trypsin-EDTA and used for experiments between passages
2-6; 293 cells were obtained from American Type Culture Collection.
F- and G-actin staining by confocal microscopy. F-actin was stained with rhodamine phalloidin, and G-actin was stained with FITC DNase I. Cells were plated on coverslips coated with gelatin. Cells were kept quiescent in EGM culture media containing 0.5% BSA overnight before experimental treatment. Immediately after experimental protocol, cells were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. Coverslips were then incubated at room temperature with rhodamine-phalloidin (0.165 μM) alone or with FITC-DNaseI (0.3 μM) for 20 min. Cells were washed three times with PBS, and the coverslips were then mounted in SlowFade mounting media and sealed. Cells were directly visualized by fluorescence microscopy or confocal microscopy and representative photographs were obtained.
F- and G-actin quantitation with fluorescent plate reader. HUVEC were cultured to reach 80-90% confluence in 96-well black plates (Corning) coated with gelatin. After treatment, cells went through the same staining procedure as described above but FITC-phalloidin was used instead of rhodamine-phalloidin. Staining with FITC-phalloidin or FITC-DNase I was performed in separate wells. After being stained, the cells were washed three times with PBS and then 100 μl of PBS were left in each well and the plate was read with Millipore Cytofluor 2300 fluorescent plate reader using filter setting for excitation wavelength at 485 nm and emission wavelength at 530 nm. In selected experiments, cells were treated with kinase inhibitors, NAC or DPI, before TGF-β exposure or transduced with adenovirus for 72 h before TGF-β exposure.
ROS visualization and quantitation. HUVEC were plated on coverslips and made quiescent overnight in 0.5% BSA before stimulation with TGF-β. Cells were loaded for 10 min with 5 μM CM-H2DCFDA in phenol-red-free medium in the dark and then treated with TGF-β in the presence or absence of various inhibitors. In separate experiments, cells were transduced with adenoviral LacZ or dominant-negative ΔNADPH Nox4 vectors 72 h before experiments. Cells were then visualized by immunofluorescence microscopy after fixation and staining of the nuclei with DAPI or examined live using confocal microscopy. DCF fluorescence was visualized at an excitation wavelength of 488 nm and emission at 515 to 540 nm. To avoid photooxidation of the indicator dye, images were collected with a single rapid scan using identical parameters for all samples.
As a quantitative index of ROS generation, the Amplex Red reagent [10-acetyl-3,7-dihydroxyphenoxazine; Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, Molecular Probes (A-22188)] was used. Amplex Red reacts with hydrogen peroxide in the presence of horseradish peroxidase (HRP) with a 1:1 stoichiometry to form resorufin. HUVEC were cultured on 96-well black plates coated with gelatin. The cells were rested overnight with 0.5% BSA in EGM medium and modulated with adenovirus or inhibitors. After the completion of pretreatment of HUVEC, Amplex Red and peroxidase in Kreb’s Ringer phosphate buffer (pH 7.4) were introduced into each well in the dark. The final reaction volume in each well was 100 μl. TGF-β at different concentrations was introduced just before reading the plate. The contents of the plate were shaken for 5 s before the commencement of the first cycle of measurement. Fluorescence intensity was kinetically recorded with excitation at 544 nm and emission of 590 nm at 37°C over a 20-cycle period with 10 flashes per well via fluorescent plate reader (POLARstar OPTIMA, BMG Labtechnologies). Measurements were made at 1-min intervals over a 20-min period. The data are reported as the mean value from each well over a 20-cycle period with n = 4-6 wells per experiment. Each experiment was repeated three times.
Immunoblot analysis of Nox4.
Whole cell lysates from 293 cells and HUVEC were prepared with lysis buffer that contained 50 mM Tris·HCl (pH 7.2), 150 mM NaCl, 1% (wt/vol) Triton X-100, 1 mM EDTA, 1 mM PMSF, and 5 μg/ml aprotonin and leupeptin. Forty micrograms of cell lysate were resolved on a 10% reducing SDSPAGE and immunoblotted with antibody to Nox4 at 1:1,500 dilution. A secondary HRP-conjugated anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:2,000 dilution. The antibody to Nox4 was raised against the COOH-terminal end of Nox4 and detected by ECL chemiluminescence as previously described (17
). Two hundred ninety-three cells were transfected with a cDNA construct for human Nox4 as previously described (6
Immunostaining for Nox4 in HUVEC. HUVEC were plated on coverslips and made quiescent overnight in 0.5% BSA before stimulation with TGF-β. After stimulation, primary antibody at 1:100 dilution (rabbit polyclonal anti-Nox4 antibody) and secondary antibody (goat anti-rabbit IgG) conjugated to Alexa-Fluor-568 at 10 μg/ml final concentration (Molecular Probes) were used to detect Nox4. Bio-Rad MRC-600 confocal laser-scanning microscope mounted on a Zeiss Axiovert 100 fluorescent microscope equipped with a ×63 objective with rhodamine filter was used. Negative control was performed by omitting the primary antibody.
Preparation of NADPH Nox4 adenoviral construct and transduction of HUVEC
. Nox4 deletion construct was generated as described previously (17
). Briefly, cDNA for dominant-negative Nox4 lacking the NADPH binding domain was generated by removing the COOH-terminal sequence from wild-type Nox4 cDNA encoding 578 amino acids (GenBank accession number AF254621) for NADPH (amino acids 1 to 383). The construct was introduced into Ad-5 adenovirus using the Adeno-X expression system (BD Biosciences, Palo Alto, CA). A recombinant adenovirus encoding β-galactosidase was used as a control. Adenoviral constructs (600 infection forming units/cell) were preincubated for 100 min at room temperature with EGM media containing 0.5% BSA and 0.5 mg polylysine/ml. Transduction efficiency as measured by percent of cells expressing β-galactosidase was 90-95%, similar to prior studies with adenoviral transduction efficiency in HUVEC (22
). After preincubation, adenovirus transduction of HUVEC was performed by incubating cells in adenovirus-containing media overnight. The next day medium was changed to fresh EGM medium with 10% FBS. Seventy-two hours posttransduction, cells were used for experiments, after being made quiescent overnight in media containing 0.5% BSA.
Statistical analysis. In all experiments, data were evaluated for significance by one-way ANOVA using Student’s t-test. P < 0.05 was considered statistically significant.