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Cellular Biology
Uncoupling Protein 2 Impacts Endothelial Phenotype via
p53-Mediated Control of Mitochondrial Dynamics
Yukio Shimasaki, Ning Pan, Louis M. Messina, Chunying Li, Kai Chen, Lijun Liu,
Marcus P. Cooper, Joseph A. Vita, John F. Keaney Jr
Rationale: Mitochondria, although required for cellular ATP production, are also known to have other important
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
functions that may include modulating cellular responses to environmental stimuli. However, the mechanisms
whereby mitochondria impact cellular phenotype are not yet clear.
Objective: To determine how mitochondria impact endothelial cell function.
Methods and Results: We report here that stimuli for endothelial cell proliferation evoke strong upregulation of
mitochondrial uncoupling protein 2 (UCP2). Analysis in silico indicated increased UCP2 expression is common
in highly proliferative cell types, including cancer cells. Upregulation of UCP2 was critical for controlling
mitochondrial membrane potential (Δψ) and superoxide production. In the absence of UCP2, endothelial growth
stimulation provoked mitochondrial network fragmentation and premature senescence via a mechanism involving
superoxide-mediated p53 activation. Mitochondrial network fragmentation was both necessary and sufficient for
the impact of UCP2 on endothelial cell phenotype.
Conclusions: These data identify a novel mechanism whereby mitochondria preserve normal network integrity
and impact cell phenotype via dynamic regulation of UCP2. (Circ Res. 2013;113:891-901.)
Key Words: angiogenesis
■
endothelium ■ endothelial function ■ ischemia
■ mitochondrial uncoupling proteins
T
■
mitochondria
■
superoxides
proteins, such as cytochrome c,9 catalase,10 and cytochrome
oxidase.11 The mitochondrial release of proteins, such as apoptosis-inducing factor and the second mitochondrial activator of
caspase/DIABLO gene product, into the cytosol also regulates
genes important for apoptosis.12 Thus, mitochondrial events are
readily communicated to the nucleus to affect gene regulation.
The cellular responses to environmental stimuli also involve
mitochondria. Proteolytic processing of cell-surface receptors,
such as epidermal growth factor receptor tyrosine kinase B4, can
release intracellular domains that trigger mitochondrial release
of proapoptotic proteins.13 Energy deprivation responses, such as
AMP kinase activation, induce mitochondrial gene upregulation
that is critical for cellular stress adaptation.14 Upregulation of
endothelial vascular endothelial growth factor involves perinuclear mitochondrial clustering and the local production of mitochondrial •O2−.15 The latter has also been linked to the control of
mitogen-activated protein kinase phosphatases and the coordination of nuclear factor κB–dependent inflammatory responses.16
Finally, mitochondrial electron transport is known to impact the
activation state of cell-surface growth factor receptors.17 Thus,
he cellular requirement for ATP as a high-energy intermediate to fuel many critical cellular reactions is universal. The
production of ATP depends on an extracellular source of carbohydrates or lipids that are initially metabolized in the cytosol to
products that are then transported into mitochondria for oxidative phosphorylation to produce a proton gradient (ie, membrane
potential, Δψ) that powers ATP generation.1 Known mitochondrial functions extend beyond ATP production to include other
cellular processes, such as apoptosis,2 heme synthesis,3 calcium
homeostasis,4 inflammation,5 and development.6 Thus, mitochondria have the capacity to impact cellular phenotype via
energy-dependent and energy-independent mechanisms.
Editorial, see p 846
One means by which mitochondria can impact cellular phenotype is through participation in signaling paradigms, and
there is ample evidence from multiple organisms that mitochondria signal to the nucleus. Respiratory deficiency induced by
either pharmacologic7 or genetic8 means is known to affect gene
expression in the nucleus. Mitochondrial-derived heme coordinates nuclear regulation of genes encoding heme-dependent
Original received March 5, 2013; revision received June 27, 2013; accepted July 2, 2013. In May 2013, the average time from submission to first decision
for all original research papers submitted to Circulation Research was 15 days.
From the Division of Cardiovascular Medicine, Department of Medicine (Y.S., N.P., C.L., K.C., L.L., M.P.C., J.F.K.); Division of Vascular Surgery,
Department of Surgery, University of Massachusetts Medical School, Worcester, MA (L.M.M.); and the Department of Medicine, Boston University
School of Medicine (J.A.V.).
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.
113.301319/-/DC1.
Correspondence to John F. Keaney Jr, MD, Division of Cardiovascular Medicine, University of Massachusetts Medical School, 55 Lake Ave N, Room
S3-855, Worcester, MA, 01655. E-mail [email protected]
© 2013 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.113.301319
891
892 Circulation Research September 13, 2013
Nonstandard Abbreviations and Acronyms
Δψ
BAEC
Drp1
Fis1
MLEC
Mfn1
Mfn2
Opa1
ROS
UCP2
mitochondrial membrane potential
bovine aortic endothelial cell
dynamin-related protein 1
fission 1
murine lung endothelial cell
Mitofusin 1
Mitofusin 2
optic atrophy 1
reactive oxygen species
uncoupling protein 2
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the mitochondrion is emerging as an important organelle for the
coordination of cellular environmental responses.
Although mitochondria clearly impact how cells respond to
the environment, the mechanisms involved in this process are
not well-understood. Many mitochondrial actions are linked to
the electron transport chain and the resultant membrane gradient, Δψ. Control of Δψ involves the relative availabilities of
substrates for electron transport (nicotinamide adenine dinucleotide and flavin adenine dinucleotide-2), respiration (O2),
and ATP synthesis (ADP),1 as well as any proton leak.18 The
latter is largely regulated via uncoupling proteins that belong
to the mitochondrial carrier superfamily.18 Herein, we report
that endothelial cell proliferation and angiogenesis involve
upregulation of mitochondrial uncoupling protein 2 (UCP2)
to reduce Δψ and limit mitochondrial •O2− that otherwise promotes p53-dependent mitochondrial fragmentation resulting
in premature senescence. These findings suggest a new function for UCP2 that has broad implications for processes that
involve the vascular endothelium.
Methods
Endothelial Cell Culture and Transfection
Bovine aortic endothelial cells (BAECs) were purchased from
Genlantis (San Diego, CA) and were cultured in endothelial basal
medium supplemented with EGM-MV Bullet Kit from Lonza
(Walkersville, MD). Before each experiment, BAECs were made
quiescent by 24-hour incubation in low-serum medium (endothelial
basal medium supplemented with 0.1% or 0.4% fetal bovine serum).
Human aortic endothelial cells were from Lonza and were cultured
as described.14 Murine lung endothelial cells (MLECs) from mice
of both sexes were isolated as described,19 and cultured on gelatinor collagen-coated plates and grown in MLEC medium containing
20% fetal bovine serum, 38% Dulbecco's modified eagle medium,
38% Ham’s F-12 with 100-μg/mL endothelial cell growth supplement (ECGS, Biomedical Technologies, Stoughton, MA), 4-mmol/L
L-glutamine, 100-μg/mL heparin, and penicillin/streptomycin.
Endothelial purity was confirmed by staining with 1,1'-dioctadecyl
– 3,3,3',3'-tetramethyl-indocarbocyanine perchlorate acetylated lowdensity lipoprotein (Biomedical Technologies, Stoughton, MA) and
anti-CD31 antibody (BD Biosciences, San Jose, CA).
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from cells and tissues with the RNeasy Mini
Kit (Qiagen) or TRIzol reagent (Invitrogen), and 1 μg of t­otal RNA was
reverse transcribed with oligo(dT) primers for cDNA synthesis. Realtime polymerase chain reaction was performed in the iQ5 real-time
polymerase chain reaction detection system (Bio-Rad Laboratories) and
the products were detected using either SYBR Green dyes (Bio-Rad
Laboratories) or TaqMan probes of the TaqMan Gene Expression
Assays for specific genes (Applied Biosystems, Foster City, CA).
Transfections
For gene overexpression, BAECs or MLECs were incubated with adenoviruses at 50 to 100 multiplicity of infection as described.14,20 For gene
silencing, BAECs were transfected with 1.3 μg of small interfering RNA
oligonucleotides (Thermo Scientific Dharmacon, Lafayette, CO) against
human UCP2 (Cat. Nos. D-005114-01, -02, -03, and -04); human superoxide dismutase 2 (SOD2; D-009784-03, -04, -19, and -20); or negative control (D-001210-02, -03, -04, and -05) as described.14 Likewise,
MLECs were transfected with mouse mitofusin 1 (Mfn1) (J-065399-09,
-10, -11, and -12); mouse mitofusin 2 (Mfn2) (J-046303-05, -06, -07, and
-08); mouse p21 (J-058636-05, -06, -07, and -08); mouse p53 (J-04064209, -10, -11, and -12); or the nontargeting pool (D-001810-10-05).
Cell Proliferation and Migration Assays
DNA synthesis was directly measured via the 3H-thymidine incorporation assay. Cell proliferation was determined with CyQUANT GR
fluorescent dye (Invitrogen Molecular Probes, Eugene, OR) to determine the relative cell number with a fluorescence microplate reader
(Gemini XPS, Molecular Devices). Cell migration was assayed with
the in vitro scratch assay in which a monolayer of quiescent cells was
uniformly scratched and the rate of cell migration to close the void
was evaluated 20 hours after wounding using ImageJ.
Mitochondrial Membrane Potential and
Superoxide Measurements
Mitochondrial membrane potential (Δψ) was determined using 2
complementary fluorescent methods. Cells were incubated with
1-μmol/L 5, 5´, 6, 6´-tetrachloro-1, 1´, 3, 3´-tetraethylbenzimidazolcarbocyanine iodide (JC-1) for 30 minutes and Δψ estimated as the
fluorescence ratio of JC-1 aggregates (red, excitation 550 nm, emission 600 nm) to monomers (green, excitation 485 nm, emission 535
nm) formed as a function of inner mitochondrial membrane potential.21 For measurement of mitochondrial superoxide (•O2−), cells were
loaded with 5-μmol/L MitoSOX Red (Invitrogen) for 10 minutes and
the fluorescence (excitation 396 nm; emission 510 nm) determined.
Live cells were labeled with 0.5-μmol/L MitoSOX for 20 minutes
and the fluorescence images at both 405- and 514-nm excitation were
captured using an Eclipse TE2000-S fluorescence microscope (Nikon,
Melville, NY) with a CCD camera using a SPOT Insight 2MP Firewire
Color Mosaic (Diagnostic Instruments, Sterling Heights, MI).
Antibodies and Immunoblotting
Antibodies against cytochrome c oxidase subunit IV, p21cip/waf,
p16ink16a, phospho-p53 (mouse Ser18), phospho-c-Jun (Ser63), phospho p38 mitogen-activated protein kinase (Thr180/Tyr182), and -rabbit or mouse IgG were purchased from Cell Signaling Technology.
Antibodies against cytochrome c were from BD Biosciences. We obtained antibodies against UCP1, UCP2, and UCP3 from Fitzgerald
Industries International (Acton, MA) and specific UCP2 antibodies (N-19 and A-19) and native p53 antibody from Santa Cruz
Biotechnology (Santa Cruz, CA). We obtained SOD2 antibody
from Millipore (Temecula, CA) and actin antibody from SigmaAldrich (St. Louis, MO). Protein extracts in DTT-containing SDS
sample buffer were separated in 10% or 12% SDS-polyacrylamide
gels and transferred to Hybond ECL nitrocellulose membranes (GE
Healthcare, Piscataway, NJ). Immunoblotting was then performed
and quantified as described.22 In cases where loading controls produced paired bands, both were used for quantification.
Oxygen Consumption and Lactate
Using a Clark-type oxygen electrode (Hansatech), respiration in
whole cells was quantified as previously described.23,24 Briefly, 1×106
cells were resuspended in 1 mL of respiration medium (Dulbecco`s
phosphate-buffered saline, 2 mmol/L glucose, 1 mmol/L pyruvate,
2% fatty acid-free bovine serum albumin). To obtain proton leak,
oligomycin was added to a final concentration of 2 µmol/L. To measure maximal respiration, carbonyl cyanide 4-(trifluoromethoxy)
Shimasaki et al UCP2 and Endothelial Function 893
phenylhydrazone was added to a final concentration of 2.4 µmol/L.
Nonmitochondrial respiration, obtained by adding myxothiazol to a
final concentration of 4 µmol/L, was subtracted from total respiration
measurements. For lactate, accumulation was determined by a lactate
fluorometric assay kit (MBL International, Woburn, MA) according
to the manufacturer’s instructions and the concentration of secreted
lactate was normalized to the cell number in each sample.
Capillary Sprouting Assay in Aortas
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Thoracic aorta was placed in endothelial basal medium-2 as described,25 periaortic tissue was carefully removed, and then the aorta
was cleaned and sliced into 1 mm-long rings. Rings were then embedded in liquid collagen gel in 48-well plates (BD Biosciences), incubated at 37°C for 1 hour to polymerize the collagen, and the solid gel
covered with MLEC medium diluted 1:2 with Dulbecco's modified eagle medium. Each aortic ring was examined daily and capillary sprouts
counted along the sample perimeter under ×100 and ×200 magnification. Vascular sprouts were distinguished from fibroblasts via morphology as described26 and CD31 staining. To infect aortic rings with
adenoviral vectors, each ring was embedded in liquid collagen gel
containing respective vectors (adenovirus–UCP2, adenovirus–SOD2,
etc) at 1.0×108 pfu before polymerized. Transfection was validated in
separate experiments with green fluorescent protein adenovirus (AdGFP) transfection and after 4 days by fixation of collagen gel-embedded tissue with 4% formaldehyde in phosphate-buffered saline for 10
minutes at 4°C. Fixed sections underwent immunofluorescence staining with anti-GFP and anti-CD31 antibody and counterstained with
4',6-diamidino-2-phenylindole before fluorescence imaging.
Animals and Hindlimb Ischemia Model
Heterozygous UCP2-null animals27 on the C57 background were obtained from Dr Bradford Lowell (Harvard Medical School) and bred
to homogeneity with age-matched controls. SOD2 heterozygous mice
were obtained from Jackson Laboratories and bred to obtain heterozygous and control animals. All experimental protocols were approved
by the Institutional Animal Care and Use Committee of University of
Massachusetts Medical School. Male mice at 8 to 12 weeks of age
were anesthetized with intraperitoneal injection of combination of
100 mg/kg ketamine hydrochloride and 5 mg/kg xylazine (Webster
Veterinary, Devens, MA) before surgery. Unilateral hindlimb ischemia
in the left leg was introduced in the mice as described.28 In selected experiments, 100 µL (2.0×108 pfu) of adenoviral vectors encoding UCP2,
or β-glactosidase (LacZ) was injected into 5 different sites of the ischemic thigh muscles, such as adductor longus, adductor magnus, and
adductor brevis muscles. Hindlimb tissue perfusion was assessed with
Moor LDI2-IR laser-Doppler imaging system (Moor Instruments,
Devon, UK). Blood flow images were obtained under conditions of
constant body temperature (36±1.0°C) and average hindlimb blood
flow was expressed as the ratio of ischemic to nonischemic foot flow
to account for minor variations in imaging conditions.
Cell Cycle Analysis
Endothelial cells at 50% confluence were synchronized in 0.4%
­serum overnight. Cells were then cultured in complete medium and
at 24 and 48 hours harvested, stained with propidium iodide and subjected to fluorescence-activated cell sorting analysis as described.29
Cellular Senescence Assay
MLECs were seeded onto 0.1% gelatin-coated 12-well plates and
maintained for 14 days under normal conditions. Senescence was
assayed as senescence-associated β-galactosidase activity using the
senescence β-galactosidase staining kit (Cell Signaling Technology).
Both bright-field and phase-contrast pictures were viewed through ×4
or ×10 objective on a microscope on the Eclipse TE2000-S microscope
as above and images processed with SPOT Advanced Version 4.6 imaging software (Diagnostic Instruments Inc). Senescence-associated βgalactosidase–positive cells were quantified with the ImageJ software.
Mitochondrial Length Measurements
Mitochondrial length was measured as an index of mitochondrial
fragmentation. Live endothelial mitochondria were labeled with
100-nmol/L MitoTracker Green FM for 30 minutes and the fluorescence images were captured using a ×100 oil immersion objective to
acquire high-resolution images of mitochondria as described.30 To determine mitochondrial length, ImageJ was applied in a blinded fashion to the measurement for each frame of a selected region of interest
as described for determining DNA contour lengths.31
Statistical Analysis
All data are expressed as mean±SE and the numbers of independent experiments are indicated. Statistical comparisons were conducted between 2 groups by use of Student t test or Mann–Whitney
U test as appropriate. Multiple groups were compared with either
1-way Kruskal–Wallis or ANOVA with a post hoc Tukey–Kramer
multiple comparisons test as indicated in legends. A P value <0.05
was considered significant. All statistics were done using StatView
version 5.0 (SAS Institute, Cary, NC) or GraphPad Prism version 5
(GraphPad Software, La Jolla, CA).
Results
Dynamic Modulation of UCP2 and Δψ With
Endothelial Proliferation
Quiescent BAEC monolayers stimulated to proliferate with
fetal bovine serum exhibited a reduction in Δψ (Figure 1A),
whereas increasing cell confluence was associated with an
increase in Δψ (Figure 1B and 1C). Because mitochondrial
UCPs are implicated in Δψ regulation,18 we probed endothelial UCP expression and observed mRNA and protein only for
UCP2 and UCP3 (Figure 1D). We then observed that UCP2
is upregulated with endothelial proliferation (Figure 1A) and
downregulated with increasing confluence, with no dynamic
regulation of UCP3 (Figure 1B and 1E). We could recapitulate the proliferation-induced changes in Δψ by molecular
manipulation of UCP2 (Figure 1F) and UCP2-null cells had
increased Δψ (Figure 1G). Thus, our data indicate that the
proliferation-induced changes in Δψ can be explained, at least
in part, by dynamic regulation of UCP2.
UCP2 and Endothelial Metabolism
Because we found that UCP2 is dynamically regulated in the
endothelium, we probed its implications for basic mitochondrial functions. We found that UCP2-null cells had a trend
for lower basal respiration rate (*P<0.07) than wild-type
cells and similar rates of basal proton leak. UCP2-null cells
also had similar maximal respiration (2.08±0.30) than wildtype cells (2.61±0.58; P=0.23 by 2-tailed t test; Figure 2A).
Proliferating UCP2-null cells also produced lesser lactate
than wild-type cells (Figure 2B), consistent with a lower rate
of glycolysis.32 Suppression of UCP2 was associated with
reduced ATP levels (Figure 2C) and endothelial ATP levels
seemed greatest in endothelium with the highest proliferation
rate (Figure 2D). Collectively, these data indicate that UCP2null cells have more prominent perturbations in glycolysis
than respiration, and that endothelial ATP levels vary as a
function of proliferation.
UCP2 Regulates Endothelial Phenotype via
Changes in Δψ
To gain insight into the potential function of UCP isoforms, we
examined UCP mRNA in silico as a function of cell type. We
found that UCP1 exhibited relatively homogeneous mRNA
expression across multiple cell types as did UCP3, with the
exception of a 3-fold higher expression in skeletal muscle
894 Circulation Research September 13, 2013
(kDa)
33
42
1
0
2
1
0
0.5 5 10
FBS (%)
D
(%)
200
*
150
100
50
0
50 60 70 80 90 100
50 60 70 80 90 100
Cell Confluence (%)
E
Cell Confluence (%)
F
(kDa) UCP2 UCP3
35
30
0.2
0.1
0
G
100
80
*
60
* *
*
40
20
0
50 60 70 80 90 100
Cell Confluence (%)
Figure 1. Regulation of endothelial uncoupling protein 2 (UCP2) and Δψ with proliferation. Bovine aortic endothelial cells (BAECs)
were (A) stimulated with fetal bovine serum (FBS) or (B) cultured to specific confluence before assessment of Δψ by JC-1 fluorescence
and expression of UCP2, UCP3, cytochrome c oxidase IV (COX IV), or actin by immunoblotting. Carbonyl cyanide m-chlorophenyl
hydrazone (CCCP) of 5-μmol/L was used as a control for the lower limit of Δψ. Data represent n=3; *P<0.05 for trend from 1-way ANOVA.
C, Mitochondrial Δψ by tetramethylrhodamine ethyl ester (TMRE) corrected for mitochondrial mass. n=3; *P<0.05 for trend from 1-way
ANOVA. D, Expression of UCP2 and UCP3 mRNA and protein in BAECs. E, Densitometric analysis of UCP2 protein by immunoblot as a
function of confluence. n=5; *P<0.05 vs 50% by ANOVA with Tukey–Kramer post hoc test. F, BAECs (control [CTL]) were transfected with
UCP2 (Ad-UCP2 at the indicated multiplicity of infection [MOI]), β-galactosidase (Ad-LacZ; 100 MOI), or the indicated small interfering
RNA (siRNA) followed by assessment for Δψ or protein levels of transfected or endogenous UCP2, COX IV, or actin by immunoblotting.
n=3; *P<0.05 vs Ad-LacZ or siRNA control (siCTL). G, Mitochondrial Δψ by TMRE as in C. n=3; *P<0.05 by unpaired t test. Ad indicates
adenovirus; GADPH indicates glyceraldehyde 3-phosphate dehydrogenase; siUCP2, small interfering UCP2; and WT, wild-type.
(Online Figure I). In contrast, UCP2 mRNA expression varied
more than 50-fold as a function of cell type with the highest transcript levels in rapidly dividing cells, including those
harboring erythroid (CD71), endothelial progenitor (CD34),
and endothelial angiogenic (CD105) markers (Online Figure
A
250
Lactate (pmol/cell)
Relative O2 uptake
2
1
200
150
100
*
50
0
0
Basal
Oligo
D
(%)
100
*
80
60
40
20
0
WT
FCCP
SiCTL
siUCP2
UCP2-/-
(%)
(Cellular ATP Level)
C
I). Based on this latter association, we tested known stimuli
for angiogenesis such as vascular endothelial growth factor
and AMP kinase33 and found UCP2 upregulation (Figure 3A).
Serum- and vascular endothelial growth factor–mediated
UCP2 upregulation was associated with c-Jun N-terminal
B
WT
UCP2-/-
3
(Cellular ATP Level)
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UCP2 / Actin (% of 50)
*
3
*
JC-1 (Δψ)
JC-1 (Δψ)
Actin
2
UCP2
UCP3
COX IV
Actin
33
34
16
UCP2
42
3
C
(kDa)
(UCPs mRNA / GAPDH mRNA)
B
(TMRE / MitoGreen)
A
100
80
60
40
20
0
*
50 60 70 80 90 100 Oligo
Cell Confluence (%)
Figure 2. Metabolic signature of uncoupling
protein 2 (UCP2)-null endothelium. A, Oxygen
consumption was determined in wild-type (WT)
and UCP2-null murine lung endothelial cells
(MLECs) in the presence or absence of oligomycin
(oligo) or carbonyl cyanide 4-(trifluoromethoxy)
phenylhydrazone (FCCP) using a Clark electrode
as described in Methods section. B, Media from
wild-type or UCP2-null MLECs in 0.1% serum
was analyzed for lactate content and expressed
as a function of cell count. n=3; *P<0.01 vs WT by
unpaired t test. C, Bovine aortic endothelial cell
(BAEC) ATP levels as a function of UCP2 status
as described in Methods section. n=3; *P<0.01 vs
siRNA control (siCTL) by unpaired t test. D, BAEC
ATP levels as a function of confluence or oligomycin
(oligo) treatment. n=4; *P<0.01 for trend by 1-way
ANOVA. siUCP2 indicates small interfering UCP2.
Shimasaki et al UCP2 and Endothelial Function 895
B
(kDa)
33
UCP2
24
SOD2
16
COX IV
15
Cyt c
42
Actin
D
Cell Migration (cell #)
400
*
300
200
*
100
0
Cells / well (x 104)
C
*
50
0
CTL VEGF Ad- Ad- Control UCP2
LacZ UCP2 siRNA siRNA
E
48
WT
UCP2-/-
40
32
24
*
16
0
1
3
5
(days)
20
16
12
*
8
4
0
0
4
5
G
6 7
(days)
8
9
UCP2-/-
UCP2-/- + Ad-UCP2
UCP2-/- + Ad-LacZ
UCP2-/-
20
16
12
*
8
Perfusion
(ischemic/non-ischemic)
WT
WT
UCP2-/-
*
100
H
(Sprouts / aorta)
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F
(Sprouts / aorta)
150
8
Ad- Ad- Control UCP2
LacZ UCP2 siRNA siRNA
*
200
Cell Migration (cell #)
Cell Proliferation (%CTL)
A
7
5
7
(days)
*
30
20
10
WT UCP2-/-
WT
UCP2-/-
1.2
1.0
0.8
0.6
*
0.4
0.2
0
Pre 0 1 3 7
14
21
(Post-Operative Day)
Pre-op
POD 0
POD 7
POD 14
N
N
N
28
POD 28
WT
UCP2
-/-
I
0
3
40
0
9
4
1
50
9
kinase activation and inhibition attenuated both UCP2 upregulation and vascular endothelial growth factor–mediated
endothelial cell proliferation (Online Figure II). We also manipulated endothelial UCP2 levels and found that endothelial
cell proliferation (Figure 3B) and migration (Figure 3C) were
directly related to UCP2. Consistent with these observations,
UCP2-null endothelium exhibited impaired proliferation
(Figure 3D) and migration (Figure 3E), without any compensatory upregulation of UCP3 (Online Figure IIIA).
Because endothelial proliferation and migration are features
of angiogenesis, we examined the impact of UCP2 in a capillary sprout formation assay, an in vitro model of early angiogenesis. Wild-type aortic segments exhibited a higher rate and
total extent of capillary sprout formation than did UCP2-null
aortic segments (Figure 3F), and reconstitution of UCP2 into
UCP2-null endothelium rescued the defect in capillary sprout
formation (Figure 3G). Similarly, capillary sprout formation
from UCP2-null adipose tissue was impaired relative to wildtype, and UCP2 reintroduction also rescued this defect (Online
I
I
N
I
Figure 3. Uncoupling protein 2 (UCP2)
modulates endothelial cell proliferation
and migration. A, Wild-type (WT) murine lung
endothelial cells (MLECs) were treated with
aminoimidazole carboxamide ribonucleotide
(AICAR; 0.5 mmol/L) or vascular endothelial
growth factor (VEGF; 25 ng/mL) as indicated
for 24 hours followed by immunoblots for the
indicated proteins. Actin, cytochrome c (Cyt
c), and cytochrome c oxidase subunit IV were
loading controls for cytosol and mitochondria.
B, Bovine aortic endothelial cell (BAEC)
proliferation by [3H]-thymidine incorporation
in response to VEGF or UCP2 manipulation
as indicated. Data are normalized to the
VEGF vehicle control. n=4 to 5; *P<0.05 vs
respective controls by Mann–Whitney U test.
C, BAECs underwent UCP2 manipulation
as indicated and migration assessed 20
hours after scratch. n=3 to 5; *P<0.01 vs
respective control by Mann–Whitney U test. D,
Proliferation of WT and UCP2−/− MLECs by cell
count. n=4; *P<0.05 vs WT by 2-way repeated
measures ANOVA. E, Migration of WT and
UCP2−/− MLECs after scratch wounding.
n=5; *P<0.01 vs WT by Mann–Whitney U
test. F, Aortic segments from the indicated
genotypes implanted in collagen gel after
7 days (bar=500 μm) with capillary sprout
counts as a function of time. n=6; *P<0.001 vs
WT by 2-way repeated-measures ANOVA. G,
Capillary sprouting in UCP2−/− aortic segments
in collagen gel containing no additions (contol
[CTL]) or the indicated adenoviral vector. n=6
per group; *P<0.01 vs vehicle or Ad-LacZ by
2-way repeated measures ANOVA. (H) Blood
flow recovery in WT and UCP2−/− mice with
unilateral femoral artery excision expressed
as a fraction ratio of the ischemic (I) vs
nonischemic (N) limbs with representative
images of laser-Doppler tissue perfusion in
hindlimbs. n=4; *P=0.02 vs WT by 2-way
repeated measures ANOVA. Ad indicates
adenovirus; POD, postoperative day; siRNA,
small interfering RNA; and SOD2, superoxide
dismutase.
Figure IIIB and IIIC). To determine the physiological relevance
of our in vitro observations, we examined hindlimb ischemiainduced angiogenesis and observed a lower rate of blood flow
recovery in UCP2-null mice compared with wild type controls
(Figure 3H) that was largely rescued by adenoviral-mediated
restoration of UCP2 expression (Online Figure IIID).
UCP2 Modulates Endothelial Proliferation Via ΔψDependent Changes in Mitochondrial •O2−
Because Δψ modulates mitochondrial •O2− and UCP2 can
impact Δψ,18 we investigated mitochondrial •O2− as a function of cell proliferation. Both BAECs (Figure 4A) and
murine endothelial cells (Figure 4B) exhibited an inverse
relation between mitochondrial •O2− and cell proliferation
rate and this response was accentuated in UCP2-null endothelium (Figure 4B). Reconstitution of UCP2 into UCP2-null
endothelium (Figure 4C) normalized mitochondrial •O2− and
forced overexpression of UCP2 in wild-type cells attenuated
mitochondrial •O2− (Figure 4C). Treatment of either wild-type
896 Circulation Research September 13, 2013
Superoxide (RFU)
50%
C
WT
UCP2-/-
80
*
60
40
20
0
50
90%
60
70
80 90 100
*
†
2
†
1
0
Ad-LacZ Ad-UCP2 Ad-SOD2
200
10
5
16
12
*
8
*
100
50
0
Ad- Ad- AdLacZ UCP2 SOD2
H
Proliferation (%CTL)
15
Superoxide (RFU)
Cells / well (x 104)
20
*
150
CTL
WT
SOD2+/–
20
6
*
4
*
2
*
*
150
100
0
50
0
4
0
1
4
7
(days)
*
*
100
Ad- Ad- Control SOD2
LacZ SOD2 siRNA siRNA
*
(Sprouts / aorta)
400
16
14
12
10
8
6
4
2
0
SOD2+/-
SOD2+/-
J
Superoxide (RFU)
I
10
K
Wild-type
SOD2+/25
20
15
10
5
*
0
CTL SOD2
siRNA siRNA
0
4
5
6
(days)
7
Perfusion
(ischemic/non-ischemic)
WT SOD2+/-
Cell Migration (cell #)
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Superoxide (RFU)
*
25
0
0
*
3
G
F
30
200
†
Cell Confluence (%)
E
300
4
D
WT
UCP2-/-
Proliferation (%CTL)
B
Superoxide (RFU)
A
1.2
WT
SOD2+/- + Ad-UCP2
SOD2+/- + Ad-LacZ
1.0
0.8
*
0.6
†
0.4
0.2
0
Pre 0 1 3
7
14
21
28
(Post-Operative Day)
Figure 4. Uncoupling protein 2 (UCP2) dictates endothelial phenotype via mitochondrial •O2−. A, Bovine aortic endothelial cells
(BAECs) stained with mitochondrial-targeted hydroethidine at the indicated level of confluence to assess mitochondrial •O2− (bar=50
μm). B, Mitochondrial •O2− in murine lung endothelial cells (MLECs) from the indicated genotype as a function of confluence expressed
as relative fluoresence units (RFU). n=4; *P<0.01 for wild-type (WT) vs UCP2−/− by 2-way factorial ANOVA. C, MLEC mitochondrial •O2− in
the indicated genotypes transfected with UCP2 (Ad-UCP2), SOD2 (Ad-SOD2), or LacZ. n=3; *P<0.05 vs UCP2−/− Ad-LacZ; †P<0.05 vs
WT Ad-LacZ. D, Proliferation of UCP2-null MLECs transfected as in C with data normalized to control (CTL). n=3; *P<0.05 vs Ad-LacZ
by Kruskal–Wallis ANOVA. E, Mitochondrial •O2− in WT vs SOD2+/− endothelium. n=4 to 8; *P<0.05 vs WT by Mann–Whitney U test. F,
Proliferation of WT and SOD2+/− MLECs as a function of time. n=4; *P<0.05 for trend by 2-way repeated measures ANOVA. Mitochondrial
•O2− (G) and proliferation (H) in SOD2+/− MLECs as a function of transfection with control (LacZ), UCP2, or SOD2 adenovirus. n=3; *P<0.05
vs LacZ by Kruskal–Wallis ANOVA. I, left, Migration of BAECs with manipulated SOD2 levels via the indicated adenovirus or small
interfering RNA (siRNA). n=6; *P=0.01 vs respective controls by Mann–Whitney U test. I, right, BAEC Mitochondrial •O2− as a function of
treatment with CTL or SOD2 siRNA. n=3; *P<0.01 vs control siRNA by Student t test. J, Capillary sprouting in aortic segments from the
indicated genotypes. n=6; *P<0.001 vs WT by 2-way repeated measures ANOVA. K, Blood flow recovery in WT and SOD2+/− mice after
unilateral hindlimb ischemia with or without hindlimb transfection with UCP2 or LacZ adenovirus. n=5 per group; †P<0.01 vs WT and
*P<0.01 vs Ad-LacZ by 2-way repeated measures ANOVA. Ad indicates adenovirus; and SOD2, superoxide dismutase 2. or UCP2-null endothelium with mitochondrial SOD (SOD2)
also reduced the mitochondrial •O2− flux (Figure 4C). Finally,
attenuation of mitochondrial •O2− in UCP2-null cells with either UCP2 or SOD2 (Figure 4C) enhanced endothelial cell
proliferation (Figure 4D). These data indicate UCP2 modulates endothelial cell proliferation via mitochondrial •O2− .
If mitochondrial •O2− explains the UCP2-null phenotype,
then independent mitochondrial •O2− manipulation should
produce qualitatively similar effects. To this end, we found
that SOD2+/− endothelium exhibited increased mitochondrial
•O2− (Figure 4E) and impaired proliferation (Figure 4F) that
were both corrected by either UCP2 or SOD2 transfection
(Figure 4G and 4H). Forced overexpression of SOD2 in BAECs
enhanced migration in the scratch assay (Figure 4I), whereas
SOD2 suppression inhibited migration and increased mitochondrial •O2− (Figure 4I). Capillary sprouting in aortic segments
from SOD2+/− animals was impaired compared with wild-type
animals (Figure 4J). In the hindlimb ischemia model, we found
less blood flow recovery in SOD2+/− mice than in wild-type
mice that was rescued via adenoviral overexpression of UCP2
(Figure 4K) in a manner that suppressed mitochondrial •O2−
(Figure 4G) and improved proliferation (Figure 4H) in the endothelium. Thus, excess mitochondrial •O2− produces impaired
endothelial cell proliferation, migration, and angiogenesis.
Shimasaki et al UCP2 and Endothelial Function 897
B
1.5
1.0
0.5
0
Cell Migration (Cell #)
C
*
250
200
*
10000
5000
0
AdAdLacZ UCP1
D
*
150
100
50
0
15000
Ad- AdLacZ UCP1
AdAdLacZ UCP1
(MitoSOX / MitoGreen)
JC-1 (Δψ)
2.0
Cell Proliferation (RFU)
A
5
4
3
*
2
1
0
AdLacZ
AdUCP1
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Figure 5. Independent manipulation of Δψ impacts
endothelial phenotype. Bovine aortic endothelial cells (BAECs)
were transfected with irrelevant (Ad-LacZ) or uncoupling
protein 1 adenovirus (Ad-UCP1) and examined for (A) Δψ,
(B) proliferation, (C) migration, and (D) mitochondrial •O2− as
described in Methods section. n=4 to 6; *P<0.05 vs Ad-LacZ by
unpaired t test.
UCP2 Does Not Limit Endothelial Antioxidants or
Impact Basal NO• Bioactivity
Because endothelial antioixdants and •O2− can impact nitric
oxide (NO•) bioactivity,34 we investigated both as a function
of UCP2. Acute suppression of UCP2 had no material impact
on BAEC enzymatic antioxidant levels (Online Figure IVA).
In addition, stimulated NO• bioactivity with acetylcholine
was not different between wild-type and UCP2-null vessels
(Online Figure IVB) and basal NO• bioactivity was actually
greater in UCP2-null vessels than wild-type vessels (Online
Figure IVC and IVD). Thus, the impact of mitochondrial •O2−
on endothelial phenotype is not dependent on reduced antioxidant levels or basal NO• bioactivity.
Limiting Δψ Is Sufficient to Explain the Impact
of UCP2
To confirm that Δψ changes can account for the impact
of UCP2 effects on endothelial phenotype, we force expressed UCP1, a protein not expressed in endothelium, to
decrease endothelial Δψ. We found that forced expression
of UCP1 decreased Δψ (Figure 5A), increased proliferation (Figure 5B), increased migration (Figure 5C), and limited mitochondrial •O2− (Figure 5D). Thus, independently
decreasing Δψ with UCP1 produced qualitatively similar
changes in endothelial cell phenotype as Δψ manipulation
with UCP2. These data suggest that the impact of UCP2 on
endothelial phenotype is because, in part, of its effect on
mitochondrial Δψ.
UCP2 Regulates Cell Cycle Progression via
Mitochondrial •O2−
Because NO• bioactivity was not impaired, we examined other mechanisms of impaired endothelial function. Proliferating
UCP2-null endothelium exhibited more cells in the G1-phase
cells than wild-type endothelium (Figure 6A), suggesting
impaired G1-S cell cycle transition. This part of the cell cycle is
controlled, in part, by cyclin-dependent kinases, and we found
that UCP2-null cells exhibited higher expression of the cyclindependent kinase inhibitors, such as p16ink4a and p21cip/waf, than
did wild-type cells (Figure 6B and 6C). Because cell cycle inhibition can lead to senescence, we examined the senescence
indicator, β-galactosidase, and found a 4.5-fold increase in senescence in UCP2-null endothelium compared with wild-type cells
(Figure 6D and 6E). Transfection of UCP2-null cells with either
UCP2 or SOD2 (Figure 6E) attenuated senescence. We observed
a similar senescence increase in SOD2+/− cells (Figure 6E) and
transfection of either UCP2 or SOD2 also inhibited the development of senescence (Figure 6E). Because p53 is both sensitive to reactive oxygen species (ROS)29 and can control p16ink4a
and p21cip/waf expression, we investigated the role of p53 in the
UCP2-null phenotype. We found that p53 suppression by small
interfering RNA rescued the proliferation defect of UCP2-null
endothelium (Figure 6F). Moreover, we observed that proliferating and hypoxic UCP2-null endothelium exhibited p53
phosphorylation at serine 18 (Figure 6G), a residue involved in
redox-sensitive p53 activation.29 Thus, UCP2-null endothelium
exhibits a dysfunctional phenotype manifest as premature senescence via excess mitochondrial •O2− in a p53-dependent manner.
UCP2 Regulates Endothelial Phenotype via
Superoxide-Dependent Control of Mitochondrial
Fragmentation
Because mitochondria undergo dynamic changes in fusion and
fission during the G1-S cell cycle transition,35 we examined endothelial mitochondrial morphology as a function of UCP2.
Compared with wild-type endothelium, proliferating UCP2null cells exhibited superoxide-dependent mitochondrial fragmentation that was rescued by transfection with either UCP2
or SOD2 (Figure 7A and 7B). Similarly, SOD2 heterozygous
cells exhibited greater mitochondrial fragmentation than wildtype cells that was largely rescued by transfection with SOD2
or UCP2 (Figure 7A and 7B). This fragmentation was linked to
p53, because p53 RNA interference attenuated fragmentation
(Figure 7C), whereas p21cip/waf forced overexpression recapitulated mitochondrial fragmentation (Figure 7D). To gain insight
into the mechanism of mitochondrial fragmentation, we examined gene expression important for normal mitochondrial morphology (Figure 7E) and found that genes required for fusion
of mitochondria, such as mitofusin 1 and 2 (Mfn1, Mfn2) and
optic atrophy 1 (Opa1), were significantly lower than genes
required for mitochondrial fission, such as fission 1 (Fis1) and
dynamin-related protein 1 (Drp1)—a pattern associated with
mitochondrial fragmentation.36 To determine whether mitochondrial fragmentation was sufficient to cause the UCP2-null
phenotype, we suppressed mitofusin 1 and 2 expression and
found that induction of mitochondrial fragmentation significantly reduced endothelial cell proliferation (Figure 7F). Taken
together, these results indicate that endothelial UCP2 is necessary for maintaining mitochondrial morphology, and that disrupting the normal balance between mitochondrial fusion and
fission has implications for endothelial function.
898 Circulation Research September 13, 2013
A
UCP2-/-
24 hr
# Cells
G1
B
WT
G1 65.0%
S 19.7%
G2 7.1%
G1 40.0%
S 37.7%
G2 12.5%
G2
48 hr
# Cells
G1 74.5%
S 6.4%
G2 10.3%
PI Content
G1 49.1%
S 20.4%
G2 14.5%
*
3
3
2
2
1
1
0
WT UCP2-/-
0
Wild type
SA-β-gal stained cells (%)
E
Cell Proliferation
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
(RFU)
10000
UCP2-/-
WT
UCP2-/-
5000
0
p21
21
Actin
42
WT UCP2-/-
*
40
30
20
‡
‡
10
0
Ad- Ad- Ad- Ad- Ad- Ad- AdGFP GFP UCP2 SOD2 GFP UCP2 SOD2
UCP2-/-
SOD2+/-
G
†
‡
Hypoxia
Normoxia
WT UCP2-/-
p-p53
*
No
Control
Treatment siRNA
*
†
†
WT UCP2-/-
**
(kDa)
16
*
WT
15000
WT UCP2-/p16
PI Content
D
F
C
p21
mRNA
p16
mRNA
(kDa)
(Ser18)
p53
53
Actin
42
Figure 6. Uncoupling protein 2 (UCP2)
and endothelial senescence. A, Cell
cycle analysis in wild-type (WT) and
UCP2-null murine lung endothelial cells
(MLECs) at the indicated time based
upon propidium iodide (PI) content.
B, Expression of WT and UCP2-null
mRNA for p16ink4a and p21cip/waf. n=3;
*P<0.05 vs WT by Mann–Whitney U
test. C, Expression of WT and UCP2null endothelial p16ink4a and p21cip/waf by
immunoblotting. D, WT and UCP2-null
senescence-associated β-galactosidase
(SA-β-gal) via x-gal staining. Bar=750
μm. E, Endothelial SA-β-gal staining as a
function of genotype and transfection with
either UCP2 or SOD2. n=3 to 6; *P<0.05
vs WT+Ad-GFP, †P<0.05 vs UCP2−/−+AdGFP, and ‡P<0.05 vs SOD2+/−+Ad-GFP
by Kruskal–Wallis ANOVA and post hoc
comparison. F, Cell proliferation in WT
and UCP2−/− MLECs treated with none,
control, or p53 small interfering RNA
(siRNA). n=4; *P<0.05 vs WT with control
siRNA; **P<0.01 vs WT with no treatment;
†P<0.01 vs WT with control siRNA; and
‡P<0.01 vs UCP2−/− with control siRNA
by 1-way ANOVA with Tukey–Kramer
test. G, MLECs of the indicated genotype
under normoxic or hypoxic (1% O2)
conditions for 16 hours were lysed and
probed for the indicated proteins by
immunoblot. Ad indicates adenovirus; and
SOD2, superoxide dismutase 2. p53
siRNA
Discussion
The principal finding of this work is that endogenous UCP2
modulates endothelial mitochondrial network morphology that
dictates, in part, endothelial cell function. Through UCP2, endothelial Δψ decreases with cell proliferation and, as endothelial growth slows, reduced UCP2 levels produce an increase in
Δψ. We found these changes in Δψ moved in parallel with mitochondrial •O2− production that, if left uncontrolled, resulted
in p53-dependent mitochondrial network fragmentation that
limited endothelial functions important for angiogenesis, including proliferation, migration, and blood flow recovery from
tissue ischemia. The central role of Δψ was supported by observations that Δψ manipulation with UCP1 could phenocopy
the effect of UCP2. Moreover, the key role of mitochondrial
•O2− was consistent with observations that SOD2+/− endothelium, with excess mitochondrial •O2−, demonstrated mitochondrial network fragmentation and impaired endothelial
cell function. Collectively, these data indicate that endogenous
UCP2 is an important modulator of endothelial cell function,
in part, via its impact on mitochondrial network integrity.
The precise biological functions of uncoupling proteins remain
a matter of debate. UCP1, the predominant uncoupling protein
in brown adipose tissue, mediates Δψ proton leak that is critical for adaptive thermogenesis.37 In contrast, UCP238 and UCP339
can mediate proton leak, but are not necessary for adaptive thermogenesis or normal energy metabolism.18,40 These latter 2 uncoupling proteins modulate Δψ to a lesser extent than UCP1,18
even in the presence of requisite coactivators, such as fatty acids and •O2− -derived alkenals.41,42 Multiple models of UCP2 or
UCP3 overexpression suggest a protective role against oxidative stress38,43,44 and endothelial dysfunction from diet-induced
obesity.45 Considering that endothelial cell proliferation can be
associated with excess •O2− and oxidative stress,46 one could argue that our data identify UCP2 as a stress-responsive protein.
However, UCP overexpression is prone to artifact from improper
membrane insertion,40 casting doubt on the ultimate role of UCPs
in limiting •O2−-mediated toxicity. Our data strongly support a
role of UCP2 to limit mitochondrial •O2− and deserve particular
attention, because we focused on endogenous UCP2 regulation.
Moreover, we used loss-of-function models with rescue by UCP2
complementation that are less prone to artifact. Consistent with
the idea that UCP2 limits mitochondrial •O2−, we found that independent manipulation of mitochondrial •O2− (via SOD2) copied
the UCP2-null phenotype, increasing confidence that endogenous UCP2 importantly regulates mitochondrial ROS.
One might expect that mitochondrial •O2− would limit NO•
bioactivity in our system to explain the UCP2-null phenotype.34
Rather, we found intact NO• bioactivity in unstressed UCP2-null
endothelium, but the cells exhibited premature senescence and
upregulation of p21cip/waf and p16ink4a. These 2 gene products have
been linked, in part, to activation of the tumor suppressor, p53.47
Our observations that correction of excess mitochondrial •O2−
prevented senescence is consistent with data that ROS can upregulate p53 to induce both cell cycle arrest48 and genes that tend
to lower cellular ROS levels.49,50 In this context, it is germane to
Shimasaki et al UCP2 and Endothelial Function 899
A
WT
UCP2-/-
UCP2-/- + AdUCP2
SOD2+/-
*
*
3
*
*
2
*
1
0
4
3
1
0
WT
4
3
UCP2-/-
SOD2+/-
E
Relative Expression to GAPDH
WT + Ad-p21
WT
UCP2-/-
2.0
1.5
1.0
*
*
**
0.5
**
0
Mfn1
Mfn2
Opa1
Fis1
Drp1
Ucp2
F
2
1
*
0
siCTL si-p21
WT
Ad- AdLacZ p21
CTL siRNA
Mfn2 siRNA
Cell Number / Field
Mitochondrial Length (μm)
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
WT + AdLacZ
siCTL si-p53 siCTL si-p53 siCTL si-p53
SOD2+/-
UCP2-/-
*
*
2
WT UCP2-/- Ad- Ad- Ad- Ad- Ad- AdLacZ UCP2 SOD2 LacZ UCP2 SOD2
D
Figure 7. Mitochondrial morphology
and endothelial phenotype. A,
Representative images of murine lung
endothelial cell (MLEC) mitochondrial
morphology by Mitotracker green
staining as a function of the indicated
genotype and transfection with the
indicated adenovirus (Ad). B, Composite
data indicating average mitochondrial
length as an index of fragmentation in
MLECs from the indicated genotype and
transfected with the indicated adenovirus.
n=13 to 16; *P<0.01 vs respective wildtype (WT) or LacZ control by Kruskal–
Wallis ANOVA. C, Mitochondrial length as
an index of fragmentation in the indicated
genotypes with and without suppression
of p53. n=12 to 16; *P<0.05 vs respective
control (CTL). D, MLEC mitochondrial
morphology and average length with
forced expression of p21cip/waf. Bar=5 μm;
n=9 to 13; *P<0.05 vs LacZ by Kruskal–
Wallis ANOVA. E, MLEC expression of
mitochondrial genes as a function of the
indicated genotype. n=3 to 4; *P<0.05,
**P<0.01 vs WT by Mann–Whitney U
test. F, WT MLEC proliferation with
suppression of mitofusin 2 (Mfn2) or both
mitofusin 1 (Mfn1) and Mfn2. n=10 each;
*P<0.05 vs CTL siRNA by 2-way repeated
measures ANOVA. Drp1 indicates
dynamin-related protein 1; Fis1, fission
1; opa1, optic atrophy 1; siCTL, siRNA
control; and SOD2 indicates superoxide
dismutase 2.
C
4
Mitochondrial Length (μm)
Mitochondrial Length (μm)
B
SOD2+/- + AdSOD2
CTL siRNA
Mfn1+2 siRNA
500
500
400
*
400
*
300
300
0
2
5
Days in Culture
note that RNA interference–­mediated suppression of prohibitin,
an inner mitochondrial protein, also upregulates mitochondrial
•O2−, producing premature senescence and impaired endothelial
proliferation.51 Studies in other cell types indicate prohibitins are
required for proliferation,52 prompting speculation that suppressing mitochondrial •O2− (perhaps to prevent p53 activation) may
be a general requirement of cellular proliferation, perhaps via
prevention of premature senescence. In this regard, we need to interpret our data with caution as we have not determined whether
senescence alone could explain all of our experimental findings.
The notion that UCP2 suppression of mitochondrial •O2−
may be important for cell proliferation is supported by our
observation that UCP2 mRNA is most prominent in very proliferative cells and tissues, such as bone marrow, lymphomas,
leukemias, erythroid precursors, T-cells, and CD105+ endothelial cells (Online Figure I). Moreover, UCP2 gene silencing
prevented epidermal cell tumor induction in a p53-dependent
manner.53 Thus, data from diverse cell types link UCP2 upregulation (and mitochondrial •O2− suppression) to rapid cell
0
2
5
Days in Culture
proliferation, suggesting that UCP2 impacts some fundamental
component of the proliferative response. Because UCP2 is a
target gene for peroxisome proliferator γ coactivator-1α, the
principal determinant of mitochondrial biogenesis,54 UCP2
may be needed to expand mitochondrial mass during cell proliferation, perhaps by limiting mitochondrial ROS-mediated
damage. Alternatively, UCP2 is known to promote oxygeninsensitive glycolysis (the Warburg effect)55 and glycolysis is
required for the high proliferation rate of many cancer cells.32
Thus, UCP2 may be required for glycolysis-dependent growth,
a finding consistent with our observations of reduced lactate
production in UCP2-null endothelium (Figure 2).
We observed excess mitochondrial fragmentation with
UCP2-null mice that was linked to mitochondrial •O2−, because
it was recapitulated in the SOD2+/− mice and attenuated with
maneuvers that reduce mitochondrial •O2−. These findings suggest a novel relation between mitochondrial redox state and
the balance between mitochondrial fusion and fission. This relation is consistent with our observations that mitochondrial
900 Circulation Research September 13, 2013
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
fragmentation in UCP2-null cells involved a p53- and p21cip/wafdependent mechanism, and literature that p53 can be activated
by ROS via p53 phosphorylation on serine 20 (mouse Ser18).29
This latter event is known to involve the ataxia-telangiectasia–
mutated kinase. Thus, one potential explanation for our data
would be ataxia-telangiectasia–mutated kinase activation by
mitochondrial •O2−. This contention is supported by observations that ataxia-telangiectasia–mutated and its p53 target
(Ser18/20) have important implications for metabolism.56,57
The findings presented here suggest a role for UCP2 in cell
proliferation. With the increased metabolic flux that occurs as
a result of growth stimulation, it is not surprising that mitochondrial Δψ and •O2− would increase. The fact that forced
expression of UCP1 limited Δψ and recapitulated the effect of
UCP2 suggests that UCP2 upregulation is an important mechanism for limiting Δψ and the resultant mitochondrial •O2− flux
that, if left unchecked, would activate pathways to limit cell
proliferation. With regards to the endothelium, this paradigm
has important therapeutic implications. For example, our findings suggest that targeting UCP2 may prove to be an important
means of limiting tumor angiogenesis. Conversely, promotion
of mitochondrial uncoupling could enhance the angiogenic response to ischemia and this strategy could prove helpful in the
setting of occlusive vascular diseases. Thus, our data highlight
the importance of mitochondrial uncoupling and its dynamic
regulation in the endothelial cell proliferative response.
Acknowledgments
We kindly thank Dr Bradford Lowell for the UCP2-null mice.
Sources of Funding
This work was supported by National Institutes of Health grants
DK089185 (M.P. Cooper), HL092122 and HL098407 (J.F. Keaney).
Informatics and imaging core services were supported by University
of Massachusetts D.E.R.C. grant DK32520.
Disclosures
None.
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Novelty and Significance
What Is Known?
• Mitochondria are known to participate in a number of cellular functions beyond that of energy production.
• The main driving force for many mitochondrial functions is the electrochemical gradient (Δψ) produced by the mitochondrial electron
transport chain.
• The regulation of Δψ involves both the electron transport chain
and uncoupling proteins that facilitate protein leak across the inner
mitochondrial membrane.
What New Information Does This Article Contribute?
• Mitochondrial Δψ is dynamically regulated in endothelial cells by uncoupling protein 2 (UCP2) in response to external stimuli such signals
to promote proliferation.
• The decrease in endothelial cell Δψ by UCP2 upregulation during
proliferation limits mitochondrial superoxide generation.
• In the absence of UCP2, excess mitochondrial superoxide leads to
p53-dependent mitochondrial fragmentation that is necessary and
sufficient to impair endothelial cell proliferation and angiogenesis.
Mitochondria have long been known to be critical for cellular
ATP production. More recently, mitochondrial have been implicated in other cellular processes, but their role in the endothelium is largely unknown. Thus, we sought to probe the role of
mitochondria in endothelial proliferative responses. We found that
endothelial cell proliferation was associated with a reduced Δψ
because of upregulation of UCP2, a protein known to regulate
Δψ. In the absence of UCP2, proliferating endothelial cells had
higher Δψ and excess mitochondrial superoxide that prevented
their proliferation, migration, and participation in angiogenesis.
Similarly, endothelium lacking mitochondrial superoxide dismutase had excess mitochondrial superoxide and mimicked the
UCP2-null phenotype. This excess mitochondrial superoxide lead
to p53-dependent mitochondrial fragmentation and endothelial
cell senescence. Correction of excess mitochondrial superoxide
corrected both the mitochondrial fragmentation and endothelial
dysfunction. These data indicate that endothelial UCP2 is important to maintain both normal mitochondrial dynamics and endothelial function.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Uncoupling Protein 2 Impacts Endothelial Phenotype via p53-Mediated Control of
Mitochondrial Dynamics
Yukio Shimasaki, Ning Pan, Louis M. Messina, Chunying Li, Kai Chen, Lijun Liu, Marcus P.
Cooper, Joseph A. Vita and John F. Keaney, Jr
Circ Res. 2013;113:891-901; originally published online July 2, 2013;
doi: 10.1161/CIRCRESAHA.113.301319
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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World Wide Web at:
http://circres.ahajournals.org/content/113/7/891
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2013/07/02/CIRCRESAHA.113.301319.DC1
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CIRCRES/2013/301319/R1
Supplemental Material
Supplemental Experimental Procedures
Bioinformatics – The gene expression profiles of human UCP1, 2, and 3 in various tissues were
examined using the BioGPS database from microarrays (Genomics Institute of the Novartis
Research Foundation; San Diego, CA).
Capillary Sprouting Assay in Adipose Tissues – Freshly harvested epididymal fat pads were
placed in Krebs-Ringer solution buffered with HEPES (KRH) as described,1 digested for 30 min
at 37°C in KRH, pH 7.4, containing 1 mg/ml collagenase type I (Worthington Biochemical;
Lakewood, NJ) and 2.5% BSA Fraction V (Sigma-Aldrich). Digested tissue was then filtered
through a 100 µm cell strainer (Fisher Scientific BD Falcon; Pittsburgh, PA) and the captured
stromal vascular fraction was cut into small (1 mm3) pieces that were embedded in liquid
collagen gel in 48-well plates (BD Biosciences), incubated at 37°C for 1h to polymerize the
collagen, and the solid gel covered with MLEC medium diluted 1:2 with DMEM. Each adipose
tissue was examined daily and capillary sprouts counted along the sample perimeter under 100
and 200 x magnification. Vascular sprouts were distinguished from fibroblasts via morphology
as described1,2 and CD31 staining.
Vascular Ring Relaxation – To study NO• bioactivity, mice aged 8-12 weeks were euthanized
and thoracic aortae were isolated, cleaned of connective tissue, and cut into 2 mm segments.
The vessel segments were mounted on stainless-steel holders in organ baths (Danish Myo
Technology, Denmark) containing physiological saline solution (PSS; 119 mM NaCl, 4.69 mM
KCl, 1.17 mM MgSO4, 1.18 mM KH2PO4, 2.5 mM CaCl2, 25 mM NaHCO3, 0.03 mM EDTA, 5.5
1
CIRCRES/2013/301319/R1
mM Glucose) and aerated with 95% O2 / 5% CO2 for isometric force recording. Preparations
were allowed to equilibrate for 30 min under constant passive force of (~1 mN) and
synchronized with KCl and phenylephrine. Basal NO• bioactivity was estimated as the
difference in contraction to phenylephrine with or without 300 µM L-NAME to inhibit endothelial
nitric oxide synthase. Evoked NO• bioactivity was estimated as the relaxation response to
acetylcholine (ACh) in phenylephrine-contracted segments. Data were analyzed with PowerLab
software (AD Instruments).
2
CIRCRES/2013/301319/R1
Online Figure I
Human UCP1
Human UCP2
Human UCP3
10000
0
Median
10
3xM
15
Median 10xM
3xM
30xM
5000
100xM
0
Median
50
3xM
100
Online Figure I. Uncoupling protein mRNA expression as a function of cell type.
Gene expression profiles of human UCP1, 2, and 3 as a function of cell type examined with the
BioGPS online database. Expression levels are indicated as a multiple of the median
expression level.
3
CIRCRES/2013/301319/R1
A
0
WT
5 15 30 60 180 0
C
UCP2-/5 15 30 60 180 min
P-c-Jun
P-p38
B
CTL
0
5’ 15’ 24h 0
SB203580
SP600125
5’ 15’ 24h
0
5’ 15’ 24h
P-c-Jun
*
150
Proliferation (% DMSO)
Actin
†
†
100
50
P-p38
UCP2
0
DM
SO
DM
SO
SP
5
0.
SP
5 )
2. (µM
VEGF
Online Figure II. c-Jun, N-terminal kinase and UCP2 upregulation. (A) Wild-type (WT) and
UCP2-null endothelium was exposed to 10% serum for the indicated number of minutes, lysed,
and immunoblots performed for actin or phosphorylated (activated) forms of c-Jun (Ser63) or
p38 MAP kinase (Thr180/Tyr182). (B) BAECs in the presence or absence of vehicle alone
(CTL), the JNK inhibitor SP600125 (0.5 µM), or the p38 MAP kinase inhibitor SB203580 (10
µM) were exposed to 10% serum for the indicated time. Cells were then lysed and examined
for UCP2 or activation of JNK or p38 MAP kinase as in (A). (C) BAECs were exposed to vehicle
(DMSO) or VEGF (25 ng/mL) with or without SP600125 as indicated for 24h followed by
assessment of proliferation by [3H]-thymidine incorporation. N=4; *P<0.05 vs DMSO; †P<0.05
vs. VEGF/DMSO by one-way ANOVA with Tukey post hoc test.
4
CIRCRES/2013/301319/R1
Supplemental Figure 3
Wild-type
UCP2-/-
2.0
Perfusion
(ischemic/non-ischemic)
1.2
C
UCP2-/- adipose tissue
1.0
0.5
Ad-LacZ
†
WT + Ad-LacZ
UCP2-/- + Ad-UCP2
UCP2-/- + Ad-LacZ
40
Ad-UCP2
1.5
*
*
30
20
10
0
0
UCP2
D
B
(Sprouts / fat)
(UCPs mRNA / GAPDH mRNA)
A
UCP3
0
2
4
(days)
6
UCP2-/- + Ad-UCP2
UCP2-/- + Ad-LacZ
1.0
*
0.8
0.6
0.4
0.2
0
Pre 0 1 3 7
14
21
(Post-Operative Day)
28
Online Figure III. Uncoupling protein manipulation and angiogenesis.
(A) UCP2 and 3 mRNA levels in mouse lung endothelial cells. Dagger = not detectable. (B)
Representative phase contrast images of capillary sprouts from UCP2-/- adipose tissue explants
taken with a 40 x objective on the day 4 culture. Scale bar, 500 µm. (C) Ex vivo capillary
sprouting in fat pad explants from the indicated genotype embedded in collagen gel 5d after in
vivo transfection with UCP2 or LacZ adenovirus. N=5/group; *P < 0.05 vs. UCP2-/- with AdLacZ by two-way ANOVA.. (D) Blood flow recovery in UCP2-/- mice with unilateral hindlimb
ischemia and treatment with control (Ad-LacZ) or UCP2 adenovirus. N=5; *P < 0.05 vs. LacZ by
two-way ANOVA.
5
CIRCRES/2013/301319/R1
B
2
(kDa)
P
L
L CT UC
T
i
i
C s
s
33
UCP2
24
SOD2
100"
22
GPx-1
65
Catalase
42
Actin
16
COX IV
%"Ini=al"Tension"
A
WT#
UCP2(/(#
50"
25"
0"
'9"
'8"
'7" '6.5" '6"
'5"
Acetylcholine"(Log"M)"
C
D
75"
100"
WT"
50"
UCP2&/&"
*"
25"
0"
&9"
&8"
&7" &6.5" &6"
&5"
Phenylephrine"(Log"M)"
%"Constric5on"
%"ConstricCon""
75"
(+) L-NAME
75"
75"
50"
25"
0"
WT"
50"
-/UCP2&/&"
25"
'9"
'8"
'7"
'6.5"
'6"
'5"
Phenylephrine"(Log"M)"
0"
&9"
&8"
&7" &6.5" &6"
Online Figure IV. UCP2, antioxidant enzymes, and NO• bioactivity.
(A) BAECs were treated with CTL or UCP2 siRNA as indicated for 24h followed by
immunoblotting for the indicated antioxidant enzymes. (B) Acetylcholine-induced relaxation of
aortic segments of the indicated genotype contracted with phenylephrine. N=6; P = NS. (C)
Phenylephrine-induced contraction of aortic segments by genotype. N=6; P < 0.05 vs. WT by
two-way repeated measures ANOVA. (D) Phenylephrine-induced contraction of aortic rings by
genotype in the presence of 300 µM L-NAME (P = NS).
6
&5"
CIRCRES/2013/301319/R1
1.
Gealekman O, Burkart A, Chouinard M, Nicoloro SM, Straubhaar J, Corvera S. Enhanced
angiogenesis in obesity and in response to PPARgamma activators through adipocyte
VEGF and ANGPTL4 production. Am J Physiol Endocrinol Metab. 2008; 295:E1056–64.
2.
Nicosia RF, Ottinetti A. Growth of microvessels in serum-free matrix culture of rat aorta. A
quantitative assay of angiogenesis in vitro. Lab Invest. 1990; 63:115–122.
7