Aikawa, Elena

Goettsch, Claudia

Rogers, Maximillian

Journal of the American Heart Association

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 Medial and Intimal Calcification in Chronic Kidney Disease:Stressing the Contributions  (Article begins on next page)The Harvard community has made this article openly available.Please share how this access benefits you. Your story matters.Citation Rogers, Maximillian, Claudia Goettsch, and Elena Aikawa. 2013.“Medial and Intimal Calcification in Chronic Kidney Disease:Stressing the Contributions.” Journal of the American HeartAssociation: Cardiovascular and Cerebrovascular Disease 2 (5):e000481. doi:10.1161/JAHA.113.000481.http://dx.doi.org/10.1161/JAHA.113.000481.Published Version doi:10.1161/JAHA.113.000481Accessed February 19, 2015 2:51:36 PM ESTCitable Link http://nrs.harvard.edu/urn-3:HUL.InstRepos:11879124Terms of Use This article was downloaded from Harvard University's DASHrepository, and is made available under the terms and conditionsapplicable to Other Posted Material, as set forth athttp://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAAMedial and Intimal Calciﬁcation in Chronic Kidney Disease:Stressing the ContributionsMaximillian Rogers, PhD; Claudia Goettsch, PhD; Elena Aikawa, MD, PhDC ardiovascular calciﬁcation is a prominent feature ofchronic inﬂammatory disorders that associate withsigniﬁcant morbidity and mortality. Vascular calciﬁcation ishighly prevalent in patients with chronic kidney disease (CKD),a multifactorial disorder. CKD often results from hypertensionand diabetes, and patients with CKD are among thehighest-risk groups for cardiovascular events. Notably, CKDaccelerates the development of atherosclerosis. Our groupand others have demonstrated that CKD causes excessivevascular inﬂammation and calciﬁcation.1 CKD is characterizedby increased serum phosphate levels, which is, in turn,associated with the progression of calciﬁcation. But phos-phate binder therapy reportedly has no effect on vascularcalciﬁcation in CKD patients,2 suggesting that targetingphosphate alone cannot improve vessel stiffness. A varietyof other therapies, which have produced mixed results at best,have been tested to prevent or hinder the progression ofvascular calciﬁcation in CKD.3 A greater understanding of themineralization process itself may therefore lead to new andeffective therapeutics for vascular calciﬁcation in CKD.In vitro and in vivo models of cardiovascular calciﬁcationhave helped generate a greater mechanistic understandingof the vascular calciﬁcation process. A common model ofCKD is apolipoprotein E-deﬁcient (ApoE/) or low-densitylipoprotein receptor-deﬁcient (ldlr/) mice subjected to 5/6 nephrectomy and fed a high-fat, high-cholesterol diet.4Similar to dysmetabolic CKD patients, both arterial intimaland medial calciﬁcation occurs in these animals. The use of5/6 nephrectomy in mice with varying diets allows for theinvestigation of medial calciﬁcation, which representspatients with renal failure undergoing hemodialysis. Eachmodel recapitulates the formation of calciﬁcation in distinctvascular layers, which may lead to different clinical out-comes (Figure).Clinically, vascular calciﬁcation occurs in the vessel intimaor media and often overlaps, particularly in a growingpopulation of patients with CKD-related atherosclerosis. Theprocess of intimal calciﬁcation associates with atheroscleroticvascular disease, observed as spotty calciﬁcations of theatherosclerotic plaques, where small hydroxyapatite mineralclefs (microcalciﬁcations) seem to associate with cholesterolcrystals observed in early lesions.5 These microcalciﬁcationsin the plaque could provoke a rupture of vulnerable plaque.6Medial calciﬁcation occurs primarily in association with CKDand diabetes, and is independent of hypercholesterolemia.The sheet-like calciﬁcations of the tunica media can lead toincreased vascular stiffness and reduced compliance ofvessels. The differences between medial and intimal calciﬁ-cation stress the importance of choosing appropriate diseasemodels for mechanistic and therapeutic studies.Pathogenesis of vascular calciﬁcation is complex—beyondjust a simple precipitation of calcium and phosphate, it isinstead an active, cell-regulated process. Several cell typeslikely play a role in the vascular calciﬁcation process,including smooth muscle cells (SMCs),7 circulating bonemarrow-derived cells,8 and macrophages.5 Vascular calciﬁca-tion mechanistic studies have focused on cellular differenti-ation, calcium and phosphate homeostasis, loss of inhibition,apoptosis, the release of calcifying vesicles, and changes inthe extracellular matrix (ECM) among several other cellularprocesses3 (Figure). Osteogenic transition of vascular SMCsor stem cells is induced by bone morphogenetic proteins,9high phosphate levels,10 inﬂammation,11,12 and oxidativestress,13 and leads to a unique molecular pattern marked byosteogenic transcription factors. Emerging evidence alsosuggests that alternative mechanisms independent of oste-ogenic differentiation, including the release of matrix vesiclesby SMCs or macrophages, may contribute to vascularcalciﬁcation in CKD.5,14 Understanding the connectionsbetween these mechanisms and signaling pathways couldThe opinions expressed in this article are not necessarily those of the editorsor of the American Heart Association.From the Centers for Interdisciplinary Cardiovascular Sciences (M.R., C.G.,E.A.) and Excellence in Vascular Biology (E.A.), Division of CardiovascularMedicine, Brigham and Women’s Hospital, Harvard Medical School, Boston,MA.Correspondence to: Elena Aikawa, MD, PhD, Brigham and Women’s Hospital,Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115.E-mail: eaikawa@partners.orgJ Am Heart Assoc. 2013;2:e000481 doi: 10.1161/JAHA.113.000481.ª 2013 The Authors. Published on behalf of the American Heart Association,Inc., by Wiley Blackwell. This is an Open Access article under the terms of theCreative Commons Attribution-NonCommercial License, which permits use,distribution and reproduction in any medium, provided the original work isproperly cited and is not used for commercial purposes.DOI: 10.1161/JAHA.113.000481 Journal of the American Heart Association 1EDITORIALSprovide novel mechanistic insight into the calciﬁcationprocess, and potentially help lead to cardiovascular diseasetherapeutics.In this issue of JAHA, Masuda et al15 build on previousstudies linking tumor necrosis factor a (TNFa) to vascularcalciﬁcation, while providing a novel link between inﬂamma-tion, endoplasmic reticulum stress, and calciﬁcation. Theirstudy found that administering TNFa to mouse vascular SMCsdose-dependently induced the protein kinase RNA-like endo-plasmic reticulum kinase (PERK)—eukaryotic initiation factor2a (eIF2a)-activating transcription factor 4 (ATF4)—C/EBPhomologous protein (CHOP) axis of the endoplasmic reticulumstress response.Suggesting that inﬂammation may regulate vascular calci-ﬁcation through an endoplasmic reticulum stress pathway, theauthors also showed short hairpin RNA-mediated knockdownsof PERK, ATF4, and CHOP led to signiﬁcant reductions in bothTNFa-induced calcium deposition and alkaline phosphataseactivity. Supporting their in vitro ﬁndings, both phospho-ATF4and total ATF4, along with CHOP expression levels, increasedsigniﬁcantly in the aortas of 5/6 nephrectomy ApoE/ miceon a high-fat, high-cholesterol diet. When treated withinﬂiximab to block TNFa, or with 2 different chemicalchaperones to reduce endoplasmic reticulum stress, calciﬁedlesions decreased signiﬁcantly in these CKD mice. Theinﬂiximab results provide further support for targetinginﬂammation as a means of blocking vascular calciﬁcation,which had been previously reported by another group usingldlr/ diabetic mice.16On a mechanistic level, treatment of these CKD mice withthe chemical chaperones also led to a signiﬁcant reduction inthe expression of a type III sodium-dependent phosphatetransporter, PiT-1, possibly mediated through a reduction ofCHOP expression. This study also demonstrated that control-ling the expression of PiT-1 through CHOP overexpressioncorrelated with the amount of inorganic phosphate uptakein SMCs. Of importance relating to this result, PiT-1 wasshown previously to participate in the vascular calciﬁcationprocess.17The ﬁndings of Masuda et al suggest that inﬂammation atleast partly regulates vascular calciﬁcation through activatingan endoplasmic reticulum stress pathway, which in turn mayincrease inorganic phosphate uptake, leading to increasedmineralized calcium deposition and osteogenic differentiation.The growing connection of endoplasmic reticulum stressto vascular calciﬁcation is intriguing, considering that thisstress response process has also been connected to boneformation.18Figure. Modeling human vascular calciﬁcation in mice. Human disease associated intimal and medial vascular calciﬁcation diagramed withplausible mechanisms and commonly used mouse models. ApoE/ indicates apolipoprotein E-deﬁcient; Ca, calcium; CKD, chronic kidneydisease; ldlr/, low-density lipoprotein receptor-deﬁcient; P, phosphate; WT, wild type.DOI: 10.1161/JAHA.113.000481 Journal of the American Heart Association 2Medial and Intimal Calciﬁcation in CKD Rogers et alEDITORIALSBesides inﬂammation, many additional mechanistic factorshave been proposed and likely play a role in vascularcalciﬁcation in combination with or independently of theinﬂammation driven process. For example, previous studies19have demonstrated that some components of the ECM(elastin, collagen) could be important factors in the regulationof calciﬁcation progression.1 Exactly how the ECM compo-nents and the factors that regulate ECM remodeling, such asmatrix metalloproteases and cathepsins, could lead to medialcalciﬁcation is not yet fully understood. Furthermore, addi-tional unreported factors may also be involved in thismechanistic pathway.A report by Purnomo et al20 in this issue of JAHA examinedthe role of ECM in the development of vascular calciﬁcation,by highlighting the role of glycosaminoglycans (GAG). Thestudy found that exostosin-like glycosyltransferase 2 (EXTL2)deﬁciency caused increased heparin sulfate and chondroitin-sulfate in the aortas of EXTL2-deﬁcienct mice. When CKD wasinduced in mice by 5/6 nephrectomy along with a high-phos-phate diet—a different CKD model than that used by Masudaet al—elevated calciﬁcation was observed in the media layerof the aorta, which is characterized by increased GAG levels.The use of 5/6 nephrectomy along with a high-phosphate dietallowed other aspects of CKD, in addition to vascularcalciﬁcation, to be modeled. EXTL2-deﬁcient CKD mice hadalso higher systolic blood pressure compared to wild-typemice, which might be due to increased vascular calciﬁcationaccompanied by increased stiffness and decreased complianceof the arteries.Importantly, CKD itself increased heparin sulfate andchondroitinsulfate in wild-type mice, which might acceleratethe progression of vascular calciﬁcation. In line with thisevidence, treatment with heparitinase and chondroitinaseprevented calciﬁcation in aorta explants. As such, changes inthe sulfation pattern of GAG could conceivably contribute tothe vascular calciﬁcation process. Further study is required,however, to dissect the full inﬂuence of speciﬁc GAG sulfationpatterns in triggering pathways involved in the development ofvascular calciﬁcation.In the EXTL2-deﬁcient mice, Runx2 and collagen 1A1 wereincreased, whereas SMC markers were decreased. Purnomoet al suggested that under high-phosphate conditions heparinsulfate and chondroitinsulfate trigger a signaling pathway thatfurther induces SMC osteogenic transition. Whether similarresults occur in EXTL2-deﬁcient mice under other disease-rel-evant conditions remains to be determined. For example,testing this pathway in dyslipidemic conditions, such as thoseused by Masuda et al, may help reveal additional informationabout the disease process (eg, whether ECM-based calciﬁca-tion effects intimal calciﬁcation).Taken together, the results of this study provide novelinsights into the role of GAG in calciﬁcation, and furthersuggest that modulation of the ECM might be a therapeutictarget in the prevention of vascular calciﬁcation in CKD.While these two studies depicting the role of twopotentially independent mechanisms of calciﬁcation in CKDhelp to connect previous ﬁndings from other groups, severalquestions remain unanswered—notably, how inﬂammatorycytokines like TNFa activate endoplasmic reticulum stress,and whether GAG are increased in the aortas of CKD patients.Just how these proposed mechanisms will ultimately translateto the human disease condition remains to be seen. Whilemouse models are useful, an important next step to furtheringthese results will be to test these working hypotheses inhuman cells and tissues.In summary, the work of Masuda et al and Purnomo et aladds to several existing working hypotheses on how vascularcalciﬁcation occurs, particularly in CKD. Of paramountimportance in interpreting the results of these and otherstudies involves understanding the model used and the typeof calciﬁcation it reﬂects. Differences between intimal andmedial calciﬁcation may also be reﬂected in the mechanismsbehind their development. Each animal and cell culture modelhas limits; no one model fully encapsulates the humandisease process. Each new study, however, incrementallyadds toward our growing understanding of the process ofvascular calciﬁcation.AcknowledgmentsThe authors thank Sara Karwacki for her excellent editorialassistance.Sources of FundingDr Aikawa is supported by grants from the National Institutesof Health (R01HL114805, R01HL109506).DisclosureNone.References1. Aikawa E, Aikawa M, Libby P, Figueiredo JL, Rusanescu G, Iwamoto Y, FukudaD, Kohler RH, Shi GP, Jaffer FA, Weissleder R. Arterial and aortic valvecalciﬁcation abolished by elastolytic cathepsin S deﬁciency in chronic renaldisease. Circulation. 2009;119:1785–1794.2. Seifert ME, de Las Fuentes L, Rothstein M, Dietzen DJ, Bierhals AJ, Cheng SC,Ross W, Windus D, Davila-Roman VG, Hruska KA. Effects of phosphate bindertherapy on vascular stiffness in early-stage chronic kidney disease. Am JNephrol. 2013;38:158–167.3. Wu M, Rementer C, Giachelli CM. Vascular calciﬁcation: an update onmechanisms and challenges in treatment. Calcif Tissue Int. 2009 Mar 1 [Epubahead of print] doi:10.1007/s00223-013-9712-z.4. Shobeiri N, Adams MA, Holden RM. Vascular calciﬁcation in animal models ofCKD: a review. Am J Nephrol. 2010;31:471–481.5. New SE, Goettsch C, Aikawa M, Marchini JF, Shibasaki M, Yabusaki K, Libby P,Shanahan CM, Croce K, Aikawa E. Macrophage-derived matrix vesicles: anDOI: 10.1161/JAHA.113.000481 Journal of the American Heart Association 3Medial and Intimal Calciﬁcation in CKD Rogers et alEDITORIALSalternative novel mechanism for microcalciﬁcation in atherosclerotic plaques.Cir Res. 2013;113:72–77.6. Kelly-Arnold A, Maldonado N, Laudier D, Aikawa E, Cardoso L, Weinbaum S.Revised microcalciﬁcation hypothesis for ﬁbrous cap rupture in humancoronary arteries. Proc Natl Acad Sci USA. 2013;110:10741–10746.7. Wada T, McKee MD, Steitz S, Giachelli CM. Calciﬁcation of vascularsmooth muscle cell cultures: inhibition by osteopontin. Cir Res. 1999;84:166–178.8. Khosla S, Eghbali-Fatourechi GZ. Circulating cells with osteogenic potential.Ann N Y Acad Sci. 2006;1068:489–497.9. Bostr€om K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. Bonemorphogenetic protein expression in human atherosclerotic lesions. J ClinInvest. 1993;91:1800–1809.10. Shuichi J, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morri H, GiachelliCM. Phosphate regulation of vascular smooth muscle cell calciﬁcation. CirRes. 2000;87:e10–e17.11. Tintut Y, Patel J, Parhami F, Demer LL. Tumor necrosis factor-alpha promotesin vitro calciﬁcation of vascular cells via the cAMP pathway. Circulation.2000;102:2636–2642.12. Aikawa E, Nahrendorf M, Figueiredo JL, Swirski FK, Shtatland T, Kohler RH,Jafferer FA, Aikawa M, Weissleder R. Osteogenesis associates with inﬂamma-tion in early-stage atherosclerosis evaluated by molecular imaging in vivo.Circulation. 2007;116:2841–2850.13. Goettsch C, Rauner M, Hamann C, Sinningen K, Hempel U, Bornstein SR,Hofbauer LC. Nuclear factor of activated T cells mediates oxidisedLDL-induced calciﬁcation of vascular smooth muscle cells. Diabetologia.2011;54:2690–2701.14. Kapustin AN, Davies JD, Reynolds JL, McNair R, Jones GT, Sidibe A, SchurgersLJ, Skepper JN, Proudfoot D, Mayr M, Shanahan CM. Calcium regulates keycomponents of vascular smooth muscle cell-derived matrix vesicles toenhance mineralization. Cir Res. 2011;109:e1–e12.15. Masuda M, Miyazaki-Anzai S, Levi M, Ting T, Miyazaki M. PERK-eIF2a-ATF4--CHOP signaling contributes to TNFa-induced vascular calciﬁcation. J Am HeartAssoc. 2013;2:e000238 doi:10.1161/JAHA.113.000238.16. Al-Aly Z, Shao JS, Lai CF, Huang E, Cai J, Behrmann A, Cheng SL, Towler DA.Aortic Msx2-Wnt calciﬁcation cascade is regulated by TNF-alpha-dependentsignals in diabetic Ldlr/ mice. Aterioscler Thromb Vasc Biol. 2007;27:2589–2596.17. Li X, Yang HY, Giachelli CM. Role of the sodium-dependent phosphatecotransporter, Pit-1, in vascular smooth muscle cell calciﬁcation. Circ Res.2006;98:905–912.18. Murakami T, Saito A, Hino S, Kondo S, Kanemoto S, Chihara K, Sekiya H,Tsumagari K, Ochiai K, Yoshinaga K, Saitoh M, Nishimura R, Yoneda T, Kou I,Furuichi T, Ikegawa S, Ikawa M, Okabe M, Wanaka A, Imaizumi K. Signalingmediated by the endoplasmic reticulum stress transducer OASIS is involved inbone formation. Nat Cell Biol. 2009;11:1205–1211.19. Pai AS, Giachelli CM. Matrix remodeling in vascular calciﬁcation associatedwith chronic kidney disease. J Am Soc Nephrol. 2010;21:1637–1640.20. Purnomo E, Emoto N, Nugrahaningsih DA, Nakayama K, Yagi K, Heiden S,Nadanaka S, Kitagawa H, Hirata K. Glycosaminoglycan overproduction in theaorta increases aortic calciﬁcation in murine chronic kidney disease. J AmHeart Assoc. 2013;2:e000405 doi:10.1161/JAHA.113.000405.Key Words: Editorials • atherosclerosis • chronickidneydis-ease • endoplasmic reticulumstress • inﬂammation • intimalandmedial calciﬁcationDOI: 10.1161/JAHA.113.000481 Journal of the American Heart Association 4Medial and Intimal Calciﬁcation in CKD Rogers et alEDITORIALS

Medial and Intimal Calcification in Chronic Kidney Disease: Stressing the Contributions

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Medial and Intimal Calcification in Chronic Kidney Disease: Stressing the Contributions

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