Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition.

Medial artery calcification, which does not accompany lipid or cholesterol deposit, preferentially occurs in elderly population, but its underlying mechanisms remain unclear. In the present study, we investigated the potential role of senescent vascular smooth muscle cells (VSMCs) in the formation of senescence-associated medial calcification. Replicative senescence was induced by the extended passages (until passages 11-13) in human primary VSMCs, and cells in early passage (passage 6) were used as control young cells. VSMC calcification was markedly enhanced in the senescent cells compared with that in the control young cells. We identified that genes highly expressed in osteoblasts, such as alkaline phosphatase (ALP) and type I collagen, were significantly upregulated in the senescent VSMCs, suggesting their osteoblastic transition during the senescence. Knockdown of either ALP or type I collagen significantly reduced the calcification in the senescent VSMCs. Of note, runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates the osteoblastic differentiation, was also upregulated in the senescent VSMCs. Knockdown of RUNX-2 significantly reduced the ALP expression and calcification in the senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of aorta from the klotho(-/-) aging mouse model demonstrated in vivo emergence of osteoblast-like cells expressing RUNX-2 exclusively in the calcified media. We also found that statin and Rho-kinase inhibitor effectively reduced the VSMC calcification by inhibiting P(i)-induced apoptosis and potentially enhancing matrix Gla protein expression in the senescent VSMCs. These findings strongly suggest an important role of senescent VSMCs in the pathophysiology of senescence-associated medial calcification, and the inhibition of osteoblastic transition could be a new therapeutic approach for the prevention of senescence-associated medial calcification.

[1]  Koji Ikeda,et al.  Identification of ARIA regulating endothelial apoptosis and angiogenesis by modulating proteasomal degradation of cIAP-1 and cIAP-2 , 2009, Proceedings of the National Academy of Sciences.

[2]  Yuhong Wang,et al.  Current understanding of klotho , 2009, Ageing Research Reviews.

[3]  J. Kappes,et al.  Oxidative Stress Induces Vascular Calcification through Modulation of the Osteogenic Transcription Factor Runx2 by AKT Signaling* , 2008, Journal of Biological Chemistry.

[4]  Daniel S Berman,et al.  Long-term prognosis associated with coronary calcification: observations from a registry of 25,253 patients. , 2007, Journal of the American College of Cardiology.

[5]  R. DePinho,et al.  Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence , 2007, Aging cell.

[6]  N. Tomita,et al.  Amelioration of progressive renal injury by genetic manipulation of Klotho gene , 2007, Proceedings of the National Academy of Sciences.

[7]  Y. Ouchi,et al.  Gas6/Axl-PI3K/Akt pathway plays a central role in the effect of statins on inorganic phosphate-induced calcification of vascular smooth muscle cells. , 2007, European journal of pharmacology.

[8]  Toshihisa Komori,et al.  Regulation of osteoblast differentiation by transcription factors , 2006, Journal of cellular biochemistry.

[9]  J. Loscalzo,et al.  Vascular Calcification: Pathobiological Mechanisms and Clinical Implications , 2006, Circulation research.

[10]  C. Shanahan,et al.  Molecular mechanisms mediating vascular calcification: Role of matrix Gla protein (Review Article) , 2006, Nephrology.

[11]  N. Chen,et al.  The mechanisms of uremic serum-induced expression of bone matrix proteins in bovine vascular smooth muscle cells. , 2006, Kidney international.

[12]  I. Komuro,et al.  Angiotensin II Induces Premature Senescence of Vascular Smooth Muscle Cells and Accelerates the Development of Atherosclerosis via a p21-Dependent Pathway , 2006, Circulation.

[13]  Y. Ouchi,et al.  Statins Protect Human Aortic Smooth Muscle Cells From Inorganic Phosphate-Induced Calcification by Restoring Gas6-Axl Survival Pathway , 2006, Circulation research.

[14]  S. Achenbach,et al.  Effect of Intensive Versus Standard Lipid-Lowering Treatment With Atorvastatin on the Progression of Calcified Coronary Atherosclerosis Over 12 Months: A Multicenter, Randomized, Double-Blind Trial , 2006, Circulation.

[15]  T. Ogihara,et al.  Anti-apoptotic and anti-senescence effects of Klotho on vascular endothelial cells. , 2006, Biochemical and biophysical research communications.

[16]  J. Liao,et al.  Rho GTPases, statins, and nitric oxide. , 2005, Circulation research.

[17]  J. Hamilton,et al.  Arsenite Regulates Cystic Fibrosis Transmembrane Conductance Regulator and P-glycoprotein: Evidence of Pathway Independence , 2005, Cellular Physiology and Biochemistry.

[18]  K. Hruska,et al.  Bone Morphogenetic Proteins in Vascular Calcification , 2005, Circulation research.

[19]  R. Prescott,et al.  A randomized trial of intensive lipid-lowering therapy in calcific aortic stenosis. , 2005, The New England journal of medicine.

[20]  M. McKee,et al.  Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. , 2005, Genes & development.

[21]  P. Moreau,et al.  Evolution and modulation of age-related medial elastocalcinosis: impact on large artery stiffness and isolated systolic hypertension. , 2005, Cardiovascular research.

[22]  Z. Massy,et al.  Uremia accelerates both atherosclerosis and arterial calcification in apolipoprotein E knockout mice. , 2004, Journal of the American Society of Nephrology : JASN.

[23]  I. Komuro,et al.  Vascular cell senescence and vascular aging. , 2004, Journal of molecular and cellular cardiology.

[24]  I. Komuro,et al.  Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21‐dependent pathway , 2004, The EMBO journal.

[25]  C. Lanctôt,et al.  Osteocrin, a Novel Bone-specific Secreted Protein That Modulates the Osteoblast Phenotype* , 2003, Journal of Biological Chemistry.

[26]  Y. Nabeshima,et al.  Mouse Snail Family Transcription Repressors Regulate Chondrocyte, Extracellular Matrix, Type II Collagen, and Aggrecan* , 2003, Journal of Biological Chemistry.

[27]  E. Mackie.  Osteoblasts: novel roles in orchestration of skeletal architecture. , 2003, The international journal of biochemistry & cell biology.

[28]  D. Rowitch,et al.  Sox9 is required for determination of the chondrogenic cell lineage in the cranial neural crest , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[29]  T. Aigner,et al.  SOX9 expression does not correlate with type II collagen expression in adult articular chondrocytes. , 2003, Matrix biology : journal of the International Society for Matrix Biology.

[30]  P. Weissberg,et al.  Osteo/Chondrocytic Transcription Factors and Their Target Genes Exhibit Distinct Patterns of Expression in Human Arterial Calcification , 2003, Arteriosclerosis, thrombosis, and vascular biology.

[31]  M. Matsuo,et al.  Molecular Identification and Characterization of a Novel Nuclear Protein Whose Expression Is Up-regulated in Insulin-resistant Animals* , 2003, The Journal of Biological Chemistry.

[32]  R. Aebersold,et al.  Smooth Muscle Cell Phenotypic Transition Associated With Calcification: Upregulation of Cbfa1 and Downregulation of Smooth Muscle Lineage Markers , 2001, Circulation research.

[33]  P. Geusens,et al.  Differential Expression of Bone Matrix Regulatory Proteins in Human Atherosclerotic Plaques , 2001, Arteriosclerosis, thrombosis, and vascular biology.

[34]  M. Engelse,et al.  Vascular calcification: expression patterns of the osteoblast-specific gene core binding factor alpha-1 and the protective factor matrix gla protein in human atherogenesis. , 2001, Cardiovascular research.

[35]  J. Skepper,et al.  Apoptosis Regulates Human Vascular Calcification In Vitro: Evidence for Initiation of Vascular Calcification by Apoptotic Bodies , 2000, Circulation research.

[36]  M. Serrano,et al.  Tumor suppressors and oncogenes in cellular senescence☆ , 2000, Experimental Gerontology.

[37]  P. Weissberg,et al.  Medial localization of mineralization-regulating proteins in association with Mönckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification. , 1999, Circulation.

[38]  Luis I. Mendez,et al.  Age-related loss of proliferative activity of human vascular smooth muscle cells in culture , 1999, Mechanisms of Ageing and Development.

[39]  Y. Nishizawa,et al.  Dexamethasone enhances In vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells. , 1999, Arteriosclerosis, thrombosis, and vascular biology.

[40]  T. Callister,et al.  Effect of HMG-CoA reductase inhibitors on coronary artery disease as assessed by electron-beam computed tomography. , 1998, The New England journal of medicine.

[41]  R. Faragher,et al.  How might replicative senescence contribute to human ageing? , 1998, BioEssays : news and reviews in molecular, cellular and developmental biology.

[42]  P. Price,et al.  Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves. , 1998, Arteriosclerosis, thrombosis, and vascular biology.

[43]  R. Naoumova,et al.  The Effect of Cholesterol Reduction with Fluvastatin on Aortic Compliance, Coronary Calcification and Carotid Intimal-Medial Thickness: A Pilot Study , 1998, Journal of cardiovascular risk.

[44]  Tadashi Kaname,et al.  Mutation of the mouse klotho gene leads to a syndrome resembling ageing , 1997, Nature.

[45]  C Roskelley,et al.  A biomarker that identifies senescent human cells in culture and in aging skin in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[46]  M. Takagi,et al.  Inductive Effects of Dexamethasone on the Gene Expression of Cbfa1, Osterix and Bone Matrix Proteins During Differentiation of Cultured Primary Rat Osteoblasts , 2004, Journal of Molecular Histology.

[47]  N. Takahashi,et al.  Bone Morphogenetic Protein-2 Converts the Differentiation Pathway of C2C12 Myoblasts into the Osteoblast Lineage , 2002 .