However, mice treated with valsartan, fenofibrate or PDTC showed alleviated progression of atherosclerosis and reductions in inflammatory parameters. plasma CRP, TNF-, nuclear factor-kappa B (NF-B) and H2O2 were determined by ELISA. CCR2 (the receptor of MCP-1), macrophages, and smooth muscle cells were Adenine sulfate detected by immunohistochemistry. P47phox, MCP-1 and eNOS were detected by RT-PCR, while P47phox, NF-B and MCP-1 were detected by Western blot assay. The aortic atherosclerotic lesions were significantly increased in AT1 peptide/AT1-AA treated mice, along with simultaneous increases in inflammatory parameters. However, mice treated with valsartan, fenofibrate or PDTC showed alleviated progression of Adenine sulfate atherosclerosis and reductions in inflammatory parameters. Thus, AT1-AA may accelerate aortic atherosclerosis in ApoE-/- mice, which is mediated, at least in part, by the inflammatory reaction involving nicotinamide-adenine dinucleotide phosphate oxidase, reactive oxygen species, and NF-B. In addition, valsartan, fenofibrate and Rabbit polyclonal to WBP2.WW domain-binding protein 2 (WBP2) is a 261 amino acid protein expressed in most tissues.The WW domain is composed of 38 to 40 semi-conserved amino acids and is shared by variousgroups of proteins, including structural, regulatory and signaling proteins. The domain mediatesprotein-protein interactions through the binding of polyproline ligands. WBP2 binds to the WWdomain of Yes-associated protein (YAP), WW domain containing E3 ubiquitin protein ligase 1(AIP5) and WW domain containing E3 ubiquitin protein ligase 2 (AIP2). The gene encoding WBP2is located on human chromosome 17, which comprises over 2.5% of the human genome andencodes over 1,200 genes, some of which are involved in tumor suppression and in the pathogenesisof Li-Fraumeni syndrome, early onset breast cancer and a predisposition to cancers of the ovary,colon, prostate gland and fallopian tubes PDTC may inhibit the AT1-AA induced atherosclerosis. access to food and water. Eight-week-old male ApoE-/- mice with C57BL/6J background were purchased from the Jackson Laboratory (Maine, NE, USA). They were bred in Peking University, China and raised in the Experimental Animal Center of Tongji Medical College. The mice were housed in a room with a 12-hour light/dark cycle and the temperature was maintained at 22C. They were given access to food and water for 16 weeks. Preparation of the AT1 peptide and AT1-AA A peptide corresponding to amino acids 165-191 of the second extracellular loop of human AT1 receptor was produced using an automated multiple solid-phase peptide synthesizer in our laboratory. The peptide purity was determined by high performance liquid chromatography (HPLC) and 95% purity was noted. The sequence of this peptide was as follow: IHRNVFFIENTNITVCAFHYESQNSTL. To prepare AT1-AA, male Japanese white rabbits weighing 2.0-2.5 kg (n = 6) were used, the AT1 peptide was conjugated and rabbits were handled as previously described and raised for 9 weeks [20]. At week 9, blood was collected from these rabbits for the determination of antibody response to this peptide by ELISA as previously described [20]. Five immunized rabbits had anti-peptide antibodies at a high titer (above 1:12000). Subsequently, the rabbits were anesthetized and sacrificed by exsanguination from the common carotid arteries, and IgG was prepared as described previously [21]. The AT1-AA activity was measured by a bioassay that is used to evaluate the beats per minute (bpm) of rat myocardial cells in vitro, as described previously [22]. Results indicated statistical significance (data not shown). Animal models and treatments The animal models of atherosclerosis were established with AT1-AA in mice in two ways. In the first group, mice were injected subcutaneously with the conjugated peptide, prepared as described previously and emulsified in Freunds complete adjuvant (FCA), at multiple sites. A booster injection (emulsified in Freunds incomplete adjuvant (FIA)) was performed at weeks 2, 4, 8, and 12. The even-aged male ApoE-/- mice were treated with Freunds adjuvant alone in the same manner and served as a negative control group. In the second group, animals were infused weekly with antibodies prepared from rabbits, and even-aged male ApoE-/- mice treated with BSA in the same manner served as a negative control group. In both groups, mice were randomly further divided into five subgroups. The number of mice and treatments used in each subgroup are shown in Table 2. Subsequently, these mice were given access to food and water Adenine sulfate for 16 weeks. Valsartan at Adenine sulfate 1 mg/kg/d (Novartis Pharma AG, Switzerland) and fenofibrate at 30 mg/kg/d (Laboratories FOURNIER, France) were intragastrically administered once daily. Pyrrolidine dithiocarbamate (PDTC) (30 mg/kg) (Sigma, USA) was intraperitoneally administered Adenine sulfate once every other day. Blood was collected at weeks 0, 4, 8, 12 and 16 after overnight fasting and processed for the detection of antibody titer by ELISA. In negative control groups, blood was collected at week 0. The antibody titer in different groups is shown in Figure 1. Open in a separate window Figure 1 Antibody titer in AT1 peptide group and AT1-AA group. Blood was collected at the end of 4th, 8th, 12th, and 16th weeks, and at 4th, 8th and 12th weeks before.