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Canadian Journal of Cardiology

Mammalian Target of Rapamycin: A Novel Pathway in Vascular Calcification

  • Augusto C. Montezano
    Affiliations
    Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, United Kingdom
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  • Rhian M. Touyz
    Correspondence
    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.
    Affiliations
    Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, United Kingdom
    Search for articles by this author
Published:March 07, 2014DOI:https://doi.org/10.1016/j.cjca.2014.03.001
      Calcification of arteries is common in advanced age, chronic kidney disease, diabetes mellitus, atherosclerosis, and hypertension and is an independent predictor of cardiovascular events.
      • Sunkara N.
      • Wong N.D.
      • Malik S.
      Role of coronary artery calcium in cardiovascular risk assessment.
      • Jablonski K.L.
      • Chonchol M.
      Vascular calcification in end-stage renal disease.
      • Kramer C.K.
      • Zinman B.
      • Gross J.L.
      • 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.
      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.
      • Sunkara N.
      • Wong N.D.
      • Malik S.
      Role of coronary artery calcium in cardiovascular risk assessment.
      • Jablonski K.L.
      • Chonchol M.
      Vascular calcification in end-stage renal disease.
      • Kramer C.K.
      • Zinman B.
      • Gross J.L.
      • 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.
      • Briet M.
      • Burns K.D.
      Chronic kidney disease and vascular remodelling: molecular mechanisms and clinical implications.
      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.
      • Massy Z.A.
      • Drüeke T.B.
      Vascular calcification.
      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.
      • Massy Z.A.
      • Drüeke T.B.
      Vascular calcification.
      • Crouthamel M.H.
      • Lau W.L.
      • Leaf E.M.
      • et al.
      Sodium-dependent phosphate cotransporters and phosphate-induced calcification of vascular smooth muscle cells: redundant roles for PiT-1 and PiT-2.
      • Shao J.S.
      • Aly Z.A.
      • Lai C.F.
      • et al.
      Vascular Bmp Msx2 Wnt signaling and oxidative stress in arterial calcification.
      Calcium uptake is also important and involves regulation by the calcium-sensing receptor by voltage-gated Ca2+ channels.
      • Shroff R.
      • Long D.A.
      • Shanahan C.
      Mechanistic insights into vascular calcification in CKD.
      Molecular mechanisms regulating these events involve upregulation of transcription factors such as core-binding factor 1α (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.
      • Massy Z.A.
      • Drüeke T.B.
      Vascular calcification.
      • Crouthamel M.H.
      • Lau W.L.
      • Leaf E.M.
      • et al.
      Sodium-dependent phosphate cotransporters and phosphate-induced calcification of vascular smooth muscle cells: redundant roles for PiT-1 and PiT-2.
      • Shao J.S.
      • Aly Z.A.
      • Lai C.F.
      • et al.
      Vascular Bmp Msx2 Wnt signaling and oxidative stress in arterial calcification.
      • Shroff R.
      • Long D.A.
      • Shanahan C.
      Mechanistic insights into vascular calcification in CKD.
      Another process contributing to vascular mineralization is loss of calcification inhibitors, such as fetuin-A, matrix Gla protein, pyrophosphate, and osteopontin.
      • Herrmann M.
      • Kinkeldey A.
      • Jahnen-Dechent W.
      Fetuin-A function in systemic mineral metabolism.
      • Luo G.
      • Ducy P.
      • McKee M.D.
      • et al.
      Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein.
      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.
      • Teplyuk N.M.
      • Galindo M.
      • Teplyuk V.I.
      • et al.
      Runx2 regulates G protein-coupled signaling pathways to control growth of osteoblast progenitors.
      • McCarthy H.S.
      • Williams J.H.
      • Davie M.W.
      • Marshall M.J.
      Platelet-derived growth factor stimulates osteoprotegerin production in osteoblastic cells.
      • Montezano A.C.
      • Zimmerman D.
      • Yusuf H.
      • et al.
      Vascular smooth muscle cell differentiation to an osteogenic phenotype involves TRPM7 modulation by magnesium.
      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.
      • 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.
      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.
      • Yang Z.
      • Ming X.F.
      mTOR signalling: the molecular interface connecting metabolic stress, aging and cardiovascular diseases.
      Dysregulation of mTOR is linked to obesity, metabolic syndrome, cardiovascular disease, and cancer.
      • Yang Z.
      • Ming X.F.
      mTOR signalling: the molecular interface connecting metabolic stress, aging and cardiovascular diseases.
      • Howell J.J.
      • Ricoult S.J.
      • Ben-Sahra I.
      • Manning B.D.
      A growing role for mTOR in promoting anabolic metabolism.
      • Gilley R.
      • Balmanno K.
      • Cope C.L.
      • Cook S.J.
      Adaptation to chronic mTOR inhibition in cancer and in aging.
      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.
      • Wrighton K.H.
      Cell signalling: where the mTOR action is.
      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.
      • Sciarretta S.
      • Volpe M.
      • Sadoshima J.
      Mammalian target of rapamycin signaling in cardiac physiology and disease.
      • Liu Y.
      • Vertommen D.
      • Rider M.H.
      • Lai Y.C.
      Mammalian target of rapamycin-independent S6K1 and 4E-BP1 phosphorylation during contraction in rat skeletal muscle.
      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-4E-binding 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) 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.
      • Yang Z.
      • Ming X.F.
      mTOR signalling: the molecular interface connecting metabolic stress, aging and cardiovascular diseases.
      • Howell J.J.
      • Ricoult S.J.
      • Ben-Sahra I.
      • Manning B.D.
      A growing role for mTOR in promoting anabolic metabolism.
      • Gilley R.
      • Balmanno K.
      • Cope C.L.
      • Cook S.J.
      Adaptation to chronic mTOR inhibition in cancer and in aging.
      • Wrighton K.H.
      Cell signalling: where the mTOR action is.
      • Sciarretta S.
      • Volpe M.
      • Sadoshima J.
      Mammalian target of rapamycin signaling in cardiac physiology and disease.
      • Liu Y.
      • Vertommen D.
      • Rider M.H.
      • Lai Y.C.
      Mammalian target of rapamycin-independent S6K1 and 4E-BP1 phosphorylation during contraction in rat skeletal muscle.
      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.
      • Pereira M.J.
      • 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.
      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 Ca2+ channels and intracellular Ca2+ mobilization, thereby influencing vasoconstriction.
      • Liu Y.
      • Vertommen D.
      • Rider M.H.
      • Lai Y.C.
      Mammalian target of rapamycin-independent S6K1 and 4E-BP1 phosphorylation during contraction in rat skeletal muscle.
      • Martín-Cano F.E.
      • Camello-Almaraz C.
      • Hernandez D.
      • Pozo M.J.
      • Camello P.J.
      mTOR pathway and Ca²+ stores mobilization in aged smooth muscle cells.
      • MacMillan D.
      FK506 binding proteins: cellular regulators of intracellular Ca2+ signalling.
      mTOR has also been shown to play a role in VSMC proliferation and when dysregulated has been implicated in vascular remodelling.
      • Goncharova E.A.
      mTOR and vascular remodeling in lung diseases: current challenges and therapeutic prospects.
      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).
      • 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.
      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.
      • Sun H.
      • Kim J.K.
      • Mortensen R.
      • et al.
      Osteoblast-targeted suppression of PPARγ increases osteogenesis through activation of mTOR signaling.
      • Kim J.K.
      • Baker J.
      • Nor J.E.
      • Hill E.E.
      mTor plays an important role in odontoblast differentiation.
      • 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.
      How these processes control mineralization processes remains to be elucidated.
      Figure thumbnail gr1
      Figure 1Putative 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.
      • 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.
      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 well-described mechanisms involving peroxisome proliferator-activated receptor (PPAR) and hypoxia-inducible factor 1 (HIF-1).
      • Yang Z.
      • Ming X.F.
      mTOR signalling: the molecular interface connecting metabolic stress, aging and cardiovascular diseases.
      • Howell J.J.
      • Ricoult S.J.
      • Ben-Sahra I.
      • Manning B.D.
      A growing role for mTOR in promoting anabolic metabolism.
      • Gilley R.
      • Balmanno K.
      • Cope C.L.
      • Cook S.J.
      Adaptation to chronic mTOR inhibition in cancer and in aging.
      Rapamycin and adiponectin inhibit mTOR-induced activation of S6K1, thereby reducing osteogenic differentiation. Dashed lines indicate possible pathways.
      Of particular interest, adiponectin was found to inhibit mTOR-induced osteogenic differentiation.
      • 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.
      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.
      • Silva T.E.
      • Colombo G.
      • Schiavon L.L.
      Adiponectin: A multitasking player in the field of liver diseases.
      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.
      • 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.
      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 Ca2+, Na+, 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 vascular calcification. Zhan et al. provide new insights into the molecular biology of vascular calcification, and more research is certainly warranted.
      • 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.
      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.

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