Vascular Calcification Mechanisms

REVIEWS J Am Soc Nephrol 15: 2959 2964, 2004 Vascular Calcification Mechanisms CECILIA M. GIACHELLI Bioengineering Department, University of Washington, Seattle, Washington. Abstract. Vascular calcification is highly correlated with cardiovascular disease mortality, especially in patients with ESRD or diabetes. In addition to the devastating effects of inappropriate biomineralization seen in cardiac valvulopathies, calciphylaxis, and idiopathic arterial calcification, vascular calcification is now recognized as a marker of atherosclerotic plaque burden as well as a major contributor to loss of arterial compliance and increased pulse pressure seen with age, diabetes, and renal insufficiency. In recent years, several mechanisms to explain vascular calcification have been identified including (1) loss of inhibition, (2) induction of bone formation, (3) circulating nucleational complexes, and (4) cell death. Alterations in calcium (Ca) and phosphorus (P) balance as seen in patients with ESRD promotes vascular calcification via multiple mechanisms and may explain the alarmingly high levels of cardiovascular disease deaths in these patients. Strategies to control Ca and P levels in patients with ESRD have met with early success in preventing progression of vascular calcification. Whether or not vascular calcification can be reversed is not yet known, but exciting new studies suggest that this may be possible in the future. Pathologic calcification of cardiovascular structures, or vascular calcification, is associated with a number of diseases including ESRD and cardiovascular disease. Calcium phosphate deposition, in the form of bioapatite, is the hallmark of vascular calcification and can occur in the blood vessels, myocardium, and cardiac valves. In blood vessels, calcified deposits are found in distinct layers of the blood vessel and are related to underlying pathology. Intimal calcification occurs in atherosclerotic lesions (1,2), whereas medial calcification (also known as Monckeberg s medial sclerosis) is associated with vascular stiffening and arteriosclerosis observed with age, diabetes, and ESRD (3,4). Intimal calcification may occur independently of medial calcification and vice versa. In patients with ESRD, a mixture of intimal and medial calcification has been observed in affected vessels (5,6). Correspondence to Dr. Cecilia M. Giachelli, Bioengineering Department, Box 351720, University of Washington, Okanogan Lane, Bagley Hall, Seattle, WA 98195. Phone: 206-543-0205; Fax: 206-616-9763; E-mail: ceci@u.washington.edu 1046-6673/1512-2959 Journal of the American Society of Nephrology Copyright 2004 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000145894.57533.C4 Clinical Consequences of Vascular Calcification Vascular calcification can lead to devastating organ dysfunction depending on its extent and the organ affected. In the heart, calcification of cardiac valve leaflets is recognized as a major mode of failure of native as well as bioprosthetic valves (7,8). In dialysis patients, vascular medial calcification is responsible for calcific uremic arteriolopathy, a necrotizing skin condition associated with extremely high mortality rates (9). Finally, a genetic deficiency in pyrophosphate levels causes idiopathic infantile arterial calcification, a disease characterized by arterial calcification, fibrosis, and stenosis that leads to premature death in affected neonates (10). In contrast, calcification of blood vessels commonly seen with aging, ESRD, diabetes, and atherosclerosis has historically been considered a benign finding. However, the introduction of new techniques to measure vascular calcification noninvasively, such as electron beam computed tomography, have revolutionized our current thinking about the risks of vascular calcification. In coronary arteries, calcification is positively correlated with atherosclerotic plaque burden (11,12), increased risk of myocardial infarction (13 15), and plaque instability (2,16). Although some of these findings may relate to the correlation of coronary calcification with extent of underlying atherosclerotic disease, it is also possible that vascular calcification itself may contribute to initiation or progression of cardiovascular disease (CVD). This possibility seems particularly plausible in the case of coronary calcification associated with ESRD (see below). Finally, vascular calcification, especially that found in the media of large arteries, leads to increased stiffening and therefore decreased compliance of these vessels. The consequent loss of the important cushioning function of these arteries is associated with increased arterial pulse wave velocity and pulse pressure, and leads to impaired arterial distensibility, increased afterload favoring left ventricular hypertrophy, and compromised coronary perfusion (17,18). Indeed, medial arterial calcification is strongly correlated with coronary artery disease and future cardiovascular events in patients with type 1 (19,20) and is a strong prognostic marker of CVD mortality in patients with ESRD (21). Thus, vascular calcification has a profound influence on cardiovascular function and health. Cardiovascular Calcification and CVD Mortality in ESRD More than half the deaths in patients with ESRD are due to CVD. In fact, the risk of CVD mortality in adult patients with ESRD is 20 to 30 times higher than that of the general popu-

2960 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 2959 2964, 2004 lation (22). Growing evidence suggests that this increased risk of CVD mortality may be partly explained by the predisposition of this population to vascular calcification. Hyperphosphatemia and elevated Ca P ion product (Ca P; prevalent in patients with ESRD) promote vascular calcification, and are significantly linked to all cause and CVD mortality in patients with ESRD (23). In a landmark study, Goodman et al. (24) found that coronary artery calcification occurred in young patients with ESRD decades before this pathology was observed in the normal population. Furthermore, progression of vascular calcification in this group was positively correlated with serum P levels, Ca P, and daily intake of Ca (24). Similar findings were observed by Eifinger et al. (25) as well as Oh et al. (26) in young adults with childhood-onset ESRD. In addition, Raggi et al. (27) found that coronary artery calcification was very common and severe in adult hemodialysis patients, and significantly correlated with ischemic CVD. Again, calcification was highly correlated with elevated serum Ca and P levels. Finally, arterial medial calcification, a strong prognostic marker for CVD mortality in patients with ESRD, was also associated with elevated serum Ca, P, Ca P, and prescribed Ca intake (18,21). Thus derangements in Ca and P balance are now considered major nontraditional risk factors for CVD in ESRD. Mechanisms of Vascular Calcification Vascular calcification is currently considered an actively regulated process that may arise by several different, non mutually exclusive mechanisms (28). As shown in Figure 1, four different mechanisms for initiating vascular calcification have been proposed. First, human and mouse genetic findings have determined that blood vessels normally express inhibitors of mineralization, such as pyrophosphate and matrix gla protein, respectively, and that lack of these molecules ( loss of inhibition ) leads to spontaneous vascular calcification and increased mortality (10,29). Likewise, fetuin/ 2-HS-glycoprotein is a major inhibitor of apatite found in the circulation, and decreased fetuin levels have recently been correlated with elevated CVD mortality in hemodialysis patients (30). Second, the presence of bone proteins such as osteopontin (31), osteocalcin (32), and BMP2 (33), matrix vesicles (34), and outright bone and cartilage formation in calcified vascular lesions (1,35) has suggested that osteogenic mechanisms may also play a role in vascular calcification. Indeed, cells derived from the vascular media undergo bone- and cartilage-like phenotypic change and calcification in vitro under various conditions, and is discussed in further detail below (36 40). Third, bone turnover leading to release of circulating nucleational complexes has been proposed to explain the link between vascular calcification and osteoporosis in postmenopausal women (41 43). Fourth, cell death can provide phospholipidrich membranous debris and apoptotic bodies that may serve to nucleate apatite, especially in diseases where necrosis and apoptosis are prevalent, such as atherosclerosis (34,44,45). Finally, via thermodynamic mechanisms (sometimes referred to as passive mechanisms), elevated Ca, P, and Ca P promote apatite nucleation and crystal growth and would be expected to exacerbate vascular calcification initiated by any of the other mechanisms described above. Furthermore, new evidence suggests that Ca and P may additionally have direct effects on vascular cells that predispose to mineralization, as described below. Roles of Ca and P in Vascular Smooth Muscle Cell Mineralization As indicated above, elevated serum P, Ca, and increased Ca burden are correlated with vascular calcification and cardiovascular mortality in patients with ESRD. To determine whether elevated Ca or P directly affect cell-mediated regulation of vascular calcification, we and others have made use of vascular smooth muscle cell culture systems. Increasing inorganic P in the culture media to those seen in hyperphosphatemia ( 2.4 mm) leads to deposition of apatite into the extracellular matrix surroundings the cells (37,46 48). Concomitant with mineralization, the cells undergo a phenotypic change characterized by loss of smooth muscle specific gene expression and upregulation of genes commonly associated with bone differentiation including osteocalcin, osteopontin and Runx2. Similar phenotypic changes have also been observed in vivo in human as well as animal models of vascular calcification (46,49,50). Elevated P-induced phenotypic transition and mineralization were shown to be dependent on a sodium-dependent phosphate cotransporter, Pit-1, on the basis of their ability to be inhibited by phosphonoformic acid (37) and Pit-1 specific small interfering RNA (Giachelli and Li, unpublished data). These data confirm the importance of inorganic P as a signaling molecule with the ability to initiate both phenotypic change and mineralization in vascular smooth muscle cells. Likewise, elevating Ca levels in the culture media to levels considered hypercalcemic ( 2.6 mm) leads to enhanced mineralization and phenotypic transition of vascular smooth muscle cells (51). Elevated calcium-induced mineralization was also dependent on the function of a sodium-dependent phosphate cotransporter. Although elevated Ca did not appear to increase P uptake acutely, prolonged exposure of smooth muscle cell cultures to elevated Ca induced Pit-1 mrna levels, suggesting that elevated Ca regulates P sensitivity of vascular smooth muscle cells. These findings were recently confirmed and extended by Proudfoot et al. (52). In those studies, elevated Ca (1.8 to 5.0 mm) stimulated human smooth muscle cell calcification in vitro. Furthermore, elevated Ca levels stimulated release of mineralization-competent matrix vesicles from human smooth muscle cells, and diltiazem and BAPTA, both inhibitors of intracellular Ca influx, blocked smooth muscle cell mineralization. A rise in intracellular Ca was also associated with altered alkaline phosphatase, decreased matrix Gla protein (MGP), and increased fetuin levels. Together with our own, these findings confirm that elevated Ca has pro-mineralizing effects beyond simply raising the Ca P, and regulates multiple systems in smooth muscle cells that promote susceptibility to matrix mineralization. A diagram summarizing the possible roles of elevated Ca and P on vascular smooth muscle calcification is shown in Figure 2.

J Am Soc Nephrol 15: 2959 2964, 2004 Vascular Calcification 2961 Figure 1. Schematic illustrating four, non mutually exclusive theories for vascular calcification: (1) loss of inhibition as a result of deficiency of constitutively expressed tissue-derived and circulating mineralization inhibitors leads to default apatite deposition, (2) induction of bone formation resulting from altered differentiation of vascular smooth muscle or stem cells (3), circulating nucleational complexes released from actively remodeling bone, and (4) cell death leading to release of apoptotic bodies and/or necrotic debris that may serve to nucleate apatite at sites of injury. Figure reprinted from (28) with permission from Elsevier. Can Vascular Calcification Be Controlled? Several recent studies suggest that vascular calcification may be slowed, and potentially even reversed, in humans as well as experimental animal models. In light of the findings that elevated serum P and Ca are strongly correlated with vascular calcification and CVD mortality in ESRD, an emphasis has been placed on the use of non Ca-containing P binders, such as sevelamer, to treat hyperphosphatemia in these patients Figure 2. Proposed model for the effects of elevated Ca and P on vascular smooth muscle cell (SMC) matrix mineralization. Elevated Ca and P are proposed to stimulate vascular matrix mineralization in two ways. First, both Ca and P increase the activity of Pit-1: elevated P stimulates P uptake via Pit-1, and elevated Ca induces expression of Pit-1 mrna. Both mechanisms are proposed to enhance P uptake into SMC as well as matrix vesicles. Elevated intracellular P than leads to SMC phenotypic modulation, which includes upregulation of osteogenic genes (Runx2, osteocalcin, and alkaline phosphatase), and generation of a mineralization-competent extracellular matrix. In addition, increased Pit-1 in matrix vesicles promotes P loading of matrix vesicles, promoting nucleation of mineral within the extracellular matrix. Second, elevated Ca and/or P lead to increased Ca P ion product, thereby promoting growth of apatite crystals in the matrix via thermodynamic mechanisms.

2962 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 2959 2964, 2004 (53). Indeed, when sevelamer was compared to commonly used Ca-based P binders in a large hemodialysis patient group it was found that patients receiving sevelamer had unchanged median coronary artery and aorta calcification scores after 1 yr as opposed to Ca-treated patients whose arterial calcification scores increased 28% over baseline (12). Significantly, although both treatments controlled P levels equivalently, treatment with Ca containing binders led to an increased frequency of hypercalcemic episodes and greater suppression of serum parathyroid hormone (PTH) levels in hemodialysis patients (54,55). Similar effects of sevelamer were also noted in a rat uremia model, where renal calcification was greatly reduced compared to Ca carbonate treatment (56). Thus, decreasing Ca and P burdens appear to be beneficial in blocking vascular calcification. Antihypertensive agents have also been implicated in control of vascular calcification. In a clinical study, the Ca channel blocker nifedipine slowed progression of coronary calcification in hypertensive patients compared to diuretics (57). Moreover, Moreau s group has developed a novel animal model of isolated systolic hypertension that is caused by arterial calcification (58,59). In this model, rats are treated with warfarin and vitamin K and this leads to calcification of the aortic wall and isolated systolic hypertension. Treatment with an endothelin (ET-1) receptor antagonist or angiotensin II blocker prevents increases in pulse pressure as well as calcification of the vessels. Remarkably, treatment of rats with ET-1 antagonist after calcification was established caused regression of vascular calcification and normalization of pulse pressure. These data support the idea that calcification of compliance vessels leads to hypertensive effects, especially increased pulse pressure as a result of increased stiffness, and identify ET-1 antagonists as major regulators of vascular calcification. Furthermore, these data suggest that regression of vascular calcification can occur and is actively regulated. Indeed, evidence for regression of coronary calcification in humans has also been obtained. In a study of 102 symptomatic patients with coronary calcification currently being treated with standard medications, 15% of patients showed evidence of regression on the basis of electron beam computed tomography calcium scores measured at baseline and approximately 6 mo later (60). Conclusions Vascular calcification is highly correlated with CVD morbidity and mortality, especially in high-risk populations such patients with ESRD or diabetes. Four non mutually exclusive mechanisms for vascular calcification are emerging, including (1) loss of inhibition, (2) induction of bone formation, (3) circulating nucleational complexes, and (4) cell death. Derangements in Ca and P metabolism effect all of these mechanisms by elevating the Ca P, and also by direct effects on smooth muscle cells that promote bonelike differentiation. The challenge remains to understand which mechanisms are active and/or predominate under various disease states, and to develop effective therapeutic strategies that may prevent and potentially reverse vascular calcification. Acknowledgments Dr. Giachelli s research is supported by NIH grants HL62329, AR48798, NSF grant EEC9529161, and a grant from Genzyme. References 1. Hunt JL, Fairman R, Mitchell ME, Carpenter JP, Golden M, Khalapyan T, Wolfe M, Neschis D, Milner R, Scoll B, Cusack A, Mohler ER 3rd: Bone formation in carotid plaques: A clinicopathological study. Stroke 33: 1214 1219, 2002 2. Burke AP, Taylor A, Farb A, Malcom GT, Virmani R: Coronary calcification: Insights from sudden coronary death victims. Z Kardiol, 89[Suppl 2]: 49 53, 2000 3. Monckeberg JG: Uber die reine mediaverkalkung der extremitaten-arteries und ihr Verhalten zur arteriosklerose. Virchows Arch 171: 141 167, 1903 4. Edmonds ME, Morrison N, Laws JW, Watkins PJ: Medial arterial calcification and diabetic neuropathy. Br Med J Clin Res Ed 284(6320): 928 930, 1982 5. Schwarz U, Buzello M, Ritz E, Stein G, Raabe G, Wiest G, Mall G, Amann K: Morphology of coronary atherosclerotic lesions in patients with end-stage renal failure. Nephrol Dial Transplant 15: 218 223, 2000 6. Ibels LS, Alfrey AC, Huffer WE, Craswell PW, Anderson JT, Weil R, 3rd: Arterial calcification and pathology in uremic patients undergoing dialysis. Am J Med 66: 790 796, 1979 7. Schoen FJ, Levy RJ: Founder s Award, 25th Annual Meeting of the Society for Biomaterials, perspectives. Providence, RI, April 28 May 2, 1999. Tissue heart valves: Current challenges and future research perspectives. J Biomed Mater Res 47: 439 465, 1999 8. O Keefe JH, Lavie CJ, Nishimura RA, Edwards WD: Degenerative aortic stenosis. One effect of the graying of America. Postgrad Med 89: 143 154, 1991 9. Coates T, Kirkland GS, Dymock RB, Murphy BF, Brealey JK, Mathew TH, Disney AP: Cutaneous necrosis from calcific uremic arteriolopathy. Am J Kidney Dis 32: 384 391, 1998 10. Rutsch F, Ruf N, Vaingankar S, Toliat MR, Suk A, Hohne W, Schauer G, Lehmann M, Roscioli T, Schnabel D, Epplen JT, Knisely A, Superti-Furga A, McGill J, Filippone M, Sinaiko AR, Vallance H, Hinrichs B, Smith W, Ferre M, Terkeltaub R, Nurnberg P: Mutations in ENPP1 are associated with idiopathic infantile arterial calcification. Nat Genet 34: 379 381, 2003 11. Rumberger JA, Simons DB, Fitzpatrick LA, Sheedy PF, Schwartz RS: Coronary artery calcium area by electron-beam computed tomography and coronary atherosclerotic plaque area: A histopathologic correlative study. Circulation 92: 2157 2162, 1995 12. Sangiorgi G, Rumberger JA, Severson A, Edwards WD, Gregoire J, Fitzpatrick LA, Schwartz RS: Arterial calcification and not lumen stenosis is highly correlated with atherosclerotic plaque burden in humans: A histologic study of 723 coronary artery segments using nondecalcifying methodology. J Am Coll Cardiol 31: 126 133, 1998 13. Beadenkopf WG, Daoud AS, Love BM: Calcification in the coronary arteries and its relationship to arteriosclerosis and myocardial infarction. Am J Roentgenol 92: 865 871, 1964 14. Locker TH, Schwartz RS, Cotta CW, Hickman JR: Fluoroscopic coronary artery calcification and asociated coronary disease in asymptomatic young men. J Am Coll Cardiol 19: 1167 1172, 1992

J Am Soc Nephrol 15: 2959 2964, 2004 Vascular Calcification 2963 15. Puentes G, Detrano R, Tang W, Wong N, French W, Narahara K, Brundage B, Baksheshi H: Estimation of coronary calcium mass using electron beam computed tomography: A promising approach for predicting coronary events? Circulation 92: I313, 1995 16. Fitzgerald PJ, Ports TA, Yock PG: Contribution of localized calcium deposits to dissection after angioplasty: An observational study using intravascular ultrasound. Circulation 86: 64 70, 1992 17. Marchais SJ, Guerin AP, London GM: Arterial calcinosis, chronic renal failure and calcium antagonism. Drugs 44[Suppl 1]: 119 122, 1992 18. Guerin AP, London GM, Marchais SJ, Metivier F: Arterial stiffening and vascular calcifications in end-stage renal disease. Nephrol Dial Transplant 15: 1014 1021, 2000 19. Olson JC, Edmundowicz D, Becker DJ, Kuller LH, Orchard TJ: Coronary calcium in adults with type 1 diabetes: A stronger correlate of clinical coronary artery disease in men than in women. Diabetes 49: 1571 1578, 2000 20. Lehto S, Niskanen L, Suhonen L, Ronnemaa T, Laakso M: Medial artery calcification. A neglected harbinger of cardiovascular complications in non-insulin-dependent diabetes mellitus. Arterioscler Thromb Vasc Biol 16: 978 983, 1996 21. London GM, Guerin AP, Marchais SJ, Metivier F, Pannier B, Adda H: Arterial media calcification in end-stage renal disease: Impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant 18: 1731 1740, 2003 22. Foley RN, Parfrey PS: Cardiovascular disease and mortality in ESRD. J Nephrol 11: 239 245, 1998 23. Block GA: Prevalence and clinical consequences of elevated Ca P product in hemodialysis patients. Clin Nephrol 54: 318 324, 2000 24. Goodman WG, Goldin J, Kuizon BD, Yoon C, Gales B, Sider D, Wang Y, Chung J, Emerick A, Greaser L, Elashoff RM, Salusky IB: Coronary-artery calcification in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med 342: 1478 1483, 2000 25. Eifinger F, Wahn F, Querfeld U, Pollok M, Gevargez A, Kriener P, Gronemeyer D: Coronary artery calcifications in children and young adults treated with renal replacement therapy. Nephrol Dial Transplant 15: 1892 1894, 2000 26. Oh J, Wunsch R, Turzer M, Bahner M, Raggi P, Querfeld U, Mehls O, Schaefer F: Advanced coronary and carotid arteriopathy in young adults with childhood-onset chronic renal failure. Circulation 106: 100 105, 2002 27. Raggi P, Boulay A, Chasan-Taber S, Amin N, Dillon M, Burke SK, Chertow GM: Cardiac calcification in adult hemodialysis patients. A link between end-stage renal disease and cardiovascular disease? J Am Coll Cardiol 39: 695 701, 2002 28. Speer MY, Giachelli CM: Regulation of cardiovascular calcification. Cardiovasc Pathol 13: 63 70, 2004 29. Luo G DP, McKee MD, Pinero GJ, Loyer E, Behringer RR, and G Karsenty: Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 386(March 6): 78 81, 1997 30. Ketteler M, Bongartz P, Westenfeld R, Wildberger JE, Mahnken AH, Bohm R, Metzger T, Wanner C, Jahnen-Dechent W, Floege J: Association of low fetuin-a (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: A crosssectional study. Lancet 361(9360): 827 833, 2003 31. Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM: Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest 92: 1686 1696, 1993 32. Levy RJ, Schoen FJ, Levy JT, Nelson AC, Howard SL, Oshry LJ: Biologic determinants of dystrophic calcification and osteocalcin deposition in glutaraldehyde-preserved porcine aortic valve leaflets implanted subcutaneously in rats. Am J Pathol 113: 143 155, 1983 33. Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL: Bone morphogenic protein expression in human atherosclerotic lesions. J Clin Invest 91: 1800 1809, 1993 34. Tanimura A, McGregor DH, Anderson HC: Matrix vesicles in atherosclerotic calcification. Proc Soc Exp Biol Med 172: 173 177, 1983 35. Mohler ER 3rd, Gannon F, Reynolds C, Zimmerman R, Keane MG, Kaplan FS: Bone formation and inflammation in cardiac valves. Circulation 103: 1522 1528, 2001 36. Jono S, Nishizawa Y, Shioi A, Morii H: 1,25-Dihydroxyvitamin D3 increases in vitro vascular calcification by modulating secretion of endogenous parathyroid hormone-related peptide. Circulation 98: 1302 1306, 1998 37. Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM: Phosphate regulation of vascular smooth muscle cell calcification. Circ Res 87: E10 E17, 2000 38. Parhami F, Basseri B, Hwang J, Tintut Y, Demer LL: Highdensity lipoprotein regulates calcification of vascular cells. Circ Res 91: 570 576, 2002 39. Tintut Y, Parhami F, Bostrom K, Jackson SM, Demer LL: camp stimulates osteoblast-like differentiation of calcifying vascular cells: Potential signaling pathway for vascular calcification. J Biol Chem 273: 7547 7553, 1998 40. Tintut Y, Patel J, Parhami F, Demer LL: Tumor necrosis factoralpha promotes in vitro calcification of vascular cells via the camp pathway. Circulation 102: 2636 2642, 2000 41. Price PA, Caputo JM, Williamson MK: Bone origin of the serum complex of calcium, phosphate, fetuin, and matrix Gla protein: Biochemical evidence for the cancellous bone-remodeling compartment. J Bone Miner Res 17: 1171 1179, 2002 42. Price PA, Faus SA, Williamson MK: Bisphosphonates alendronate and ibandronate inhibit artery calcification at doses comparable to those that inhibit bone resorption. Arterioscler Thromb Vasc Biol 21: 817 824, 2001 43. Price PA, June HH, Buckley JR, Williamson MK: Osteoprotegerin inhibits artery calcification induced by warfarin and by vitamin D. Arterioscler Thromb Vasc Biol 21: 1610 1616, 2001 44. Proudfoot D, Skepper JN, Hegyi L, Bennett MR, Shanahan CM, Weissberg PL: Apoptosis regulates human vascular calcification in vitro: Evidence for initiation of vascular calcification by apoptotic bodies. Circ Res 87: 1055 1062, 2000 45. Schoen FJ, Tsao JW, Levy RJ: Calcification of bovine pericardium used in cardiac valve bioprostheses. Am J Pathol 123: 134 145, 1986 46. Steitz SA, Speer MY, Curinga G, Yang HY, Haynes P, Aebersold R, Schinke T, Karsenty G, Giachelli CM: Smooth muscle cell phenotypic transition associated with calcification: Upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res 89: 1147 1154, 2001 47. Wada T, McKee MD, Stietz S, Giachelli CM: Calcification of vascular smooth muscle cell cultures: Inhibition by osteopontin. Circ Res 84: 1 6, 1999 48. Chen NX, O Neill KD, Duan D, Moe SM: Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells. Kidney Int 62: 1724 1731, 2002

2964 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 2959 2964, 2004 49. Moe SM, Duan D, Doehle BP, O Neill KD, Chen NX: Uremia induces the osteoblast differentiation factor Cbfa1 in human blood vessels. Kidney Int 63: 1003 1011, 2003 50. Moe SM, O Neill KD, Duan D, Ahmed S, Chen NX, Leapman SB, Fineberg N, Kopecky K: Medial artery calcification in ESRD patients is associated with deposition of bone matrix proteins. Kidney Int 61: 638 647, 2002 51. Yang H, Curinga G, Giachelli CM: Elevated extracellular calcium levels induce smooth muscle cell matrix mineralization in vitro. Kidney Int, 2004, in press 52. Proudfoot D, Skepper JN, McNair R, Johannides A, Weissberg PL, Shanahan CM: MIneral ion-induced release of mineralization-competent matrix vesicles from human vascular smooth muscle cells. Cardiovasc Pathol 13[3 Suppl]: S186, 2004 53. Chertow GM: Slowing the progression of vascular calcification in hemodialysis. J Am Soc Nephrol 14[9 Suppl 4]: S310 S314, 2003 54. Chertow GM, Burke SK, Raggi P: Sevelamer attenuates the progression of coronary and aortic calcification in hemodialysis patients. Kidney Int 62: 245 252, 2002 55. Chertow GM, Raggi P, McCarthy JT, Schulman G, Silberzweig J, Kuhlik A, Goodman WG, Boulay A, Burke SK, Toto RD: The effects of sevelamer and calcium acetate on proxies of atherosclerotic and arteriosclerotic vascular disease in hemodialysis patients. Am J Nephrol 23: 307 314, 2003 56. Cozzolino M, Dusso AS, Liapis H, Finch J, Lu Y, Burke SK, Slatopolsky E: The effects of sevelamer hydrochloride and calcium carbonate on kidney calcification in uremic rats. JAmSoc Nephrol 13: 2299 2308, 2002 57. Motro M, Shemesh J: Calcium channel blocker nifedipine slows down progression of coronary calcification in hypertensive patients compared with diuretics. Hypertension 37: 1410 1413, 2001 58. Essalihi R, Dao HH, Yamaguchi N, Moreau P: A new model of isolated systolic hypertension induced by chronic warfarin and vitamin K(1) treatment. Am J Hypertens 16: 103 110, 2003 59. Dao HH, Essalihi R, Graillon JF, Lariviere R, De Champlain J, Moreau P: Pharmacological prevention and regression of arterial remodeling in a rat model of isolated systolic hypertension. J Hypertens 20: 1597 1606, 2002 60. Schmermund A, Baumgart D, Mohlenkamp S, Kriener P, Pump H, Gronemeyer D, Seibel R, Erbel R: Natural history and topographic pattern of progression of coronary calcification in symptomatic patients: An electron-beam CT study. Arterioscler Thromb Vasc Biol 21: 421 426, 2001