Mthfr deficiency induces endothelial progenitor cell senescence via
uncoupling of eNOS and downregulation of SIRT1
Catherine A. Lemarié,1Layla Shbat,1Chiara Marchesi,1Orlando J. Angulo,1Marie-Eve Deschênes,1
Mark D. Blostein,1,2Pierre Paradis,1and Ernesto L. Schiffrin1,2
1Lady Davis Institute for Medical Research and2Department of Medicine, Sir Mortimer B. Davis-Jewish General Hospital,
McGill University, Montreal, Quebec, Canada
Submitted 29 March 2010; accepted in final form 1 December 2010
Lemarié CA, Shbat L, Marchesi C, Angulo OJ, Deschênes
ME, Blostein MD, Paradis P, Schiffrin EL. Mthfr deficiency
induces endothelial progenitor cell senescence via uncoupling of
eNOS and downregulation of SIRT1. Am J Physiol Heart Circ
Physiol 300: H745–H753, 2011. First published December 17,
(HHcy) has been shown to induce endothelial dysfunction in part as a
result of enhanced oxidative stress. Function and survival of endothe-
lial progenitor cells (EPCs, defined as sca1?c-kit?flk-1?bone
marrow-derived cells), which significantly contribute to neovascular-
ization and endothelial regeneration, depend on controlled production
of reactive oxygen species (ROS). Mice heterozygous for the gene
deletion of methylenetetrahydrofolate reductase (Mthfr?/?) have a
1.5- to 2-fold elevation in plasma homocysteine. This mild HHcy
significantly reduced the number of circulating EPCs as well as their
differentiation. Mthfr deficiency was also associated with increased
ROS production and reduced nitric oxide (NO) generation in
Mthfr?/?EPCs. Treatment of EPCs with sepiapterin, a precursor of
tetrahydrobiopterin (BH4), a cofactor of endothelial nitric oxide syn-
thase (eNOS), significantly reduced ROS and improved NO produc-
tion. mRNA and protein expression of eNOS and the relative amount
of eNOS dimer compared with monomer were decreased by Mthfr
deficiency. Impaired differentiation of EPCs induced by Mthfr defi-
ciency correlated with increased senescence, decreased telomere
length, and reduced expression of SIRT1. Addition of sepiapterin
maintained cell senescence and SIRT1 expression at levels compara-
ble to the wild type. Taken together, these results demonstrate that
Mthfr deficiency impairs EPC formation and increases EPC senes-
cence by eNOS uncoupling and downregulation of SIRT1.
homocysteine; methylenetetrahydrofolate reductase; endothelium;
HYPERHOMOCYSTEINEMIA (HHcy) has been recognized as a major
risk factor of cardiovascular disease (20). Methylenetetrahy-
drofolate reductase (MTHFR) plays a key role in the remethy-
lation cycle, converting homocysteine to methionine (37). A
common variant in Mthfr, C677T, is associated with decreased
enzyme activity leading to HHcy in humans (10, 17). The lack
of this enzyme leads to the accumulation of homocysteine and
to HHcy. Although the mechanism by which HHcy injures the
vessel wall and induces atherosclerosis is poorly understood, a
growing body of evidence has suggested that endothelial dys-
function plays a major role (40).
Endothelial dysfunction ultimately induces an imbalance
between the severity of injury and the capacity for repair (13).
A variety of evidence has suggested that circulating endothelial
progenitor cells (EPCs) constitute one aspect of the repair
process (13, 35). Indeed, EPCs are regarded as having a key
role in the maintenance of endothelial integrity and the replace-
ment of apoptotic or damaged endothelial cells in response to
various cardiovascular risk factors (2, 26). EPCs are bone
marrow-derived mononuclear cells that are lineage negative
and express the surface marker stem cell antigen (Sca1) and the
endothelial marker VEGF receptor 2 (flk-1 in mice) or CD31.
Additionally, EPCs take up low-density lipoprotein and bind
lectin. EPCs contribute to vascular repair, although their exact
mechanism of action is still controversial. Most likely EPCs
enhance the reparative capacity of the endothelium adjacent to
the injury by releasing paracrine factors (14, 25, 41).
Patients with coronary artery disease exhibit reduced levels
and functional impairment of EPCs (12, 13, 32, 38), severe
endothelial dysfunction, and reduced bioavailability of endo-
thelium-derived nitric oxide (NO), due to the presence of
cardiovascular risk factors, advanced age, or both (4, 24, 34,
42). We previously showed (33) that mild HHcy was associ-
ated with increased generation of reactive oxygen species
(ROS) in the aortic wall, which could not be reduced by
treatment with antioxidants such as vitamin C. The oxidative
environment has been shown to be critical for the differentia-
tion and the mobilization of EPCs (25). Moreover, ROS are
important mediators of DNA damage that leads to cellular
In the present study, we hypothesized that mild HHcy would
affect EPC numbers and function by inducing premature se-
nescence, which could account for HHcy-induced endothelial
Animal experiments. All experimental procedures were approved
by the Animal Care Committee of the Lady Davis Institute for
Medical Research, McGill University, and followed the recommen-
dations of the Canadian Council of Animal Care. Mice heterozygous
for disruption of the Mthfr gene presenting mild HHcy were generated
at the Montreal Children’s Hospital Research Institute as reported
previously (7). Heterozygous Mthfr-deficient (Mthfr?/?) mice and
wild-type control mice were obtained by mating Mthfr?/?mice with
wild-type BALB/cAnNCrlBR mice (Charles River). Female Mthfr?/?
mice and littermate wild-type control mice aged 12–14 wk were
studied. We have already shown (7, 33) that Mthfr?/?mice have a
1.5- to 2-fold elevation in plasma homocysteine (7.7 ? 0.5 mmol/l)
compared with wild-type mice (4.3 ? 0.3 mmol/l).
Blood collection and mononuclear cell extraction. Blood from
wild-type and Mthfr?/?mice was collected from the heart in EDTA
tubes. RPMI was added to the blood (1:1 vol/vol), and mononuclear
cells were isolated by centrifugation on a Ficoll gradient. Mononu-
clear cells then underwent flow cytometry analysis.
Address for reprint requests and other correspondence: E. L. Schiffrin, Lady
Davis Inst. for Medical Research, Sir Mortimer B. Davis-Jewish General
Hosp., 3755 Côte-Ste-Catherine Rd., #B-127, Montreal, PQ, Canada H3T 1E2
Am J Physiol Heart Circ Physiol 300: H745–H753, 2011.
First published December 17, 2010; doi:10.1152/ajpheart.00321.2010.
0363-6135/11 Copyright © 2011 the American Physiological Societyhttp://www.ajpheart.orgH745
Bone marrow mononuclear cell culture. Bone marrow mononu-
clear cells (BM-MNCs) were obtained by flushing tibia and femur of
wild-type and Mthfr?/?mice. Low-density mononuclear cells were
then isolated by centrifugation on a Ficoll gradient. BM-MNCs (1.5 ?
106/ ml) were seeded on 11-mm cell culture dishes coated with bovine
plasma vitronectin (Calbiochem) and 0.1% gelatin (Sigma) and main-
tained in endothelial growth medium (EGM2; Cambrex). When indi-
cated, BM-MNCs were treated with or without sepiapterin (10
?mol/l) for 7 days. Nonadherent cells were then removed, and
adherent cells underwent analysis.
Flow cytometry analysis. Flow cytometry (Becton Coulter) was
used to characterize and quantify EPCs, either extracted from blood or
as cultured BM-MNCs, by the expression of fluorescein isothiocya-
nate (FITC)-conjugated monoclonal antibody against c-kit (2B8) and
phycoerythrin (PE)-conjugated monoclonal antibody against Sca1
(D7) or PE-conjugated monoclonal antibody against flk-1 (BD
Immunofluorescence. EPCs from wild-type mice were also charac-
Cruz) and a polyclonal flk-1 (VEGF receptor-2) antibody (Abcam). As
well, the uptake of 1,1=-dioctadecyl-3,3,3=,3=-tetramethylindocarbocya-
nine-labeled acetylated low-density lipoprotein (AcLDL-Dil) was evalu-
ated. For this evaluation, cells were incubated in medium containing
AcLDL-Dil (Invitrogen, Carlsbad, CA) at 37°C for 1 h. Cells were then
fixed with 2% paraformaldehyde and incubated with FITC-labeled
BS-1 lectin (Sigma). Dual-positive staining for both AcLDL-Dil and
BS-1 lectin characterized EPCs. EPC numbers were counted and
expressed in number of cells per well. Five replicates were counted for
each experimental condition. Three independent investigators evalu-
ated the number of EPCs per well by counting three randomly selected
high-power fields under epifluorescence microscopy. Results are ex-
pressed as percentages of total cell numbers.
Western blot analysis. For Western blot analysis, EPCs were
homogenized in lysis buffer (final concentrations in PBS: 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/l PMSF, 1
?g/ml leupeptin, 1 ?g/ml aprotinin, 1 ?g/ml pepstatin, and 1 mmol/l
NaVO4). Lysates from EPCs containing 20 ?g of protein were
separated by electrophoresis on polyacrylamide gels and transferred to
nitrocellulose membranes (Bio-Rad). Membranes were incubated with
anti-SIRT1 polyclonal antibodies (Upstate Biotech) overnight at 4°C.
A monoclonal anti-GAPDH antibody (Novus Biologicals) was used to
reprobe blots to confirm equal loading in lanes. Signals were revealed
by chemiluminescence (SuperSignal West Pico chemiluminescent
signal, Thermo Scientific, Rockford, IL) with the Molecular Imager
Chemidoc XRS system (Bio-Rad, Mississauga, ON, Canada) and
quantified by densitometry with Quantity one software (Bio-Rad).
Low-temperature SDS-PAGE. EPC extracts were prepared with
sodium dodecyl sulfate sample buffer in low-temperature conditions.
Samples were loaded on 7.5% polyacrylamide gels and subjected to
electrophoresis. Buffers and gels were cooled to 4°C, and the buffer
tank was placed in an ice bath during electrophoresis. eNOS mono-
mers and dimers were detected by Western blotting analysis using
eNOS monoclonal antibody (Transduction Lab).
Superoxide/nitric oxide measurements. To evaluate superoxide
generation, cells were incubated in the dark for 30 min at 37°C with
dihydroethidium (DHE; 2 ?M). The fluorescence (red signal) of
oxidized DHE products was detected with a fluorescent microscope
(Leica). Absolute numbers of DHE-positive cells were counted and
expressed as DHE-positive cells per total number of cells. Intracellu-
lar superoxide levels were evaluated with 5-(and 6)-chloromethyl-2=,7=-
dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Invit-
rogen), which becomes fluorescent when oxidized to dichlorofluorescein.
Briefly, BM-MNCs were seeded on black 96-well plates with or without
sepiapterin (10 ?M) for 7 days. After wash with PBS, cells were
incubated with CM-H2DCFDA and fluorescence was read every 2 min
for 60 min with a Fluoskan FL (Thermo Fisher). Data are expressed as
relative fluorescence units per 400,000 cells. NO production was evalu-
ated with 4-amino-5-methylamino-2=,7=-difluorofluorescein diacetate
(DAF-FM diacetate; Invitrogen). Absolute numbers of DAF-FM-positive
cells were counted and expressed as DAF-FM-positive cells per total
number of cells.
NADPH oxidase activity. The lucigenin-derived chemilumines-
cence assay was used to determine NADPH oxidase activity in EPC
homogenates after 7 days of culture as previously described (15).
NADPH (100 ?mol/l) was added to the suspension (100 ?l) contain-
ing lucigenin (5 ?mol/l). Chemiluminescence was measured for 1 s
every ?6 s for 3 min in an Orion II microplate luminometer (Berthold
Detection Systems, Pforzheim, Germany) and activity expressed as
relative light units per minute per microgram of protein.
Determination of cell viability by thiazolyl blue tetrazolium bro-
mide test. Four hundred thousand BM-MNCs were seeded in 96-well
plates and incubated 7 days at 37°C and 5% CO2 with or without
sepiapterin. On the day of the experiment, cells were incubated 6 h at
37°C and 5% CO2 with thiazolyl blue tetrazolium bromide (MTT;
Sigma) at 5 mg/ml after shaking for 5 min at 150 rpm. The metabolic
product of MTT (formozan) was resuspended in DMSO. Optical
density was read at 560 nm.
Quantitative RT-PCR. Expression at the mRNA level was deter-
mined by reverse transcription (RT) followed by quantitative real-
time PCR (qPCR). RNA was isolated with TRIzol reagent (Invitro-
gen). One microgram of total RNA was reverse transcribed with a
Quantitect RT kit (Qiagen, Mississauga, ON, Canada). qPCR was
performed with a QuantiTect SYBR Green PCR Kit (Qiagen) with the
Mx3005P real-time PCR cycler (Stratagene, La Jolla, CA). qPCR
results were normalized with small ribosomal protein 16 (S16) and
expressed as change relative to control. Primers were designed with
Fig. 1. Mthfr deficiency reduced circulating endothelial progenitor cells
(EPCs) in vivo. A: EPCs were characterized by flow cytometry analysis for the
late markers of EPC differentiation, Sca1 and flk-1. The number of double-
positive cells extracted from blood was significantly reduced in methylenetet-
rahydrofolate reductase (MTHFR)-deficient (Mthfr?/?; ?/?) compared with
wild-type (WT) mice (n ? 4/group). CTRL, control. B: reverse transcription
(RT)-quantitative real-time PCR (qPCR) showed that mRNA expression of
MTHFR is significantly reduced in Mthfr?/?compared with wild-type mice
(n ? 5/group). S16, small ribosomal protein. Data are presented as means ?
SE. *P ? 0.05 vs. wild-type mice.
HYPERHOMOCYSTEINEMIA AND EPCs
AJP-Heart Circ Physiol • VOL 300 • MARCH 2011 • www.ajpheart.org
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