Methotrexate-mediated activation of an AMPK-CREB-dependent pathway

a novel mechanism for vascular protection in chronic systemic inflammation

C. C. Thornton, F. Al-Rashed, D. Calay, G. M. Birdsey, A. Bauer, H. Mylroie, B. J. Morley, A. M. Randi, D. O. Haskard, J. J. Boyle, J. C. Mason

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Aims: Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used diseasemodifying drug for rheumatoid arthritis (RA), reduces disease-associated cardiovascular mortality. MTX increases intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4- carboxamide ribonucleotide which activates AMPactivated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPKregulated protective genes. 

Methods/results: In the (NZW×BXSB)F1 murine model of inflammatory vasculopathy, MTX 1 mg/kg/week significantly reduced intramyocardial vasculopathy and attenuated end-organ damage. Studies of human umbilical vein endothelial cells (HUVEC) and arterial endothelial cells (HAEC) showed that therapeutically relevant concentrations of MTX phosphorylate AMPKαThr172, and induce cytoprotective genes including manganese superoxide dismutase (MnSOD) and haem oxygenase-1 (HO-1). These responses were preserved when HUVECs were pretreated with tumour necrosis factor-α to mimic dysfunctional endothelium. Furthermore, MTX protected against glucose deprivationinduced endothelial apoptosis. Mechanistically, MTX treatment led to cyclic AMP response element-binding protein (CREB)Ser133 phosphorylation, while AMPK depletion attenuated this response and the induction of MnSOD and HO-1. CREB siRNA inhibited upregulation of both cytoprotective genes by MTX, while chromatin immunoprecipitation demonstrated CREB binding to the MnSOD promoter in MTX-treated EC. Likewise, treatment of (NZW×BXSB)F1 mice with MTX enhanced AMPKαThr172 phosphorylation and MnSOD, and reduced aortic intercellular adhesion molecule-1 expression. 

Conclusions: These data suggest that MTX therapeutically conditions vascular endothelium via activation of AMPK-CREB. We propose that this mechanism contributes to the protection against cardiovascular events seen in patients with RA treated with MTX.

Original languageEnglish
Pages (from-to)439-448
Number of pages10
JournalAnnals of the Rheumatic Diseases
Volume75
Issue number2
Early online date30 Dec 2014
DOIs
Publication statusPublished - 1 Feb 2016

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Cyclic AMP Response Element-Binding Protein
Methotrexate
Protein Kinases
Blood Vessels
Chemical activation
Inflammation
Superoxide Dismutase
Heme Oxygenase (Decyclizing)
Phosphorylation
Genes
Endothelial cells
Vascular Endothelium
Rheumatoid Arthritis
Ribonucleotides
Chromatin Immunoprecipitation
Human Umbilical Vein Endothelial Cells
Response Elements
Intercellular Adhesion Molecule-1
Adenosine Monophosphate
Protein Binding

ASJC Scopus subject areas

  • Rheumatology
  • Immunology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Allergy

Cite this

Methotrexate-mediated activation of an AMPK-CREB-dependent pathway : a novel mechanism for vascular protection in chronic systemic inflammation. / Thornton, C. C.; Al-Rashed, F.; Calay, D.; Birdsey, G. M.; Bauer, A.; Mylroie, H.; Morley, B. J.; Randi, A. M.; Haskard, D. O.; Boyle, J. J.; Mason, J. C.

In: Annals of the Rheumatic Diseases, Vol. 75, No. 2, 01.02.2016, p. 439-448.

Research output: Contribution to journalArticle

Thornton, CC, Al-Rashed, F, Calay, D, Birdsey, GM, Bauer, A, Mylroie, H, Morley, BJ, Randi, AM, Haskard, DO, Boyle, JJ & Mason, JC 2016, 'Methotrexate-mediated activation of an AMPK-CREB-dependent pathway: a novel mechanism for vascular protection in chronic systemic inflammation', Annals of the Rheumatic Diseases, vol. 75, no. 2, pp. 439-448. https://doi.org/10.1136/annrheumdis-2014-206305
Thornton, C. C. ; Al-Rashed, F. ; Calay, D. ; Birdsey, G. M. ; Bauer, A. ; Mylroie, H. ; Morley, B. J. ; Randi, A. M. ; Haskard, D. O. ; Boyle, J. J. ; Mason, J. C. / Methotrexate-mediated activation of an AMPK-CREB-dependent pathway : a novel mechanism for vascular protection in chronic systemic inflammation. In: Annals of the Rheumatic Diseases. 2016 ; Vol. 75, No. 2. pp. 439-448.
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AU - Thornton, C. C.

AU - Al-Rashed, F.

AU - Calay, D.

AU - Birdsey, G. M.

AU - Bauer, A.

AU - Mylroie, H.

AU - Morley, B. J.

AU - Randi, A. M.

AU - Haskard, D. O.

AU - Boyle, J. J.

AU - Mason, J. C.

PY - 2016/2/1

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N2 - Aims: Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used diseasemodifying drug for rheumatoid arthritis (RA), reduces disease-associated cardiovascular mortality. MTX increases intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4- carboxamide ribonucleotide which activates AMPactivated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPKregulated protective genes. Methods/results: In the (NZW×BXSB)F1 murine model of inflammatory vasculopathy, MTX 1 mg/kg/week significantly reduced intramyocardial vasculopathy and attenuated end-organ damage. Studies of human umbilical vein endothelial cells (HUVEC) and arterial endothelial cells (HAEC) showed that therapeutically relevant concentrations of MTX phosphorylate AMPKαThr172, and induce cytoprotective genes including manganese superoxide dismutase (MnSOD) and haem oxygenase-1 (HO-1). These responses were preserved when HUVECs were pretreated with tumour necrosis factor-α to mimic dysfunctional endothelium. Furthermore, MTX protected against glucose deprivationinduced endothelial apoptosis. Mechanistically, MTX treatment led to cyclic AMP response element-binding protein (CREB)Ser133 phosphorylation, while AMPK depletion attenuated this response and the induction of MnSOD and HO-1. CREB siRNA inhibited upregulation of both cytoprotective genes by MTX, while chromatin immunoprecipitation demonstrated CREB binding to the MnSOD promoter in MTX-treated EC. Likewise, treatment of (NZW×BXSB)F1 mice with MTX enhanced AMPKαThr172 phosphorylation and MnSOD, and reduced aortic intercellular adhesion molecule-1 expression. Conclusions: These data suggest that MTX therapeutically conditions vascular endothelium via activation of AMPK-CREB. We propose that this mechanism contributes to the protection against cardiovascular events seen in patients with RA treated with MTX.

AB - Aims: Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used diseasemodifying drug for rheumatoid arthritis (RA), reduces disease-associated cardiovascular mortality. MTX increases intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4- carboxamide ribonucleotide which activates AMPactivated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPKregulated protective genes. Methods/results: In the (NZW×BXSB)F1 murine model of inflammatory vasculopathy, MTX 1 mg/kg/week significantly reduced intramyocardial vasculopathy and attenuated end-organ damage. Studies of human umbilical vein endothelial cells (HUVEC) and arterial endothelial cells (HAEC) showed that therapeutically relevant concentrations of MTX phosphorylate AMPKαThr172, and induce cytoprotective genes including manganese superoxide dismutase (MnSOD) and haem oxygenase-1 (HO-1). These responses were preserved when HUVECs were pretreated with tumour necrosis factor-α to mimic dysfunctional endothelium. Furthermore, MTX protected against glucose deprivationinduced endothelial apoptosis. Mechanistically, MTX treatment led to cyclic AMP response element-binding protein (CREB)Ser133 phosphorylation, while AMPK depletion attenuated this response and the induction of MnSOD and HO-1. CREB siRNA inhibited upregulation of both cytoprotective genes by MTX, while chromatin immunoprecipitation demonstrated CREB binding to the MnSOD promoter in MTX-treated EC. Likewise, treatment of (NZW×BXSB)F1 mice with MTX enhanced AMPKαThr172 phosphorylation and MnSOD, and reduced aortic intercellular adhesion molecule-1 expression. Conclusions: These data suggest that MTX therapeutically conditions vascular endothelium via activation of AMPK-CREB. We propose that this mechanism contributes to the protection against cardiovascular events seen in patients with RA treated with MTX.

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DO - 10.1136/annrheumdis-2014-206305

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