representative of only those patients whose arteries might calcify in the presence of uremic concentrations of phosphate and not of all sHPT patients. Further the cell passage number and differentiation media used in the studies limit the ability to directly compare these results with other reports that used different cell growth conditions. Although the passage 7 cells, used in accordance with earlier studies of human SMCs [Steitz et al., 2001], responded to phosphate and vitamin D sterols, earlier passages were not assessed for calciﬁcation potential or gene expression proﬁles. Therefore we cannot rule out de-differentiation of the cells during expansion. The current experiments used medium containing AA and Dex (10 9 M), making comparisons with other studies, performed with EM þ BGP BGP alone, difﬁcult due to possible confounding by the effects of these agents on VSMCs. Dexamethasone was used to shorten the mineralization time-line by several days due to its reported enhancement of SMC and human osteoblast mineralization [Cheng et al., 1994; Mori et al., 1999]. However, the inclusion of AA þ Dex could, in part, explain differences between CASMC and bovine aorta SMC results [Shalhoub et al., 2006] due to known effects of Dex on differentiation, migration, proliferation, and gene expression proﬁles in VSMCs [Mori et al., 1999]. Although these differences may also be explained, in part, by species differences or use of different passage numbers, some bovine aorta SMC genes modulated after 7 days treatment [Shalhoub et al., 2006] (MGP, FRZB, IL-6, COLXIA1) were changed in the same direction in this study, albeit some at earlier times (Supplemental Tables I and II). In vivo validation of these in vitro ﬁndings is required for regulated genes not previously reported at sites of vitamin D-induced calciﬁcation. Results presented here provide novel insights regarding mechan- isms that may be involved in regulating vitamin D sterol-mediated calciﬁcation of human CASMC. In CASMCs expressing VDR, vitamin D sterol-enhanced calciﬁcation is associated with altered expression of genes whose promoters contain VDRE. Thus, vitamin D sterols likely inﬂuence transcription in these non-classical vitamin D target cells via genomic pathways with enhancement or suppression of transcription rates reﬂecting recruitment of different co-regulators to the VDR transcriptional complex [Dusso et al., 2005]. However, non-genomic pathways cannot be ruled out and future studies will be required to determine if VDR interacts with the putative VDREs. The effect of vitamin D sterols on gene expression in the absence of added phosphate was not addressed in this study due to the lack of calciﬁcation using these culture conditions in our laboratory (previous unpublished observations). Others have demonstrated that in resting human CASMCs (no added phosphate), vitamin D modulates genes including some identiﬁed in this study (ENPP1, THBD, and THBS1) [Wu-Wong et al., 2007b], but association with calciﬁcation was not examined. Future studies will be focused on determining whether vitamin D sterol regulation of cardiovascular and osteoblastic genes is dependent upon existing mineral disturbances or prior phenotypic switch. In conclusion, we identiﬁed altered gene expression patterns accompanying vitamin D sterol-induced exacerbation of human CASMC calciﬁcation that were representative of multiple pathways implicated in VC. Further studies are required to determine the precise roles of these genes in vitamin D sterol associated VC. ACKNOWLEDGMENTS We thank William Richards, PhD (Amgen, Inc.) (review), William W. Stark, Jr, PhD (Amgen, Inc.) (review and editing), and Jon Nilsen, PhD (Amgen, Inc.) (review and editing). The study and the writing of this manuscript were funded by Amgen, Inc. REFERENCES Alam MU, Kirton JP, Wilkinson FL, Towers E, Sinha S, Rouhi M, Vizard TN, Sage AP, Martin D, Ward DT, Alexander MY, Riccardi D, Canﬁeld AE. 2009. Calciﬁcation is associated with loss of functional calcium-sensing receptor in vascular smooth muscle cells. Cardiovasc Res 81:260–268. Blacher J, Guerin AP, Pannier B, Marchais SJ, London GM. 2001. Arterial calciﬁcations, arterial stiffness, and cardiovascular risk in end-stage renal disease. Hypertension 38:938–942. Block GA, Martin KJ, de Francisco AL, Turner SA, Avram MM, Suranyi MG, Hercz G, Cunningham J, Abu-Alfa AK, Messa P, Coyne DW, Locatelli F, Cohen RM, Evenepoel P, Moe SM, Fournier A, Braun J, McCary LC, Zani VJ, Olson KA, Drueke TB, Goodman WG. 2004. Cinacalcet for secondary hyper- parathyroidism in patients receiving hemodialysis. N Engl J Med 350:1516– 1525. Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, Demer LL. 1993. Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest 91:1800–1809. Cardus A, Panizo S, Parisi E, Fernandez E, Valdivielso JM. 2007. Differential effects of vitamin D analogs on vascular calciﬁcation. J Bone Miner Res 22:860–866. Cheng SL, Yang JW, Rifas L, Zhang SF, Avioli LV. 1994. Differentiation of human bone marrow osteogenic stromal cells in vitro: Induction of the osteoblast phenotype by dexamethasone. Endocrinology 134:277–286. Dusso AS, Brown AJ, Slatopolsky E. 2005. Vitamin D. Am J Physiol Renal Physiol 289:F8–F28. Farzaneh-Far A, Proudfoot D, Weissberg PL, Shanahan CM. 2000. Matrix gla protein is regulated by a mechanism functionally related to the calcium- sensing receptor. Biochem Biophys Res Commun 277:736–740. Favus MJ. 2006. Does denosumab improve low BMD in postmenopausal women? Nat Clin Pract Endocrinol Metab 2:600–601. Goodman WG, Frazao JM, Goodkin DA, Turner SA, Liu W, Coburn JW. 2000a. A calcimimetic agent lowers plasma parathyroid hormone levels in patients with secondary hyperparathyroidism. Kidney Int 58:436–445. Goodman WG, Goldin J, Kuizon BD, Yoon C, Gales B, Sider D, Wang Y, Chung J, Emerick A, Greaser L, Elashoff RM, Salusky IB. 2000b. Coronary-artery calciﬁcation in young adults with end-stage renal disease who are under- going dialysis. N Engl J Med 342:1478–1483. Haffner D, Hocher B, Muller D, Simon K, Konig K, Richter CM, Eggert B, Schwarz J, Godes M, Nissel R, Querfeld U. 2005. Systemic cardiovascular disease in uremic rats induced by 1,25(OH)2D3. J Hypertens 23:1067–1075. Henley C, Colloton M, Cattley RC, Shatzen E, Towler DA, Lacey D, Martin D. 2005. 1,25-Dihydroxyvitamin D3 but not cinacalcet HCl (Sensipar/Mimpara) treatment mediates aortic calciﬁcation in a rat model of secondary hyper- parathyroidism. Nephrol Dial Transplant 20:1370–1377. Ivanovski O, Nikolov IG, Joki N, Caudrillier A, Phan O, Mentaverri R, Maizel J, Hamada Y, Nguyen-Khoa T, Fukagawa M, Kamel S, Lacour B, Drueke TB, Massy ZA. 2009. The calcimimetic R-568 retards uremia-enhanced vascular calciﬁcation and atherosclerosis in apolipoprotein E deﬁcient (apoE-/-) mice. Atherosclerosis 205:55–62. Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM. 2000. Phosphate regulation of vascular smooth muscle cell calciﬁcation. Circ Res 87:E10–E17. 920 VITAMIN D STEROLS AFFECT CORONARY ASMC GENES JOURNAL OF CELLULAR BIOCHEMISTRY