(Carlsbad, CA). Human peripheral blood mononuclear cells (PBMC) were isolated and purified KPT-330 cost from blood (Red Cross Blood Bank) by density gradient centrifugation and adherence as described by us previously (Liao et al., 1994). PBMC were then cultured in serum-free macrophage media (37 °C, 5% CO2) overnight with lipopolysaccharide (Escherichia coli, 100 ng mL−1) or vehicle alone. Doxycycline was added at final concentrations ranging from 0.1 to 20 μM. Conditioned media were analyzed for the cytokines [tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)] and MMP-9 by enzyme-linked immunosorbent
assay (ELISA). In separate assays, PBMC at 5 × 105 cells mL−1 were cultured with macrophage medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and containing 100 U mL−1 penicillin and 100 μg mL−1 streptomycin in Teflon beakers for 7 days with different concentrations of doxycycline. At the end of the 7-day incubation, conditioned media were analyzed by gelatin zymography as
described by us previously (Golub et al., 1995). Western blot, gelatinase and collagenase activity assays were carried out as described below. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)/fluorography of [3H]-labeled type I collagen was scanned using a laser densitometer to quantify the effect of doxycycline on the collagenase activity, the latter assessed by the production of [3H]-labeled collagen degradation learn more fragments as described by us previously (Yu et al., 1993). R22 rat heart smooth muscle Selleckchem Sirolimus cells were cultured in minimum essential medium supplemented with FBS, tryptose phosphate broth and cefotaxime (Gu et al., 2005). The R22 cells were plated onto multiwell tissue culture
plates at an initial density of 2.5 × 104 cells cm−2 and were maintained at 37 °C in 5% CO2. At confluence, the medium was supplemented with [3H]-fucose, which were incorporated into a complete interstitial ECM elaborated by the cells. Every 4 days, 50 μg mL−1 ascorbic acid was added to ensure maximal formation of an insoluble collagen-rich ECM. After culturing for at least 1 week in radiolabeled medium, cells were lysed by brief exposure to 25 mM NH4OH without disrupting the ECM. The wells were washed three times with sterile H2O and once in phosphate-buffered saline (PBS) containing 0.02% NaN3. Excess PBS was then removed and plates were stored at 4 °C until use. Before use, the ECM was rehydrated by rinsing three times with sterile buffer. PBMC in serum-free media were applied to R22 ECM-coated wells of microplates at a density of 5 × 105 cells mL−1 and incubated for 2 days at 37 °C in 5% CO2 in the presence or absence of doxycycline. After the 2-day incubation, the supernatants were collected and the remaining undegraded ECM in each well was solubilized by overnight incubation with 2 M NaOH. The radioactivity in the supernatants and in the NaOH was determined in an LKB liquid scintillation counter (Gu et al., 2005).