Abstract
In CKD patients, arteriosclerotic lesions, including calcification, can occur in vascular smooth muscle cells in a process called Moenckeberg’s medial arteriosclerosis. Iron overload induces several complications, including the acceleration of arteriosclerosis. However, the relationship between Moenckeberg’s arteriosclerosis in vascular smooth muscle cells and iron accumulation has remained unknown. We tested the accelerated effect of iron on calcification in cultured human aortic vascular smooth muscle cells (HASMCs). After establishment of this model, we performed a microarray analysis using mRNA from early stage culture HASMCs after iron stimulation with or without TNF-alpha stimulation. The role of interleukin-24 (IL-24) was confirmed from candidate genes that might contribute to calcification. HASMCs demonstrated calcification induced by iron and TNF-alpha. Calcification of HASMCs was synergistically enhanced by stimulation with both iron and TNF-alpha. In the early phase of calcification, microarray analysis revealed up-regulation of IL-24. Stimulation of HASMCs by IL-24 instead of iron induced calcification. The anti-IL-24 antibody reversed the effect of IL-24, supporting the important role of IL-24 in HASMCs calcification. In conclusion, iron-induced calcification in vascular smooth muscle cells occurred via IL-24, IL-24 was increased during the calcification process induced by iron, and IL-24 itself caused calcification in the absence of iron.
Highlights
The concept of chronic kidney disease (CKD) was established because of a high incidence of cardiovascular events; arteriosclerosis diseases have occupied an important part of CKD1,2
Human aortic vascular smooth muscle cells (HASMCs), in which Moenckeberg’s arteriosclerosis is induced in CKD patients, demonstrated calcification induced by iron and pro-inflammatory cytokine stimulation (TNF-alpha)
In the early phase of calcification, microarray analysis revealed up-regulation of IL-24, which was confirmed at the mRNA level by real-time PCR and at the protein level by enzyme-linked immunosorbent assay (ELISA)
Summary
The concept of chronic kidney disease (CKD) was established because of a high incidence of cardiovascular events; arteriosclerosis diseases have occupied an important part of CKD1,2. Our hypothesis is that iron accumulation under uremic conditions with hyperphosphatemia might be related to calcification of vascular smooth muscle cells (Moenckeberg’s arteriosclerosis). To induce human aortic smooth muscle cells (HASMCs) calcification, the cells were incubated with the calcification medium for 15–21 days, supplemented with holo-transferrin (holo-Tf) (0, 30, or 100 μg/mL) and TNF-alpha (0, 1, or 10 ng/mL). Both 100 μg/mL iron (holo-transferrin) and 1 ng/mL TNF-alpha synergistically induced HASMCs calcification These experiments used two cell lines of HASMCs. In this study, we tested the accelerated effect of iron on calcification in cultured vascular smooth muscle cells. We tested the accelerated effect of iron on calcification in cultured vascular smooth muscle cells After establishment of this model, we performed a microarray analysis using mRNA from early stage culture vascular media cells after iron stimulation with or without TNF-alpha stimulation. The role of interleukin-24 (IL-24) was confirmed from the candidate genes that might contribute to calcification in vascular media cells
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