1. Iron citrate reduces high phosphate-induced vascular calcification by inhibiting apoptosis 
Paola Ciceri, Francesca Elli, Paola Braidotti, Monica Falleni, Delfina Tosi, Gaetano Bulfamante, Geoffrey A. Block, Mario Cozzolino 
Atherosclerosis 
Volume 254, Pages (November 2016)
DOI: /j.atherosclerosis
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2. Fig. 1
Experimental protocol of prophylactic and therapeutic iron addiction. (A) Experimental scheme of prophylactic addiction of iron with evaluation of Ca deposition at day 7. (B) Experimental scheme of therapeutic addiction of iron at different days after the beginning of 5 mM Pi challenge and Ca deposition evaluation at day 12.
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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3. Fig. 2
Effect of ferric citrate on high Pi-induced extracellular calcium deposition and iron storage. (A) Increasing concentration of ferric citrate in the calcification medium induces a decrease in high Pi-induced Ca++ deposition. Ca++ was measured and normalized by cellular protein content (day 7). Data were presented as mean ± SE of four different experiments. (B) Ca++ deposition was visualized at light microscopic level by Alizarin Red staining. No deposit was found in the control culture. Red color indicates high Pi-dependent Ca++deposits. The figure shows the inhibitory effect of 50 μM Fe3+ on high Pi-induced VSMC Ca++ deposition (day 7). Magnification 200×. Representative result of one of three different experiments. (C) Serial sections were processed to visualize iron deposition by Perls' blue Prussian staining as blue granules (image 1 and 3) and calcium deposition by Von Kossa staining as black granules (image 2 and 4) at day 14. In the high Pi treated sample, no iron deposits are detectable (image 1), but abundant extra- and intra-cellular Ca deposits are present (image 2). In the Pi+50 μM Fe3+ sample, some finely iron blue granules are present (image 3), and black deposits appear greatly reduced (image 4); blue and black deposits can be observed in the same areas (insets 3a and 4a). (D) Seven-day treated Pi+50 μM Fe3+ sample. Images A–D: ultrathin sections without OsO4 post-fixation and very light counterstaining (see Materials and methods). Image E: well routinely counterstained section. A: cell with an autophagic vacuole; scale bar = 500 nm. B: enlargement of boxed area in A showing dark small dots referable to Fe3+ particles; scale bar = 500 nm. C: intracytoplasmic autophagic vacuole; scale bar = 200 nm. D: enlargement of boxed area in C; scale bar = 200 nm. E: a well counterstained section showing an autophagic vacuole very similar to that shown in B; scale bar = 200 nm. (*p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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4. Fig. 3
Effect of ferric citrate on high Pi-induced apoptosis. (A) Morphological aspects of VSMCs were visualized at light microscopy level by haematoxylin and eosin staining, 7 days after challenge with high Pi. Mitotic and apoptotic figures (arrow and circle, respectively) detected in all experimental conditions. Panel 1: mitotic and apoptotic figures appear to be balanced in control; Panel 2: in high Pi samples, an unpaired increase in apoptotis is observed compared to mitosis; Panel 3: mitotic and apoptotic figures appear to be balanced in Pi+50 μM Fe3+. (B) Rat VSMCs were cultured in calcification medium and challenged with 5 mM Pi for 12 days. The effect of 50 μM Fe3+ on nuclear fragmentation is represented as enrichment factor of apoptotic fragments. Data are presented as mean ± SE (*p < 0.01). (C) Rat VSMCs were cultured in calcification medium and challenged with 5 mM Pi for 9 days. Gas-6 and Axl protein expression was analyzed by Western blot. Lane 1: control; lane 2: 5 mM Pi; lane 3: 5 mM Pi+50 μM Fe3+. Representative result of one of three different experiments. (D and E) Analysis of the intensity of the immunoblot bands of Gas6 and AXL are shown. Data are presented as mean ± SE of three different experiments (*p < 0.01).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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5. Fig. 4
Effect of ferric citrate on high Pi-induced autophagy. (A) Image A: electron microscopy (EM) micrograph of an ultra-thin section from 7-day treated 5 mM Pi sample; arrows point to Ca deposits. Scale bar = 200 nm. Image B: enlargement of red boxed area in A, arrows point to calcified mitochondria, scale bar = 500 nm. Image C: enlargement of yellow boxed area in A, yellow arrows point to intracytoplasmic completely calcified mitochondria, black arrows point to mitochondria in extra cellular matrix (ECM). Scale bar = 500 nm. (B) Image A: ultra-thin section from 7-day 5 mM Pi+50 μM Fe3+ sample shows two autophagic vacuoles. Scale bar = 200 nm. Image B: enlargement of the boxed area in A, arrows point to partly calcified mitochondria; scale bar = 500 nm. Image C: another autophagic vacuole with 4 completely calcified mitochondria; scale bar = 200 nm. Image D: enlargement of boxed area in C; scale bar = 500 nm. (C) VSMCs were challenged with 5 mM Pi for 9 days and LC3IIB protein expression was detected by immunoblotting analysis in absence or in presence of ON treatment with 25 μM chloroquine at day 8. Lane 1: control; lane 2: 5 mM Pi; lane 3: 5 mM Pi+50 μM Fe3+; lane 4: control + chloroquine; lane 5: 5 mM Pi + chloroquine; lane 6: Pi+50 μM Fe3++chloroquine. Representative result of one of three different experiments. (D) Analysis of the intensity of the immunoblot bands is shown, empty rectangles indicate the autophagic flux. Data are presented as mean ± SE (*p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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6. Fig. 5
Effect of ferric citrate on high Pi-induced smooth muscle lineage markers regulation. (A) α-actin and SM-22α protein expression was detected by immunoblotting at day 9. Lane 1: control; lane 2: 5 mM Pi; lane 3: 5 mM Pi+50 μM Fe3+. (B and C) Alpha-actin and SM-22α analysis of the intensity of the immunoblot bands is shown. Data are presented as mean ± SE (*p < 0.05). Representative result of one of three different experiments. (D) α-actin immunoreactivity is visualized by light microscopy. Image 1: Control cells, strong cytoplasmic positivity with cell membrane enhancement sometimes associated with longitudinal streaking (arrows). Image 2 and 3: In Pi and Pi + Fe3+ treated cells, there is a comparable reduction in α-actin immunoreactivity with disappearance of membrane enhancement and cytoplasmic streaking. However, a cell membrane enhanced immunoreactivity can be appreciated in small groups of Pi + Fe3+ treated cells (inset).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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7. Fig. 6
Effect of ferric citrate on high Pi-induced VSMC simil-osteoblatic differentiation. (A) BMP2 mRNA expression was measured by RT-PCR at day 8, with a significant inhibitory effect of iron treatment on high Pi-induced gene expression. (B) OPN mRNA expression was measured by RT-PCR at day 8, with a significant effect of iron treatment that increases gene expression respect high Pi treated samples. (C) RUNX2 mRNA expression was measured by RT-PCR at day 5, with no significant effect of iron treatment on high Pi-induced gene expression. (D) RUNX2 protein expression was detected by immunoblotting at day 7. Lane 1: control; lane 2: 5 mM Pi; lane 3: 5 mM Pi+50 μM Fe3+. (E) RUNX2 analysis of the intensity of the immunoblot bands is shown. Data are presented as mean ± SE (*p < 0.01).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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8. Fig. 7
Therapeutic effect of ferric citrate on the progression of high Pi-induced calcium deposition. Rat VSMCs were cultured with 5 mM Pi in the calcification medium for 12 days ( ). The presence of 50 μM Fe3+ from day 0 up to day 12 completely inhibits high Pi-induced calcium deposition ( ). The addiction of 50 μM Fe3+ during the progression of calcium deposition (: from day 2 to day 12;: from day 5 to day 12;: from day 7 to day 12;: from day 9 to day 12) is able to block calcification at the same level that results before its addiction. Ca++ deposition was measured and normalized by cellular protein content. Data were presented as mean ± SE of three experiments in triplicate (*p < 0.01 Pi + Fe3+vs. Pi at day 12 for every Fe3+ treatment tested).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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