Pathophysiology of vascular calcification: Pivotal role of cellular senescence in vascular smooth muscle cells

Experimental Gerontology 45 (2010) 819–824

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Experimental Gerontology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / ex p g e r o

Review

Pathophysiology of vascular calcification: Pivotal role of cellular senescence in vascular smooth muscle cells D.G.A. Burton a,⁎, H. Matsubara b, K. Ikeda b a b

Division of Gerontology and Geriatric Medicine, Department of Medicine, University of Miami, Miami, FL 33125, USA Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto, Japan

a r t i c l e

i n f o

Article history: Received 18 May 2010 Received in revised form 10 July 2010 Accepted 14 July 2010 Available online 18 July 2010 Section Editor: O. Pereira-Smith Keywords: Cellular senescence Vascular calcification Vascular smooth muscle Ageing RUNX-2 SASP CESP

a b s t r a c t The accumulation of senescent cells within tissues can potentially lead to biological dysfunction and manifestation of disease associated with ageing. The majority of senescent cells display a commonly altered secretome similar to a wound healing response (termed the senescence-associated secretory phenotype or SASP), which could have deleterious implications on the tissue microenvironment. However, senescent cells also appear to have a cell-type (or even cell-strain) exclusive senescent phenotype (CESP), an area of research that is underexplored. One such CESP is the pro-calcificatory phenotype recently reported in senescent vascular smooth muscle cells (VSMCs). Senescent VSMCs have been shown to overexpress genes and proteins (including RUNX-2, alkaline phosphatase (ALP), type I collagen and BMP-2) associated with osteoblasts, leading to partial osteoblastic transdifferentiation. As such, it has been suggested that senescent VSMCs contribute to cardiovascular dysfunction through induction of vascular calcification. This review discusses recent findings on VSMC senescence and their potential role in the pathophysiology of vascular calcification. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Senescent vascular smooth muscle cells (VSMCs) have recently been shown to exhibit a pro-calcificatory/osteoblastic phenotype (Burton et al., 2009; Nakano-Kurimoto et al., 2009). Microarray analysis of senescent VSMC shows a differential regulation of genes associated with vascular calcification, including matrix Gla protein (MGP), bone morphogenetic protein-2 (BMP-2), osteoprotegerin (OPG), osteopontin (OPN) and decorin (DCN) (Burton et al., 2009). Of particular note is the repression of MGP, an inhibitor of calcification and the upregulation (~ 3-fold) of BMP-2, a promoter of calcification. In addition, genes and proteins highly expressed in osteoblasts such as alkaline phosphatase (ALP), type I collagen and runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates osteoblastic differentiation, are significantly upregulated in senescent VSMCs (Nakano-Kurimoto et al., 2009). Moreover, knockdown of RUNX-2 significantly reduced ALP expression and calcification in senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of the aorta from the klotho−/− ageing mouse model demonstrated an in vivo emergence of osteoblast-like cells expressing

⁎ Corresponding author. Tel.: +1 305 243 3456. E-mail address: [email protected] (D.G.A. Burton). 0531-5565/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2010.07.005

RUNX-2 exclusively in the calcified media (Nakano-Kurimoto et al., 2009). These findings thus suggest that the pro-calcificatory phenotype of senescent VSMCs could play a major role in the pathophysiology of age-related vascular calcification (VC). This review intends to bridge the divide between research into cellular senescence and research specifically focused on understanding the processes governing VC. 2. Cellular senescence Cellular senescence, the irreversible growth arrest of mitotic cells was first proposed as a potential mechanism of ageing by Leonard Hayflick in 1965 after demonstrating that normal human diploid cells have a limited replicative capacity (Hayflick, 1965). These findings were contrary to a 50-year-old dogma, which stated that cells in culture can divide indefinitely if properly maintained (Parker, 1938; Witkowski, 1990). For the next 30 years, research into the relationship between cellular senescence and ageing was somewhat limited, partly due to a limited understanding of the phenotypic changes associated with the onset of cellular senescence and a lack of a biomarker which could detect senescent cells in vivo. These limitations slowly began to unravel with time and advances in science. Building upon findings by West et al. (1989) who first demonstrated that cellular senescence in cultured fibroblasts resulted

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in the loss of regulation and overexpression of collagenase activity, a greater in-depth understanding of the senescent phenotype was gradually unfolding, if only initially in fibroblasts. Additionally, Dimri et al. found that modification of the beta-galactosidase assay could be used to detect the presence of senescent cells in culture and in tissues (Dimri et al., 1995). However, it soon became apparent that this assay should be used with several caveats in mind (Severino et al., 2000). More recently, improved understanding of the molecular changes associated with cellular senescence has yielded other, more robust biomarkers for detecting senescence cells, such as p53/p21cip1/waf1 and/or p16INK4a overexpression, accumulation of hypo-phosphorylated H2AX, senescence-associated heterochromatin foci (SAHF), telomere dysfunction-induced foci (TIF) and markers for lack of proliferation such as Ki67 (Takai et al., 2003; Narita et al., 2003; d'Adda et al., 2003; Ressler et al., 2006; Lawless et al., 2010). With increasing understanding of the senescent phenotype and the ability to detect senescent cells in vivo, there has been an emergence of definitive research focusing on the relationship between cellular senescence, ageing and age-related disease (Faragher et al., 2009; Burton, 2009). Cellular senescence can be triggered by a number of mechanisms; telomere-shortening (replicative senescence, RS) (Harley et al., 1990; Allsopp et al., 1992), p16INK4a expression (telomere-independent senescence, TIS) (Rheinwald et al., 2002), oxidative stress/DNA damaging agents (stress-induced premature senescence, SIPS) (Toussaint et al., 2000) and activated oncogenes such as Ras (oncogene-induced senescence, OIS) (Bartkova et al., 2006). To what extent each of these mechanisms plays in inducing cellular senescence in tissues is currently speculative. 2.1. Biological impact of the senescent phenotype Senescent cells have the potential to detrimentally impact tissue function by a variety of mechanisms. An increase in the senescent cell fraction means a decrease in the fraction of cells capable of cellular division and this may lead to an impaired regenerative capacity (Satyanarayana et al., 2003). This is particularly important to postmitotic tissues that rely on mitotic cells for maintenance and cellular replacement, such as satellite cells with muscle fibres and astrocytes with neuronal cells (Corbu et al., 2010; Pertusa et al., 2007). In addition to this, the degree of global transcriptional alteration during senescence appears to be similar to that observed when cells are induced to differentiate (Burton et al., 2005). A senescent cell should thus be considered as a completely different cell-type and as such can no longer carry out its original function effectively. A consequence of this altered transcriptome is an altered secretome, termed the senescence-associated secretory phenotype (SASP) (Coppé et al., 2008). RS, SIPS and OIS have all been shown to exhibit the SASP (Coppé et al., 2008; Young and Narita, 2009). However, it has been suggested that cells induced to senesce by overexpression of p16 do not express a SASP (Coppé et al., 2010), but this is in need of further investigations. The SASP includes an increase in the secretion of proinflammatory cytokines, proteases and growth factors (Kuilman and Peeper, 2009; Coppé et al., 2010). This phenotype appears to be similar to a wound healing response (Traversa and Sussman, 2001; Adams, 2009), suggesting it may function in aiding the removal of senescent cells from tissues (Xue et al., 2007; Krizhanovsky et al., 2008; Burton, 2009). However, age-related impairment of the wound healing response (Reed et al., 2003) would likely cause senescent cells to persist in tissues, potentially altering the behaviour and function of cells through autocrine/paracrine signaling due to the SASP. Secretion of proteases from senescent cells can also result in the degradation of the extracellular matrix (ECM), which is important in providing structural support and for intercellular communication. Further, senescence-associated secretion of growth factors has been reported to stimulate cell proliferation and tumour formation (Krtolica et al., 2001; Bavik et al., 2006; Chuaire-Noack et al., 2010).

2.2. Cell-type exclusive senescent phenotype (CESP) The SASP is a collection of commonly secreted factors observed in a wide range of senescent cells types investigated to date. However, in addition to the SASP, there appears to be a cell-type (or even cellstrain) exclusive senescent phenotype (CESP) that occurs but is not commonly reported. Microarray data of senescent cells show a differential expression of genes unrelated to SASP, which is nevertheless cell-type/strain specific (Shelton et al., 1999; Zhang et al., 2003; Burton et al., 2009, unpublished data). These transcriptional changes likely do not perform a specific physiological function (unlike the potential wound healing response of the SASP) and may be caused by random changes associated with alterations to the chromatin structure during senescence (Zhang et al., 2003; Funayama and Ishikawa, 2007). However, these altered transcriptional profiles may manifest into changes associated with altered morphology, behaviour and function and such changes could have a detrimental impact on tissue. Additionally, aspects of the CESP may provide cell-type specific senescent biomarkers for investigating senescence in ageing tissues. An example of a CESP is the decrease in nitric oxide synthase (NOS) activity observed in senescent vascular endothelial cells (Matsushita et al., 2001; Minamino et al., 2002). The synthesis of nitric oxide (NO) by NOS in vascular endothelial cells is important for maintaining vascular homeostasis and a decrease in NO has been suggested to be a potential risk factor in cardiovascular disease (Cannon, 1998). Similarly, senescence in pancreatic beta cells has been shown to result in insufficient insulin release (Sone and Kagawa, 2005). Due to the importance of insulin release in regulating glucose metabolism, an impairment in this function is associated with the pathogenesis of type II diabetes. The osteoblastic phenotype of senescent VSMCs (Burton et al., 2009; Nakano-Kurimoto et al., 2009) also appears to be an example of a CESP, since no other senescent cell-type to date has demonstrated such a change. VSMCs play an important role in the contraction and relaxation of blood vessels, a mechanism that is responsible for the redistribution of the blood within the body to areas where it is needed. The pro-calcificatory phenotype of senescent VSMCs could impair their normal function, thus contributing to cardiovascular dysfunction. 3. VSMCs and vascular calcification Vascular calcification (VC) is a well-known major risk factor for the development of cardiovascular diseases (Rennenberg et al., 2009; Thompson and Partridge, 2004; Adragao et al., 2004; Mitsutake et al., 2006). Furthermore, VC is also an independent risk factor for all-cause mortality (Rennenberg et al., 2009; Blaha et al., 2009; Budoff et al., 2007). There are three types of VC; (1) calcification in tunica media (medial calcification), (2) atherosclerotic intimal calcification and (3) valvular calcification. Medial calcification, also known as Monckeberg's sclerosis, is often observed in the elderly population, as well as in patients with diabetes mellitus and chronic kidney disease (Blumenthal et al., 1944; Chen and Moe, 2003; Lansing et al., 1948; Leskinen et al., 2002). Medial calcification increases throughout ageing, and accumulation of calcium in the elastin-rich layer of the media is ≥30-times more in the thoracic aorta at 90 years of age than that at 20 years of age (Elliott and McGrath, 1994). Nevertheless, medial calcification has long been regarded as a commonly occurring age-related change in blood vessels rather than a clinically significant lesion, because it does not encroach the vessel lumen. However, studies have revealed that medial calcification is an independent risk factor for cardiovascular events, as well as for peripheral artery occlusive disease (Niskanen et al., 1994; Fuessl et al., 1985). The underlying mechanisms that lead to the development of VC currently remain elusive. This is largely due to VC long being

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considered, until recently, a consequence of passive precipitation of mineral crystals into the vascular wall and not as an actively regulated process. Furthermore, research has focused predominantly on understanding the altered molecular pathways governing calcification. The physiological changes which may have resulted in these alterations in the first place (the underlying ageing mechanism) are far less studied. Investigations of the molecular pathways which regulate VC have shown that the process appears to be similar to that observed with bone calcification. During bone calcification, hydroxyapatite crystals, which contain calcium and inorganic phosphate, precipitate within the bone collagen fibrils (Murshed et al., 2005). Bone calcification is elaborately governed by several regulatory factors such as matrix Gla protein (MGP), alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2). Inorganic pyrophosphate, a small molecule made of two phosphate ions, as well as MGP, prevents the incorporation of mineral crystals into the collagen fibrils (Murshed et al., 2005; Proudfoot and Shanahan, 2006; Price, 1989). In contrast, ALP promotes bone calcification by cleaving pyrophosphate (Murshed et al., 2005). BMP-2 is a potent inducer for osteoblastic differentiation of mesenchymal precursor cells through initiating RUNX-2, a core transcription factor for osteoblastic genes (Lee et al., 2000). Osteoblasts play a central role in bone calcification through producing ALP as well as collagenous and non-collagenous matrices, where mineral crystals precipitate (Murshed et al., 2005). These bonecalcification regulatory factors have been identified in blood vessels, particularly at sites of medial calcification, and their expressions are differentially regulated between non-diseased and diseased vessels with higher concentrations found in diseased vessels (Shanahan et al., 1999; Tyson et al., 2003; Bostrom et al., 1993). Calcification in the media usually occurs in the absence of macrophages and lipid, and is associated with α-smooth muscle actin-positive VSMCs, suggesting that VSMCs are the primary key player in medial calcification. Moreover, MGP-deficient mice demonstrated massive medial calcification, indicating a critical role of MGP in VC (Luo et al., 1997). Of note, VSMC-specific re-expression of MGP (via the SM22α promotor) was sufficient to prevent medial calcification in MGP-deficient mice, indicating that MGP locally produced by VSMCs is essential for preventing ectopic calcification on tunica media (Murshed et al., 2004). In addition, some stimuli such as inorganic phosphate, uremic serum and hydrogen peroxide promote osteogenic differentiation in VSMCs through induction of RUNX-2 and downstream osteogenic programs (Reynolds et al., 2004; Byon et al., 2008; Chen et al., 2006). However, it is possible that some factors which have been shown to promote osteogenic differentiation in VSMCs, such as hydrogen peroxide, may do so by triggering premature senescence which consequently leads to the senescence-mediated osteoblastic transition. High levels of reactive oxygen species (ROS) such as hydrogen peroxide induce DNA damage which triggers senescence (Chen and Ames, 1994; Zdanov et al., 2006; Rai et al., 2009). Alternatively, ROS has been shown to play a role as physiological regulators of signal transduction and gene expression (Hancock et al., 2001), suggesting that these functions of ROS may have some involvement in the osteoblastic transition of VSMCs, possibly by influencing RUNX-2 expression (Byon et al., 2008). 4. Regulation of physiological osteoblast differentiation Osteoblasts are derived from mesenchymal stem cells through elaborately regulated machinery (Komori, 2006), with RUNX-2 considered to be the most important transcription factor for osteoblast differentiation, since ossification in RUNX-2 deficient mice has been completely absent throughout the body (Shapiro, 1999). RUNX-2 potently initiates the expression of major bone matrix protein genes at early stages of osteoblast differentiation for the

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purpose of engaging the transition of mesenchymal stem cells down the osteoblast lineage. However, overexpression of RUNX-2 leads to osteopenia (reduced bone density) through inhibiting the maturation and the transition of osteoblasts into osteocytes (Liu et al., 2001). Thus, RUNX-2 appears to play a role in maintaining the supply of immature osteoblasts, and appropriate gene expression regulation of the various osteogenic factors is essential for proper bone formation. Immature osteoblasts embed into the bone matrix where they become osteocytes. Another critical transcription factor for osteoblast differentiation is osterix, given that osterix-knockout mice demonstrate a lack of osteoblast formation (Nakashima et al., 2002). Since RUNX-2 expression is detected in osterix−/− mice, whereas osterix expression is absent in RUNX-2−/− mice, osterix is presumably a downstream gene for RUNX-2 (Komori, 2006). Canonical Wnt signaling also plays a key role in the osteoblast differentiation through inhibiting the phosphorylation of β-catenin, which leads to its nuclear translocation to initiate the transcription of target osteoblastic genes. The beta-catenin pathway has been shown to regulate osterix expression independently of RUNX-2 and so may also be an essential mediator in osteoblast differentiation (Komori, 2006). Genetic inactivation of β-catenin in mesenchymal progenitor cells completely abolishes osteoblast differentiation (Komori, 2006). 4.1. Role of cytokines/growth factors in osteoblast differentiation Cytokines/growth factors are soluble proteins which act as local signaling molecules to control and co-ordinate cellular behaviour and function and thus play an important role in osteoblast differentiation (reviewed in detail by Hughes et al., 2006). For example, the cytokine, interleukin 1 (IL-1) appears to have functions both in bone resorption and in bone formation. Konig et al. showed that tumour necrosis factor α (TNFα) and IL1 stimulate bone resorption in vitro (König et al., 1988). Wei et al. reported that IL1 mediates TNF-induced osteoclastogenesis by influencing the expression of RANKL and directly stimulating the differentiation of osteoclast precursors (Wei et al., 2005). In contrast to these findings, Hanazawa et al. demonstrated that purified IL-1 is effective at inducing the differentiation of clonal osteoblastic cells (MC3T3-E1) (Hanazawa et al., 1986). IL-1 inhibited DNA synthesis and cell growth, but enhanced alkaline phosphatase activity. It has further been shown that IL-1β regulates the formation of bone nodules in cell culture, thus demonstrating the importance of IL-1β on osteoblast differentiation (Lin et al., 2010). Short-term treatment (2 days) of calvarial cells with IL-1β significantly increases the occurrence of mineralised nodules compared to control. However, longer exposure to IL-1β (6 days) results in significantly fewer nodules. This study not only demonstrates the importance of IL-1β in bone formation, but also the importance of cytokine exposure time as a determinant of cellular phenotype. Interleukin 6 (IL-6) also appears to play a role in osteoblast formation. IL-6 and its soluble receptor (sIL-6R) are known to influence osteoblast proliferation and/or differentiation, possibly in an indirect manner by controlling the production of local factors such as insulin-like growth factor (IGF)-I and BMP-6 (Franchimont et al., 2005). IL-6 in MC3T3 osteoblastic cells has been shown to decrease proliferation and enhanced expression of RUNX-2 and osteocalcine (Li et al., 2008). Iwasaki et al. have also shown that IL-6/sIL-6R enhances both RUNX-2 expression and ALP activity through IGF-I production in periodontal ligament cells (Iwasaki et al., 2008). The secretion of BMPs plays a pivotal role in osteoblast differentiation largely through inducing RUNX-2 expression (Lee et al., 2000; Matsubara et al., 2008; Katagiri et al., 1994). Although many BMPs have been identified (BMP-1 to -13), BMP-2 is the most extensively studied BMP with respect to osteoblast differentiation. BMP-2 has been shown to regulate osterix through Msx2 and RUNX-2 during osteoblast differentiation (Matsubara et al., 2008). Additionally,

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BMP-2 appears to control ALP expression and osteoblast mineralization by the induction of a Wnt autocrine/paracrine loop (Rawadi et al., 2003). Altered expression of Wnt genes at senescence (Adams and Enders, 2008) may thus have some involvement in the senescence-mediated osteoblastic transition in VSMCs, but this needs to be investigated further. 5. Osteoblastic transition of senescent VSMCs In senescent VSMCs, RUNX-2 expression is enhanced, whereas the expression of osterix is unexpectedly reduced (Nakano-Kurimoto et al., 2009). Moreover, senescent VSMCs still express VSMC markers and do not become fully matured osteoblasts. It thus appears that the senescence-mediated osteoblastic transition shares common features with the early stages of osteoblast differentiation, but cannot progress beyond this point, due perhaps in part to lack of osterix expression. This senescence-mediated osteoblastic transition appears to be unique to VSMCs, at least compared to other cell-types investigated to date. So why do senescent VSMCs adopt this osteoblastic phenotype while others do not? The answer to this question may lie in the autocrine/paracrine effects of the SASP as well as the phenotypic plasticity in VSMCs. The persistence of senescent cells in tissues would result in continuous exposure to their own SASP and this may activate pathways leading to VSMCs partly transdifferentiating into osteoblasts. In this regard, factors associated with the SASP may directly influence changes associated with the CESP. Thus, different cell-types may respond differently to the commonly secreted components of the SASP. Since cytokines and growth factors are important in osteoblast differentiation, they may play an important role in initiating the osteoblastic phenotype of senescent VSMCs. Interleukin 1 (IL-1) is a possible secretory candidate that may influence such a transition. IL-1 is commonly elevated in senescent cells (Coppé et al., 2010) and ILβ expression is highly upregulated in senescent VSMCs (Minamino et al., 2003; Burton et al., 2009). This increased expression of IL-1β may stimulate the expression of BMP-2. Fukui et al. have shown that IL-1β and TNFα increase BMP-2 mRNA and protein levels by 8-fold and 15-fold respectively in cultured chondrocytes (Fukui et al., 2003). This suggests that IL-1β produced by senescent VSMCs could potentially stimulate BMP-2 expression, which in turn enhances the expression of RUNX-2. Interestingly, it has recently been reported that dedifferentiated VSMCs play a role in atherosclerotic intimal calcification by giving rise to osteogenic signals via BMP-2 (Nakagawa et al., 2010). However, there appears to be conflicting results in regard to BMP-2 expression in senescent VSMCs. Microarray analysis of senescent VSMCs showed BMP-2 expression to be increased 3-fold (Burton et al., 2009), while findings by Nakano-Kurimoto et al. demonstrated no difference between early and late passage VSMCs (Nakano-Kurimoto et al., 2009). Despite these conflicting results, both studies showed that MGP, a BMP-2 inhibitor, is repressed in senescent VSMCs, suggesting that if BMP-2 is upregulated, it may be due to lack of MGP. Further study is thus needed to determine the state of BMP-2 in senescent VSMCs. IL-6 is also commonly associated with the SASP and Minamino et al. have shown that Ras-induced VSMC senescence upregulates IL-6 12-fold (Minamino et al., 2003). The role of IL-6 in osteoblast differentiation has previously been discussed and so like IL-1, further study is needed to determine if IL-6 has a role in VSMC calcification. Alternatively, transcriptional changes associated with the CESP (independent of changes influenced by the SASP) of VSMCs may directly be responsible for the senescence-mediated osteoblastic transition or indirectly by making the cells more receptive to calcification by other mechanisms. It has been shown that senescent VSMCs are highly prone to be calcified when cultured in calcification medium (Nakano-Kurimoto et al., 2009). Knockdown of osteoblastic genes such as type I collagen and ALP significantly reduces

calcification in senescent VSMCs (Nakano-Kurimoto et al., 2009), indicating that the senescence-mediated osteoblastic transition is directly involved in the higher susceptibility of senescent VSMCs to calcification. Therefore, further research into the changes that occur during senescence in VSMCs is required in order to understand the molecular pathways governing the observed pro-calcificatory phenotype. For those wishing to investigate the CESP of VSMCs for themselves, Affymetrix microarray data for transcripts differentially regulated 2-fold or more can be perused at www.madras.cf.ac.uk/ vsmc (Burton et al., 2009). 6. Concluding remarks The link between VSMC senescence and calcification provides a valuable model for investigating the relationship between cellular senescence and tissue dysfunction/diseases associated with calcification (not necessarily limited to VSMCs). Such tissue dysfunction/ diseases may include aortic valve, atherosclerotic, renal, cerebral and articular cartilage calcification. Our recent findings on the osteoblastic phenotype of senescent VSMCs suggest that future work should focus not just on the impact of the SASP, but also the CESP of cell-types linked to age-related pathology. Such investigations may provide invaluable insight for the development of new therapeutic applications for preventing and treating disease. Acknowledgement Dr Priyamvada Rai is thanked for her support and guidance. References Adams, P.D., 2009. Healing and hurting: molecular mechanisms, functions, and pathologies of cellular senescence. Mol. Cell 36 (1), 2–14 (Oct 9). Adams, P.D., Enders, G.H., 2008. Wnt-signaling and senescence: a tug of war in early neoplasia? Cancer Biol. Ther. 7 (11), 1706–1711 (Nov Epub 2008 Nov 7). Adragao, T., Pires, A., Lucas, C., Birne, R., Magalhaes, L., Goncalves, M., Negrao, A.P., 2004. A simple vascular calcification score predicts cardiovascular risk in haemodialysis patients. Nephrol. Dial. Transplant. 19 (6), 1480–1488. Allsopp, R.C., Vaziri, H., Patterson, C., Goldstein, S., Younglai, E.V., Futcher, A.B., Greider, C.W., Harley, C.B., 1992. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl Acad. Sci. USA 89 (21), 10114–10118 (Nov 1). Bartkova, J., Rezaei, N., Liontos, M., Karakaidos, P., Kletsas, D., Issaeva, N., Vassiliou, L.V., Kolettas, E., Niforou, K., Zoumpourlis, V.C., Takaoka, M., Nakagawa, H., Tort, F., Fugger, K., Johansson, F., Sehested, M., Andersen, C.L., Dyrskjot, L., Ørntoft, T., Lukas, J., Kittas, C., Helleday, T., Halazonetis, T.D., Bartek, J., Gorgoulis, V.G., 2006. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 444 (7119), 633–637 (Nov 30). Bavik, C., Coleman, I., Dean, J.P., Knudsen, B., Plymate, S., Nelson, P.S., 2006. The gene expression program of prostate fibroblast senescence modulates neoplastic epithelial cell proliferation through paracrine mechanisms. Cancer Res. 66 (2), 794–802 (2006 Jan 15). Blaha, M., Budoff, M.J., Shaw, L.J., Khosa, F., Rumberger, J.A., Berman, D., Callister, T., Raggi, P., Blumenthal, R.S., Nasir, K., 2009. Absence of coronary artery calcification and all-cause mortality. Jacc 2 (6), 692–700. Blumenthal, H.T., Lansing, A.I., Wheeler, P.A., 1944. Calcification of the media of the human aorta and its relation to intimal arteriosclerosis, ageing and disease. Am. J. Pathol. 20 (4), 665–687. Bostrom, K., Watson, K.E., Horn, S., Wortham, C., Herman, I.M., Demer, L.L., 1993. Bone morphogenetic protein expression in human atherosclerotic lesions. J. clin. investig. 91 (4), 1800–1809. Budoff, M.J., Shaw, L.J., Liu, S.T., Weinstein, S.R., Mosler, T.P., Tseng, P.H., Flores, F.R., Callister, T.Q., Raggi, P., Berman, D.S., 2007. Long-term prognosis associated with coronary calcification: observations from a registry of 25,253 patients. J. Am. Coll. Cardiol. 49 (18), 1860–1870. Burton, D.G.A., 2009. Cellular senescence, ageing and disease. Age (Dordr) 31 (1), 1–9 (Mar). Burton, D.G.A., Allen, M.C., Bird, J.L., Faragher, R.G., 2005. Bridging the gap: ageing, pharmacokinetics and pharmacodynamics. J. Pharm. Pharmacol. 57 (6), 671–679 (2005 Jun). Burton, D.G.A., Giles, P.J., Sheerin, A.N., Smith, S.K., Lawton, J.J., Ostler, E.L., RhysWilliams, W., Kipling, D., Faragher, R.G., 2009. Microarray analysis of senescent vascular smooth muscle cells: a link to atherosclerosis and vascular calcification. Exp. Gerontol. 44 (10), 659–665 (Oct). Byon, C.H., Javed, A., Dai, Q., Kappes, J.C., Clemens, T.L., Darley-Usmar, V.M., McDonald, J.M., Chen, Y., 2008. Oxidative stress induces vascular calcification through modulation of

D.G.A. Burton et al. / Experimental Gerontology 45 (2010) 819–824 the osteogenic transcription factor RUNX-2 by AKT signaling. J. Biol. Chem. 283 (22), 15319–15327. Cannon III, R.O., 1998. Role of nitric oxide in cardiovascular disease: focus on the endothelium. Clin. Chem. 44 (8 Pt 2), 1809–1819 (Aug). Chen, Q., Ames, B.N., 1994. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc. Natl Acad. Sci. USA 91 (10), 4130–4134 (May 10). Chen, N.X., Moe, S.M., 2003. Arterial calcification in diabetes. Curr. Diab. Rep. 3 (1), 28–32. Chen, N.X., Duan, D., O'Neill, K.D., Wolisi, G.O., Koczman, J.J., Laclair, R., Moe, S.M., 2006. The mechanisms of uremic serum-induced expression of bone matrix proteins in bovine vascular smooth muscle cells. Kidney Int. 70 (6), 1046–1053. Chuaire-Noack, L., Sánchez-Corredor, M.C., Ramírez-Clavijo, S., 2010. The dual role of senescence in tumorigenesis. Int. J. Morphol. 28 (1), 37–50. Coppé, J.P., Patil, C.K., Rodier, F., Sun, Y., Muñoz, D.P., Goldstein, J., Nelson, P.S., Desprez, P.Y., Campisi, J., 2008. Senescence-associated secretory phenotypes reveal cellnonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 6 (12), 2853–2868 (Dec 2). Coppé, J.P., Desprez, P.Y., Krtolica, A., Campisi, J., 2010. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99–118. Corbu, A., Scaramozza, A., Badiali-DeGiorgi, L., Tarantino, L., Papa, V., Rinaldi, R., D'Alessandro, R., Zavatta, M., Laus, M., Lattanzi, G., Cenacchi, G., 2010. Satellite cell characterization from aging human muscle. Neurol. Res. 32 (1), 63–72 (Feb). d'Adda di Fagagna, F., Reaper, P.M., Clay-Farrace, L., Fiegler, H., Carr, P., Von Zglinicki, T., Saretzki, G., Carter, N.P., Jackson, S.P., 2003. A DNA damage checkpoint response in telomere-initiated senescence. Nature 426 (6963), 194–198 (Nov 13). Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., Pereira-Smith, O., et al., 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92 (20), 9363–9367 (Sep 26). Elliott, R.J., McGrath, L.T., 1994. Calcification of the human thoracic aorta during aging. Calcif. Tissue Int. 54 (4), 268–273 (Apr). Faragher, R.G., Sheerin, A.N., Ostler, E.L., 2009. Can we intervene in human ageing? Expert Rev. Mol. Med. 7 (11), e27 (Sep). Franchimont, N., Wertz, S., Malaise, M., 2005. Interleukin-6: an osteotropic factor influencing bone formation? Bone 37 (5), 601–606 Nov. Fuessl, H.S., Schalzky, H., Schewe, S., Frey, K.W., Goebel, F.D., 1985. Pathogenesis and clinical significance of Monckeberg medial calcinosis. Klinische Wochenschrift 63 (5), 211–216. Fukui, N., Zhu, Y., Maloney, W.J., Clohisy, J., Sandell, L.J., 2003. Stimulation of BMP-2 expression by pro-inflammatory cytokines IL-1 and TNF-alpha in normal and osteoarthritic chondrocytes. J. Bone Joint Surg. Am. 85-A (Suppl 3), 59–66 (2003). Funayama, R., Ishikawa, F., 2007. Cellular senescence and chromatin structure. Chromosoma 116 (5), 431–440 (Oct). Hanazawa, S., Ohmori, Y., Amano, S., Hirose, K., Miyoshi, T., Kumegawa, M., Kitano, S., 1986. Human purified interleukin-1 inhibits DNA synthesis and cell growth of osteoblastic cell line (MC3T3-E1), but enhances alkaline phosphatase activity in the cells. FEBS Lett. 203 (2), 279–284 (Jul 28). Hancock, J.T., Desikan, R., Neill, S.J., 2001. Role of reactive oxygen species in cell signalling pathways. Biochem. Soc. Trans. 29 (Pt 2), 345–350 2001 May. Harley, C.B., Futcher, A.B., Greider, C.W., 1990. Telomeres shorten during ageing of human fibroblasts. Nature 345 (6274), 458–460 (1990 May 31). Hayflick, L., 1965. The limited in vitro lifespan of human diploid cell strains. Exp. Cell Res. 37, 614–636. Hughes, F.J., Turner, W., Belibabasakis, G., Martuscelli, G., 2006. Effects of growth factors and cytokines on osteoblast differentiation. Periodontol. 2000 41, 48–72 (2006). Iwasaki, K., Komaki, M., Mimori, K., Leon, E., Izumi, Y., Ishikawa, I., 2008. IL-6 induces osteoblastic differentiation of periodontal ligament cells. J. Dent. Res. 87 (10), 937–942 (Oct). Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J.M., Fujisawa-Sehara, A., Suda, T., 1994. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J. Cell Biol. 127 (6 Pt 1), 1755–1766. Komori, T., 2006. Regulation of osteoblast differentiation by transcription factors. J. Cell. Biochem. 99 (5), 1233–1239. König, A., Mühlbauer, R.C., Fleisch, H., 1988. Tumor necrosis factor alpha and interleukin-1 stimulate bone resorption in vivo as measured by urinary [3H] tetracycline excretion from prelabeled mice. J. Bone Miner. Res. 3 (6), 621–627 (Dec). Krizhanovsky, V., Xue, W., Zender, L., Yon, M., Hernando, E., Lowe, S.W., 2008. Implications of cellular senescence in tissue damage response, tumor suppression, and stem cell biology. Cold Spring Harb. Symp. Quant. Biol. 73, 513–522. Krtolica, A., Parrinello, S., Lockett, S., Desprez, P.Y., Campisi, J., 2001. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc. Natl Acad. Sci. USA 98 (21), 12072–12077 (Oct 9). Kuilman, T., Peeper, D.S., 2009. Senescence-messaging secretome: SMS-ing cellular stress. Nat. Rev. Cancer 9 (2), 81–94 (Feb Epub 2009 Jan 9). Lansing, A.I., Blumenthal, H.T., Gray, S.H., 1948. Aging and calcification of the human coronary artery. J. Gerontol. 3 (2), 87–97. Lawless, C., Wang, C., Jurk, D., Merz, A., Zglinicki, T.V., Passos, J.F., 2010. Quantitative assessment of markers for cell senescence. Exp. Gerontol. (Feb 1. [Epub ahead of print]). Lee, K.S., Kim, H.J., Li, Q.L., Chi, X.Z., Ueta, C., Komori, T., Wozney, J.M., Kim, E.G., Choi, J.Y., Ryoo, H.M., Bae, S.C., 2000. RUNX-2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between RUNX-2

823

and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol. Cell. Biol. 20 (23), 8783–8792. Leskinen, Y., Salenius, J.P., Lehtimaki, T., Huhtala, H., Saha, H., 2002. The prevalence of peripheral arterial disease and medial arterial calcification in patients with chronic renal failure: requirements for diagnostics. Am. J. Kidney Dis. 40 (3), 472–479. Li, Y., Bäckesjö, C.M., Haldosén, L.A., Lindgren, U., 2008. IL-6 receptor expression and IL6 effects change during osteoblast differentiation. Cytokine 43 (2), 165–173 (Aug). Lin, F.H., Chang, J.B., McGuire, M.H., Yee, J.A., Brigman, B.E., 2010. Biphasic effects of interleukin-1beta on osteoblast differentiation in vitro. J. Orthop. Res. 28 (7), 958–964. Liu, W., Toyosawa, S., Furuichi, T., Kanatani, N., Yoshida, C., Liu, Y., Himeno, M., Narai, S., Yamaguchi, A., Komori, T., 2001. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J. Cell Biol. 155 (1), 157–166 (Oct 1). Luo, G., Ducy, P., McKee, M.D., Pinero, G.J., Loyer, E., Behringer, R.R., Karsenty, G., 1997. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 386 (6620), 78–81. Matsubara, T., Kida, K., Yamaguchi, A., Hata, K., Ichida, F., Meguro, H., Aburatani, H., Nishimura, R., Yoneda, T., 2008. BMP2 regulates Osterix through Msx2 and RUNX-2 during osteoblast differentiation. J. Biol. Chem. 283 (43), 29119–29125. Matsushita, H., Chang, E., Glassford, A.J., Cooke, J.P., Chiu, C.P., Tsao, P.S., 2001. eNOS activity is reduced in senescent human endothelial cells: preservation by hTERT immortalization. Circ. Res. 89, 793–798. Minamino, T., Miyauchi, H., Yoshida, T., Ishida, Y., Yoshida, H., Komuro, I., 2002. Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation 105, 1541–1544. Minamino, T., Yoshida, T., Tateno, K., Miyauchi, H., Zou, Y., Toko, H., Komuro, I., 2003. Ras induces vascular smooth muscle cell senescence and inflammation in human atherosclerosis. Circulation 108 (18), 2264–2269 (Nov 4). Mitsutake, R., Niimura, H., Miura, S., Zhang, B., Iwata, A., Nishikawa, H., Kawamura, A., Kumagai, K., Shirai, K., Matsunaga, A., Saku, K., 2006. Clinical significance of the coronary calcification score by multidetector row computed tomography for the evaluation of coronary stenosis in Japanese patients. Circ. J. 70 (9), 1122–1127. Murshed, M., Schinke, T., McKee, M.D., Karsenty, G., 2004. Extracellular matrix mineralization is regulated locally; different roles of two gla-containing proteins. J. Cell Biol. 165 (5), 625–630. Murshed, M., Harmey, D., Millan, J.L., McKee, M.D., Karsenty, G., 2005. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev. 19 (9), 1093–1104. Nakagawa, Y., Ikeda, K., Akakabe, Y., Koide, M., Uraoka, M., Yutaka, K., KurimotoNakano, R., Takahashi, T., Matoba, S., Yamada, H., Okigaki, M., Matsubara, H., 2010. Paracrine Osteogenic Signals via Bone Morphogenetic Protein-2 Accelerate the Atherosclerotic Intimal Calcification in Vivo. ATVB published July 22. doi:10.1161/ ATVBAHA.110.206185. Nakano-Kurimoto, R., Ikeda, K., Uraoka, M., Nakagawa, Y., Yutaka, K., Koide, M., Takahashi, T., Matoba, S., Yamada, H., Okigaki, M., Matsubara, H., 2009. Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition. Am. J. Physiol. Heart Circ. Physiol. 297 (5), H1673–H1684 (Nov). Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J.M., Behringer, R.R., de Crombrugghe, B., 2002. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108 (1), 17–29. Narita, M., Nũnez, S., Heard, E., Narita, M., Lin, A.W., Hearn, S.A., Spector, D.L., Hannon, G.J., Lowe, S.W., 2003. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 113 (6), 703–716 (Jun 13). Niskanen, L., Siitonen, O., Suhonen, M., Uusitupa, M.I., 1994. Medial artery calcification predicts cardiovascular mortality in patients with NIDDM. Diab. Care 17 (11), 1252–1256. Parker, R.C., 1938. Methods of Tissue Culture. Paul B. Hoebner Inc., New York. Pertusa, M., Garcia-Matas, S., Rodriguez-Farre, E., Sanfeliu, C., Cristofol, R., 2007. Astrocytes aged in vitro show a decreased neuroprotective capacity. J. Neurochem. 101, 794–805. Price, P.A., 1989. Gla-containing proteins of bone. Connect. Tissue Res. 21 (1–4), 51–57 (discussion 57–60). Proudfoot, D., Shanahan, C.M., 2006. Molecular mechanisms mediating vascular calcification: role of matrix Gla protein. Nephrology (Carlton) 11 (5), 455–461. Rai, P., Onder, T.T., Young, J.J., McFaline, J.L., Pang, B., Dedon, P.C., Weinberg, R.A., 2009. Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence. Proc. Natl Acad. Sci. USA 106 (1), 169–174 (2009 Jan 6). Rawadi, G., Vayssière, B., Dunn, F., Baron, R., Roman-Roman, S., 2003. BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J. Bone Miner. Res. 18 (10), 1842–1853 (2003 Oct). Reed, M.J., Koike, T., Puolakkainen, P., 2003. Wound repair in aging. A review. Meth. Mol. Med. 78, 217–237. Rennenberg, R.J., Kessels, A.G., Schurgers, L.J., van Engelshoven, J.M., de Leeuw, P.W., Kroon, A.A., 2009. Vascular calcifications as a marker of increased cardiovascular risk: a meta-analysis. Vasc. Health Risk Manage. 5 (1), 185–197. Ressler, S., Bartkova, J., Niederegger, H., Bartek, J., Scharffetter-Kochanek, K., JansenDürr, P., Wlaschek, M., 2006. p16INK4A is a robust in vivo biomarker of cellular aging in human skin. Aging Cell 5 (5), 379–389 (Oct;5 Epub 2006 Aug 15). Reynolds, J.L., Joannides, A.J., Skepper, J.N., McNair, R., Schurgers, L.J., Proudfoot, D., Jahnen-Dechent, W., Weissberg, P.L., Shanahan, C.M., 2004. Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J. Am. Soc. Nephrol. 15 (11), 2857–2867.

824

D.G.A. Burton et al. / Experimental Gerontology 45 (2010) 819–824

Rheinwald, J.G., Hahn, W.C., Ramsey, M.R., Wu, J.Y., Guo, Z., Tsao, H., De Luca, M., Catricalà, C., O'Toole, K.M., 2002. A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status. Mol. Cell. Biol. 22 (14), 5157–5172 (Jul). Satyanarayana, A., Wiemann, S.U., Buer, J., Lauber, J., Dittmar, K.E., Wüstefeld, T., Blasco, M.A., Manns, M.P., Rudolph, K.L., 2003. Telomere shortening impairs organ regeneration by inhibiting cell cycle re-entry of a subpopulation of cells. EMBO J. 22 (15), 4003–4013 (Aug 1). Severino, J., Allen, R.G., Balin, S., Balin, A., Cristofalo, V.J., 2000. Is beta-galactosidase staining a marker of senescence in vitro and in vivo? Exp. Cell Res. 257 (1), 162–171 (2000 May 25). Shanahan, C.M., Cary, N.R., Salisbury, J.R., Proudfoot, D., Weissberg, P.L., Edmonds, M.E., 1999. Medial localization of mineralization-regulating proteins in association with Monckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation 100 (21), 2168–2176. Shapiro, I.M., 1999. Discovery: Osf2/Cbfa1, a master gene of bone formation. Clin. Orthod. Res. 2 (1), 42–46. Shelton, D.N., Chang, E., Whittier, P.S., Choi, D., Funk, W.D., 1999. Microarray analysis of replicative senescence. Curr. Biol. 9 (17), 939–945 (Sep 9). Sone, H., Kagawa, Y., 2005. Pancreatic beta cell senescence contributes to the pathogenesis of type 2 diabetes in high-fat diet-induced diabetic mice. Diabetologia 48 (1), 58–67 (Jan Epub 2004 Dec 29). Takai, H., Smogorzewska, A., de Lange, T., 2003. DNA damage foci at dysfunctional telomeres. Curr. Biol. 13 (17), 1549–1556 (2003 Sep 2). Thompson, G.R., Partridge, J., 2004. Coronary calcification score: the coronary-risk impact factor. Lancet 363 (9408), 557–559.

Toussaint, O., Medrano, E.E., von Zglinicki, T., 2000. Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes. Exp. Gerontol. 35 (8), 927–945 (Oct). Traversa, B., Sussman, G., 2001. The role of growth factors, cytokines and proteases in wound management. Prim. Intention 9 (4), 161–167. Tyson, K.L., Reynolds, J.L., McNair, R., Zhang, Q., Weissberg, P.L., Shanahan, C.M., 2003. Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler. Thromb. Vasc. Biol. 23 (3), 489–494. Wei, S., Kitaura, H., Zhou, P., Ross, F.P., Teitelbaum, S.L., 2005. IL-1 mediates TNFinduced osteoclastogenesis. J. Clin. Invest. 115 (2), 282–290 (Feb). West, M.D., Pereira-Smith, O.M., Smith, J.R., 1989. Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity. Exp. Cell Res. 184 (1), 138–147 (1989 Sep). Witkowski, J.A., 1990. Carrel's cultures. Science 247, 1385–1386. Xue, W., Zender, L., Miething, C., Dickins, R.A., Hernando, E., Krizhanovsky, V., CordonCardo, C., Lowe, S.W., 2007. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 445 (7128), 656–660 (2007 Feb 8). Young, A.R., Narita, M., 2009. SASP reflects senescence. EMBO Rep. 10 (3), 228–230 (Mar). Zdanov, S., Remacle, J., Toussaint, O., 2006. Establishment of H2O2-induced premature senescence in human fibroblasts concomitant with increased cellular production of H2O2. Ann. NY Acad. Sci. 1067, 210–216 (May). Zhang, H., Pan, K.H., Cohen, S.N., 2003. Senescence-specific gene expression fingerprints reveal cell-type-dependent physical clustering of up-regulated chromosomal loci. Proc. Natl Acad. Sci. USA 100 (6), 3251–3256 (Mar 18).