Advanced glycation end products promote human aortic smooth muscle cell calcification in vitro via activating NF-KB and down-regulating IGFIR expression  

作　　者：Yi WANG Zhen-yu ZHANG Xiao-qing CHEN Xiang WANG Heng CAO Shao-wen LIU 

机构地区：[1]Department of Cardiology, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080,China

出　　处：《Acta Pharmacologica Sinica》2013年第4期480-486,共7页中国药理学报（英文版）

摘　　要：Aim： To investigate the effects of advanced glycation end products （AGEs） on calcification in human aortic smooth muscle cells （HASMCs） in vitro and the underlying mechanisms. Methods： AGEs were artificially prepared. Calcification of HASMCs was induced by adding inorganic phosphate （Pi, 2 mmol/L） in the media, and observed with Alizarin red staining. The calcium content in the supernatant was measured using QuantiChrome Calcium Assay Kit. Expression of the related mRNAs and proteins was analyzed using real-time PCR and Western blot, respectively. Chromatin immunoprecipitation （CHIP） assay was used to detect the binding of NF-KB to the putative IGFIR promoter. Results： AGEs （100 pg/mL） significantly enhanced Pi-induced calcification and the levels of osteocalcin and Cbfal in HASMCs. Further- more, the treatment decreased the expression of insulin-like growth factor I receptor （IGFIR）. Over-expression of IGFIR in HASMCs suppressed the AGEs-induced increase in calcium deposition. When IGFIR expression was knocked down in HASMCs, AGEs did not enhance the calcium deposition. Meanwhile, AGEs time-dependently decreased the amounts of IκBa and Flag-tagged p65 in the cytoplasmic extracts, and increased the amount of nuclear p65 in HASMCs. In the presence of NF-κB inhibitor PDTC （50 pmol/L）, the AGEs-induced increase in calcium deposition was blocked. Over-expression of p65 significantly enhanced Pi-induced mineralization, but suppressed IGFIR mRNA level. Knockdown of p65 suppressed the AGEs-induced increase in calcium deposition, and rescued the IGFIR expression. The ChIP analysis revealed that NF-κB bound the putative IGFIR promoter at position -230 to -219 bp. The inhibition of IGF1R by NF-κB was abolished when IGFIR reporter plasmid contained mutated binding sequence for NF-κB or an NF-κB reporter vector. Conclusion： The results demonstrate that AGEs promote calcification of human aortic smooth muscle cells in vitro via activation of NF-κB and down-regulation of IGFIR expressAim： To investigate the effects of advanced glycation end products （AGEs） on calcification in human aortic smooth muscle cells （HASMCs） in vitro and the underlying mechanisms. Methods： AGEs were artificially prepared. Calcification of HASMCs was induced by adding inorganic phosphate （Pi, 2 mmol/L） in the media, and observed with Alizarin red staining. The calcium content in the supernatant was measured using QuantiChrome Calcium Assay Kit. Expression of the related mRNAs and proteins was analyzed using real-time PCR and Western blot, respectively. Chromatin immunoprecipitation （CHIP） assay was used to detect the binding of NF-KB to the putative IGFIR promoter. Results： AGEs （100 pg/mL） significantly enhanced Pi-induced calcification and the levels of osteocalcin and Cbfal in HASMCs. Further- more, the treatment decreased the expression of insulin-like growth factor I receptor （IGFIR）. Over-expression of IGFIR in HASMCs suppressed the AGEs-induced increase in calcium deposition. When IGFIR expression was knocked down in HASMCs, AGEs did not enhance the calcium deposition. Meanwhile, AGEs time-dependently decreased the amounts of IκBa and Flag-tagged p65 in the cytoplasmic extracts, and increased the amount of nuclear p65 in HASMCs. In the presence of NF-κB inhibitor PDTC （50 pmol/L）, the AGEs-induced increase in calcium deposition was blocked. Over-expression of p65 significantly enhanced Pi-induced mineralization, but suppressed IGFIR mRNA level. Knockdown of p65 suppressed the AGEs-induced increase in calcium deposition, and rescued the IGFIR expression. The ChIP analysis revealed that NF-κB bound the putative IGFIR promoter at position -230 to -219 bp. The inhibition of IGF1R by NF-κB was abolished when IGFIR reporter plasmid contained mutated binding sequence for NF-κB or an NF-κB reporter vector. Conclusion： The results demonstrate that AGEs promote calcification of human aortic smooth muscle cells in vitro via activation of NF-κB and down-regulation of IGFIR express

关 键 词：vascular calcification advanced glycation end products human aortic smooth muscle cell OSTEOCALCIN CBFAL insulin-likegrowth factor I receptor （IGFIR） nuclear factor-kappa B PDTC 

分 类 号：Q513.5[生物学—生物化学] Q463