Role Of Antibodies Against The Ectodomains Of AT1 Receptor In The Pathogenesis Of Atherosclerosis

Aims: Use synthetic ectodomains of AT1 receptor immunized rabbits ,to prepare polyclonal antibody .Purified and identified it's biological activity.To know the functions of the antibody in the pathogenesis of atherosclerosis which provides the diagnosis and treatment of this disease.Methods and Results: According to the human AT1 receptor second extracellular loop amino acid sequences of peptides, synthetic peptides, used chemical methods with the carrier protein bovine serum albumin (BSA) coupled to immunize New Zealand white rabbits, Detected antibody titers used the method ELISA.Found that the synthetic peptides have good immunogenicity, one week after the second immunization the antibodies began to increase ,after the fifth immunization the immune response of antibody titers raised to1:409 600,and then harvested the rabbit antiserum. Immunoaffinity chromatography purification of anti-serum, SDS-PAGE detected the purity of the purified antibody showed a single clear protein band; Western blot results were detected:the anti-AT1 receptor antibody with the AT1 receptor protein binding at the molecular weight of 40KD and forms the unique band, immunofluorescence observed the antibody with the AT1 receptor on the surface of the cell.Conclusions: The rabbit anti-human polyclonal antibody we preparaed can specifically recognize the AT1 receptor. Made a good foundation. for the follow-up cellular and molecular level for experimental study. Aims: To study the effects of the rabbit anti-human AT1 receptor second extracellular loop peptide antibodies (AT1-AA) on human umbilical vein endothelial cells (HUVECs) express MCP-1 (monocyte chemotactic factor -1),and the associated signaling mechanisms.Methods and Results: The AT1-AA was incubated with HUVECs, with different dilutions and incubation times detected the expression of MCP-1, took the maximum expression of MCP-1,dilution 1:20 dilution, incubated with HUVECs for 12 hours. Use Western blot and PT-PCR method to detect the expression of cell MCP-1 protein and mRNA. Results showed that: After AT1-AA incubated with HUVECs cell MCP-1 expression was significantly increased, Losartan can significantly inhibited the AT1-AA induced endothelial cell expression of MCP-1.PDTC was a specific inhibitor of NF-κB P65, with different concentration of PDTC (5,10,20μmo l / L) pretreatment with endothelial cells for 1 hour, then plused 1:20 dilution of the AT1-AA incubated with HUVECs for 12 hours. Harvested the cells.Used the Western blot and PT-PCR method to detect cell MCP-1 and P65 protein and mRNA expression. Results showed that: compared with control group, PDTC treatment group with PDTC concentration increased MCP-1 protein and mRNA expression levels decreased (P <0.05) , P65 protein and mRNA expression was also decreased (P <0.05).Linear correlation analysis showed that: MCP-1 expression and PDTC concentration was significantly negatively correlated, P65 expression and the PDTC concentration was significantly negatively correlated,Conclusions: AT1-AA can up-regulated the expression of MCP-1 by NF-κB pathway promote monocyte adhered with endothelial cells may be one of the mechanisms of AT1 receptor autoantibodies involved in the development of atherosclerosis Aims: Through the study of the effects of AT1-AA on the foam cells derived from THP-1 cells express ABCA1 (ATP binding cassette transporter A1) to know the effects of AT1-AA on the expression of lipid transfer receptor . To clarify their place and relationship in the development of atherosclerosis.Methods and Results: PMA induced THP-1 differentiated into adherent marophages, given ox-LDL to induce the cells into foam cells .Under the microscope we could see cells had irregular morphology, fused, oil red O staining could see a large number of lipid droplets in cells, Fluorescent detected showed a large number of cholesterol in cells, showed that foam cell model constructed successfully.AT1-AA was incubated with foam cells. Western blot, and PT-PCR method was used to detect cell expression of ABCA1 mRNA and protein expression. Results showed that: compared with the control group, AT1-AA significantly inhibited the expression of ABCA1 protein and mRNA, losartan could significantly reduce the AT1-AA on the expression of ABCA1 protein and mRNA but it was still significantly lower than the negative control group.Conclusions: AT1-AA reduce THP-1 derived foam cells express ABCA1, impede the reverse cholesterol transport of the cells, may be one of the mechanisms AT1 receptor autoantibodies participated in the development of atherosclerosis.