Ferryl Hemoglobin Inhibits Osteoclastic Differentiation of Macrophages in Hemorrhaged Atherosclerotic Plaques

Abstract

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-Β 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 (tartrateresistant 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-Β). 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 NFκB (nuclear factor kappa-Β) 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.

Liquid PRF Reduces the Inflammatory Response and Osteoclastogenesis in Murine Macrophages

“…Searching for possible explanations, we show here that also hemoglobin alone can suppress osteoclastogenesis. These findings are supported by observations that oxidized ferryl hemoglobin greatly abolished osteoclastogenesis in vitro (37). In vivo, upon hemolysis hemoglobin is released from red blood cells to the circulation and give a rise to heme which has pro-inflammatory properties (38).…”

Identification of Potential Key Genes Involved in the Carotid Atherosclerosis

“…GO analysis result showed that cellar component (CC) and molecular function (MF) were significantly enriched in “extracellular matrix”, “plasma membrane protein complex”, “collagen-containing extracellular matrix”, “cell adhesion molecule binding” “actin binding” and “amide binding” ( Figure 3B and C ). The KEGG enrichment analysis result showed that some pathways were related to carotid atherosclerosis, such as PI3K-Akt signaling pathway, 18 Chemokine signaling pathway, 19 Cell adhesion molecules, 20 MAPK signaling pathway, 21 Calcium signaling pathway, 22 NOD-like receptor signaling pathway 23 and Fluid shear stress and atherosclerosis 24 ( Figure 3D ). Furthermore, we found that the change of BPs was significantly “leukocyte migration”, “leukocyte proliferation”, “myeloid leukocyte migration”, “phagocytosis”, “regulation of lymphocyte activation”, “response to molecule of bacterial origin” and “T cell activation” in atheroma plaque compared with control samples based on GSEA result ( Figure 3E ).…”

Role of NFAT in the Progression of Diabetic Atherosclerosis

“…In addition, the mechanism by which iron hemoglobin FHb inhibits the osteoclast differentiation of macrophages in atherosclerotic plaques is also closely related to NFAT, Zavaczki et al found that FHb (rather than iron hemoglobin) reduced bone resorption activity induced by RANKL (nuclear factor-κ receptor activator) and inhibited osteoclast-specific gene expression (anti-tartaric acid phosphatase, calcitonin receptor and dendritic cell-specific transmembrane protein). Increased osteoblasts and decreased osteoclast activities may contribute to intimal calcification, FHb inhibits NFATc1 nuclear translocation, and thus inhibit RANKL—induced macrophage osteoclast differentiation, FHb plays a certain role in inhibiting the calcification of atherosclerotic plaques ( 62 ).…”

“…The formation of RANK (NF-κB receptor activator)- TRAF6 (tumor necrosis factor receptor-related factor 6) complexes activates NF-κB and AP-1, By regulating NFATc1, these complexes promote osteoclast formation and affect vascular calcification ( 74 ). FHb inhibits RANKL—induced osteoclast differentiation in macrophages, suggesting that FHb accumulation in calcified areas of atherosclerotic lesions may delay OLC (osteoclast-like cell) formation and could impair calcium absorption, thereby promoting vascular calcification ( 62 ). Moreover, CD137 signals can regulate vascular calcification through the CD137-NFATc1 axis.…”