Canadian Journal of Cardiology 30 (2014) 482e484
Editorial
Mammalian Target of Rapamycin: A Novel Pathway in Vascular Calcification Augusto C. Montezano, PhD, and Rhian M. Touyz, MD, PhD Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, United Kingdom
See article by Zhan et al., pages 568-575 of this issue. Calcification of arteries is common in advanced age, chronic kidney disease, diabetes mellitus, atherosclerosis, and hypertension and is an independent predictor of cardiovascular events.1-3 Pathologically, arterial calcification contributes to advanced atherosclerosis (calcification of plaques) and increased vascular stiffness, leading to target organ injury in the heart, kidney, and brain through secondary microvascular damage.1-4 Vascular calcification is associated with accumulation of calcium deposits in the vessel wall and mineralization of the internal elastic lamina and elastic fibres in the vascular media. This is a highly controlled process similar to events that regulate osteogenesis in bone cells. Factors that promote calcification include abnormalities in mineral metabolism, particularly hyperphosphatemia and hypercalcemia, which stimulate vascular smooth muscle cell (VSMC) differentiation to an osteoblastic phenotype.5 Vascular calcification is an active cell-mediated response involving VSMC apoptosis and vesicle release, a shift in the balance of inhibitors and promoters of vascular calcification, and VSMC differentiation from a contractile to osteochondrogenic phenotype. This phenotypic shift requires phosphate, and the uptake of phosphate by the sodium-dependent phosphate cotransporters PiT-1 and PiT-2, which are upregulated by proinflammatory cytokines.5-7 Calcium uptake is also important and involves regulation by the calcium-sensing receptor by voltage-gated Ca2þ channels.8 Molecular mechanisms regulating these events involve upregulation of transcription factors such as core-binding factor 1a (cbfa1)/runt-related transcription factor 2 (Runx2), msh homeobox 2 (MSX-2), and bone morphogenetic protein 2 (BMP-2), which are critically important in normal bone development and control the expression of osteogenic proteins, including osteocalcin, osteonectin, alkaline phosphatase, collagen-1, and bone sialoprotein.5-8 Another process contributing to vascular Received for publication March 4, 2014. Accepted March 4, 2014. Corresponding author: Dr Rhian M. Touyz, Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, United Kingdom G12 8TA. Tel.: 44(0)141 330 7775; fax: 44(0)141 330 3360. E-mail: [email protected] See page 484 for disclosure information.
mineralization is loss of calcification inhibitors, such as fetuinA, matrix Gla protein, pyrophosphate, and osteopontin.9,10 Factors that trigger these events remain elusive, although multiple kinases including Src, phospholipase C (PLC), ERK1/2, p38 mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) may be important.11-13 To add to the complex kinase network, Zhan et al. report in this issue of the Canadian Journal of Cardiology, a putative role of mammalian target of rapamycin (mTOR) in vascular osteogenesis.14 mTOR is an intriguing candidate as a regulator of osteogenesis, because it is characteristically associated with cellular metabolism and longevity through its effects on cell growth, proliferation, and survival rather than on bone metabolism.15 Dysregulation of mTOR is linked to obesity, metabolic syndrome, cardiovascular disease, and cancer.15-17 The finding that it is also instrumental in molecular processes associated with calcification of vessels is novel and may provide an interesting link between cardiovascular disease and metabolic diseases, because vascular calcification occurs in both pathologic processes. Signalling through mTOR is complex. It is a serine/threonine kinase that belongs to the phosphatidylinositol 3-kinase (PI3K)-related kinase family and functions as an intracellular sensor for energy metabolism, nutrient availability, and cellular stresses and regulates cell growth and metabolism to adapt to environmental changes.18 mTOR forms a catalytic core of 2 multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2. mTORC1 and mTORC2 enzymatic activity is regulated by accessory proteins raptor and rictor, respectively.19,20 These adaptor proteins act as scaffold proteins to recruit mTOR substrates and regulators. mTOR is sensitive to the antifungal macrolide rapamycin, which forms a complex with FK506-binding protein that specifically inhibits mTORC1. Multiple upstream factors regulate mTOR, including growth factors, DNA damage, nutrients, and hypoxia. mTOR is a major activator of cell growth and proliferation. Once activated, mTORC1 phosphorylates p70 S6 kinase 1 (S6K1) and inhibits eIF-4Ebinding protein 1 (4E-BP1), leading to activation of ribosomal protein S6 and eIF2E, protein synthesis, and cell proliferation. mTORC1 also activates transcription factors SREP1, peroxisome proliferator-activated receptor (PPAR)
0828-282X/$ - see front matter Ó 2014 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cjca.2014.03.001
Montezano and Touyz mTOR: Novel Pathway in Vascular Calcification
483
Figure 1. Putative molecular mechanisms whereby mammalian target of rapamycin (mTOR) may influence vascular smooth muscle cell (VSMC) differentiation to an osteogenic phenotype that may contribute to vascular calcification.14 mTOR activation induces activation of S6 kinase (S6K1), which induces protein synthesis of osteogenic factors that promote calcification. mTOR also regulates lipid and glucose metabolism through welldescribed mechanisms involving peroxisome proliferator-activated receptor (PPAR) and hypoxia-inducible factor 1 (HIF-1).15-17 Rapamycin and adiponectin inhibit mTOR-induced activation of S6K1, thereby reducing osteogenic differentiation. Dashed lines indicate possible pathways.
gamma, and hypoxia-inducible factor 1 (HIF-1), thereby regulating lipid biogenesis and glycolysis. Because of the essential role of mTOR in anabolic metabolism, energy storage, and cell growth/survival it is not surprising that this pathway is important in adipogenesis and glucose and lipid metabolism.15-20 In obesity and metabolic disorders, mTORC1 is hyperactivated and may be associated with insulin resistance. In contrast, immunosuppressive drugs that inhibit mTOR, including cyclosporine A, tacrolimus, and rapamycin, enhance lipolysis and inhibit lipid storage and expression of lipogenic genes in adipose tissue, which may contribute to the development of dyslipidemia and insulin resistance associated with immunosuppressive therapy.21 Although mTOR is now considered a major player in lipid and glucose metabolism, its role in the vasculature is still largely unknown. There is some evidence that it regulates vascular Ca2þ channels and intracellular Ca2þ mobilization, thereby influencing vasoconstriction.20,22,23 mTOR has also been shown to play a role in VSMC proliferation and when dysregulated has been implicated in vascular remodelling.24 Zhan et al. advance the field of mTOR in the vasculature by showing that mTOR, through S6K1, promotes osteogenic differentiation, possibly predisposing to vascular calcification (Fig. 1).14 Exact mechanisms whereby mTOR induces mineralization are unclear, but increased synthesis of osteogenic factors, such as bone morphogenetic proteins, may be important. Studies investigating the PI3K/AKT-mTOR axis
in bone biological processes and mineralization are rare, although there is some evidence that this system influences chondrocyte maturation, endochondral ossification, and osteoblast differentiation through effects on activation of p70S6K and BMP expression.25-27 How these processes control mineralization processes remains to be elucidated. Of particular interest, adiponectin was found to inhibit mTOR-induced osteogenic differentiation.14 Adiponectin is the most abundant adipokine produced by adipocytes and has been implicated as a key player in adiposity, insulin resistance, and inflammation through its effects on lipid and glucose metabolism.28 As such, this mechanism may further link vascular calcification to metabolic disorders. Although the study under discussion in this issue of the Canadian Journal of Cardiology presents an interesting paradigm, there are some limitations in the study that warrant further consideration.14 First, studies were performed in isolated VSMCs in which only a few markers of osteoblastic differentiation were identified. This may not necessarily translate into vascular calcification in vivo. Second, although the study identifies a novel putative signalling pathway through mTOR, the exact molecular processes whereby mTOR-S6K1 influences activation of ion channels important in cellular Ca2þ, Naþ, and phosphate regulation, such as PiT-1 and PiT-2, are not addressed. However, despite these limitations, the study presents an interesting paradigm in which mTOR may be a link between metabolic disorders and
484
vascular calcification. Zhan et al. provide new insights into the molecular biology of vascular calcification, and more research is certainly warranted.14 It will be particularly interesting to know in whole animals whether inhibition of mTOR with adiponectin or rapamycin, which have been shown to influence adiposity and insulin resistance, would also ameliorate vascular calcification. Funding Sources Work from the authors’ laboratory was supported by grants 44018 and 57886, from the Canadian Institutes of Health Research, and grants from the British Heart Foundation. R.M.T. is supported through a British Heart Foundation Chair, and A.C.M. is supported through a Leadership Fellowship from the University of Glasgow. Disclosures The authors have no conflicts of interest to disclose. References 1. Sunkara N, Wong ND, Malik S. Role of coronary artery calcium in cardiovascular risk assessment. Expert Rev Cardiovasc Ther 2014;12(1): 87-94. 2. Jablonski KL, Chonchol M. Vascular calcification in end-stage renal disease. Hemodial Int 2013;17:S17-21. 3. Kramer CK, Zinman B, Gross JL, et al. Coronary artery calcium score prediction of all cause mortality and cardiovascular events in people with type 2 diabetes: systematic review and meta-analysis. BMJ 2013;346: f1654. 4. Briet M, Burns KD. Chronic kidney disease and vascular remodelling: molecular mechanisms and clinical implications. Clin Sci (Lond) 2012;123:399-416. 5. Massy ZA, Drüeke TB. Vascular calcification. Curr Opin Nephrol Hypertens 2013;22:405-12. 6. Crouthamel MH, Lau WL, Leaf EM, et al. Sodium-dependent phosphate cotransporters and phosphate-induced calcification of vascular smooth muscle cells: redundant roles for PiT-1 and PiT-2. Arterioscler Thromb Vasc Biol 2013;33:2625-32. 7. Shao JS, Aly ZA, Lai CF, et al. Vascular Bmp Msx2 Wnt signaling and oxidative stress in arterial calcification. Ann N Y Acad Sci 2007;1117: 40-50. 8. Shroff R, Long DA, Shanahan C. Mechanistic insights into vascular calcification in CKD. J Am Soc Nephrol 2013;24:179-89. 9. Herrmann M, Kinkeldey A, Jahnen-Dechent W. Fetuin-A function in systemic mineral metabolism. Trends Cardiovasc Med 2012;22:197-201. 10. 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.
Canadian Journal of Cardiology Volume 30 2014 11. Teplyuk NM, Galindo M, Teplyuk VI, et al. Runx2 regulates G proteincoupled signaling pathways to control growth of osteoblast progenitors. J Biol Chem 2008;283:27585-97. 12. McCarthy HS, Williams JH, Davie MW, Marshall MJ. Platelet-derived growth factor stimulates osteoprotegerin production in osteoblastic cells. J Cell Physiol 2009;218:350-4. 13. Montezano AC, Zimmerman D, Yusuf H, et al. Vascular smooth muscle cell differentiation to an osteogenic phenotype involves TRPM7 modulation by magnesium. Hypertension 2010;56:453-62. 14. Zhan J-K, Wang Y-J, Wang Y, et al. The mammalian target of rapamycin signalling pathway is involved in osteoblastic differentiation of vascular smooth muscle cells. Can J Cardiol 2014;30:568-75. 15. Yang Z, Ming XF. mTOR signalling: the molecular interface connecting metabolic stress, aging and cardiovascular diseases. Obes Rev 2012: 58-68. 16. Howell JJ, Ricoult SJ, Ben-Sahra I, Manning BD. A growing role for mTOR in promoting anabolic metabolism. Biochem Soc Trans 2013;41: 906-12. 17. Gilley R, Balmanno K, Cope CL, Cook SJ. Adaptation to chronic mTOR inhibition in cancer and in aging. Biochem Soc Trans 2013;41: 956-61. 18. Wrighton KH. Cell signalling: where the mTOR action is. Nat Rev Mol Cell Biol 2013;14:191. 19. Sciarretta S, Volpe M, Sadoshima J. Mammalian target of rapamycin signaling in cardiac physiology and disease. Circ Res 2014;114:549-64. 20. Liu Y, Vertommen D, Rider MH, Lai YC. Mammalian target of rapamycin-independent S6K1 and 4E-BP1 phosphorylation during contraction in rat skeletal muscle. Cell Signal 2013;25:1877-86. 21. Pereira MJ, Palming J, Rizell M, et al. The immunosuppressive agents rapamycin, cyclosporin A and tacrolimus increase lipolysis, inhibit lipid storage and alter expression of genes involved in lipid metabolism in human adipose tissue. Mol Cell Endocrinol 2013;365:260-9. 22. Martín-Cano FE, Camello-Almaraz C, Hernandez D, Pozo MJ, Camello PJ. mTOR pathway and Ca2þ stores mobilization in aged smooth muscle cells. Aging 2013;5:339-46. 23. MacMillan D. FK506 binding proteins: cellular regulators of intracellular Ca2þ signalling. Eur J Pharmacol 2013;700:181-93. 24. Goncharova EA. mTOR and vascular remodeling in lung diseases: current challenges and therapeutic prospects. FASEB J 2013;27:1796-807. 25. Sun H, Kim JK, Mortensen R, et al. Osteoblast-targeted suppression of PPARg increases osteogenesis through activation of mTOR signaling. Stem Cells 2013;31:2183-92. 26. Kim JK, Baker J, Nor JE, Hill EE. mTor plays an important role in odontoblast differentiation. J Endod 2011;37:1081-5. 27. Pantovic A, Krstic A, Janjetovic K, et al. Coordinated time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells. Bone 2013;52:524-31. 28. Silva TE, Colombo G, Schiavon LL. Adiponectin: A multitasking player in the field of liver diseases. Diabetes Metab 2014:S1262-3636.
Editorial
Mammalian Target of Rapamycin: A Novel Pathway in Vascular Calcification Augusto C. Montezano, PhD, and Rhian M. Touyz, MD, PhD Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, United Kingdom
See article by Zhan et al., pages 568-575 of this issue. Calcification of arteries is common in advanced age, chronic kidney disease, diabetes mellitus, atherosclerosis, and hypertension and is an independent predictor of cardiovascular events.1-3 Pathologically, arterial calcification contributes to advanced atherosclerosis (calcification of plaques) and increased vascular stiffness, leading to target organ injury in the heart, kidney, and brain through secondary microvascular damage.1-4 Vascular calcification is associated with accumulation of calcium deposits in the vessel wall and mineralization of the internal elastic lamina and elastic fibres in the vascular media. This is a highly controlled process similar to events that regulate osteogenesis in bone cells. Factors that promote calcification include abnormalities in mineral metabolism, particularly hyperphosphatemia and hypercalcemia, which stimulate vascular smooth muscle cell (VSMC) differentiation to an osteoblastic phenotype.5 Vascular calcification is an active cell-mediated response involving VSMC apoptosis and vesicle release, a shift in the balance of inhibitors and promoters of vascular calcification, and VSMC differentiation from a contractile to osteochondrogenic phenotype. This phenotypic shift requires phosphate, and the uptake of phosphate by the sodium-dependent phosphate cotransporters PiT-1 and PiT-2, which are upregulated by proinflammatory cytokines.5-7 Calcium uptake is also important and involves regulation by the calcium-sensing receptor by voltage-gated Ca2þ channels.8 Molecular mechanisms regulating these events involve upregulation of transcription factors such as core-binding factor 1a (cbfa1)/runt-related transcription factor 2 (Runx2), msh homeobox 2 (MSX-2), and bone morphogenetic protein 2 (BMP-2), which are critically important in normal bone development and control the expression of osteogenic proteins, including osteocalcin, osteonectin, alkaline phosphatase, collagen-1, and bone sialoprotein.5-8 Another process contributing to vascular Received for publication March 4, 2014. Accepted March 4, 2014. Corresponding author: Dr Rhian M. Touyz, Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, United Kingdom G12 8TA. Tel.: 44(0)141 330 7775; fax: 44(0)141 330 3360. E-mail: [email protected] See page 484 for disclosure information.
mineralization is loss of calcification inhibitors, such as fetuinA, matrix Gla protein, pyrophosphate, and osteopontin.9,10 Factors that trigger these events remain elusive, although multiple kinases including Src, phospholipase C (PLC), ERK1/2, p38 mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) may be important.11-13 To add to the complex kinase network, Zhan et al. report in this issue of the Canadian Journal of Cardiology, a putative role of mammalian target of rapamycin (mTOR) in vascular osteogenesis.14 mTOR is an intriguing candidate as a regulator of osteogenesis, because it is characteristically associated with cellular metabolism and longevity through its effects on cell growth, proliferation, and survival rather than on bone metabolism.15 Dysregulation of mTOR is linked to obesity, metabolic syndrome, cardiovascular disease, and cancer.15-17 The finding that it is also instrumental in molecular processes associated with calcification of vessels is novel and may provide an interesting link between cardiovascular disease and metabolic diseases, because vascular calcification occurs in both pathologic processes. Signalling through mTOR is complex. It is a serine/threonine kinase that belongs to the phosphatidylinositol 3-kinase (PI3K)-related kinase family and functions as an intracellular sensor for energy metabolism, nutrient availability, and cellular stresses and regulates cell growth and metabolism to adapt to environmental changes.18 mTOR forms a catalytic core of 2 multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2. mTORC1 and mTORC2 enzymatic activity is regulated by accessory proteins raptor and rictor, respectively.19,20 These adaptor proteins act as scaffold proteins to recruit mTOR substrates and regulators. mTOR is sensitive to the antifungal macrolide rapamycin, which forms a complex with FK506-binding protein that specifically inhibits mTORC1. Multiple upstream factors regulate mTOR, including growth factors, DNA damage, nutrients, and hypoxia. mTOR is a major activator of cell growth and proliferation. Once activated, mTORC1 phosphorylates p70 S6 kinase 1 (S6K1) and inhibits eIF-4Ebinding protein 1 (4E-BP1), leading to activation of ribosomal protein S6 and eIF2E, protein synthesis, and cell proliferation. mTORC1 also activates transcription factors SREP1, peroxisome proliferator-activated receptor (PPAR)
0828-282X/$ - see front matter Ó 2014 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cjca.2014.03.001
Montezano and Touyz mTOR: Novel Pathway in Vascular Calcification
483
Figure 1. Putative molecular mechanisms whereby mammalian target of rapamycin (mTOR) may influence vascular smooth muscle cell (VSMC) differentiation to an osteogenic phenotype that may contribute to vascular calcification.14 mTOR activation induces activation of S6 kinase (S6K1), which induces protein synthesis of osteogenic factors that promote calcification. mTOR also regulates lipid and glucose metabolism through welldescribed mechanisms involving peroxisome proliferator-activated receptor (PPAR) and hypoxia-inducible factor 1 (HIF-1).15-17 Rapamycin and adiponectin inhibit mTOR-induced activation of S6K1, thereby reducing osteogenic differentiation. Dashed lines indicate possible pathways.
gamma, and hypoxia-inducible factor 1 (HIF-1), thereby regulating lipid biogenesis and glycolysis. Because of the essential role of mTOR in anabolic metabolism, energy storage, and cell growth/survival it is not surprising that this pathway is important in adipogenesis and glucose and lipid metabolism.15-20 In obesity and metabolic disorders, mTORC1 is hyperactivated and may be associated with insulin resistance. In contrast, immunosuppressive drugs that inhibit mTOR, including cyclosporine A, tacrolimus, and rapamycin, enhance lipolysis and inhibit lipid storage and expression of lipogenic genes in adipose tissue, which may contribute to the development of dyslipidemia and insulin resistance associated with immunosuppressive therapy.21 Although mTOR is now considered a major player in lipid and glucose metabolism, its role in the vasculature is still largely unknown. There is some evidence that it regulates vascular Ca2þ channels and intracellular Ca2þ mobilization, thereby influencing vasoconstriction.20,22,23 mTOR has also been shown to play a role in VSMC proliferation and when dysregulated has been implicated in vascular remodelling.24 Zhan et al. advance the field of mTOR in the vasculature by showing that mTOR, through S6K1, promotes osteogenic differentiation, possibly predisposing to vascular calcification (Fig. 1).14 Exact mechanisms whereby mTOR induces mineralization are unclear, but increased synthesis of osteogenic factors, such as bone morphogenetic proteins, may be important. Studies investigating the PI3K/AKT-mTOR axis
in bone biological processes and mineralization are rare, although there is some evidence that this system influences chondrocyte maturation, endochondral ossification, and osteoblast differentiation through effects on activation of p70S6K and BMP expression.25-27 How these processes control mineralization processes remains to be elucidated. Of particular interest, adiponectin was found to inhibit mTOR-induced osteogenic differentiation.14 Adiponectin is the most abundant adipokine produced by adipocytes and has been implicated as a key player in adiposity, insulin resistance, and inflammation through its effects on lipid and glucose metabolism.28 As such, this mechanism may further link vascular calcification to metabolic disorders. Although the study under discussion in this issue of the Canadian Journal of Cardiology presents an interesting paradigm, there are some limitations in the study that warrant further consideration.14 First, studies were performed in isolated VSMCs in which only a few markers of osteoblastic differentiation were identified. This may not necessarily translate into vascular calcification in vivo. Second, although the study identifies a novel putative signalling pathway through mTOR, the exact molecular processes whereby mTOR-S6K1 influences activation of ion channels important in cellular Ca2þ, Naþ, and phosphate regulation, such as PiT-1 and PiT-2, are not addressed. However, despite these limitations, the study presents an interesting paradigm in which mTOR may be a link between metabolic disorders and
484
vascular calcification. Zhan et al. provide new insights into the molecular biology of vascular calcification, and more research is certainly warranted.14 It will be particularly interesting to know in whole animals whether inhibition of mTOR with adiponectin or rapamycin, which have been shown to influence adiposity and insulin resistance, would also ameliorate vascular calcification. Funding Sources Work from the authors’ laboratory was supported by grants 44018 and 57886, from the Canadian Institutes of Health Research, and grants from the British Heart Foundation. R.M.T. is supported through a British Heart Foundation Chair, and A.C.M. is supported through a Leadership Fellowship from the University of Glasgow. Disclosures The authors have no conflicts of interest to disclose. References 1. Sunkara N, Wong ND, Malik S. Role of coronary artery calcium in cardiovascular risk assessment. Expert Rev Cardiovasc Ther 2014;12(1): 87-94. 2. Jablonski KL, Chonchol M. Vascular calcification in end-stage renal disease. Hemodial Int 2013;17:S17-21. 3. Kramer CK, Zinman B, Gross JL, et al. Coronary artery calcium score prediction of all cause mortality and cardiovascular events in people with type 2 diabetes: systematic review and meta-analysis. BMJ 2013;346: f1654. 4. Briet M, Burns KD. Chronic kidney disease and vascular remodelling: molecular mechanisms and clinical implications. Clin Sci (Lond) 2012;123:399-416. 5. Massy ZA, Drüeke TB. Vascular calcification. Curr Opin Nephrol Hypertens 2013;22:405-12. 6. Crouthamel MH, Lau WL, Leaf EM, et al. Sodium-dependent phosphate cotransporters and phosphate-induced calcification of vascular smooth muscle cells: redundant roles for PiT-1 and PiT-2. Arterioscler Thromb Vasc Biol 2013;33:2625-32. 7. Shao JS, Aly ZA, Lai CF, et al. Vascular Bmp Msx2 Wnt signaling and oxidative stress in arterial calcification. Ann N Y Acad Sci 2007;1117: 40-50. 8. Shroff R, Long DA, Shanahan C. Mechanistic insights into vascular calcification in CKD. J Am Soc Nephrol 2013;24:179-89. 9. Herrmann M, Kinkeldey A, Jahnen-Dechent W. Fetuin-A function in systemic mineral metabolism. Trends Cardiovasc Med 2012;22:197-201. 10. 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.
Canadian Journal of Cardiology Volume 30 2014 11. Teplyuk NM, Galindo M, Teplyuk VI, et al. Runx2 regulates G proteincoupled signaling pathways to control growth of osteoblast progenitors. J Biol Chem 2008;283:27585-97. 12. McCarthy HS, Williams JH, Davie MW, Marshall MJ. Platelet-derived growth factor stimulates osteoprotegerin production in osteoblastic cells. J Cell Physiol 2009;218:350-4. 13. Montezano AC, Zimmerman D, Yusuf H, et al. Vascular smooth muscle cell differentiation to an osteogenic phenotype involves TRPM7 modulation by magnesium. Hypertension 2010;56:453-62. 14. Zhan J-K, Wang Y-J, Wang Y, et al. The mammalian target of rapamycin signalling pathway is involved in osteoblastic differentiation of vascular smooth muscle cells. Can J Cardiol 2014;30:568-75. 15. Yang Z, Ming XF. mTOR signalling: the molecular interface connecting metabolic stress, aging and cardiovascular diseases. Obes Rev 2012: 58-68. 16. Howell JJ, Ricoult SJ, Ben-Sahra I, Manning BD. A growing role for mTOR in promoting anabolic metabolism. Biochem Soc Trans 2013;41: 906-12. 17. Gilley R, Balmanno K, Cope CL, Cook SJ. Adaptation to chronic mTOR inhibition in cancer and in aging. Biochem Soc Trans 2013;41: 956-61. 18. Wrighton KH. Cell signalling: where the mTOR action is. Nat Rev Mol Cell Biol 2013;14:191. 19. Sciarretta S, Volpe M, Sadoshima J. Mammalian target of rapamycin signaling in cardiac physiology and disease. Circ Res 2014;114:549-64. 20. Liu Y, Vertommen D, Rider MH, Lai YC. Mammalian target of rapamycin-independent S6K1 and 4E-BP1 phosphorylation during contraction in rat skeletal muscle. Cell Signal 2013;25:1877-86. 21. Pereira MJ, Palming J, Rizell M, et al. The immunosuppressive agents rapamycin, cyclosporin A and tacrolimus increase lipolysis, inhibit lipid storage and alter expression of genes involved in lipid metabolism in human adipose tissue. Mol Cell Endocrinol 2013;365:260-9. 22. Martín-Cano FE, Camello-Almaraz C, Hernandez D, Pozo MJ, Camello PJ. mTOR pathway and Ca2þ stores mobilization in aged smooth muscle cells. Aging 2013;5:339-46. 23. MacMillan D. FK506 binding proteins: cellular regulators of intracellular Ca2þ signalling. Eur J Pharmacol 2013;700:181-93. 24. Goncharova EA. mTOR and vascular remodeling in lung diseases: current challenges and therapeutic prospects. FASEB J 2013;27:1796-807. 25. Sun H, Kim JK, Mortensen R, et al. Osteoblast-targeted suppression of PPARg increases osteogenesis through activation of mTOR signaling. Stem Cells 2013;31:2183-92. 26. Kim JK, Baker J, Nor JE, Hill EE. mTor plays an important role in odontoblast differentiation. J Endod 2011;37:1081-5. 27. Pantovic A, Krstic A, Janjetovic K, et al. Coordinated time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells. Bone 2013;52:524-31. 28. Silva TE, Colombo G, Schiavon LL. Adiponectin: A multitasking player in the field of liver diseases. Diabetes Metab 2014:S1262-3636.
