Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the

Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of atherosclerosis and it can stimulate the expression of Cyt387 a variety of inflammatory signals. mechanism we investigated the effect of different concentrations of Ox-LDL (50 100 150 μg/mL) on endothelial cell proliferation and apoptosis. Stimulation with Ox-LDL increased OX40L protein 1.44-fold and mRNA 4.0-fold in endothelial cells and these effects were inhibited by blocking LOX-1. These results indicate that LOX-1 plays an important role in the chronic inflammatory process in blood vessel walls. Inhibiting LOX-1 may reduce blood vessel inflammation and provide a therapeutic option to limit atherosclerosis progression. model of endothelial cell injury. In addition we investigated the underlying mechanisms involved in order to provide a new theoretical basis and targets for drug therapy for prevention and treatment of atherosclerosis. Material and Methods Cell lines and reagents Human umbilical vein endothelial cells (HUVECs) were obtained from Chongqing Medical University (Chongqing China) and cultured in RPMI-1640 (Gibco USA) supplemented with 10% fetal bovine serum (Gibco). For Ox-LDL injury HUVECs were treated with various concentrations of Ox-LDL (XieSheng Bio China) and Poly I (Santa Cruz USA) for 24 h. Cells were cultivated in a humidified atmosphere with 5% CO2 at 37°C. Cell proliferation assays The cell counting kit (CCK-8 Beyotime Institute of Biotechnology China) assay was used to determine cell proliferation. Increasing concentrations of Ox-LDL (50 100 and 150 μg/mL) were added to cell cultures which were then incubated for 24 h. Absorbance was detected with a microplate reader at a wavelength of 450 nm using a 96-well multichannel auto reader (Biotech Instruments USA). The percentage inhibition of cell proliferation was determined by comparing the absorbance of treated with untreated controls as follows: Inhibition (%) = [1-(A of the experimental sample/A of the control)]×100%]. Assessment of cell cycle and apoptosis Proliferating HUVECs were serum-starved overnight and treated with Ox-LDL (100 μg/mL) in complete media for 24 h. Following treatment the cells were harvested and fixed in 70% ice-cold ethanol. The percentages of cells in the G0/G1- S- Cyt387 G2- and M-phases were quantitated by flow cytometry. The extent of apoptosis was evaluated by Annexin-V staining. HUVECs were incubated in the presence of Ox-LDL (100 μg/mL) for 24 h and stained with Annexin-V-fluorescein isothyocyanate (FITC) and propidium iodide (PI). Samples were analyzed by flow cytometry. The data shown are representative of at least three impartial experimental sets. Immunoblotting HUVECs were plated in a culture flask 1 day before the experiment. The cells were then incubated Cyt387 for 24 h under the following conditions: a) no Ox-LDL b) 100 μg/mL Ox-LDL or c) 250 μg/μL Poly I plus 100 μg/mL Ox-LDL (14). The cells were washed three times with ice-cold PBS lysed with RIPA lysis buffer (Beyotime) and placed on ice for 30 min. Proteins were separated by SDS-PAGE (12% gels) and subsequently transferred to a PVDF membrane (Millipore USA). The membrane was blocked with 5% BSA in Tris-buffered saline and Tween-20 (10 mM Tris pH 7.5 140 mM NaCl 0.05% Tween-20) for 2 h at room temperature. A rabbit polyclonal Lum antibody against OX40L (1:1000 Santa Cruz) and a rabbit polyclonal antibody against LOX-1 (1:1000 Abcam Hong Kong) were used as the primary antibodies and horseradish peroxidase-conjugated goat anti-rabbit IgG was used as a secondary antibody. BeyoECL Plus (Beyotime) was used for antibody detection according to the manufacturer’s instructions. Immunocytochemistry Following the same stimulation conditions described above HUVECs were fixed in 4 paraformaldehyde for 20 min and washed three times in PBS. A rabbit polyclonal primary antibody against OX40L (1:1000 Santa Cruz) was added at 4°C overnight. Cells were washed three times in PBS and incubated with the blocking solution which included FITC-conjugated goat Cyt387 anti-rabbit IgG (CWBio China) at 37 for 2 h. Cells were washed three times in PBS and incubated with PI at 37°C for 5 min. Cells were observed with a laser confocal microscope and the average fluorescence value of eight cells from a random.