Calcified Tissue International

, Volume 79, Issue 5, pp 326–339 | Cite as

Hypercholesterolemia Accelerates Vascular Calcification Induced by Excessive Vitamin D via Oxidative Stress

  • F. T. Tang
  • S. R. Chen
  • X. Q. Wu
  • T. Q. Wang
  • J. W. Chen
  • J. Li
  • L. P. Bao
  • H. Q. Huang
  • P. Q. LiuEmail author


Hypercholesterolemia plays an important role in the initiation and progression of atherosclerosis and has a positive correlation with cardiovascular disease. Calcification is a common feature of atherosclerotic lesions and contributes to cardiovascular dysfunctions. The present study investigated the role of hypercholesterolemia in vascular calcification and its potential mechanism. Models of vascular calcification were established by administering vitamin D2 (VD) to rats alone or combined with a high-cholesterol diet (HCD) and by treating rat aorta smooth muscle cells (RASMCs) with β-glycerophosphate (GP) alone or combined with oxidized low-density lipoprotein (oxLDL) in vitro. In rats, the combination of VD with HCD significantly enhanced vessel calcium deposition and the activity and mRNA expression of vessel alkaline phosphatase (ALP) compared to treatment with VD alone. This combination also enhanced serum levels of total cholesterol, oxLDL, and malondialdehyde as well as vascular production of superoxide anion, while it reduced the vascular activity of superoxide dismutase. Both simvastatin, a cholesterol-lowering agent, and antioxidant vitamin E antagonized the effects of the above combination. In RASMCs, oxLDL accumulation dependently accelerated calcium deposition in cell layers initiated by GP alone. Also, oxLDL stimulated ALP activity and mRNA expression in RASMCs in a concentration-dependent manner. Taken together, these results suggest that acceleration of vascular calcification by hypercholesterolemia might be attributed to oxidative stress and such calcification may be another target of statin or antioxidant action in antiatherosclerosis.


Hypercholesterolemia Vascular calcification model Oxidized low-density lipoprotein Simvastatin Vitamin E 



This work was supported by the Major Program in Key Field of People’s Government of Guangdong Province, PR China (2003A30904); Key Program of the Ministry of Education, PR China (104146); Key Natural Science Fund of Guangdong Province, PR China (04105349); Key Program of Guangdong Province, PR China (2003B31713); and Key Program of Guangzhou City, PR China (2003Z1-E5011). We thank Ms. Liu Jie and Mr. Mo Mingcong for excellent technical support. We thank Professor Tao Liang for critical reading of the manuscript.


  1. 1.
    Proudfoot D, Davies JD, Skepper JN, Weissberg PL, Shanahan CM (2002) Acetylated low-density lipoprotein stimulates human vascular smooth muscle cell calcification by promoting osteoblastic differentiation and inhibiting phagocytosis. Circulation 106:3044–3050PubMedCrossRefGoogle Scholar
  2. 2.
    Bostrom K (2001) Insights into the mechanism of vascular calcification. Am J Cardiol 88:20E–22EPubMedCrossRefGoogle Scholar
  3. 3.
    Wallin R, Wajih N, Greenwood GT, Sane DC (2001) Arterial calcification: a review of mechanisms, animal models, and the prospects for therapy. Med Res Rev 21:274–301PubMedCrossRefGoogle Scholar
  4. 4.
    Laroche M, Delmotte A (2005) Increased arterial calcification in Paget’s disease of bone. Calcif Tissue Int 77:129–133PubMedCrossRefGoogle Scholar
  5. 5.
    Hsu HH (2003) In vitro effect of cholesterol on calcifying activity of vesicles isolated from rabbit aortas. Biochim Biophys Acta 1638:235–240PubMedGoogle Scholar
  6. 6.
    Kramsch DM, Chan CT (1978) The effect of agents interfering with soft tissue calcification and cell proliferation on calcific fibrous-fatty plaques in rabbits. Circ Res 42:562–571PubMedGoogle Scholar
  7. 7.
    Sarig S, Weiss TA, Katz I, Kahana F, Azoury R, Okon E, Kruth HS (1994) Detection of cholesterol associated with calcium mineral using confocal fluorescence microscopy. Lab Invest 71:782–787PubMedGoogle Scholar
  8. 8.
    Hsu HH, Camacho NC, Tawfik O, Sun F (2002) Induction of calcification in rabbit aortas by high cholesterol diets: roles of calcifiable vesicles in dystrophic calcification. Atherosclerosis 161:85–94PubMedCrossRefGoogle Scholar
  9. 9.
    Rattazzi M, Bennett BJ, Bea F, Kirk EA, Ricks JL, Speer M, Schwartz SM, Giachelli CM, Rosenfeld ME (2005) Calcification of advanced atherosclerotic lesions in the innominate arteries of ApoE-deficient mice: potential role of chondrocyte-like cells. Arterioscler Thromb Vasc Biol 25:1420–1425PubMedCrossRefGoogle Scholar
  10. 10.
    O’Brien KD, Reichenbach DD, Marcovina SM, Kuusisto J, Alpers CE, Otto CM (1996) Apolipoproteins B, (a), and E accumulate in the morphologically early lesion of “degenerative” valvular aortic stenosis. Arterioscler Thromb Vasc Biol 16:523–532PubMedGoogle Scholar
  11. 11.
    Parhami F, Morrow AD, Balucan J, Leitinger N, Watson AD, Tintut Y, Berliner JA, Demer LL (1997) Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler Thromb Vasc Biol 17:680–687PubMedGoogle Scholar
  12. 12.
    Mody N, Parhami F, Sarafian TA, Demer LL (2001) Oxidative stress modulates osteoblastic differentiation of vascular and bone cells. Free Radic Biol Med 31:509–519PubMedCrossRefGoogle Scholar
  13. 13.
    Kitagawa S, Yamaguchi Y, Kunitomo M, Imaizumi N, Fujiwara M (1992) Impairment of endothelium-dependent relaxation in aorta from rats with arteriosclerosis induced by excess vitamin D and high-cholesterol diet. Jpn J Pharmacol 59:339–347PubMedGoogle Scholar
  14. 14.
    Yagi K (1984) Assay for blood plasma or serum. Methods Enzymol 105:328–331PubMedGoogle Scholar
  15. 15.
    Szocs K, Lassegue B, Sorescu D, Hilenski LL, Valppu L, Couse TL, Wilcox JN, Quinn MT, Lambeth JD, Griendling KK (2002) Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury. Arterioscler Thromb Vasc Biol 22:21–27PubMedCrossRefGoogle Scholar
  16. 16.
    Oyanatui Y (1984) Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. Anal Biochem 142:290–296CrossRefGoogle Scholar
  17. 17.
    Parhami F, Morrow AD, Balucan J, Leitinger N, Watson AD, Tintut Y, Berliner JA, Demer LL (1997) Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler Thromb Vasc Biol 17:680–687PubMedGoogle Scholar
  18. 18.
    Ross R (1971) The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol 50:172–186PubMedCrossRefGoogle Scholar
  19. 19.
    Wada T, McKee MD, Steitz S, Giachelli CM (1999) Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin. Circ Res 84:166–178PubMedGoogle Scholar
  20. 20.
    Jono S, Nishizawa Y, Shioi A, Morii H (1997) Parathyroid hormone-related peptide as a local regulator of vascular calcification. Its inhibitory action on in vitro calcification by bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 17:1135–1142PubMedGoogle Scholar
  21. 21.
    Demer LL, Tintut Y (2003) Mineral exploration: search for the mechanism of vascular calcification and beyond. The 2003 Jeffrey M. Hoeg Award lecture. Arterioscler Thromb Vasc Biol 23:1739–1743PubMedCrossRefGoogle Scholar
  22. 22.
    Parhami F, Basseri B, Hwang J, Tintut Y, Demer LL (2002) High-density lipoprotein regulates calcification of vascular cells. Circ Res 91:570–576PubMedCrossRefGoogle Scholar
  23. 23.
    Meisinger C, Baumert J, Khuseyinova N, Loewel H, Koenig W (2005) Plasma oxidized low-density lipoprotein, a strong predictor for acute coronary heart disease events in apparently healthy, middle-aged men from the general population. Circulation 112:651–657PubMedCrossRefGoogle Scholar
  24. 24.
    Steinberg D, Lewis A (1997) Conner Memorial Lecture. Oxidative modification of LDL and atherogenesis. Circulation 95:1062–1071PubMedGoogle Scholar
  25. 25.
    Berliner JA, Navab M, Fogelman AM, Frank JS, Demer LL, Edwards PA, Watson AD, Lusis AJ (1995) Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation 91:2488–2496PubMedGoogle Scholar
  26. 26.
    Garabedian M, Tanaka Y, Holick MF, Deluca HF (1974) Response of intestinal calcium transport and bone calcium mobilization to 1,25-dihydroxyvitamin D3 in thyroparathyroidectomized rats. Endocrinology 94:1022–1027PubMedCrossRefGoogle Scholar
  27. 27.
    Krohn K, Haffner D, Hügel U, Himmele R, Klaus G, Mehls O, Schaefer1 F (2003) 1,25(OH)2D3 and dihydrotestosterone interact to regulate proliferation and differentiation of epiphyseal chondrocyte. Calcif Tissue Int 73:400–410PubMedCrossRefGoogle Scholar
  28. 28.
    Hass GM, Trueheart RE, Hemmens A (1960) Experimental arteriosclerosis due to hypervitaminosis D. Am J Pathol 37:521–549PubMedGoogle Scholar
  29. 29.
    Porta R, Conz A, Conto A, Pescador R, Mantovani M, Ferro L (1994) Comparable beneficial effects of defibrotide and nifedipine in calcium induced atherosclerosis. Life Sci 54:799–812PubMedCrossRefGoogle Scholar
  30. 30.
    Parhami F, Demer LL (1997) Arterial calcification in face of osteoporosis in ageing: can we blame oxidized lipids? Curr Opin Lipidol 8:312–314PubMedCrossRefGoogle Scholar
  31. 31.
    Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G (1997) Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 386:78–81PubMedCrossRefGoogle Scholar
  32. 32.
    Shearer MJ (1995) Vitamin K. Lancet 345:229–234PubMedCrossRefGoogle Scholar
  33. 33.
    Fang X, Weintraub NL, Rios CD, Chappell DA, Zwacka RM, Engelhardt JF, Oberley LW, Yan T, Heistad DD, Spector AA (1998) Overexpression of human superoxide dismutase inhibits oxidation of low-density lipoprotein by endothelial cells. Circ Res 82:1289–1297PubMedGoogle Scholar
  34. 34.
    Heinecke JW, Baker L, Rosen H, Chait A (1986) Superoxide-mediated modification of low density lipoprotein by arterial smooth muscle cells. J Clin Invest 77:757–761PubMedCrossRefGoogle Scholar
  35. 35.
    Steinbrecher UP (1988) Role of superoxide in endothelial-cell modification of low-density lipoproteins. Biochim Biophys Acta 959:20–30PubMedGoogle Scholar
  36. 36.
    Guo Z, Van Remmen H, Yang H, Chen X, Mele J, Vijg J, Epstein CJ, Ho YS, Richardson A (2001) Changes in expression of antioxidant enzymes affect cell-mediated LDL oxidation and oxidized LDL-induced apoptosis in mouse aortic cells. Arterioscler Thromb Vasc Biol 21:113Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • F. T. Tang
    • 1
  • S. R. Chen
    • 1
  • X. Q. Wu
    • 1
  • T. Q. Wang
    • 1
  • J. W. Chen
    • 1
  • J. Li
    • 1
  • L. P. Bao
    • 1
  • H. Q. Huang
    • 1
  • P. Q. Liu
    • 1
    Email author
  1. 1.Laboratory of Pharmacology and Toxicology, School of Pharmaceutical SciencesSun Yat-sen UniversityGuangzhouPeople’s Republic of China

Personalised recommendations