HAS-UD Vascular Biology and Myocardial Pathophysiology Research Group, Hungarian Academy of Sciences, Debrecen, Hungary.
Department of Internal Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Oxid Med Cell Longev. 2020 Feb 27;2020:3721383. doi: 10.1155/2020/3721383. eCollection 2020.
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 NFB (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.
斑块内出血常发生于动脉粥样硬化斑块，导致无细胞血红蛋白释放，在高度氧化环境中被氧化为高铁血红蛋白（ferryl hemoglobin，FHb）。破骨细胞样细胞（osteoclast-like cells，OLCs）来源于巨噬细胞，是动脉粥样硬化中钙沉积的平衡机制。我们的目的是研究氧化血红蛋白是否改变破骨细胞形成，从而影响矿化动脉粥样硬化病变中钙的清除。通过评估骨吸收活性、破骨细胞特异性基因的表达和信号通路的激活，研究 RANKL（核因子κB 受体激活物配体）诱导的 RAW264.7 细胞破骨细胞生成和破骨细胞活性对氧化血红蛋白的反应。通过免疫组织化学评估疾病人颈动脉中的 OLCs。FHb 而非亚铁血红蛋白降低了骨吸收活性并抑制了 RANKL 诱导的破骨细胞特异性基因表达（抗酒石酸酸性磷酸酶、降钙素受体和树突状细胞特异性跨膜蛋白）。此外，FHb 抑制了 RANK（核因子κB 受体激活物）下游的破骨细胞生成信号通路。它阻止了 TRAF6（肿瘤坏死因子（TNF）受体相关因子 6）和 c-Fos 的诱导、p-38 和 JNK（c-Jun N-末端激酶）的磷酸化以及 NFKB（核因子 kappa-）和 NFATc1（激活 T 细胞的核因子，细胞质 1）的核易位。这些作用与 RAW264.7 细胞和小鼠中 HO-1 基因敲低证明的血红素加氧酶-1 无关。重要的是，FHb 与 RANK 竞争 RANKL 结合，提示 FHb 损害破骨细胞分化的可能机制。在疾病人颈动脉中，OLCs 大量存在于钙化斑块中，并与钙沉积区域共定位，而在存在 FHb 积累的出血性病变中，这些细胞的数量较低，尽管有钙沉积。我们得出结论，FHb 抑制了 RANKL 诱导的巨噬细胞破骨细胞分化，并表明 FHb 在伴有出血的动脉粥样硬化病变的钙化区域积累会延迟 OLCs 的形成，从而可能损害钙的吸收。
