单纯收缩期高血压动物模型建立及其早期干预的实验研究
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摘要
目的:建立单纯收缩期高血压大鼠模型,研究基质金属蛋白酶抑制剂强力霉素是否能干预单纯收缩期高血压形成,为治疗和延缓单纯收缩期高血压提供新的思路。
方法:选用8周龄wirstar雄性大鼠30只作为研究对象,随机分成三组,强力霉素(dox)干预组(n=10)、单纯造模组(n=10)及空白对照组(n=10)。应用华法林和维生素K(WVK)诱导动脉中层钙化,4周及8周后右侧颈动脉插管进行有创血压和心室内压力的检测,以及取主动脉进行钙含量、免疫组化和形态学检测。强力霉素干预组同时给于强力霉素灌胃,研究强力霉素是否能干预单纯收缩期高血压形成。采用弹性纤维染色法观察组织中弹性纤维形状;Von Kossa染色分析动脉钙化程度;应用免疫组织化学和western-blot检测组织中MMP-9的表达水平;采用原子吸收光谱法测定血管组织中钙含量。
结果:ISH动物造模组大鼠血压与空白对照组相比明显增高(P<0.01),同时也高于强力霉素干预组(P<0.05);空白对照组大鼠血压与强力霉素干预组相比也有统计学差异(P<0.05);而各组间平均心室内压无明显变化。ISH大鼠血压变化的同时伴有动脉形态结构的改变,主动脉和颈动脉中层钙化明显,弹力纤维断裂变直,失去波浪形状;而强力霉素干预组动脉中层钙化程度轻,弹力纤维保持固有形状。ISH动物造模组动脉钙含量明显高于强力霉素干预组(P<0.05)和空白对照组(P<0.01)。western-blot分析ISH动物造模组MMP-9蛋白表达较空白对照组明显提高,同时强力霉素干预组MMP-9活性受到抑制。
结论:明胶酶可促使弹力蛋白降解和钙在弹力纤维薄层的沉积,强力霉素可能通过抑制动脉组织中MMP-9活性而阻止动脉硬化和单纯收缩期高血压的形成。
Objective: To generate an animal model of isolated systolic hypertension in rat and investigate whether doxycycline, a matrix metalloproteinase inhibitor, could interfere in the formation of isolated systolic hypertension so as to provide a new idea for isolated systolic hypertension deference even therapy.
Methods: 8 weeks age-matched male Wistar rats were selected to study, all 30 the rats were randomly divided into intervention group of doxycycline(n=10), pure modeling group(n=10) and blank control group(n=10). Medial elastocalcinosis were induced by warfarin and vitamin K1. 4 weeks and 8 weeks later, blood pressure and left intraventricular pressure were measured by right carotid arterial cannulation; and the segments from each aorta was processed for histological analysis, immunohistochemistry, and Calcilium analysis. Rats in intervention group of doxycycline were done with intragastric administration at the same time to investigate whether doxycycline could interfere in the formation of isolated systolic hypertension. Elatic/collagen ratio and calcification degree were measured according to different coloration methods. MMP-9 was explored by immunohistochemistry and western-bloting. Aortic calcilium contents were calculated in each group with atom-spectrum.
Results: The blood pressures in the pure modeling group were higher than in the intervention group of doxycycline (p<0.05) and blank control group (p<0.01); the blood pressures in the intervention group of doxycycline were also increased compared with the blank control group (p<0.05), but the mean left intraventricular pressure donot show an obviously change in the study group. The blood pressure of ISH animal changed with morphological aspect of each aorta, which was associated with extensive arterial medial elastocalcinosis, and the elastic lamellae was flatten, accompanied loss of the natural waviness. The degree of Medial elastocalcinosis was decreased in the intervention group of doxycycline, which followed the natural wavy aspect of the elastic lamellae. calcilium contents were increased in the pure modeling group compared with the intervention group of doxycycline (p<0.05)and the blank control group (p<0.01). Western-bloting analysis showed that protein synthesis of MMP-9 in the pure modeling group increased obviously as compared with that of the blank control group while MMP-9 activity was inhibited in the intervention group of doxycycline.
Conclusion: The presence of MMP-9 is required for elastin degeneration and calcification in the aortic elastic fibers, and doxycycline may prevent the formation of aortic stiffening and isolated systolic hypertension by inhibiting MMP-9 activity in vessel.
方法:选用8周龄wirstar雄性大鼠30只作为研究对象,随机分成三组,强力霉素(dox)干预组(n=10)、单纯造模组(n=10)及空白对照组(n=10)。应用华法林和维生素K(WVK)诱导动脉中层钙化,4周及8周后右侧颈动脉插管进行有创血压和心室内压力的检测,以及取主动脉进行钙含量、免疫组化和形态学检测。强力霉素干预组同时给于强力霉素灌胃,研究强力霉素是否能干预单纯收缩期高血压形成。采用弹性纤维染色法观察组织中弹性纤维形状;Von Kossa染色分析动脉钙化程度;应用免疫组织化学和western-blot检测组织中MMP-9的表达水平;采用原子吸收光谱法测定血管组织中钙含量。
结果:ISH动物造模组大鼠血压与空白对照组相比明显增高(P<0.01),同时也高于强力霉素干预组(P<0.05);空白对照组大鼠血压与强力霉素干预组相比也有统计学差异(P<0.05);而各组间平均心室内压无明显变化。ISH大鼠血压变化的同时伴有动脉形态结构的改变,主动脉和颈动脉中层钙化明显,弹力纤维断裂变直,失去波浪形状;而强力霉素干预组动脉中层钙化程度轻,弹力纤维保持固有形状。ISH动物造模组动脉钙含量明显高于强力霉素干预组(P<0.05)和空白对照组(P<0.01)。western-blot分析ISH动物造模组MMP-9蛋白表达较空白对照组明显提高,同时强力霉素干预组MMP-9活性受到抑制。
结论:明胶酶可促使弹力蛋白降解和钙在弹力纤维薄层的沉积,强力霉素可能通过抑制动脉组织中MMP-9活性而阻止动脉硬化和单纯收缩期高血压的形成。
Objective: To generate an animal model of isolated systolic hypertension in rat and investigate whether doxycycline, a matrix metalloproteinase inhibitor, could interfere in the formation of isolated systolic hypertension so as to provide a new idea for isolated systolic hypertension deference even therapy.
Methods: 8 weeks age-matched male Wistar rats were selected to study, all 30 the rats were randomly divided into intervention group of doxycycline(n=10), pure modeling group(n=10) and blank control group(n=10). Medial elastocalcinosis were induced by warfarin and vitamin K1. 4 weeks and 8 weeks later, blood pressure and left intraventricular pressure were measured by right carotid arterial cannulation; and the segments from each aorta was processed for histological analysis, immunohistochemistry, and Calcilium analysis. Rats in intervention group of doxycycline were done with intragastric administration at the same time to investigate whether doxycycline could interfere in the formation of isolated systolic hypertension. Elatic/collagen ratio and calcification degree were measured according to different coloration methods. MMP-9 was explored by immunohistochemistry and western-bloting. Aortic calcilium contents were calculated in each group with atom-spectrum.
Results: The blood pressures in the pure modeling group were higher than in the intervention group of doxycycline (p<0.05) and blank control group (p<0.01); the blood pressures in the intervention group of doxycycline were also increased compared with the blank control group (p<0.05), but the mean left intraventricular pressure donot show an obviously change in the study group. The blood pressure of ISH animal changed with morphological aspect of each aorta, which was associated with extensive arterial medial elastocalcinosis, and the elastic lamellae was flatten, accompanied loss of the natural waviness. The degree of Medial elastocalcinosis was decreased in the intervention group of doxycycline, which followed the natural wavy aspect of the elastic lamellae. calcilium contents were increased in the pure modeling group compared with the intervention group of doxycycline (p<0.05)and the blank control group (p<0.01). Western-bloting analysis showed that protein synthesis of MMP-9 in the pure modeling group increased obviously as compared with that of the blank control group while MMP-9 activity was inhibited in the intervention group of doxycycline.
Conclusion: The presence of MMP-9 is required for elastin degeneration and calcification in the aortic elastic fibers, and doxycycline may prevent the formation of aortic stiffening and isolated systolic hypertension by inhibiting MMP-9 activity in vessel.
引文
1. Franklin SS, Khan SA, Wong ND, et al. Is pulse pressure useful in predicting risk for coronary heart disease? Circulation, 1999, 100: 354-360.
2. Dao HH, Essalihi R, Moreau P, et al. Evolution and modulation of Age-related medial elastocalcinosis: Impact on large artery stiffness and isolated systolic hypertension. Cardiovas Res, 2005,66:307-317.
3. Elliott RJ, McGrath LT. Calcification of the human thoracic aorta during aging. Calcif Tissue Int, 1994, 54: 268–73.
4. Davies MR, Hruska KA. Pathophysiological mechanisms of vascular calcification in end-stage renal disease. Kidney Int, 2001, 60: 472-479.
5. Gillessen T, Gillessen F, Sieberth H,et al. Age-related changes in the elastic properties of the aortic tree in normotensive properties of the aortic tree in normotensive patients: Investigation by intravascular ultrasound. Eur J Med Res, 1995, 1: 144–148.
6. Guerin AP, London GM, Marchais SJ. Arterial stiffening and vascular calcifications in end-stage renal disease. Nephrol Dial Transphlant, 2000, 15: 1014–1021.
7. London GM, Guerin AP, Marchais SJ. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant, 2003,18: 1731-1740
8. Shao JS, Cai J, Towler DA. Molecular Mechanisms of Vascular Calcification: Lessons Learned From Aorta. Arterioscler Thromb Vasc Biol, 2006, 26: 1423-1430.
9. Shanahan CM. Vascular calcification: a matter of damage limitation? Nephrol Dial Transphlant, 2006, 21: 1166-1169.
10. Price PA, Faus SA, Williamson MK, et al. Warfarin-Induced artery calcification is accelerated by growth and vitamin D. Arterioscler Thromb Vasc Biol, 2000, 20:317-327.
11. Price PA, Faus SA, Williamson MK, et al. Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves. Arterioscler Thromb Vasc Biol, 1998, 18:1400-1407.
12. Luo G, Ducy P, McKee MD et al. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature, 1997, 386:78–81.
13. Essalihi R, Dao HH, Yamaguchi N, et al. A new model of isolated systolic hypertension induced by chronic warfarin and vitamin k1 treatment. Am J Hypertens, 2003, 16:103- 110.
14. Safar ME. Hypothesis on isolated systolic hypertension in the elderly. J Hum Hypertens, 1999, 13:813-815.
15. Dhore CR, Cleutjens JP, Lutgens E, et al. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol, 2001, 21:1998 –2003.
16. Giachelli CM. Vascular Calcification Mechanisms. J Am Soc Nephrol, 2004, 15: 2959-2964.
17. Vattikuti R, Towler DA. Osteogenic regulation of vascular calcification: an early perspective .Am J Physiol Endocrinol Metab, 2004, 286: E686-E696.
18. Golub LM, Lee HM, RyanME et al. Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms. Adv Dent Res, 1998, 12 : 12-26.
19. Qin X, Corriere MA, Matrisian LM, et al. Matrix metalloproteinase inhibition attenuates aortic calcification. Arterioscler Thromb Vasc Biol, 2006, 26: 1510-1516.
20. Basalyga DM, Simionescu DT, Xiong w, et al. Elastin degradation and calcification in an abdominal aorta injury model :role of matrix metalloproteinases. Circulation, 2004, 110:3480-3487.
21. Bailey M, Xiao H, Ogle M, et al. Aluminum chloride pretreatment of elastin inhibits elastolysis by matrix metalloproteinases and leads to inhibition of elastin-oriented calcification. Am J Pathol, 2001, 159:1981-1986.
22. Sato N, Maehara N, Su GH, et al. Effects of 5-Aza-2′-deoxycytidine on matrix metalloproteinase expression and pancreatic cancer cell invasiveness. Natl Cancer Inst, 2003, 95: 27 -330.
23. Bendeck MP, Irvin C, Reidy M, et al. Smooth muscle cell matrix metalloproteinase production is stimulated via αvβ3 integrin. Arterioscler Thromb Vasc Biol, 2000, 20: 1467-1474.
24. Steitz SA, Speer MY, Mckee MD, et al. Osteopontin inhibits minera deposition and promotes regression of ectopic calcification. Am J Pathol, 2002, 161: 2035-2046.
1. Franklin SS, Khan SA, Wong ND, et al. Is pulse pressure useful in predicting risk for coronary heart disease? Circulation, 1999, 100: 354-360.
2. Dao HH, Essalihi R, Moreau P, et al. Evolution and modulation of Age-related medial elastocalcinosis: Impact on large artery stiffness and isolated systolic hypertension. Cardiovas Res, 2005,66:307-317.
3. Elliott RJ, McGrath LT. Calcification of the human thoracic aorta during aging. Calcif Tissue Int, 1994, 54: 268–73.
4. Davies MR, Hruska KA. Pathophysiological mechanisms of vascular calcification in end-stage renal disease. Kidney Int, 2001,60:472-479.
5. Essalihi R, Dao HH, Yamaguchi N, et al. A new model of isolated systolic hypertension induced by chronic warfarin and vitamin k1 treatment. Am J Hypertens, 2003, 16:103-110.
6. Guerin AP, London GM, Marchais SJ. Arterial stiffening and vascular calcifications in end-stage renal disease. Nephrol Dial Transphlant, 2000, 15: 1014–1021.
7. London GM, Guerin AP, Marchais SJ. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant, 2003,18: 1731-1740
8. Niederhoffer N, Lartaud-Idjouadiene I, Atkinson J. Calcification of medial elastic fibers and aortic elasticity. Hypertension, 1997; 29: 999– 1006.
9. Shao JS, Cai J, Towler DA. Molecular Mechanisms of Vascular Calcification: Lessons Learned From Aorta. Arterioscler Thromb Vasc Biol, 2006,26:1423-1430.
10. Shanahan CM. Vascular calcification: a matter of damage limitation? Nephrol Dial Transphlant, 2006, 21: 1166-1169.
11. Iyemere VP, Proudfoot D, Weissberg PL, Vascular smooth muscle cell phenotypic plasticity and the regulation of vascular calcification. J Intern Med, 2006, 260: 192–210.
12. Vattikuti R, Towler DA. Osteogenic regulation of vascular calcification: an early perspective .Am J Physiol Endocrinol Metab, 2004, 286: E686-E696.
13. Giachelli CM, Speer MY, Li X, et al. Regulation of vascular calcification: Roles of phosphate and osteopontin. Circ Res, 2005, 96: 717-722.
14. Dhore CR, Cleutjens JP, Lutgens E, et al. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol, 2001, 21:1998 –2003.
15. Giachelli CM. Vascular Calcification Mechanisms. J Am Soc Nephrol, 2004, 15: 2959-2964.
16. Price PA, Faus SA, Williamson MK, et al. Warfarin-Induced artery calcification is accelerated by growth and vitamin D. Arterioscler Thromb Vasc Biol, 2000, 20:317-327.
17. Basalyga DM, Simionescu DT, Xiong w, et al. Elastin degradation and calcification in an abdominal aorta injury model :role of matrix metalloproteinases. Circulation, 2004, 110:3480-3487.
18. Price PA, Faus SA, Williamson MK, et al. Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves. Arterioscler Thromb Vasc Biol, 1998, 18:1400-1407.
19. Gericke A, Qin C, Spevak L, et al. Importance of phosphorylation for osteopontin regulation of biomineraliztioan. Calcif Tissue Int, 2005, 77: 45-54.
20. Bendeck MP, Irvin C, Reidy M, et al. Smooth muscle cell matrix metalloproteinase production is stimulated via αvβ3 integrin. Arterioscler Thromb Vasc Biol, 2000, 20: 1467-1474.
21. Qin X, Corriere MA, Matrisian LM, et al. Matrix metalloproteinase inhibition attenuates aortic calcification. Arterioscler Thromb Vasc Biol, 2006, 26: 1510-1516.
2. Dao HH, Essalihi R, Moreau P, et al. Evolution and modulation of Age-related medial elastocalcinosis: Impact on large artery stiffness and isolated systolic hypertension. Cardiovas Res, 2005,66:307-317.
3. Elliott RJ, McGrath LT. Calcification of the human thoracic aorta during aging. Calcif Tissue Int, 1994, 54: 268–73.
4. Davies MR, Hruska KA. Pathophysiological mechanisms of vascular calcification in end-stage renal disease. Kidney Int, 2001, 60: 472-479.
5. Gillessen T, Gillessen F, Sieberth H,et al. Age-related changes in the elastic properties of the aortic tree in normotensive properties of the aortic tree in normotensive patients: Investigation by intravascular ultrasound. Eur J Med Res, 1995, 1: 144–148.
6. Guerin AP, London GM, Marchais SJ. Arterial stiffening and vascular calcifications in end-stage renal disease. Nephrol Dial Transphlant, 2000, 15: 1014–1021.
7. London GM, Guerin AP, Marchais SJ. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant, 2003,18: 1731-1740
8. Shao JS, Cai J, Towler DA. Molecular Mechanisms of Vascular Calcification: Lessons Learned From Aorta. Arterioscler Thromb Vasc Biol, 2006, 26: 1423-1430.
9. Shanahan CM. Vascular calcification: a matter of damage limitation? Nephrol Dial Transphlant, 2006, 21: 1166-1169.
10. Price PA, Faus SA, Williamson MK, et al. Warfarin-Induced artery calcification is accelerated by growth and vitamin D. Arterioscler Thromb Vasc Biol, 2000, 20:317-327.
11. Price PA, Faus SA, Williamson MK, et al. Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves. Arterioscler Thromb Vasc Biol, 1998, 18:1400-1407.
12. Luo G, Ducy P, McKee MD et al. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature, 1997, 386:78–81.
13. Essalihi R, Dao HH, Yamaguchi N, et al. A new model of isolated systolic hypertension induced by chronic warfarin and vitamin k1 treatment. Am J Hypertens, 2003, 16:103- 110.
14. Safar ME. Hypothesis on isolated systolic hypertension in the elderly. J Hum Hypertens, 1999, 13:813-815.
15. Dhore CR, Cleutjens JP, Lutgens E, et al. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol, 2001, 21:1998 –2003.
16. Giachelli CM. Vascular Calcification Mechanisms. J Am Soc Nephrol, 2004, 15: 2959-2964.
17. Vattikuti R, Towler DA. Osteogenic regulation of vascular calcification: an early perspective .Am J Physiol Endocrinol Metab, 2004, 286: E686-E696.
18. Golub LM, Lee HM, RyanME et al. Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms. Adv Dent Res, 1998, 12 : 12-26.
19. Qin X, Corriere MA, Matrisian LM, et al. Matrix metalloproteinase inhibition attenuates aortic calcification. Arterioscler Thromb Vasc Biol, 2006, 26: 1510-1516.
20. Basalyga DM, Simionescu DT, Xiong w, et al. Elastin degradation and calcification in an abdominal aorta injury model :role of matrix metalloproteinases. Circulation, 2004, 110:3480-3487.
21. Bailey M, Xiao H, Ogle M, et al. Aluminum chloride pretreatment of elastin inhibits elastolysis by matrix metalloproteinases and leads to inhibition of elastin-oriented calcification. Am J Pathol, 2001, 159:1981-1986.
22. Sato N, Maehara N, Su GH, et al. Effects of 5-Aza-2′-deoxycytidine on matrix metalloproteinase expression and pancreatic cancer cell invasiveness. Natl Cancer Inst, 2003, 95: 27 -330.
23. Bendeck MP, Irvin C, Reidy M, et al. Smooth muscle cell matrix metalloproteinase production is stimulated via αvβ3 integrin. Arterioscler Thromb Vasc Biol, 2000, 20: 1467-1474.
24. Steitz SA, Speer MY, Mckee MD, et al. Osteopontin inhibits minera deposition and promotes regression of ectopic calcification. Am J Pathol, 2002, 161: 2035-2046.
1. Franklin SS, Khan SA, Wong ND, et al. Is pulse pressure useful in predicting risk for coronary heart disease? Circulation, 1999, 100: 354-360.
2. Dao HH, Essalihi R, Moreau P, et al. Evolution and modulation of Age-related medial elastocalcinosis: Impact on large artery stiffness and isolated systolic hypertension. Cardiovas Res, 2005,66:307-317.
3. Elliott RJ, McGrath LT. Calcification of the human thoracic aorta during aging. Calcif Tissue Int, 1994, 54: 268–73.
4. Davies MR, Hruska KA. Pathophysiological mechanisms of vascular calcification in end-stage renal disease. Kidney Int, 2001,60:472-479.
5. Essalihi R, Dao HH, Yamaguchi N, et al. A new model of isolated systolic hypertension induced by chronic warfarin and vitamin k1 treatment. Am J Hypertens, 2003, 16:103-110.
6. Guerin AP, London GM, Marchais SJ. Arterial stiffening and vascular calcifications in end-stage renal disease. Nephrol Dial Transphlant, 2000, 15: 1014–1021.
7. London GM, Guerin AP, Marchais SJ. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant, 2003,18: 1731-1740
8. Niederhoffer N, Lartaud-Idjouadiene I, Atkinson J. Calcification of medial elastic fibers and aortic elasticity. Hypertension, 1997; 29: 999– 1006.
9. Shao JS, Cai J, Towler DA. Molecular Mechanisms of Vascular Calcification: Lessons Learned From Aorta. Arterioscler Thromb Vasc Biol, 2006,26:1423-1430.
10. Shanahan CM. Vascular calcification: a matter of damage limitation? Nephrol Dial Transphlant, 2006, 21: 1166-1169.
11. Iyemere VP, Proudfoot D, Weissberg PL, Vascular smooth muscle cell phenotypic plasticity and the regulation of vascular calcification. J Intern Med, 2006, 260: 192–210.
12. Vattikuti R, Towler DA. Osteogenic regulation of vascular calcification: an early perspective .Am J Physiol Endocrinol Metab, 2004, 286: E686-E696.
13. Giachelli CM, Speer MY, Li X, et al. Regulation of vascular calcification: Roles of phosphate and osteopontin. Circ Res, 2005, 96: 717-722.
14. Dhore CR, Cleutjens JP, Lutgens E, et al. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques. Arterioscler Thromb Vasc Biol, 2001, 21:1998 –2003.
15. Giachelli CM. Vascular Calcification Mechanisms. J Am Soc Nephrol, 2004, 15: 2959-2964.
16. Price PA, Faus SA, Williamson MK, et al. Warfarin-Induced artery calcification is accelerated by growth and vitamin D. Arterioscler Thromb Vasc Biol, 2000, 20:317-327.
17. Basalyga DM, Simionescu DT, Xiong w, et al. Elastin degradation and calcification in an abdominal aorta injury model :role of matrix metalloproteinases. Circulation, 2004, 110:3480-3487.
18. Price PA, Faus SA, Williamson MK, et al. Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves. Arterioscler Thromb Vasc Biol, 1998, 18:1400-1407.
19. Gericke A, Qin C, Spevak L, et al. Importance of phosphorylation for osteopontin regulation of biomineraliztioan. Calcif Tissue Int, 2005, 77: 45-54.
20. Bendeck MP, Irvin C, Reidy M, et al. Smooth muscle cell matrix metalloproteinase production is stimulated via αvβ3 integrin. Arterioscler Thromb Vasc Biol, 2000, 20: 1467-1474.
21. Qin X, Corriere MA, Matrisian LM, et al. Matrix metalloproteinase inhibition attenuates aortic calcification. Arterioscler Thromb Vasc Biol, 2006, 26: 1510-1516.