ARTICLE Journal of Cellular Biochemistry 111:911–921 (2010) Journal of Cellular Biochemistry Chondro/Osteoblastic and Cardiovascular Gene Modulation in Human Artery Smooth Muscle Cells That Calcify in the Presence of Phosphate and Calcitriol or Paricalcitol V. Shalhoub,1* E.M. Shatzen,1 S.C. Ward,1 J-I. Young,1 M. Boedigheimer,2 L. Twehues,2 J. McNinch,4 S. Scully,4 B. Twomey,3 D. Baker,3 P. Kiaei,3 M.A. Damore,3 Z. Pan,5 K. Haas,1 and D. Martin1 1Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, California 2Department of Computational Biology, Amgen, Inc., Thousand Oaks, California 3Department of Molecular Sciences, Amgen, Inc., Thousand Oaks, California 4Department of Pathology, Amgen, Inc., Thousand Oaks, California 5Department of Lead Discovery, Amgen, Inc., Thousand Oaks, California ABSTRACT Vitamin D sterol administration, a traditional treatment for secondary hyperparathyroidism, may increase serum calcium and phosphorus, and has been associated with increased vascular calciﬁcation (VC). In vitro studies suggest that in the presence of uremic concentrations of phosphorus, vitamin D sterols regulate gene expression associated with trans-differentiation of smooth muscle cells (SMCs) to a chondro/ osteoblastic cell type. This study examined effects of vitamin D sterols on gene expression proﬁles associated with phosphate-enhanced human coronary artery SMC (CASMC) calciﬁcation. Cultured CASMCs were exposed to phosphate-containing differentiation medium (DM) with and without calcitriol, paricalcitol, or the calcimimetic R-568 (10 11–10 7 M) for 7 days. Calciﬁcation of CASMCs, determined using colorimetry following acid extraction, was dose dependently increased (1.6- to 1.9-fold) by vitamin D sterols þ DM. In contrast, R-568 did not increase calciﬁcation. Microarray analysis demonstrated that, compared with DM, calcitriol (10 8 M) þ DM or paricalcitol (10 8 M) þ DM similarly and signiﬁcantly ( P < 0.05) regulated genes of various pathways including: metabolism, CYP24A1; mineralization, ENPP1; apoptosis, GIP3; osteo/chondrogenesis, OPG, TGFB2, Dkk1, BMP4, BMP6; cardiovascular, HGF, DSP1, TNC; cell cycle, MAPK13; and ion channels, SLC22A3 KCNK3. R-568 had no effect on CASMC gene expression. Thus, SMC calciﬁcation observed in response to vitamin D sterol þ DM may be partially mediated through targeting mineralization, apoptotic, osteo/chondrocytic, and cardiovascular pathway genes, although some gene changes may protect against calciﬁcation. Further studies to determine precise roles of these genes in development of, or protection against VC and cardiovascular disease are required. J. Cell. Biochem. 111: 911–921, 2010. 2010 Wiley-Liss, Inc. KEY WORDS: CARDIOVASCULAR DISEASES; SMOOTH MUSCLE; APOPTOSIS; PHOSPHATE V ascular calciﬁcation (VC) is widespread in chronic kidney disease-mineral and bone disorder (CKD-MBD) patients with secondary hyperparathyroidism (HPT) receiving dialysis [Moe et al., 2007], and has been associated with cardiovascular disease and mortality [Blacher et al., 2001; Raggi et al., 2002]. VC in large and medium sized vessels is a highly regulated, cell mediated, multi- factorial process [Ketteler and Giachelli, 2006] associated with a repertoire of chondro/osseous proteins [Jono et al., 2000; Tyson et al., 2003]. Therapies used to control secondary HPT, including vitamin D sterols and calcimimetics, have varying direct and indirect effects on VC [Wolisi and Moe, 2005]. Although vitamin D sterols lower serum parathyroid hormone (PTH) levels, they have been associated with serum calcium (Ca) and phosphorus (P) elevations in patients receiving dialysis [Tentori et al., 2006]. This increased serum mineral burden can increase the risk for VC and mortality [Kalantar-Zadeh et al., 2006; Moe et al., 2007]. In addition to serum Ca and P mineral imbalance [Goodman et al., 2000b], contributing factors to VC may include dysregulation Additional Supporting Information may be found in the online version of this article. Grant sponsor: Amgen, Inc.. *Correspondence to: V. Shalhoub, MS 15-2-A, One Amgen Center Dr., Thousand Oaks, CA 91320. E-mail: shalhoub@amgen.com Received 6 November 2010; Accepted 13 July 2010 DOI 10.1002/jcb.22779 Published online 27 July 2010 in Wiley Online Library (wileyonlinelibrary.com). 2010 Wiley-Liss, Inc. 911