Methotrexate-mediated activation of an AMPK-CREB-dependent pathway: a novel mechanism for vascular protection in chronic systemic inflammation

Aims Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used disease-modifying 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 AMP-activated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPK-regulated 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 deprivation-induced 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.

image of the whole section was generated using a JVC KY-F1030V digital camera. The area of fibrotic infarct was calculated by thresholding using Image J software to analyse the area of red pixels representing collagen deposition. In addition, acute infarct area was quantified using Image J and expressed as a percentage of the whole section. The two values were added together to give total infarct area as a proportion of myocardial section area.

Quantification of vasculopathy
Cardiac sections stained with periodic acid-Schiff (PAS) were examined blind by two investigators. A modified scoring system for the vasculopathy of the intramyocardial arteries and arterioles based on Berden et al 3 was constructed. The peri-adventitial leukocytic infiltrate was quantified by counting the number of cells in the adventitia of each intramyocardial artery and arteriole in one section for each animal. Deposits of PAS-positive material within the arterial wall were scored as follows: 0 = normal staining, 1 = increased staining of the basement membrane, 2 = increased basement membrane staining and presence of nodular deposits.

Aortic immunofluorescence
Transverse frozen sections of murine aorta were blocked in 3% BSA/PBS, then incubated with rat anti-mouse ICAM-1 monoclonal antibody YN-1 (American Type Culture Collection, Manassas, VA) or rat IgG 2a isotype control (BD Pharmingen), followed by an Alexa Fluor 546-conjugated goat anti-rat IgG secondary antibody (Invitrogen, Carlsbad, CA). A directly conjugated Alexa Fluor 488 rat antimouse CD31 antibody (MEC13.3; Biolegend, San Diego, CA) was used as an endothelial marker, and DRAQ5 (Biostatus, Loughborough, UK) as a nuclear stain. Slides were mounted with Fluoromount-G (Southern Biotech, Birmingham, AL) and visualized and quantified by confocal microscopy (LSM 510 Meta, Carl Zeiss, Oberkochen, Germany). Scan settings were set to optimize the signal/noise ratio for each emission wavelength and there was no detectable crossover between channels. The endothelial and adventitial areas were first identified using the anti-CD31 stained colocalized image and then, to measure ICAM-1, the mean fluorescence intensity of the area in the red channel (corresponding to AlexaFluor 546 staining) was recorded. Processing and quantification was performed with the Zeiss LSM Image Browser using the histogram function.

Endothelial Cell Culture
The study of human umbilical vein (HUVEC) and human arterial endothelial cells (HAEC; Promocell, Heidelberg, Germany) was carried out as previously described 4 . HAEC were studied at passage 7

Western Blotting
HUVEC or murine aortic tissue lysates were prepared in RIPA buffer (Thermoscientific, Waltham, MA) supplemented with complete protease and phosphatase inhibitors (Roche Diagnostics Ltd, West Sussex, UK). Following quantification of protein concentration by the Bradford assay (Bio-Rad, Hercules, CA), 10-20µg protein was loaded into 4-12% gels (Invitrogen, Carlsbad, CA) and run at 150V for 1.5h, followed by semi-dry transfer to PVDF membrane at 0.1A per membrane for 1-2h.
Membranes were blocked for 1h and incubated with the primary antibody at 4°C overnight. Following incubation with an appropriate HRP-conjugated secondary antibody and development with ECL chemiluminescence (GE Healthcare, Little Chalfont, UK), films were scanned using a Canoscan (Canon, Tokyo, Japan) and pixel density of bands quantified in Image J (National Institute of Health, USA) (antibodies are listed in Table 1).

Target Antigen Species Supplier Concentration
AMPKα

Quantitative real-time RT-PCR
RNA extraction from HUVEC was carried out using an RNEasy mini kit (Qiagen, Venlo, Limburg, Netherlands) including incubation with DNAse (Qiagen), and RNA concentration was measured using a Nanodrop 2000 (Thermoscientific). cDNA was synthesised using Q-script supermix (Quanta Biosciences, Gaithersburg, MD) on a T3 Thermocycler (Biometra GmBH, Göttingen, Germany). The real-time PCR was carried out in triplicate using a CFX96 Real-Time System C1000 Thermal Cycler (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were used as housekeeping genes. Primer sequences are shown in Table 2. Relative mRNA abundances were calculated using the comparative cycle threshold method.  Table 2. Primer sequences used for quantitative real-time RT-PCR.

RNA Interference
Transfection of HUVEC with short interfering RNA (siRNA) was performed using GeneFECTOR lipid transfection reagent (VennNOVA, Pompano Beach, FL). HUVEC were incubated in Optimem serumfree medium (Gibco) for 6h, along with scrambled control (Dharmacon, Epsom, UK) or specific siRNA (Qiagen) complexed with GeneFECTOR (sequences are available in Table 3 Table 3. siRNA sequences used for RNA interference.

Induction of apoptosis and detection by flow cytometry
Apoptosis of human umbilical vein endothelial cells (

Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express kit (Active Motif, Carlsbad, CA) as previously described 5 . HUVEC stimulated for 48h with MTX were fixed for 5 minutes in 1% formaldehyde, then detached by scraping. Following nuclear lysis, chromatin was sheared by sonication at 4°C for 4 minutes in 30s pulses using a Bioruptor (Diagenode, Denville, NJ).
Immunoprecipitation was performed at 4°C overnight using 2µg anti-CREB-1 antibody or IgG control (Cell Signaling Technologies, Danvers, MA). Following retrieval of chromatin, quantitative real-time PCR was performed using primers designed to isolate the two predicted CREB binding sites in the manganese superoxide dismutase (MnSOD; SOD2) promoter (MatInspector by Genomatix), and a negative control downstream site 2kbp from the transcription start (see Table 4 for sequences).
Results were analyzed using the comparative cycle threshold method, normalizing to input and IgG control for each condition.  Table 4. ChIP primer sequences.

Statistical Analyses
All data were analyzed using GraphPad Prism 4 (GraphPad Software, La Jolla, CA). Numerical data are presented as mean and standard error. Results were tested for normality using a d'Agostino and Pearson omnibus normality test and data that passed this test were analysed using paired or unpaired Student's t-tests or one-way ANOVA as appropriate. Normalized data were analysed using the single sample t-test. Data that were not normally distributed were analyzed using a Mann-Whitney test. Differences were considered significant at p<0.05.