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Runx2-upregulated receptor activator of nuclear factor ?B ligand in calcifying smooth muscle cells promotes migration and osteoclastic differentiation of macrophages.

ABSTRACT: Clinical and experimental studies demonstrate the important roles of vascular smooth muscle cells (VSMC) in the pathogenesis of atherosclerosis. We have previously determined that the osteogenic transcription factor Runx2 is essential for VSMC calcification. The present study characterized Runx2-regulated signals and their potential roles in vascular calcification.In vivo studies with atherogenic apolipoprotein E(-/-) mice demonstrated that increased oxidative stress was associated with upregulation of Runx2 and receptor activator of nuclear factor ?B ligand (RANKL), which colocalized in the calcified atherosclerotic lesions and were juxtaposed to infiltrated macrophages and osteoclast-like cells that are positively stained for an osteoclast marker, tartrate-resistant acid phosphatase. Mechanistic studies using RNA interference, a luciferase reporter system, chromatin immunoprecipitation, and electrophoretic mobility shift assays indicated that Runx2 regulated the expression of RANKL via a direct binding to the 5'-flanking region of the RANKL. Functional characterization revealed that RANKL did not induce VSMC calcification, nor was RANKL required for oxidative stress-induced VSMC calcification. Using a coculture system, we demonstrated that VSMC-expressed RANKL induced migration as well as differentiation of bone marrow-derived macrophages into multinucleated, tartrate-resistant acid phosphatase-positive osteoclast-like cells. These effects were inhibited by the RANKL antagonist osteoprotegerin and with VSMC deficient in Runx2 or RANKL.We demonstrate that Runx2 directly binds to the promoter and controls the expression of RANKL, which mediates the crosstalk between calcifying VSMC and migration and differentiation of macrophages into osteoclast-like cells in the atherosclerotic lesions. Our studies provide novel mechanistic insights into the regulation and function of VSMC-derived RANKL in the pathogenesis of atherosclerosis and vascular calcification.

SUBMITTER: Byon CH

PROVIDER: S-EPMC3098301 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Runx2-upregulated receptor activator of nuclear factor κB ligand in calcifying smooth muscle cells promotes migration and osteoclastic differentiation of macrophages.

Byon Chang Hyun CH Sun Yong Y Chen Jianfeng J Yuan Kaiyu K Mao Xia X Heath Jack M JM Anderson Peter G PG Tintut Yin Y Demer Linda L LL Wang Deli D Chen Yabing Y

Arteriosclerosis, thrombosis, and vascular biology 20110331 6

<h4>Objective</h4>Clinical and experimental studies demonstrate the important roles of vascular smooth muscle cells (VSMC) in the pathogenesis of atherosclerosis. We have previously determined that the osteogenic transcription factor Runx2 is essential for VSMC calcification. The present study characterized Runx2-regulated signals and their potential roles in vascular calcification.<h4>Methods and results</h4>In vivo studies with atherogenic apolipoprotein E(-/-) mice demonstrated that increased ...[more]

PMID: 21454810

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Project description:AIMS/HYPOTHESIS: Our aims were to compare osteoclastic activity between patients with acute Charcot's osteoarthropathy and diabetic and healthy controls, and to determine the effect of the receptor activator of nuclear factor-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG). METHODS: Peripheral blood monocytes isolated from nine diabetic Charcot patients, eight diabetic control and eight healthy control participants were cultured in the presence of macrophage-colony stimulating factor (M-CSF) alone, M-CSF and RANKL, and also M-CSF and RANKL with excess concentrations of OPG. Osteoclast formation was assessed by expression of tartrate-resistant acid phosphatase on glass coverslips and resorption on dentine slices. RESULTS: In cultures with M-CSF, there was a significant increase in osteoclast formation in Charcot patients compared with healthy and diabetic control participants (p=0.008). A significant increase in bone resorption was also seen in the former, compared with healthy and diabetic control participants (p<0.0001). The addition of RANKL to the cultures with M-CSF led to marked increase in osteoclastic resorption in Charcot (from 0.264+/-0.06% to 41.6+/-8.1%, p<0.0001) and diabetic control (0.000+/-0.00% to 14.2+/-16.5%, p<0.0001) patients, and also in healthy control participants (0.004+/-0.01% to 10.5+/-1.9%, p<0.0001). Although the addition of OPG to cultures with M-CSF and RANKL led to a marked reduction of resorption in Charcot patients (41.6+/-8.1% to 5.9+/-2.4%, p=0.001), this suppression was not as complete as in diabetic control patients (14.2+/-16.5% to 0.45+/-0.31%, p=0.001) and in healthy control participants (from 10.5+/-1.9% to 0.00+/-0.00%, p<0.0001). CONCLUSIONS/INTERPRETATION: These results indicate that RANKL-mediated osteoclastic resorption occurs in acute Charcot's osteoarthropathy. However, the incomplete inhibition of RANKL after addition of OPG also suggests the existence of a RANKL-independent pathway.

Project description:ObjectivePlasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that promotes and inhibits cell migration, plays a complex and important role in adverse vascular remodeling. Little is known about the effects of pharmacological PAI-1 inhibitors, an emerging drug class, on migration of vascular smooth muscle cells (SMCs) and endothelial cells (ECs), crucial mediators of vascular remodeling. We investigated the effects of PAI-039 (tiplaxtinin), a specific PAI-1 inhibitor, on SMC and EC migration in vitro and vascular remodeling in vivo.Approach and resultsPAI-039 inhibited SMC migration through collagen gels, including those supplemented with vitronectin and other extracellular matrix proteins, but did not inhibit migration of PAI-1-deficient SMCs, suggesting that its antimigratory effects were PAI-1-specific and physiologically relevant. However, PAI-039 did not inhibit EC migration. PAI-039 inhibited phosphorylation and nuclear translocation of signal transducers and activators of transcription-1 in SMCs, but had no discernable effect on signal transducer and activator of transcription-1 signaling in ECs. Expression of low-density lipoprotein receptor-related protein 1, a motogenic PAI-1 receptor that activates Janus kinase/signal transducers and activators of transcription-1 signaling, was markedly lower in ECs than in SMCs. Notably, PAI-039 significantly inhibited intimal hyperplasia and inflammation in murine models of adverse vascular remodeling, but did not adversely affect re-endothelialization after endothelium-denuding mechanical vascular injury.ConclusionsPAI-039 inhibits SMC migration and intimal hyperplasia, while having no inhibitory effect on ECs, which seems to be because of differences in PAI-1-dependent low-density lipoprotein receptor-related protein 1/Janus kinase/signal transducer and activator of transcription-1 signaling between SMCs and ECs. These findings suggest that PAI-1 may be an important therapeutic target in obstructive vascular diseases characterized by neointimal hyperplasia.

Project description:Intraplaque hemorrhage frequently occurs in atherosclerotic plaques resulting in cell-free hemoglobin, which is oxidized to ferryl hemoglobin (FHb) in the highly oxidative environment. Osteoclast-like cells (OLCs) derived from macrophages signify a counterbalance mechanism for calcium deposition in atherosclerosis. Our aim was to investigate whether oxidized hemoglobin alters osteoclast formation, thereby affecting calcium removal from mineralized atherosclerotic lesions. RANKL- (receptor activator of nuclear factor kappa-? ligand-) induced osteoclastogenic differentiation and osteoclast activity of RAW264.7 cells were studied in response to oxidized hemoglobin via assessing bone resorption activity, expression of osteoclast-specific genes, and the activation of signalization pathways. OLCs in diseased human carotid arteries were assessed by immunohistochemistry. FHb, but not ferrohemoglobin, decreased bone resorption activity and inhibited osteoclast-specific gene expression (tartrate-resistant acid phosphatase, calcitonin receptor, and dendritic cell-specific transmembrane protein) induced by RANKL. In addition, FHb inhibited osteoclastogenic signaling pathways downstream of RANK (receptor activator of nuclear factor kappa-?). It prevented the induction of TRAF6 (tumor necrosis factor (TNF) receptor-associated factor 6) and c-Fos, phosphorylation of p-38 and JNK (c-Jun N-terminal kinase), and nuclear translocation of NF?B (nuclear factor kappa-?) and NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1). These effects were independent of heme oxygenase-1 demonstrated by knocking down HO-1 gene in RAW264.7 cells and in mice. Importantly, FHb competed with RANK for RANKL binding suggesting possible mechanisms by which FHb impairs osteoclastic differentiation. In diseased human carotid arteries, OLCs were abundantly present in calcified plaques and colocalized with regions of calcium deposition, while the number of these cells were lower in hemorrhagic lesions exhibiting accumulation of FHb despite calcium deposition. We conclude that FHb inhibits RANKL-induced osteoclastic differentiation of macrophages and suggest that accumulation of FHb in a calcified area of atherosclerotic lesion with hemorrhage retards the formation of OLCs potentially impairing calcium resorption.

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Runx2-upregulated receptor activator of nuclear factor ?B ligand in calcifying smooth muscle cells promotes migration and osteoclastic differentiation of macrophages.

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Runx2-upregulated receptor activator of nuclear factor κB ligand in calcifying smooth muscle cells promotes migration and osteoclastic differentiation of macrophages.

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