Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis
Identifieur interne : 000C49 ( Ncbi/Merge ); précédent : 000C48; suivant : 000C50Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis
Auteurs : Paola Ciceri ; Monica Falleni ; Delfina Tosi ; Carla Martinelli ; Stefania Cannizzo ; Giulia Marchetti ; Antonella D Rminio Monforte ; Gaetano Bulfamante ; Geoffrey A. Block ; Piergiorgio Messa ; Mario Cozzolino [Italie]Source :
- International Journal of Molecular Sciences [ 1422-0067 ] ; 2019.
Abstract
In chronic kidney disease (CKD), the first cause of mortality is cardiovascular disease induced mainly by vascular calcification (VC). Recently, iron-based phosphate binders have been proposed in advanced CKD to treat hyperphosphatemia. We studied the effect of iron citrate (iron) on the progression of calcification in high-phosphate (Pi) calcified VSMC. Iron arrested further calcification when added on days 7–15 in the presence of high Pi (1.30 ± 0.03 vs 0.61 ± 0.02; OD/mg protein; day 15; Pi vs Pi + Fe,
Url:
DOI: 10.3390/ijms20235925
PubMed: 31775364
PubMed Central: 6928632
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis</title><author><name sortKey="Ciceri, Paola" sort="Ciceri, Paola" uniqKey="Ciceri P" first="Paola" last="Ciceri">Paola Ciceri</name><affiliation><nlm:aff id="af1-ijms-20-05925">Renal Research Laboratory, Department of Nephrology, Dialysis and Renal Transplant, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico and Fondazione D’Amico per la Ricerca sulle Malattie Renali, 20100 Milan, Italy;<email>p.ciceri@hotmail.it</email> (P.C.);<email>piergiorgio.messa@unimi.it</email> (P.M.)</nlm:aff></affiliation></author><author><name sortKey="Falleni, Monica" sort="Falleni, Monica" uniqKey="Falleni M" first="Monica" last="Falleni">Monica Falleni</name><affiliation><nlm:aff id="af2-ijms-20-05925">Department of Health Sciences, Division of Pathology, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>monica.falleni@unimi.it</email> (M.F.);<email>delfina.tosi@unimi.it</email> (D.T.);<email>carla.martinelli@unimi.it</email> (C.M.);<email>gaetano.bulfamante@unimi.it</email> (G.B.)</nlm:aff></affiliation></author><author><name sortKey="Tosi, Delfina" sort="Tosi, Delfina" uniqKey="Tosi D" first="Delfina" last="Tosi">Delfina Tosi</name><affiliation><nlm:aff id="af2-ijms-20-05925">Department of Health Sciences, Division of Pathology, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>monica.falleni@unimi.it</email> (M.F.);<email>delfina.tosi@unimi.it</email> (D.T.);<email>carla.martinelli@unimi.it</email> (C.M.);<email>gaetano.bulfamante@unimi.it</email> (G.B.)</nlm:aff></affiliation></author><author><name sortKey="Martinelli, Carla" sort="Martinelli, Carla" uniqKey="Martinelli C" first="Carla" last="Martinelli">Carla Martinelli</name><affiliation><nlm:aff id="af2-ijms-20-05925">Department of Health Sciences, Division of Pathology, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>monica.falleni@unimi.it</email> (M.F.);<email>delfina.tosi@unimi.it</email> (D.T.);<email>carla.martinelli@unimi.it</email> (C.M.);<email>gaetano.bulfamante@unimi.it</email> (G.B.)</nlm:aff></affiliation></author><author><name sortKey="Cannizzo, Stefania" sort="Cannizzo, Stefania" uniqKey="Cannizzo S" first="Stefania" last="Cannizzo">Stefania Cannizzo</name><affiliation><nlm:aff id="af3-ijms-20-05925">Department of Health Sciences, Clinic of Infectious Diseases, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>stefania.cannizzo@unimi.it</email> (S.C.);<email>giulia.marchetti@unimi.it</email> (G.M.);<email>antonella.darminio@unimi.it</email> (A.D.M.)</nlm:aff></affiliation></author><author><name sortKey="Marchetti, Giulia" sort="Marchetti, Giulia" uniqKey="Marchetti G" first="Giulia" last="Marchetti">Giulia Marchetti</name><affiliation><nlm:aff id="af3-ijms-20-05925">Department of Health Sciences, Clinic of Infectious Diseases, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>stefania.cannizzo@unimi.it</email> (S.C.);<email>giulia.marchetti@unimi.it</email> (G.M.);<email>antonella.darminio@unimi.it</email> (A.D.M.)</nlm:aff></affiliation></author><author><name sortKey="D Rminio Monforte, Antonella" sort="D Rminio Monforte, Antonella" uniqKey="D Rminio Monforte A" first="Antonella" last="D Rminio Monforte">Antonella D Rminio Monforte</name><affiliation><nlm:aff id="af3-ijms-20-05925">Department of Health Sciences, Clinic of Infectious Diseases, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>stefania.cannizzo@unimi.it</email> (S.C.);<email>giulia.marchetti@unimi.it</email> (G.M.);<email>antonella.darminio@unimi.it</email> (A.D.M.)</nlm:aff></affiliation></author><author><name sortKey="Bulfamante, Gaetano" sort="Bulfamante, Gaetano" uniqKey="Bulfamante G" first="Gaetano" last="Bulfamante">Gaetano Bulfamante</name><affiliation><nlm:aff id="af2-ijms-20-05925">Department of Health Sciences, Division of Pathology, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>monica.falleni@unimi.it</email> (M.F.);<email>delfina.tosi@unimi.it</email> (D.T.);<email>carla.martinelli@unimi.it</email> (C.M.);<email>gaetano.bulfamante@unimi.it</email> (G.B.)</nlm:aff></affiliation></author><author><name sortKey="Block, Geoffrey A" sort="Block, Geoffrey A" uniqKey="Block G" first="Geoffrey A" last="Block">Geoffrey A. Block</name><affiliation><nlm:aff id="af4-ijms-20-05925">Nephrology, Denver Nephrolgysts PC, Denver, CO 80230, USA;<email>geoff.block@reatapharma.com</email></nlm:aff></affiliation></author><author><name sortKey="Messa, Piergiorgio" sort="Messa, Piergiorgio" uniqKey="Messa P" first="Piergiorgio" last="Messa">Piergiorgio Messa</name><affiliation><nlm:aff id="af1-ijms-20-05925">Renal Research Laboratory, Department of Nephrology, Dialysis and Renal Transplant, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico and Fondazione D’Amico per la Ricerca sulle Malattie Renali, 20100 Milan, Italy;<email>p.ciceri@hotmail.it</email> (P.C.);<email>piergiorgio.messa@unimi.it</email> (P.M.)</nlm:aff></affiliation></author><author><name sortKey="Cozzolino, Mario" sort="Cozzolino, Mario" uniqKey="Cozzolino M" first="Mario" last="Cozzolino">Mario Cozzolino</name><affiliation wicri:level="1"><nlm:aff id="af5-ijms-20-05925">Department of Health Sciences, Renal Division, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy</nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea>Department of Health Sciences, Renal Division, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan</wicri:regionArea><placeName><settlement type="city">Milan</settlement><region nuts="2">Lombardie</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31775364</idno><idno type="pmc">6928632</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928632</idno><idno type="RBID">PMC:6928632</idno><idno type="doi">10.3390/ijms20235925</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000620</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000620</idno><idno type="wicri:Area/Pmc/Curation">000620</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000620</idno><idno type="wicri:Area/Pmc/Checkpoint">000292</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000292</idno><idno type="wicri:Area/Ncbi/Merge">000C49</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis</title><author><name sortKey="Ciceri, Paola" sort="Ciceri, Paola" uniqKey="Ciceri P" first="Paola" last="Ciceri">Paola Ciceri</name><affiliation><nlm:aff id="af1-ijms-20-05925">Renal Research Laboratory, Department of Nephrology, Dialysis and Renal Transplant, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico and Fondazione D’Amico per la Ricerca sulle Malattie Renali, 20100 Milan, Italy;<email>p.ciceri@hotmail.it</email> (P.C.);<email>piergiorgio.messa@unimi.it</email> (P.M.)</nlm:aff></affiliation></author><author><name sortKey="Falleni, Monica" sort="Falleni, Monica" uniqKey="Falleni M" first="Monica" last="Falleni">Monica Falleni</name><affiliation><nlm:aff id="af2-ijms-20-05925">Department of Health Sciences, Division of Pathology, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>monica.falleni@unimi.it</email> (M.F.);<email>delfina.tosi@unimi.it</email> (D.T.);<email>carla.martinelli@unimi.it</email> (C.M.);<email>gaetano.bulfamante@unimi.it</email> (G.B.)</nlm:aff></affiliation></author><author><name sortKey="Tosi, Delfina" sort="Tosi, Delfina" uniqKey="Tosi D" first="Delfina" last="Tosi">Delfina Tosi</name><affiliation><nlm:aff id="af2-ijms-20-05925">Department of Health Sciences, Division of Pathology, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>monica.falleni@unimi.it</email> (M.F.);<email>delfina.tosi@unimi.it</email> (D.T.);<email>carla.martinelli@unimi.it</email> (C.M.);<email>gaetano.bulfamante@unimi.it</email> (G.B.)</nlm:aff></affiliation></author><author><name sortKey="Martinelli, Carla" sort="Martinelli, Carla" uniqKey="Martinelli C" first="Carla" last="Martinelli">Carla Martinelli</name><affiliation><nlm:aff id="af2-ijms-20-05925">Department of Health Sciences, Division of Pathology, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>monica.falleni@unimi.it</email> (M.F.);<email>delfina.tosi@unimi.it</email> (D.T.);<email>carla.martinelli@unimi.it</email> (C.M.);<email>gaetano.bulfamante@unimi.it</email> (G.B.)</nlm:aff></affiliation></author><author><name sortKey="Cannizzo, Stefania" sort="Cannizzo, Stefania" uniqKey="Cannizzo S" first="Stefania" last="Cannizzo">Stefania Cannizzo</name><affiliation><nlm:aff id="af3-ijms-20-05925">Department of Health Sciences, Clinic of Infectious Diseases, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>stefania.cannizzo@unimi.it</email> (S.C.);<email>giulia.marchetti@unimi.it</email> (G.M.);<email>antonella.darminio@unimi.it</email> (A.D.M.)</nlm:aff></affiliation></author><author><name sortKey="Marchetti, Giulia" sort="Marchetti, Giulia" uniqKey="Marchetti G" first="Giulia" last="Marchetti">Giulia Marchetti</name><affiliation><nlm:aff id="af3-ijms-20-05925">Department of Health Sciences, Clinic of Infectious Diseases, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>stefania.cannizzo@unimi.it</email> (S.C.);<email>giulia.marchetti@unimi.it</email> (G.M.);<email>antonella.darminio@unimi.it</email> (A.D.M.)</nlm:aff></affiliation></author><author><name sortKey="D Rminio Monforte, Antonella" sort="D Rminio Monforte, Antonella" uniqKey="D Rminio Monforte A" first="Antonella" last="D Rminio Monforte">Antonella D Rminio Monforte</name><affiliation><nlm:aff id="af3-ijms-20-05925">Department of Health Sciences, Clinic of Infectious Diseases, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>stefania.cannizzo@unimi.it</email> (S.C.);<email>giulia.marchetti@unimi.it</email> (G.M.);<email>antonella.darminio@unimi.it</email> (A.D.M.)</nlm:aff></affiliation></author><author><name sortKey="Bulfamante, Gaetano" sort="Bulfamante, Gaetano" uniqKey="Bulfamante G" first="Gaetano" last="Bulfamante">Gaetano Bulfamante</name><affiliation><nlm:aff id="af2-ijms-20-05925">Department of Health Sciences, Division of Pathology, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>monica.falleni@unimi.it</email> (M.F.);<email>delfina.tosi@unimi.it</email> (D.T.);<email>carla.martinelli@unimi.it</email> (C.M.);<email>gaetano.bulfamante@unimi.it</email> (G.B.)</nlm:aff></affiliation></author><author><name sortKey="Block, Geoffrey A" sort="Block, Geoffrey A" uniqKey="Block G" first="Geoffrey A" last="Block">Geoffrey A. Block</name><affiliation><nlm:aff id="af4-ijms-20-05925">Nephrology, Denver Nephrolgysts PC, Denver, CO 80230, USA;<email>geoff.block@reatapharma.com</email></nlm:aff></affiliation></author><author><name sortKey="Messa, Piergiorgio" sort="Messa, Piergiorgio" uniqKey="Messa P" first="Piergiorgio" last="Messa">Piergiorgio Messa</name><affiliation><nlm:aff id="af1-ijms-20-05925">Renal Research Laboratory, Department of Nephrology, Dialysis and Renal Transplant, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico and Fondazione D’Amico per la Ricerca sulle Malattie Renali, 20100 Milan, Italy;<email>p.ciceri@hotmail.it</email> (P.C.);<email>piergiorgio.messa@unimi.it</email> (P.M.)</nlm:aff></affiliation></author><author><name sortKey="Cozzolino, Mario" sort="Cozzolino, Mario" uniqKey="Cozzolino M" first="Mario" last="Cozzolino">Mario Cozzolino</name><affiliation wicri:level="1"><nlm:aff id="af5-ijms-20-05925">Department of Health Sciences, Renal Division, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy</nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea>Department of Health Sciences, Renal Division, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan</wicri:regionArea><placeName><settlement type="city">Milan</settlement><region nuts="2">Lombardie</region></placeName></affiliation></author></analytic><series><title level="j">International Journal of Molecular Sciences</title><idno type="eISSN">1422-0067</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>In chronic kidney disease (CKD), the first cause of mortality is cardiovascular disease induced mainly by vascular calcification (VC). Recently, iron-based phosphate binders have been proposed in advanced CKD to treat hyperphosphatemia. We studied the effect of iron citrate (iron) on the progression of calcification in high-phosphate (Pi) calcified VSMC. Iron arrested further calcification when added on days 7–15 in the presence of high Pi (1.30 ± 0.03 vs 0.61 ± 0.02; OD/mg protein; day 15; Pi vs Pi + Fe, <italic>p</italic> < 0.01). We next investigated apoptosis and autophagy. Adding iron to high-Pi-treated VSMC, on days 7–11, decreased apoptotic cell number (17.3 ± 2.6 vs 11.6 ± 1.6; Annexin V; % positive cells; day 11; Pi vs Pi + Fe; <italic>p</italic> < 0.05). The result was confirmed thorough analysis of apoptotic nuclei both in VSMCs and aortic rings treated on days 7–15 (3.8 ± 0.2 vs 2.3 ± 0.3 and 4.0 ± 0.3 vs 2.2 ± 0.2; apoptotic nuclei; arbitrary score; day 15; Pi vs Pi + Fe; VSMCs and aortic rings; <italic>p</italic> < 0.05). Studying the prosurvival axis GAS6/AXL, we found that iron treatment on days 9–14 counteracted protein high-Pi-stimulated down-regulation and induced its de novo synthesis. Moreover, iron added on days 9–15 potentiated autophagy, as detected by an increased number of autophagosomes with damaged mitochondria and an increase in autophagic flux. Highlighting the effect of iron on apoptosis, we demonstrated its action in blocking the H<sub>2</sub>O<sub>2</sub>-induced increase in calcification added both before high Pi treatment and when the calcification was already exacerbated. In conclusion, we demonstrate that iron arrests further high Pi-induced calcium deposition through an anti-apoptotic action and the induction of autophagy on established calcified VSMC.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Bover, J" uniqKey="Bover J">J. Bover</name></author><author><name sortKey="Evenepoel, P" uniqKey="Evenepoel P">P. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Int J Mol Sci</journal-id><journal-id journal-id-type="iso-abbrev">Int J Mol Sci</journal-id><journal-id journal-id-type="publisher-id">ijms</journal-id><journal-title-group><journal-title>International Journal of Molecular Sciences</journal-title></journal-title-group><issn pub-type="epub">1422-0067</issn><publisher><publisher-name>MDPI</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31775364</article-id><article-id pub-id-type="pmc">6928632</article-id><article-id pub-id-type="doi">10.3390/ijms20235925</article-id><article-id pub-id-type="publisher-id">ijms-20-05925</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Ciceri</surname><given-names>Paola</given-names></name><xref ref-type="aff" rid="af1-ijms-20-05925">1</xref></contrib><contrib contrib-type="author"><name><surname>Falleni</surname><given-names>Monica</given-names></name><xref ref-type="aff" rid="af2-ijms-20-05925">2</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0002-6130-2665</contrib-id><name><surname>Tosi</surname><given-names>Delfina</given-names></name><xref ref-type="aff" rid="af2-ijms-20-05925">2</xref></contrib><contrib contrib-type="author"><name><surname>Martinelli</surname><given-names>Carla</given-names></name><xref ref-type="aff" rid="af2-ijms-20-05925">2</xref></contrib><contrib contrib-type="author"><name><surname>Cannizzo</surname><given-names>Stefania</given-names></name><xref ref-type="aff" rid="af3-ijms-20-05925">3</xref></contrib><contrib contrib-type="author"><name><surname>Marchetti</surname><given-names>Giulia</given-names></name><xref ref-type="aff" rid="af3-ijms-20-05925">3</xref></contrib><contrib contrib-type="author"><name><surname>D’Arminio Monforte</surname><given-names>Antonella</given-names></name><xref ref-type="aff" rid="af3-ijms-20-05925">3</xref></contrib><contrib contrib-type="author"><name><surname>Bulfamante</surname><given-names>Gaetano</given-names></name><xref ref-type="aff" rid="af2-ijms-20-05925">2</xref></contrib><contrib contrib-type="author"><name><surname>Block</surname><given-names>Geoffrey A</given-names></name><xref ref-type="aff" rid="af4-ijms-20-05925">4</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0002-1512-559X</contrib-id><name><surname>Messa</surname><given-names>Piergiorgio</given-names></name><xref ref-type="aff" rid="af1-ijms-20-05925">1</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0002-8494-6252</contrib-id><name><surname>Cozzolino</surname><given-names>Mario</given-names></name><xref ref-type="aff" rid="af5-ijms-20-05925">5</xref><xref rid="c1-ijms-20-05925" ref-type="corresp">*</xref></contrib></contrib-group><aff id="af1-ijms-20-05925"><label>1</label>Renal Research Laboratory, Department of Nephrology, Dialysis and Renal Transplant, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico and Fondazione D’Amico per la Ricerca sulle Malattie Renali, 20100 Milan, Italy;<email>p.ciceri@hotmail.it</email> (P.C.);<email>piergiorgio.messa@unimi.it</email> (P.M.)</aff><aff id="af2-ijms-20-05925"><label>2</label>Department of Health Sciences, Division of Pathology, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>monica.falleni@unimi.it</email> (M.F.);<email>delfina.tosi@unimi.it</email> (D.T.);<email>carla.martinelli@unimi.it</email> (C.M.);<email>gaetano.bulfamante@unimi.it</email> (G.B.)</aff><aff id="af3-ijms-20-05925"><label>3</label>Department of Health Sciences, Clinic of Infectious Diseases, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy;<email>stefania.cannizzo@unimi.it</email> (S.C.);<email>giulia.marchetti@unimi.it</email> (G.M.);<email>antonella.darminio@unimi.it</email> (A.D.M.)</aff><aff id="af4-ijms-20-05925"><label>4</label>Nephrology, Denver Nephrolgysts PC, Denver, CO 80230, USA;<email>geoff.block@reatapharma.com</email></aff><aff id="af5-ijms-20-05925"><label>5</label>Department of Health Sciences, Renal Division, University of Milan, ASST Santi Paolo e Carlo, 20142 Milan, Italy</aff><author-notes><corresp id="c1-ijms-20-05925"><label>*</label>Correspondence: <email>mario.cozzolino@unimi.it</email>; Tel.: +02-8184-4381; Fax: +02-8184-4473</corresp></author-notes><pub-date pub-type="epub"><day>25</day><month>11</month><year>2019</year></pub-date><pub-date pub-type="collection"><month>12</month><year>2019</year></pub-date><volume>20</volume><issue>23</issue><elocation-id>5925</elocation-id><history><date date-type="received"><day>17</day><month>10</month><year>2019</year></date><date date-type="accepted"><day>23</day><month>11</month><year>2019</year></date></history><permissions><copyright-statement>© 2019 by the authors.</copyright-statement><copyright-year>2019</copyright-year><license license-type="open-access"><license-p>Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>).</license-p></license></permissions><abstract><p>In chronic kidney disease (CKD), the first cause of mortality is cardiovascular disease induced mainly by vascular calcification (VC). Recently, iron-based phosphate binders have been proposed in advanced CKD to treat hyperphosphatemia. We studied the effect of iron citrate (iron) on the progression of calcification in high-phosphate (Pi) calcified VSMC. Iron arrested further calcification when added on days 7–15 in the presence of high Pi (1.30 ± 0.03 vs 0.61 ± 0.02; OD/mg protein; day 15; Pi vs Pi + Fe, <italic>p</italic> < 0.01). We next investigated apoptosis and autophagy. Adding iron to high-Pi-treated VSMC, on days 7–11, decreased apoptotic cell number (17.3 ± 2.6 vs 11.6 ± 1.6; Annexin V; % positive cells; day 11; Pi vs Pi + Fe; <italic>p</italic> < 0.05). The result was confirmed thorough analysis of apoptotic nuclei both in VSMCs and aortic rings treated on days 7–15 (3.8 ± 0.2 vs 2.3 ± 0.3 and 4.0 ± 0.3 vs 2.2 ± 0.2; apoptotic nuclei; arbitrary score; day 15; Pi vs Pi + Fe; VSMCs and aortic rings; <italic>p</italic> < 0.05). Studying the prosurvival axis GAS6/AXL, we found that iron treatment on days 9–14 counteracted protein high-Pi-stimulated down-regulation and induced its de novo synthesis. Moreover, iron added on days 9–15 potentiated autophagy, as detected by an increased number of autophagosomes with damaged mitochondria and an increase in autophagic flux. Highlighting the effect of iron on apoptosis, we demonstrated its action in blocking the H<sub>2</sub>O<sub>2</sub>-induced increase in calcification added both before high Pi treatment and when the calcification was already exacerbated. In conclusion, we demonstrate that iron arrests further high Pi-induced calcium deposition through an anti-apoptotic action and the induction of autophagy on established calcified VSMC.</p></abstract><kwd-group><kwd>iron</kwd><kwd>vascular calcification</kwd><kwd>apoptosis</kwd><kwd>VSMC</kwd><kwd>phosphate</kwd><kwd>autophagy</kwd></kwd-group></article-meta></front><floats-group><fig id="ijms-20-05925-f001" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Effect of therapeutic addition of ferric citrate on high Pi-induced progression of calcification. Rat VSMCs were cultured with 5 mM Pi in a calcification medium for up to 15 days. (<bold>A</bold>) The addition of 50 µM Fe to the already calcified VSMCs from days 7 to day 15 was able to completely block additional high Pi calcium deposition (* <italic>p</italic> < 0.01). Calcium deposition was measured and normalized by cellular protein content. Data are presented as mean ±SE of five experiments in triplicate. (<bold>B</bold>) The addition of 50 µM Fe on already calcified VSMCs from days 7 to day 15 was able to improve VSMC viability, measured as the total protein content (# <italic>p</italic> < 0.05). Data are presented as the mean ±SE of five experiments in triplicate. (<bold>C</bold>) Ca deposition was visualized at a light microscopic level by Alizarin Red staining. Red indicates Ca deposits. The figure shows that treatment from days 7 to 15 of calcification with 50 µM Fe blocks calcium deposition that is comparable to seven. Magnification 200×. Representative results of one of the three different experiments.</p></caption><graphic xlink:href="ijms-20-05925-g001"></graphic></fig><fig id="ijms-20-05925-f002" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Effect of the therapeutic addition of ferric citrate on high Pi-induced progression of apoptosis. Rat VSMCs were cultured with 5 mM Pi in a calcification medium for up to 11 days. Apoptosis was evaluated by flow cytometry detection by annexin V and 7-amino-actinomycin D (7-AAD) staining. (<bold>A</bold>) Representative dot plots of 7-AAD versus annexin-V-PE. VSMC were left untreated (control) or were treated with Pi or Pi + Fe. The lower left quadrant contains the vital (double negative) population; the lower right contains the early apoptotic (annexin V+/7-AAD−) population; the upper right contains the late apoptotic/necrotic (annexin V+/7-AAD+) population; and the upper left contains the prenecrotic (annexin V−/7-AAD+) population. (<bold>B</bold>) Average percentage of early apoptotic and late apoptotic/necrotic cells. Treatment with 50 µM Fe from days 7 to 11 of calcification was able to significantly block the increase in high Pi-induced Annexin V positive cells (# <italic>p</italic> < 0.05, ns: No significance). Data are presented as mean ±SE of four different experiments. (<bold>C</bold>) The addition of 50 µM Fe on already calcified VSMCs from days 7 to day 11 was able to block further high Pi-induced calcium deposition (* <italic>p</italic> < 0.01). Calcium deposition was measured and normalized by cellular protein content. Data are presented as the mean ±SE of four experiments in triplicate. (<bold>D</bold>) Ca deposition was visualized at a light microscopic level by Alizarin Red staining. Red indicates high Pi-dependent Ca deposits. The images show that treatment from days 7 to 11 of calcification with 50 µM Fe blocked calcium deposition with results comparable to those of day 7. Magnification 200×. Representative result of one of four different experiments.</p></caption><graphic xlink:href="ijms-20-05925-g002"></graphic></fig><fig id="ijms-20-05925-f003" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Effect of therapeutic addition of ferric citrate on 15 days of high Pi-induced progression of apoptosis in aortic rings and VSMCs. Rat VSMCs and aortic rings were cultured with high Pi (5 and 3 mM, respectively) in a calcification medium for up to day 15. (<bold>A</bold>,<bold>D</bold>) Apoptosis was visualized by TUNEL staining; (<bold>B</bold>,<bold>C</bold>): Semi-quantitative analysis of staining data. (<bold>A</bold>,<bold>D</bold>): Therapeutic addition of 50 µM Fe was able to block high Pi-induced nuclear apoptotic red signal progressive increase from day 7 to day 14, both in VSMCs and within the aortic wall (inset). (<bold>B</bold>,<bold>C</bold>) The addition of 50 µM Fe to already calcified VSMCs and aortic rings was able to protect the vessel from the high Pi-induced nuclei fragmentation detectable as red signals. Data are presented as the mean ±SE of at least two experiments in triplicate. (# <italic>p</italic> < 0.05). Magnification: Panel A 40×, panel D 20×, inset 100×.</p></caption><graphic xlink:href="ijms-20-05925-g003"></graphic></fig><fig id="ijms-20-05925-f004" orientation="portrait" position="float"><label>Figure 4</label><caption><p>Effect of prophylactic addition of ferric citrate on H<sub>2</sub>O<sub>2</sub> worsening of high Pi-induced calcification. Rat VSMCs were cultured with 4.5 mM Pi in the calcification medium for up to 7 days. (<bold>A</bold>) Pretreatment with H<sub>2</sub>O<sub>2</sub> was able to worsen high Pi-induced calcium deposition. Iron addition (2 and 5 μM) completely prevents H<sub>2</sub>O<sub>2</sub> worsening of high Pi calcification. Calcium deposition was measured and normalized by cellular protein content. Data are presented as the mean ±SE of three experiments in triplicate. (# <italic>p</italic> < 0.05; * <italic>p</italic> < 0.01). (<bold>B</bold>) Ca deposition was visualized at light microscopic level by Alizarin Red staining. Red color indicates Ca deposits. The figure shows images of calcium deposition modulation by iron in the presence of H<sub>2</sub>O<sub>2</sub> in the experimental conditions indicated. Magnification 200×. Representative result of one of three different experiments.</p></caption><graphic xlink:href="ijms-20-05925-g004"></graphic></fig><fig id="ijms-20-05925-f005" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Effect of therapeutic addition of ferric citrate on H<sub>2</sub>O<sub>2</sub> worsening of high Pi-induced calcification. Rat VSMCs were cultured with 4.5 mM Pi in a calcification medium for up to 14 days. (<bold>A</bold>) The addition of 20 µM Fe on already calcified VSMCs from day 7 to day 14 was able to completely block additional high Pi calcium deposition induced by proapoptotic molecule H<sub>2</sub>O<sub>2</sub> (0.8 mM) treatment during the same period. Calcium deposition was measured and normalized by cellular protein content. Data are presented as the mean ±SE of three experiments in triplicate. (# <italic>p</italic> < 0.05; * <italic>p</italic> < 0.01). (<bold>B</bold>) Ca deposition was visualized at a light microscopic level by Alizarin Red staining. Red indicates Ca deposits. The figure shows images of calcium deposition modulation by iron in the presence of H<sub>2</sub>O<sub>2</sub> in the experimental conditions indicated. Magnification 200×. Representative result of one of three different experiments.</p></caption><graphic xlink:href="ijms-20-05925-g005"></graphic></fig><fig id="ijms-20-05925-f006" orientation="portrait" position="float"><label>Figure 6</label><caption><p>Effect of therapeutic addition of ferric citrate on the expression of the GAS6/AXL pathway during the progression of high Pi-induced calcification. Rat VSMCs were cultured with 5 mM Pi in a calcification medium for up to 15 days. <bold>A</bold> and <bold>C</bold>: GAS6 and AXL protein expression were analyzed by western blot. Lane 1: Control day 9; lane 2: 5 mM Pi day 9; Lane 3: Control day 15; lane 4: 5 mM Pi day 15; lane 5: 5 mM Pi + 50 µM Fe from day 9 to day 15. Representative result of one of three different experiments. <bold>B</bold> and <bold>D</bold>: Analysis of the intensity of the immunoblot bands of GAS6 and AXL normalized for beta tubulin bands are shown. Therapeutic addition of 50 µM Fe stimulates the synthesis of both GAS6 and the AXL protein, thereby inducing this prosurvival pathway. Data are presented as the mean ±SE of three different experiments (# <italic>p</italic> < 0.05).</p></caption><graphic xlink:href="ijms-20-05925-g006"></graphic></fig><fig id="ijms-20-05925-f007" orientation="portrait" position="float"><label>Figure 7</label><caption><p>Effect of the therapeutic addition of ferric citrate on autophagosome formation during the progression of high Pi-induced calcification. (<bold>A</bold>) Image a: Electron microscopy micrograph of an ultra-thin section from control VSMCs on day 9; scale bar 2 µm. Image b: Enlargement of red boxed area in image a showing normal mitochondria (arrows); scale bar 200 nm. (<bold>B</bold>) Image a: Electron microscopy micrograph of an ultra-thin section from 5 mM Pi treated VSMCs for 9 days; scale bar 1 µm. Image b: Enlargement of red boxed area in image a, arrows point to calcified mitochondria; scale bar 500 nm. (<bold>C</bold>) Electron microscopy micrograph of an ultra-thin section from control VSMCs on day 14 showing normal mitochondria (arrows); scale bar 200 nm. (<bold>D</bold>) Image a: Electron microscopy micrograph of an ultra-thin section from 5 mM Pi treated VSMCs for 14 days; scale bar 1 µm. Image b: Enlargement of red boxed area in image a, arrows point to completely calcified mitochondria; scale bar 500 nm. (<bold>E</bold>) Image a: Ultra-thin section from 5 mM Pi + 50 µM Fe treated VSMCs from day 9 to day 14, arrows point to autophagic vacuoles; scale bar 200 nm. Image b: Enlargement of the red boxed area in image a shows an autophagic vacuole with completely calcified mitochondria inside; scale bar 100 nm. Image c: Arrowhead points to an autophagic vacuole with completely calcified mitochondria inside, whereas arrows point to calcified mitochondria; scale bar 400 nm.</p></caption><graphic xlink:href="ijms-20-05925-g007"></graphic></fig><fig id="ijms-20-05925-f008" orientation="portrait" position="float"><label>Figure 8</label><caption><p>Effect of therapeutic addition of ferric citrate on autophagic flux during the progression of high Pi-induced calcification. Rat VSMCs were challenged with 5 mM Pi for up to 14 days. (<bold>A</bold>) LC3IIB protein expression was detected by immunoblotting analysis in the absence or presence of ON treatment with 25 µM chloroquine ‘C’ from day 8 or 13. Lane 1: Control day 9; lane 2: Control day 9 + chloroquine; lane 3: 5 mM Pi day 9; lane 4: 5 mM Pi day 9 + chloroquine; lane 5: Control day 14; lane 6: Control day 14+chloroquine; lane 7: 5 mM Pi day 14; lane 8: 5 mM Pi day 14+chloroquine; lane 9: 5 mM Pi + 50 µM Fe from day 9 to 14; lane 10: 5 mM Pi + 50 µM Fe from day 9 to 14+chloroquine. Representative result of one of three different experiments. (<bold>B</bold>) Graph of the immunoblot band intensity is shown normalized for beta tubulin area intensity. Shaded bars represent different conditions in presence of ON treatment with chloroquine. Empty rectangles indicate the autophagic flux. Data are presented as the mean ±SE (# <italic>p</italic> < 0.05).</p></caption><graphic xlink:href="ijms-20-05925-g008"></graphic></fig></floats-group></pmc><affiliations><list><country><li>Italie</li></country><region><li>Lombardie</li></region><settlement><li>Milan</li></settlement></list><tree><noCountry><name sortKey="Block, Geoffrey A" sort="Block, Geoffrey A" uniqKey="Block G" first="Geoffrey A" last="Block">Geoffrey A. Block</name><name sortKey="Bulfamante, Gaetano" sort="Bulfamante, Gaetano" uniqKey="Bulfamante G" first="Gaetano" last="Bulfamante">Gaetano Bulfamante</name><name sortKey="Cannizzo, Stefania" sort="Cannizzo, Stefania" uniqKey="Cannizzo S" first="Stefania" last="Cannizzo">Stefania Cannizzo</name><name sortKey="Ciceri, Paola" sort="Ciceri, Paola" uniqKey="Ciceri P" first="Paola" last="Ciceri">Paola Ciceri</name><name sortKey="D Rminio Monforte, Antonella" sort="D Rminio Monforte, Antonella" uniqKey="D Rminio Monforte A" first="Antonella" last="D Rminio Monforte">Antonella D Rminio Monforte</name><name sortKey="Falleni, Monica" sort="Falleni, Monica" uniqKey="Falleni M" first="Monica" last="Falleni">Monica Falleni</name><name sortKey="Marchetti, Giulia" sort="Marchetti, Giulia" uniqKey="Marchetti G" first="Giulia" last="Marchetti">Giulia Marchetti</name><name sortKey="Martinelli, Carla" sort="Martinelli, Carla" uniqKey="Martinelli C" first="Carla" last="Martinelli">Carla Martinelli</name><name sortKey="Messa, Piergiorgio" sort="Messa, Piergiorgio" uniqKey="Messa P" first="Piergiorgio" last="Messa">Piergiorgio Messa</name><name sortKey="Tosi, Delfina" sort="Tosi, Delfina" uniqKey="Tosi D" first="Delfina" last="Tosi">Delfina Tosi</name></noCountry><country name="Italie"><region name="Lombardie"><name sortKey="Cozzolino, Mario" sort="Cozzolino, Mario" uniqKey="Cozzolino M" first="Mario" last="Cozzolino">Mario Cozzolino</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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