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Increased calcification in osteoprotegerin-deficient smooth muscle cells: Dependence on receptor activator of NF-?B ligand and interleukin 6.

ABSTRACT: Vascular calcification is highly correlated with cardiovascular disease morbidity and mortality. Osteoprotegerin (OPG) is a secreted decoy receptor for receptor activator of NF-?B ligand (RANKL). Inactivation of OPG in apolipoprotein E-deficient (ApoE-/-) mice increases lesion size and calcification. The mechanism(s) by which OPG is atheroprotective and anticalcific have not been entirely determined. We investigated whether OPG-deficient vascular smooth muscle cells (VSMCs) are more susceptible to mineralization and whether RANKL mediates this process.Lesion-free aortas from 12-week-old ApoE-/-OPG-/- mice had spotty calcification, an appearance of osteochondrogenic factors and a decrease of smooth muscle markers when compared to ApoE-/-OPG+/+ aortas. In osteogenic conditions, VSMCs isolated from ApoE-/-OPG-/- (KO-VSMC) mice deposited more calcium than VSMCs isolated from ApoE-/-OPG+/+ (WT-VSMC) mice. Gene expression and biochemical analysis indicated accelerated osteochondrogenic differentiation. Ablation of RANKL signaling in KO-VSMCs rescued the accelerated calcification. While WT-VSMCs did not respond to RANKL treatment, KO-VSMCs responded with enhanced calcification and the upregulation of osteochondrogenic genes. RANKL strongly induced interleukin 6 (IL-6), which partially mediated RANKL-dependent calcification and gene expression in KO-VSMCs.OPG inhibits vascular calcification by regulating the procalcific effects of RANKL on VSMCs and is thus a possible target for therapeutic intervention.

SUBMITTER: Callegari A

PROVIDER: S-EPMC4057981 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Increased calcification in osteoprotegerin-deficient smooth muscle cells: Dependence on receptor activator of NF-κB ligand and interleukin 6.

Callegari Andrea A Coons Matthew L ML Ricks Jerry L JL Rosenfeld Michael E ME Scatena Marta M

Journal of vascular research 20140307 2

<h4>Objective</h4>Vascular calcification is highly correlated with cardiovascular disease morbidity and mortality. Osteoprotegerin (OPG) is a secreted decoy receptor for receptor activator of NF-κB ligand (RANKL). Inactivation of OPG in apolipoprotein E-deficient (ApoE-/-) mice increases lesion size and calcification. The mechanism(s) by which OPG is atheroprotective and anticalcific have not been entirely determined. We investigated whether OPG-deficient vascular smooth muscle cells (VSMCs) are ...[more]

PMID: 24642764

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Project description:The RANK (receptor activator of nuclear factor κB)/RANKL (RANK ligand)/OPG (osteoprotegerin) axis is activated after myocardial infarction (MI), but its pathophysiological role is not well understood. Here, we investigated how global and cell compartment-selective inhibition of RANKL affects cardiac function and remodeling after MI in mice. Global RANKL inhibition was achieved by treatment of human RANKL knock-in (huRANKL-KI) mice with the monoclonal antibody AMG161. huRANKL-KI mice express a chimeric RANKL protein wherein part of the RANKL molecule is humanized. AMG161 inhibits human and chimeric but not murine RANKL. To dissect the pathophysiological role of RANKL derived from hematopoietic and mesenchymal cells, we selectively exchanged the hematopoietic cell compartment by lethal irradiation and across-genotype bone marrow transplantation between wild-type and huRANKL-KI mice, exploiting the specificity of AMG161. After permanent coronary artery ligation, mice were injected with AMG161 or an isotype control antibody over 4 weeks post-MI. MI increased RANKL expression mainly in cardiomyocytes and scar-infiltrating cells 4 weeks after MI. Only inhibition of RANKL derived from hematopoietic cellular sources, but not global or mesenchymal RANKL inhibition, improved post-infarct survival and cardiac function. Mechanistically, hematopoietic RANKL inhibition reduced expression of the pro-inflammatory cytokine IL-1ß in the cardiac cellular infiltrate. In conclusion, inhibition of RANKL derived from hematopoietic cellular sources is beneficial to maintain post-ischemic cardiac function by reduction of pro-inflammatory cytokine production. KEY MESSAGES: Experimental myocardial infarction (MI) augments cardiac RANKL expression in mice. RANKL expression is increased in cardiomyocytes and scar-infiltrating cells after MI. Global or mesenchymal cell RANKL inhibition has no influence on cardiac function after MI. Inhibition of RANKL derived from hematopoietic cells improves heart function post-MI. Hematopoietic RANKL inhibition reduces pro-inflammatory cytokines in scar-infiltrating cells.

Project description:AimTo investigate the effects of advanced glycation end products (AGEs) on calcification in human aortic smooth muscle cells (HASMCs) in vitro and the underlying mechanisms.MethodsAGEs 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-κB to the putative IGF1R promoter.ResultsAGEs (100 μg/mL) significantly enhanced Pi-induced calcification and the levels of osteocalcin and Cbfα1 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 IκBα 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 μmol/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-κB bound the putative IGF1R promoter at position -230 to -219 bp. The inhibition of IGF1R by NF-κB was abolished when IGF1R reporter plasmid contained mutated binding sequence for NF-κB or an NF-κB reporter vector.ConclusionThe results demonstrate that AGEs promote calcification of human aortic smooth muscle cells in vitro via activation of NF-κB and down-regulation of IGF1R expression.

Dataset Information

Increased calcification in osteoprotegerin-deficient smooth muscle cells: Dependence on receptor activator of NF-?B ligand and interleukin 6.

Publications

Increased calcification in osteoprotegerin-deficient smooth muscle cells: Dependence on receptor activator of NF-κB ligand and interleukin 6.

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