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Advanced glycation end products promote human aortic smooth muscle cell calcification in vitro via activating NF-?B and down-regulating IGF1R expression.

ABSTRACT: 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.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-?B to the putative IGF1R promoter.AGEs (100 ?g/mL) significantly enhanced Pi-induced calcification and the levels of osteocalcin and Cbf?1 in HASMCs. Furthermore, the treatment decreased the expression of insulin-like growth factor 1 receptor (IGF1R). Over-expression of IGF1R in HASMCs suppressed the AGEs-induced increase in calcium deposition. When IGF1R expression was knocked down in HASMCs, AGEs did not enhance the calcium deposition. Meanwhile, AGEs time-dependently decreased the amounts of I?B? and Flag-tagged p65 in the cytoplasmic extracts, and increased the amount of nuclear p65 in HASMCs. In the presence of NF-?B inhibitor PDTC (50 ?mol/L), the AGEs-induced increase in calcium deposition was blocked. Over-expression of p65 significantly enhanced Pi-induced mineralization, but suppressed IGF1R mRNA level. Knockdown of p65 suppressed the AGEs-induced increase in calcium deposition, and rescued the IGF1R expression. The ChIP analysis revealed that NF-?B bound the putative IGF1R promoter at position -230 to -219 bp. The inhibition of IGF1R by NF-?B was abolished when IGF1R reporter plasmid contained mutated binding sequence for NF-?B or an NF-?B reporter vector.The results demonstrate that AGEs promote calcification of human aortic smooth muscle cells in vitro via activation of NF-?B and down-regulation of IGF1R expression.

SUBMITTER: Wang Y

PROVIDER: S-EPMC4002782 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Advanced glycation end products promote human aortic smooth muscle cell calcification in vitro via activating NF-κB and down-regulating IGF1R expression.

Wang Yi Y Zhang Zhen-yu ZY Chen Xiao-qing XQ Wang Xiang X Cao Heng H Liu Shao-wen SW

Acta pharmacologica Sinica 20130218 4

<h4>Aim</h4>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.<h4>Methods</h4>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 protein ...[more]

PMID: 23416929

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Project description:Diabetes is a complex disease characterized by hyperglycemia, dyslipidemia, and insulin resistance. Plasma advanced glycation end products (AGEs) activated the receptor for advanced glycation end products (RAGE) and the activation of RAGE is implicated to be the pathogenesis of type 2 diabetic mellitus (T2DM) patient vascular complications. Sitagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, is a new oral hypoglycemic agent for the treatment of T2DM. However, the beneficial effects on vascular calcification remain unclear. In this study, we used a high-fat diet (HFD)-fed low-density lipoprotein receptor deficiency (LDLR-/-) mice model to investigate the potential effects of sitagliptin on HFD-induced arterial calcification. Mice were randomly divided into 3 groups: (1) normal diet group, (2) HFD group and (3) HFD + sitagliptin group. After 24 weeks treatment, we collected the blood for chemistry parameters and DPP4 activity measurement, and harvested the aorta to evaluate calcification using immunohistochemistry and calcium content. To determine the effects of sitagliptin, tumor necrosis factor (TNF)-α combined with S100A12 was used to induce oxidative stress, activation of nicotinamide adenine dinucleotide phosphate (NADPH), up-regulation of bone markers and RAGE expression, and cell calcium deposition on human aortic smooth muscle cells (HASMCs). We found that sitagliptin effectively blunted the HFD-induced artery calcification and significantly lowered the levels of fasting serum glucose, triglyceride (TG), nitrotyrosine and TNF-α, decreased the calcium deposits, and reduced arterial calcification. In an in-vitro study, both S100A12 and TNF-α stimulated RAGE expression and cellular calcium deposits in HASMCs. The potency of S100A12 on HASMCs was amplified by the presence of TNF-α. Sitagliptin and Apocynin (APO), an NADPH oxidase inhibitor, inhibited the TNF-α + S100A12-induced NADPH oxidase and nuclear factor (NF)-κB activation, cellular oxidative stress, RAGE expression, osteo transcription factors expression and calcium deposition. In addition, treatment with sitagliptin, knockdown of RAGE or TNF-α receptor blunted the TNF-α + S100A12-induced RAGE expression. Our findings suggest that sitagliptin may suppress the initiation and progression of arterial calcification by inhibiting the activation of NADPH oxidase and NF-κB, followed by decreasing the expression of RAGE.

Dataset Information

Advanced glycation end products promote human aortic smooth muscle cell calcification in vitro via activating NF-?B and down-regulating IGF1R expression.

Publications

Advanced glycation end products promote human aortic smooth muscle cell calcification in vitro via activating NF-κB and down-regulating IGF1R expression.

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