Iron-induced calcification in human aortic vascular smooth muscle cells through interleukin-24 (IL-24), with/without TNF-alpha

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.

To confirm the safety of iron in human aortic smooth muscle cells (HASMCs), the cells were cultured with the calcification medium for 15-21 days, supplemented with holo-transferrin (holo-Tf) (0, 100, 1000 or 10000 µg/mL) and TNF-alpha (0, 1, or 10 ng/mL). Mineralized cell nodules were stained with Alizarin red, and typical calcification images of HASMCs are shown. The high concentration (10000 µg/mL) stimulation induced cell death and suppressed calcification.
Supplemental Figure 2 Time course of BMP2 mRNA expression levels following iron, TNFalpha or both iron and TNF-alpha stimulation. The time course of IL-24 gene expression was evaluated by real-time PCR on days 1, 3, 6, 9, and 12 after the addition of 100 µg/mL of iron (holo-transferrin) and/or 1 ng/mL of TNFalpha to the calcification medium. The gene BMP2 expression level was enhanced by iron and TNF-alpha stimulation at day 1, and the BMP2 gene expression level decreased to basal levels after day 3. These experiments used one cell lines of HASMCs.
Supplemental Figure 3 Time course of Runx2 mRNA expression levels following iron, TNFalpha or both iron and TNF-alpha stimulation. The time course of Runx2 gene expression was evaluated by real-time PCR on days 1, 3, 6, 9, and 12 after the addition of 100 µg/mL iron (holo-transferrin) and/or 1 ng/mL TNF-alpha to the calcification medium. The gene expression level of Runx2 seemed to be enhanced by iron and/or TNF-alpha stimulation at day 6, and the Runx2 gene expression level decreased to basal levels after day 9. These experiments used one cell lines of HASMCs.
Supplemental Figure 4 Time course of MSX2 mRNA expression levels following iron, TNFalpha or both iron and TNF-alpha stimulation. The time course of MSX2 gene expression was evaluated by real-time PCR on days 1, 3, 6, 9, and 12 after the addition of 100 µg/mL iron (holo-transferrin) and/or 1 ng/mL TNF-alpha to the calcification medium. The gene expression level of MSX2 seemed to be enhanced by iron and/or TNF-alpha stimulation on day 6 without statistical significance, and the MSX2 gene expression level was seemed return to the basal level after day 9. These experiments used one cell lines of HASMCs.
Supplemental Figure 5 Time course of RANKL mRNA expression levels following iron, TNFalpha or both Iron and TNF-alpha stimulation. The time course of RANKL gene expression was evaluated by real-time PCR on days 1, 3, 6, 9, and 12 after the addition of 100 µg/mL iron (holo-transferrin) and/or 1 ng/mL TNF-alpha to the calcification medium. The gene expression level of RANKL was significantly enhanced by iron and/or TNF-alpha stimulation after day 9, and the RANKL gene expression level was enhanced by TNF-alpha stimulation on day 12. These experiments used one cell lines of HASMCs.
Supplemental Figure 6 Time course of human osteoprotegrin (hOPG) mRNA expression levels following iron, TNF-alpha or both iron and TNF-alpha stimulation. The time course of hOPG gene expression was evaluated by real-time PCR on days 1, 3, 6, 9, and 12 after the addition of 100 µg/mL iron (holo-transferrin) and/or 1 ng/mL TNF-alpha to the calcification medium. The gene expression level of hOPG seemed to increase gradually without statistical significance. These experiments used one cell lines of HASMCs. Figure 7 Time course of alkaline phosphatase activity/protein following iron, TNF-alpha or both iron and TNF-alpha stimulation. The time course of alkaline phosphatase activity/protein was evaluated by enzymatic activity/protein on days 1, 3, 6, 9, 12 and 21 after the addition of 100 µg/mL iron (holotransferrin) and/or 1 ng/mL TNF-alpha to the calcification medium. The alkaline phosphatase activity/protein l seemed to increase on day 3 and return to the basal level without statistical significance. These experiments used one cell lines of HASMCs.