s to investigate whether TLR4 activation, due to increased RAS activity, contributes to hypertension and the functional vascular alterations observed in this pathology. The specific objectives were to investigate the following: 1) the alteration of TLR4 expression in hypertension and the contribution of Ang II to this alteration; 2) the role of TLR4 in hypertension occurrence, as well as in the associated vascular function alterations; and 3) the involvement of the TLR4-activated ROS production in the vascular dysfunction associated to this pathology. Materials and Methods Ethics statement and Animals All experiments were approved by the Ethical Commission for the Use of Animals of Universidade Federal do Espirito Santo, Brazil and by the Ethical Committee of Research of the Universidad Autonoma de Madrid, Spain. This study was carried out in strict accordance with the recommendations for biomedical research as stated by the Brazilian Societies of Experimental Biology, the guidelines for ethical care of experimental animals of the European Community, the current Spanish and European laws, and the International Guiding Principles for Biomedical Research Involving Animals. Adults male spontaneously hypertensive and Wistar rats were used for these studies. Rats were housed under a 12 h light/ 12 h dark cycle, they had free access to water and were fed a standard rat chow ad libitum. In one set of experiments, we analyzed if hypertension alters TLR4 expression and its dependence on RAS activity. For this, we used Wistar rats and SHRs untreated and treated with the AT1 receptor antagonist losartan. Systolic arterial pressure was measured by tail plethysmography. In another set of experiments, we investigated whether the TLR4 receptor plays a role in the occurrence of hypertension and TLR4 and Endothelial Dysfunction in Hypertension the associated vascular alterations. For this, we used SHRs treated with an anti-TLR4 antibody and Wistar rats and SHRs treated with a non-specific IgG and acetylcholine in endothelium-intact aortic Aphrodine chemical information segments from Wistar and SHRs treated with a non-specific IgG and SHRs treated with anti-TLR4 antibody. The results are the mean6SEM. P,0.05 vs Wistar, P,0.05 vs. SHR using two-way ANOVA and Bonferroni post-test. The number of animals used is shown in parentheses. doi:10.1371/journal.pone.0104020.g003 3 TLR4 and Endothelial Dysfunction in Hypertension 1 mg/day, saline-diluted, intraperitoneal injection, 15 days; sc2026, Santa Cruz Biotechnology Inc.) to rule out non-specific effects of the anti-TLR4 antibody treatment. Hemodynamic parameters and vascular function in aortic rings were evaluated. To further elucidate the role of TLR4 in the Ang II effects, cell culture experiments using VSMCs from Wistar and SHR were used. Rats were euthanized by CO2, and all efforts were made to minimize suffering. Then, the aortas were removed and placed in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19660899 cold Krebs-Henseleit solution aerated with a 95% O2-5% CO2 mixture. Aortic segments were dissected free of fat and connective tissue and maintained in KHS. Segments used for gene expression studies were immediately frozen in liquid nitrogen and kept at 270uC until the day of the experiment. The hearts were removed to assess cardiac hypertrophy. For this, the ratio between the heart dry weight and the length of the tibia was calculated. TLR4 and Endothelial Dysfunction in Hypertension Hemodynamic parameters At the end of the treatment, body weight was recorded and the rats were anest