the fluorescent dye SyBR Green or using Taqman Gene purchase GW 501516 Expression Assays. The primer sequences used are: TLR4; p22phox; NOX-2. Cyclophilin D and b2-microglobulin were used as normalizing internal controls. All PCRs were performed in duplicate. qRT-PCR was carried out in an ABI PRISM 7000 Sequence Detection System using the following conditions: 2 min at 50uC; 10 min at 95uC; and 40 cycles of 15 s at 95uC and 1 min at 60uC. At the end of the SyBR Green PCR, a final stage with a melting curve analysis was added to show the specificity of the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19657297 product. To calculate the relative index of gene expression, we employed the 22DDCt method using the untreated samples for calibration. Cell Culture To obtain primary cultures of VSMCs, thoracic aortas from SHRs or Wistar rats were aseptically removed, cleaned of fat tissue and blood cells and placed in cold Dulbecco’s modified Eagle’s medium containing 0.1% BSA, 200 U/ml penicillin and 200 mg/ml streptomycin. The aortas were digested in the same medium containing 2 mg/ml collagenase type II and incubated for 30 min at 37uC in a humidified atmosphere of CO2. After the adventitia was carefully removed, VSMCs were obtained using the explant method. Cells were identified as VSMCs by morphological and growth characteristics and by positive immunocytochemical staining with a specific monoclonal anti-a-actin antibody. For experiments, cells from passages 2 to 5 were rendered quiescent by incubation in DMEM containing 0.2% FBS for 24 h. The cells were stimulated with 100 nM Ang II, with or without pretreatment for Detection of ROS The oxidative fluorescent dye dihydroethidium was used to evaluate in situ superoxide anion production in VSMCs. Hydroethidine freely permeates cells, and in the presence of superoxide anions it is oxidized to ethidium bromide, which is trapped by intercalation with DNA. Ethidium bromide is excited at 546 nm and has an emission 
wavelength of 610 nm. Briefly, VSMCs were plated onto glass coverslips placed in 6-well plates TLR4 and Endothelial Dysfunction in Hypertension and cultured as described above. Subconfluent cells were stimulated with 100 nM Ang II for 2 h in the absence or presence of 1 mM CLI-095, which was added 1 h prior Ang II. The cells were then incubated with 10 mM DHE in cell culture medium for 30 min at 37uC. The images were then acquired using a fluorescence microscope, captured using a digital spot camera and processed using the Metamorph image analysis software. Non-stimulated VSMCs were imaged daily in parallel, using the same image settings throughout the course of the study. DHE fluorescence was 8 TLR4 and Endothelial Dysfunction in Hypertension quantified in individual cell nuclei. At least 5 independent experiments were performed. Then, we expressed the effects of the different drugs as fold increases over the control. NADH oxidase activity The superoxide anion generated by NADH oxidase activity was determined using a chemiluminescence assay using 5 mM lucigenin and 100 mM NADH. VSMCs treated as described for the ROS detection experiments were homogenized in a lysis buffer. The reaction was initiated with the addition of a mixture of lucigenin and NADH to the protein sample in a final volume of 300 ml. Chemiluminescence was determined every 2.4 s for 5 min in a plate luminometer. A buffer blank was subtracted from each reading, and the value of the area under the curve was used to quantify chemiluminescence. The data obtained were expressed as fold increases ov