Stock solution (12.5 mM) of resveratrol (Sigma Chemical Co.) was prepared in DMSO and stored at -20°C. For treatment, the resveratrol was diluted in RPMI 1640 and added to cultures to give the desired final concentrations. Untreated cultures received the same amount of the carrier solvent (0.2% DMSO). The rhodamine-conjugated phalloidin, used to detect F-actin, was purchased from Molecular Probes, Eugene, OR, and stored at 4°C in methanol. Before use, the solvent methanol was evaporated using N2 gas and the F-actin probe was resolubilized in TBS (Tris-buffered saline).
Cell culture and treatment with resveratrol
The BPAEC isolated from the distal main intrapulmonary artery of calf lungs were cultured in minimal essential medium (MEM) supplemented with 15% fetal bovine serum and containing D-valine as previously described [56
]. Plastic coverslips on which the cells were cultured were sterilized by washing in ethanol, followed by rinsing three times in MEM and 15 min exposure to UV radiation. The coverslips were placed in 6-well tissue culture plates, and 2 ml of the cells, at an initial density of 1 × 105
cells/ml, were added to each of the wells. Twenty-four h after seeding, the coverslips were treated with 0,10, 25, 50, and 100 μM resveratrol and maintained for 2 days at 37°C until they were fixed and stained for confocal microscopy.
Preparation and staining of slides
Coverslips containing adhered BPAEC were washed for 5 min in TBS, fixed with 5% glutaraldehyde in 0.1 M cacodylate (pH 7.4) for 15 min, washed again for 5 min in TBS, then stored in 0.1 M cacodylate/7% sucrose at 4°C until staining. Cells were permeabilized with 0.1% Triton X-100/TBS for 3 min, washed twice with TBS, blocked twice for 3 min with 0.1% bovine serum albumin in TBS, and incubated with rhodamine-conjugated phalloidin for 20 min. Routinely, 5 μl of rhodamine-phalloidin, dissolved in 200 μl TBS, was used for each coverslip stained. The coverslips were wash with 0.1% BSA/TBS for 3 min, then mounted in an inverted position onto TBS-containing beveled glass slides and sealed with clear nail polish.
A parallel plate perfusion chamber as described previously [57
] was used to generate simulated arterial flow conditions. The chamber consisted of three pieces: two rectangular slabs and a central knob with a depression that fits 18 by 21 mm coverslips. The depression containing the coverslip was placed perpendicularly to the flow of perfusate. A laminar arterial shear rate of 650 s-1
was achieved based on the dimensions of the flow chamber slit and a constant flow rate of 10 ml/min. After preincubation at 37°C, minimal medium was drawn directly by an eight roller peristaltic pump through the perfusion chamber containing the BPAEC-covered coverslip positioned 70 mm from the inlet valve at a rate of 10 ml/min for either 2 min or 5 min. A recirculating system was employed to recycle the medium after passing through the chamber.
Prepared slides containing adhered BPAEC were examined using a BioRad confocal microscope. The cells were imaged following excitation of rhodamine-phalloidin with an argon laser beam at a wavelength of 554 nm using either an inverted- or an uprighted-staged microscope equipped with either a 10X or a 20X oil-free objective and an epifluorescent illumination attachment. Cells viewed in representative field screen captures were counted manually for cell morphology and classified as either normal or elongated.
Passages 4-5 BPAEC were seeded on plastic coverslips as described above. Inhibitor concentrations were the same as in [28
]. Stock solutions were prepared by dissolving the inhibitors in DMSO. The inhibitors used and their stock solutions were as follows: quin2-AM (Sigma, 2 mM), herbimycin A (GibcoBRL, 0.175 mM), chelerythrine (Sigma, 0.4 mM), cytochalasin D (Sigma, 8 μM), nocodazole (Sigma, 0.66 mM). Quin2-AM, herbimycin A, chelerythrine, and cytochalasin D were stored at -20°C, and nocodazole was stored at 4°C. The stock solutions were diluted 1:200 in MEM to give the following final concentrations: quin2-AM (10 μM), herbimycin A (875 nM), chelerythrine (2 μM), cytochalasin D (40 nM), nocodazole (3.3 μM). Inhibitors were applied 24 h prior to the addition of resveratrol, except for quin2-AM, which was applied 1 h prior to resveratrol treatment. Since cells treated with various inhibitors grew less well, compared to controls, treatment with 100 μM resveratrol was reduced to approximately 40 h. Fixing and staining of cells for confocal microscopy was as described above.
Western blot analysis
Control and treated cells were lysed by repeated freeze-thaw cycles with buffer containing 10 mM HEPES (pH 7.5), 1.5 mM Mg(OAc)2, 1 mM dithiothreitol (DTT), 0.5% NP40, 5% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, and 10 μg/ml each of the protease inhibitors aprotinin, pepstatin, and leupeptin. Cell-free extracts were obtained by centrifugation in a microcentrifuge. Lysates (7-10 μg) from control and treated cells were separated on 10% SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes, and the membranes were incubated with the respective primary and secondary antibodies. The monoclonal primary antibodies used and their dilutions were as follows: Phosphorylated, active ERK1/2-P (RBI/Sigma, 1:1000), total ERK1/2 (RBI/Sigma, 1:1000), eIF4E (Santa Cruz, 1:250), eNOS (Sigma 1:1000). Membranes were probed with alkaline phosphatase-conjugated IgG (Santa Cruz, 1:1500) or horseradish peroxidase-conjugated IgG (Santa Cruz, 1:2000). Specific immunoreactive bands were identified by color reaction or enhanced chemiluminescence, respectively.