Download Laminar Shear Stress Inhibits Endothelial Cell Metabolism via KLF2

Laminar Shear Stress Inhibits Endothelial Cell
Metabolism via KLF2-Mediated Repression of PFKFB3
Anuradha Doddaballapur, Katharina M. Michalik, Yosif Manavski, Tina Lucas,
Riekelt H. Houtkooper, Xintian You, Wei Chen, Andreas M. Zeiher, Michael Potente,
Stefanie Dimmeler, Reinier A. Boon
Downloaded from http://atvb.ahajournals.org/ by guest on August 3, 2017
Objective—Cellular metabolism was recently shown to regulate endothelial cell phenotype profoundly. Whether the
atheroprotective biomechanical stimulus elicited by laminar shear stress modulates endothelial cell metabolism is not known.
Approach and Results—Here, we show that laminar flow exposure reduced glucose uptake and mitochondrial content in
endothelium. Shear stress–mediated reduction of endothelial metabolism was reversed by silencing the flow-sensitive
transcription factor Krüppel-like factor 2 (KLF2). Endothelial-specific deletion of KLF2 in mice induced glucose uptake
in endothelial cells of perfused hearts. KLF2 overexpression recapitulates the inhibitory effects on endothelial glycolysis
elicited by laminar flow, as measured by Seahorse flux analysis and glucose uptake measurements. RNA sequencing
showed that shear stress reduced the expression of key glycolytic enzymes, such as 6-phosphofructo-2-kinase/fructose2,6-biphosphatase-3 (PFKFB3), phosphofructokinase-1, and hexokinase 2 in a KLF2-dependent manner. Moreover, KLF2
represses PFKFB3 promoter activity. PFKFB3 knockdown reduced glycolysis, and overexpression increased glycolysis
and partially reversed the KLF2-mediated reduction in glycolysis. Furthermore, PFKFB3 overexpression reversed KLF2mediated reduction in angiogenic sprouting and network formation.
Conclusions—Our data demonstrate that shear stress–mediated repression of endothelial cell metabolism via KLF2 and
PFKFB3 controls endothelial cell phenotype. (Arterioscler Thromb Vasc Biol. 2015;35:137-145. DOI: 10.1161/
ATVBAHA.114.304277.)
Key Words: angiogenesis ◼ endothelium ◼ glycolysis ◼ hemodynamics ◼ metabolism ◼ shear stress down
regulated gene-1 protein, human
E
ndothelial cells form the inner lining of all blood vessels
and not only regulate transport of nutrients to the underlying tissue but also coordinate the formation of new blood
vessels, a process termed angiogenesis. Therefore, endothelial
cells are highly plastic cells that are capable of switching from
a resting quiescent state in normal conduit blood vessels to
a highly proliferative and migratory state when angiogenesis
takes place. Resting quiescent endothelial cells are termed
phalanx cells,1 whereas migratory angiogenic endothelial cells
are referred to as tip cells, which are followed by proliferating
so-called stalk cells.2 Although the mechanisms regulating tip
and stalk cell behavior have been extensively studied, relatively little is known about the control of the phalanx state.
Shear stress, the force that laminar blood flow exerts on
endothelial cells, is thought to be one of the factors that determine the quiescent state of endothelial cells.3 This biomechanical stimulus induces the expression of the transcription factor
Krüppel-like factor 2 (KLF2), which orchestrates a network
of genes that elicit a quiescent endothelial cell phenotype.4,5
Among the factors that are upregulated by KLF2 are antiinflammatory and antithrombotic proteins, whereas proinflammatory and prothrombotic factors are downregulated by
KLF2.4 Although not all effects of shear stress on endothelial
cells are mediated by KLF2, KLF2 coordinates approximately
half of the gene expression programs evoked by shear stress.5,6
See accompanying editorial on page 13
Recent studies have highlighted the importance of cellular
metabolism for the control of endothelial cell phenotype.7,8
Particularly, it was shown that angiogenic endothelial cells
rely heavily on glycolysis for migration and proliferation.9
The enzyme PFKFB3 is a key regulator of glycolysis in
endothelial cells that has been shown to promote angiogenic
sprouting.9–11 However, how resting endothelial cells control
Received on: July 7, 2014; final version accepted on: October 16, 2014.
From the Institute for Cardiovascular Regeneration, Center of Molecular Medicine, Goethe University, Frankfurt am Main, Germany (A.D., K.M.M.,
Y.M., T.L., S.D., R.A.B.); The Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, The Netherlands (R.H.H.); The MaxDelbrück-Center, Berlin, Germany (X.Y., W.C.); Department of Cardiology, Internal Medicine III, Goethe University Hospital Frankfurt, Frankfurt am
Main, Germany (A.M.Z.); Angiogenesis and Metabolism Laboratory, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany (M.P.);
and German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Frankfurt am Main, Germany (A.M.Z., S.D.).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304277/-/DC1.
Correspondence to Reinier A. Boon, PhD, Institute for Cardiovascular Regeneration, Center of Molecular Medicine, Theodor-Stern-Kai 7, 60590
Frankfurt am Main, Germany. E-mail [email protected]
© 2014 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
137
DOI: 10.1161/ATVBAHA.114.304277
138 Arterioscler Thromb Vasc Biol January 2015
Nonstandard Abbreviations and Acronyms
HUVEC
KLF2
PFKFB
human umbilical vein endothelial cells
Krüppel-like factor 2
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase
their metabolic activity and whether this affects the functional
properties of the phalanx phenotype is unclear.
Here, we show that the biomechanical signal shear stress,
through the upregulation of KLF2, reduces endothelial metabolic
activity by repressing PFKFB3 expression, and thereby maintains
a metabolic quiescent phenotype reminiscent of phalanx cells.
Materials and Methods
Materials and Methods are available in the online-only Data
Supplement.
Downloaded from http://atvb.ahajournals.org/ by guest on August 3, 2017
Results
Shear Stress Reduces Endothelial Glucose
Uptake and Mitochondrial Content
in a KLF2-Dependent Manner
To assess the effects of laminar shear stress on endothelial cell
metabolism, human umbilical vein endothelial cells (HUVECs)
were exposed to laminar shear stress (20 dynes/cm2) for 72
hours to achieve steady-state quiescence or left under static
conditions. Glucose uptake and mitochondrial content analysis
revealed that shear stress reduces basal endothelial cell glucose
uptake and the relative quantity of mitochondria per endothelial
cell (Figure 1A and 1B). These results were substantiated by
fluorescence microscopy–based analysis of glucose uptake of
individual HUVECs exposed to shear stress, which showed that
cellular alignment to the flow direction inversely correlates with
glucose uptake (Figure IA in the online-only Data Supplement).
Because the transcription factor KLF2 is known to be responsible
for many shear stress–induced effects on endothelial cells, we
determined whether reduction in glucose uptake by shear stress
is dependent on KLF2. To this end, we transduced HUVECs with
a lentiviral short hairpin RNA construct to silence KLF2 and subsequently subjected the cells to laminar shear stress for 72 hours
(Figure 1C), which completely abrogates shear stress–mediated
induction of KLF2. Silencing of KLF2 abolished shear stress–
mediated reduction of glucose uptake, indicating that regulation
of glucose uptake by shear stress is KLF2 dependent (Figure 1D).
To substantiate whether KLF2 regulates metabolic activity
of endothelial cells in vivo, we analyzed glucose uptake in
endothelial cells of mice lacking endothelial KLF2 (Cdh5CreERT2;KLF2fl/fl) and wild-type controls (KLF2fl/fl and
KLF2fl/+; Figure IB in the online-only Data Supplement).
Specifically, hearts of these mice were subjected to
Langendorff-mediated perfusion with 2-N-7-nitrobenz-2oxa-1,3-diazol-4-yl-amino-2-deoxyglucose and simultaneous digestion of the extracellular matrix to obtain a single-cell
suspension of cardiac cells. Then, endothelial cells were
labeled, and glucose uptake in these cells was quantified
using flow cytometry (Figure 1E–1G). Endothelial-specific
deletion of KLF2 in mice significantly induces glucose
uptake by cardiac endothelial cells (Figure 1G).
KLF2 Reduces Endothelial Metabolic Activity
Overexpression of KLF2 in endothelial cells recapitulates
many aspects of shear stress stimulation, including induction
of cellular quiescence.4,12 Lentiviral overexpression of KLF2
(Figure 2A) in endothelial cells mimics the induction of KLF2
after shear stress stimulation (Figure 1C) and indeed reduces
glucose uptake (Figure 2B). Furthermore, using a transwell
assay to model glucose availability to underlying tissue in
vitro, we measured an increase in available glucose underneath
endothelial monolayers that overexpress KLF2 (Figure 2C),
indicating that the reduction of glucose consumption mediated
by KLF2 results in more bioavailability of glucose underneath
the endothelium. Using Seahorse Flux analysis, we determined that the extracellular acidification rate, an indicator
of lactate production, is also lower in KLF2-overexpressing
endothelial cells (Figure 2D). Not only basal acidification rate
but also glucose-induced glycolysis and maximal glycolytic
capacity were reduced in KLF2-transduced cells when compared with mock-transduced control cells (Figure 2E).
Next, we determined whether mitochondrial content and
function are also regulated by KLF2. KLF2 overexpression
reduces mitochondrial content (Figure 2F), metabolic activity
(measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Figure 2G), and ATP levels (Figure 2H),
which is similar to the effects of shear stress on endothelial
cells (Figure 1). Importantly, mitochondrial membrane potential is significantly enhanced after KLF2 overexpression, suggesting that KLF2 reduces mitochondrial activity, rather than
affecting mitochondrial integrity (Figure 2I). Assessment of
cellular oxygen consumption revealed that KLF2 overexpression also reduces basal mitochondrial respiration and ATP
production (Figure 2J and 2K), but does not affect maximal respiration or spare respiratory capacity, further corroborating that
KLF2 does not affect mitochondrial integrity. Because both
oxygen consumption and lactate production are lower in KLF2overexpressing cells, the ratio of oxygen consumption over lactate production rate is not altered by KLF2, arguing against a
potential KLF2-mediated shift between aerobic respiration and
glycolysis (Figure IC in the online-only Data Supplement).
KLF2 Does Not Induce Senescence or Apoptosis
To exclude the possibility that the KLF2-mediated reduction
in metabolic activity is because of induction of senescence,
we analyzed proliferation (Figure 3A), acidic β-galactosidase
activity (Figure 3B), and p21 expression (Figure 3C) in KLF2transduced and mock control cells. Whereas KLF2 slightly
reduces the number of cells in G2/M-phase, KLF2 overexpression reduced β-galactosidase activity and p21 expression, indicating that reduction in proliferation by KLF2 is not
because of senescence. Furthermore, KLF2 reduces apoptosis,
as measured by caspase 3/7 activation (Figure 3D) and annexin
V staining (Figure ID in the online-only Data Supplement).
KLF2-Mediated Suppression of Metabolic Activity
Is Not Mediated via AMPK or Nitric Oxide
To delineate how KLF2 reduces endothelial cell metabolic
activity, we first performed phospho-kinase proteome profiling (Figure IIA in the online-only Data Supplement), which
Doddaballapur et al Shear Stress Inhibits Endothelial Cell Metabolism 139
A
B
Glucose uptake
Mitochondrial DNA content
15000
102
103
104 105
2-NBD-Glucose
C
5000
0
Static
Static
Shear
Glucose uptake
Static
10000
***
8
Shear
Static
Shear
6
4
2
0
*
5000
shKLF2
KLF2
37 KDa
α-Tubulin
55 KDa
0
-
+
-
shCon
shKLF2
+
shKLF2
shCon
F
G
Counts
CD31+
103
104
CD31
105
2-NBD-Glucose
Fluorescence (% Control)
E
Counts
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shCon
102
*
0.5
***
10
CD31-
1.0
0.0
Shear
D
2-NBDG (MFI)
KLF2 mRNA (fold change)
*
10000
KLF2 knockdown
Shear
Relative DNA content
(mtDNA vs. nDNA)
2-NBDG (MFI)
Counts
Unstained
Static
Shear
1.5
102
103
104
2-NBD-Glucose
Unstained
WT (KLF2fl/fl)
EC-KO (Cdh5-CreERT2;KLF2fl/fl)
160
140
120
100
80
60
40
20
0
*
CD31+
CD31WT (KLF2fl/fl)
EC-KO (Cdh5-CreERT2;KLF2fl/fl)
Figure 1. Laminar shear stress reduces glucose uptake in endothelial cells (ECs) in a Krüppel-like factor 2 (KLF2)–dependent manner.
A, Representative flow cytometry histogram overlay (left) and quantification of 2-N-7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino-2-deoxyglucose (2-NBD)-glucose uptake (right) in human umbilical vein endothelial cells (HUVECs) after 72 hours of flow exposure (20 dyn/cm2
shear stress) compared with static control measured by flow cytometry analysis of 2-NBD-glucose fluorescence (n=4). B, Mitochondrial
DNA content in shear exposed HUVECs (20 dyn/cm2 for 72 hours) vs static control measured by quantitative real-time polymerase chain
reaction of mitochondrial encoded gene ND1 and nuclear encoded gene ribosomal phosphoprotein, large P0 subunit. Relative levels
were normalized to median values (n=4). C, KLF2 mRNA expression (top) in HUVECs after lentiviral-mediated shRNA knockdown of KLF2
and flow exposure (20 dyn/cm2 for 72 hours). mRNA expression was normalized to GAPDH mRNA and shown as fold change relative to
static shCon-transduced cells (n=3). Representative Western blot analysis (bottom) of total cell protein lysates from HUVECs after lentiviral-mediated shRNA knockdown of KLF2 and flow exposure (12 dyn/cm2 for 48 hours) in a cone-plate viscometer, probed against antibodies for KLF2 and α-tubulin, which served as a loading control. D, 2-NBD-glucose uptake in HUVECs after lentiviral-mediated shRNA
knockdown of KLF2 and flow exposure (20 dyn/cm2 for 72 hours; n=3). E–G, Ex vivo 2-NBD-glucose uptake in heart ECs of wild-type
(WT; KLF2 fl/fl) and EC-knockout (KO; Cdh5-CreERT2;KLF2fl/fl) mice. Representative flow cytometry histogram showing CD31+ endothelial
cell population (E) from Langendorff perfused hearts, which were subsequently analyzed for 2-NBD-glucose mean fluorescence (F) and
quantified (G; n=5). In all graphs, data represent mean±SEM *P<0.05, ***P<0.001. MFI indicates mean fluorescence intensity.
showed that KLF2 overexpression reduces phosphorylation of
the 5′ adenosine monophosphate–activated protein kinase α
1 subunit (Figure IIB in the online-only Data Supplement).
However, silencing 5′ adenosine monophosphate–activated
protein kinase α 1 (Figure IIC in the online-only Data
Supplement) did not recapitulate any of the metabolic effects
observed after KLF2 overexpression (Figure IID–IIG in the
online-only Data Supplement). Conversely, KLF2 overexpression induces endothelial nitric oxide synthase expression and phosphorylation (Figure IIA in the online-only Data
Supplement). Because nitric oxide has been shown to inhibit
mitochondrial respiration,13 increased nitric oxide production
could potentially account for the inhibition of mitochondrial
activity by KLF2. However, inhibition of nitric oxide synthesis did not affect the inhibitory effect of KLF2 on respiration or glycolysis (Figure IIH and III in the online-only Data
Supplement).
KLF2 and Shear Stress Inhibit PFKFB3 Expression
To gain a broad unbiased insight into the regulation of gene
expression by shear stress, HUVECs were subjected to laminar shear stress for 72 hours or kept under static control conditions, and we performed next-generation sequencing with
RNA isolated from these cells (RNAseq). These experiments
140 Arterioscler Thromb Vasc Biol January 2015
B
Monolayer
8000
KLF2
37 kDa
α-Tubulin
55 kDa
*
6000
4000
2000
0
D
0.003
Mock
KLF2
0.002
0.001
0.000
30
1.0
*
0.5
0.0
J
KLF2
Mock
G
MTT reduction (Fold change)
1.5
*
0.5
Mock
0.002
30
60
Time (min)
90
120
OCR (pMoles/min/RFU)
Mock
KLF2
0.004
0.000
Mock
KLF2
**
0.001
**
**
*
H
Glycolysis
Glycolytic
reserve
Mitochondrial
membrane potential
25000
200
20000
150
*
100
*
15000
10000
50
0
Glycolytic
capacity
I
ATP levels
Mock
K
Antimycin A
Oligomycin FCCP & Rotenone
0.006
KLF2
0.002
KLF2
Mitochondrial respiration
0.008
Mock
Glycolytic function
Basal
ECAR
Mitochondrial activity
0.0
0
0.003
1.5
1.0
35
0.000
120
60
90
Time (min)
Mitochondrial content
Relative DNA content
(mtDNA vs. nDNA)
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ECAR (mpH/min/RFU)
2-Deoxy-D
Glucose Oligomycin glucose
***
40
KLF2
E
Glycolytic function
0.004
OCR (pMoles/min/RFU)
Mock
45
2-NBDG/TMR-Dextran
fluorescence
Mock
Measure
fluorescence
103
104 105
2-NBD-Glucose
∆ψ (TMRM MFI)
102
ECAR (mpH/min/RFU)
0
KLF2
F
2-NBDG
TMR-Dextran
4
2
Glucose transport
Unstained
Mock
KLF2
Counts
6
C
Glucose uptake
***
8
2-NBDG (MFI)
KLF2 mRNA fold change
KLF2 overexpression
10
Luminescence(RLU)
A
5000
0
KLF2
Mock
KLF2
Mitochondrial respiration
Mock
KLF2
0.005
0.004
0.003
0.002
*
0.001
*
0.000
Basal
OCR
ATP
production
Spare
Maximal
respiration respiratory
capacity
Figure 2. Krüppel-like factor 2 (KLF2) overexpression reduces endothelial metabolic activity. A, Total cell mRNA was harvested from
mock- and KLF2-transduced human umbilical vein endothelial cells (HUVECs), KLF2 mRNA expression levels relative to ribosomal
phosphoprotein, large P0 subunit (RPLP0) mRNA was measured by quantitative real-time polymerase chain reaction (n=10). Representative image of a Western blot performed with total cell lysates of mock- and KLF2-transduced cells. Blots were probed with antibodies
raised against KLF2 and α-tubulin as a loading control. B, Representative flow cytometry histogram overlay (top) and quantification of
2-N-7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino-2-deoxyglucose (2-NBD)-glucose uptake (bottom) in HUVECs on KLF2 lentiviral transduction compared with mock-transduced control measured by flow cytometry analysis of 2-NBD-glucose fluorescence (n=6). C, Graphical
representation of glucose transwell setup (top) and ratio of 2-NBDG to tetramethylrhodamine (TMR)-Dextran fluorescence (bottom),
as measured in the compartment underneath the cells, to represent glucose transport through mock- and KLF2-transduced cells on
fibronectin-coated transwell inserts (n=8). D, Extracellular acidification rate (ECAR) profile showing glycolytic function in mock- and KLF2transduced cells. Vertical lines indicate the time of addition of glucose (10 mmol/L), oligomycin (3 μmol/L), and 2-deoxy-D glucose (100
mmol/L). E, Quantification of glycolytic function parameters from C, values are normalized to DNA content (n=3). F, Mitochondrial DNA
content in KLF2-transduced cells vs mock-transduced control measured by quantitative real-time polymerase chain reaction (PCR) of
mitochondrial encoded gene ND1 and nuclear encoded gene RPLP0. Relative levels were normalized to median values (n=3). G, Mitochondrial activity in KLF2-transduced cells vs mock-transduced control measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) reduction. Absorbance levels at 550 nm levels were normalized to median values (n=6). H, ATP produced in KLF2-transduced cells vs mock-transduced control analyzed by quantification of luminescence signal from triplicate wells of each condition (n=3).
I, Mitochondrial membrane potential in KLF2-transduced cells vs mock-transduced controls assessed by flow cytometry analysis of
TMRM fluorescence (n=3). J, Oxygen consumption rate (OCR) profile showing mitochondrial respiration function in mock- and KLF2transduced cells. Vertical lines indicate the time of addition of oligomycin (3 μmol/L), Carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP; 1 μmol/L), antimycin A (1.5 μmol/L), and rotenone (3 μmol/L). K, Quantification of mitochondrial respiration function
parameters from I, values are normalized to DNA content (n=3). In all graphs, data represent mean±SEM *P<0.05, **P<0.01, ***P<0.001.
Doddaballapur et al Shear Stress Inhibits Endothelial Cell Metabolism 141
Cell cycle phase (%)
100
G0 /G1
S
G2 +M
80
60
40
20
0
Mock
KLF2
p21 mRNA expression
2.0
1.5
1.0
0.5
0.0
D
Caspase-3/7 activity (RFU)
p21 mRNA (fold change)
C
B
Cell cycle analysis
β-gal positive cells (% control)
A
Mock
KLF2
Senescence
125
100
75
50
25
0
Mock
KLF2
Caspase- 3/7 activity
6000
4000
**
2000
0
Mock
KLF2
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Figure 3. Krüppel-like factor 2 (KLF2) reduces senescence and
apoptosis. A, Proliferation of mock- and KLF2-transduced human
umbilical vein endothelial cells (HUVECs) measured by flow
cytometric bromodeoxyuridine (BrdU) incorporation analysis at
45 minutes after addition of BrdU (n=3). B, β-gal positive stained
cells counted after 24-hour incubation of acidic β-gal staining
mixture with mock- and KLF2-transduced cells to analyze senescence. Five random image fields were analyzed per condition
(n=3). C, Total RNA from mock- and KLF2-transduced HUVECs
were isolated and analyzed for p21 mRNA expression normalized to ribosomal phosphoprotein, large P0 subunit mRNA (n=4).
D, Apoptosis measured by Caspase-3/7 activity in mock- and
KLF2-transduced HUVECs (n=3). In all graphs, data represent
mean±SEM **P<0.01.
showed that shear stress stimulation downregulates the
expression of many genes involved in glycolysis (Figure 4A),
including the genes encoding hexokinase, phosphofructokinase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase
(PFKFB). Hexokinase converts glucose into glucose 6-phosphate, phosphofructokinase converts fructose 6-phosphate into
fructose 1,6-bisphosphate, and PFKFB converts 6-phosphate
into fructose 2,6-biphosphate, a strong allosteric activator
of phosphofructokinase (Figure 4A). Because the inhibition
of expression of these proteins could explain the inhibitory
effects of KLF2 on glycolysis and metabolic activity, we next
focused on the regulation of expression of these proteins.
To substantiate the observations from our RNAseq experiments (Figure 4A), we confirmed the downregulation of hexokinase-2, PFKFB3, and phosphofructokinase platelet isoform
(PFK1) mRNA expression by quantitative real-time polymerase chain reaction after 72-hour laminar shear stress stimulation (Figure 4B). KLF2 overexpression similarly inhibited
the expression of hexokinase-2, PFKFB3, and PFK1 mRNA
when compared with mock-transduced cells, as measured by
quantitative real-time polymerase chain reaction (Figure 4C).
Western blotting confirmed that hexokinase-2, PFKFB3, and
PFK1 are also significantly inhibited by KLF2 on protein level
(Figure 4D–4F). Next, we determined whether the inhibition
of PFKFB3 and PFK1 expression by shear stress is dependent on KLF2. To this end, we exposed HUVECs transduced
with shKLF2 lentivirus or control lentivirus to shear stress for
72 hours or kept the cells under static conditions and measured the expression of PFKFB3 and PFK1 (Figure 4G).
These experiments show that in the absence of KLF2, shear
stress stimulation does not repress the expression of PFKFB3
and PFK1. Because KLF2 can either activate or repress transcription by binding to specificity protein 1/KLF sites,14 we
examined the PFK1 and PFKFB3 promoters for consensus
specificity protein 1/KLF sites and identified a potential KLF2
binding site 14 bp upstream of the PFKFB3 transcriptional
start site (Figure III in the online-only Data Supplement). We
then placed the 300 bp conserved part upstream of the PFKFB3
promoter in front of a luciferase transporter construct and
measured luciferase activity after transfection into HUVECs
that were either mock or KLF2 transduced (Figure 4H). KLF2
overexpression markedly reduced PFKFB3 promoter activity and mutation of the specificity protein 1/KLF site in the
PFKFB3 promoter abolished the KLF2-mediated repression
of promoter activity. Together, these data indicate that shear
stress represses the expression of key glycolytic enzymes, in
particular, PFKFB3 via direct inhibition of promoter activity
by KLF2.
KLF2 Controls Endothelial Cell Glycolysis
and Angiogenic Phenotype Partly via
PFKFB3 Inhibition
PFKFB3 was recently shown to be an important regulator of
endothelial cell glycolysis,9 and we therefore hypothesized
that inhibition of PFKFB3 could be the underlying mechanism by which KLF2 reduces endothelial cell glycolysis.
First, we set out to confirm the role of PFKFB3 in endothelial cell glycolysis. To this end, we transfected HUVECs with
control small interfering RNA or 2 distinct small interfering
RNAs targeting PFKFB3, which both significantly reduced
PFKFB3 levels (Figure 5A). Seahorse Flux analysis of these
cells showed that robust knockdown of PFKFB3 in HUVECs
significantly reduces acidification rate in the presence of glucose (glycolysis) and also reduces maximal glycolytic capacity of HUVECs (Figure 5B and 5C). Interestingly, glycolytic
reserve capacity (lactate production in the absence of glucose
and mitochondrial function) and cellular oxygen consumption (indicative of mitochondrial activity) are not affected by
PFKFB3 depletion (Figure 5B–5D), indicating that PFKFB3
preferentially controls glycolysis.
Next, we tested whether a reduction in PFKFB3 levels is the
cause of KLF2-mediated inhibition of glycolysis. Hence, we
overexpressed KLF2 in combination with lentiviral overexpression of PFKFB3 in endothelial cells, resulting in PFKFB3
levels in KLF2 overexpressing cells that are comparable with
mock-transduced HUVECs (Figure IV in the online-only Data
Supplement). Seahorse Flux analysis showed that PFKFB3
overexpression in KLF2 overexpressing cells augmented glycolysis but did not affect basal extracellular acidification rate
(in the absence of glucose) (Figure 5E–5H). Consistent with
PFKFB3 depletion experiments, overexpression of PFKFB3
did not affect glycolytic reserve capacity or oxygen consumption (Figure 5I and 5J). These experiments suggest that
KLF2 reduces endothelial cell glycolytic function, in part, via
repression of PFKFB3 expression.
KLF2 has been proposed to repress angiogenic behavior
of endothelial cells, via its ability to induce endothelial cell
142 Arterioscler Thromb Vasc Biol January 2015
A
B
Glycolytic gene expression
1.5
Glucose
mRNA (fold change)
HK1
HK2
HK3
PFKFB1
GCK
PFKFB2
PFKFB3
Glucose-6P
Shear
Static
1.0
*
**
0.5
**
PFKFB4
GPI
0.0
Fructose 6P
C
Fructose-2,6BP
PFKL
Expression levels (RPKM)
PFKP
Fructose-1,6BP
1000
100
10
1.5
**
0.5
0.0
Mock
KLF2
HK2
α-Tubulin
G
1.0
*
0.5
0.0
Mock
**
0.5
Mock
KLF2
60 KDa
PFK-1
80 kDa
55 kDa
α-Tubulin
55 KDa
α-Tubulin
55 kDa
H
PFK1
**
1
0
shKLF2
shCon
shKLF2
Luciferase reporter assay
1.5
Static
Shear
**
**
shCon
1.0
PFKFB3
Glycolytic gene expression
***
1.5
102 kDa
PFKFB3
2
HK2 PFKFB3 PFK1
PFK1 protein expression
0.0
KLF2
Firefly/Renilla
1.0
1.5
**
*
0.5
F
PFKFB3 protein expression
KLF2
**
PFK1 protein levels
E
HK2 protein expression
PFKFB3 protein levels
HK2 protein levels
D
Mock
1.0
0.0
mRNA (fold change)
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<1
<0.25
0.5
1
2
>4
Fold change (shear stress/static)
Glycolytic gene expression
1.5
mRNA (fold change)
PFKM
HK2 PFKFB3 PFK1
1.0
*
0.5
0.0
PFKFB3 promotor
construct
KLF2
WT
WT
Mut
Mut
-
+
-
+
Figure 4. Shear stress represses glycolytic gene expression in a Krüppel-like factor 2 (KLF2)–dependent manner. A, RNA sequencing
profiling of shear stress–stimulated human umbilical vein endothelial cells (HUVECs; 20 dyn/cm2 for 72 hours) and static controls showing
expression levels and regulation of genes in the glycolytic pathway. B, mRNA expression of hexokinase-2 (HK2), 6-phosphofructo-2-­
kinase/fructose-2,6-biphosphatase-3 (PFKFB3), and phosphofructokinase-1 (PFK1) normalized to GAPDH expression in shear stress–
stimulated HUVECs (20 dyn/cm2 for 72 hours) vs static controls, fold repression by shear stress stimulation is indicated (n≥6).
C, mRNA expression of HK2, PFKFB3, and PFK1 normalized to ribosomal phosphoprotein, large P0 subunit expression in KLF2-transduced HUVECs vs mock-transduced controls, fold repression by KLF2 is indicated (n≥3). D–F, Total cell protein lysates from mock- and
KLF2-transduced cells were analyzed by Western blot and probed against antibodies for (D) HK2 (n=4), (E) PFKFB3 (n=4), (F) PFK1 (n=8),
and α-tubulin, which served as a loading control. Representative blot images are shown here and respective protein bands were quantified and normalized to α-tubulin signal. G, mRNA expression of PFKFB3 and PFK1 normalized to GAPDH expression from HUVECs
after lentiviral-mediated shRNA knockdown of KLF2 and flow exposure (20 dyn/cm2 for 72 hours; n=3). H, Luciferase promoter activity
assessed 24 hours after transfection of luciferase plasmid with PFKFB3 promoter region bearing wild-type (WT) KLF2 binding site or
mutated (mut) version of KLF2 binding site. Data are represented as a ratio of firefly luciferase and Renilla (control) activity (n=6). In all
graphs, data represent mean±SEM. *P<0.05, **P<0.01, ***P<0.001.
Doddaballapur et al Shear Stress Inhibits Endothelial Cell Metabolism 143
A
B
siRNA knockdown
1
3
3
FB
si
P
Glycolytic function
0.004
**
0.004
0.002
30
0.005
Oligomycin
2-Deoxy-D
glucose
G
Basal ECAR
Mock
PFKFB3
KLF2
KLF2+
PFKFB3
0.002
**
0.0010
0.0005
I
Glycolytic capacity
0.004
120
0.002
0.001
0.001
+
+
KLF2
-
-
+
+
PFKFB3
-
+
-
+
PFKFB3
-
+
-
+
J
Basal OCR
0.004
0.0006
0.0004
0.0002
0.0000
0.000
*
-
Glycolytic reserve
ECAR (mpH/min/RFU)
0.003
0.002
-
0.0008
***
0.003
KLF2
OCR (pMoles/min/RFU)
H
60
90
Time (min)
***
*
0.000
0.0000
30
Glycolysis
0.004
0.0015
0.004
0.000
siNeg siPFKFB3 siPFKFB3
1
2
ECAR (mpH/min/RFU)
Glucose
ECAR (mpH/min/RFU)
0.006
ECAR (mpH/min/RFU)
0.010
Glycolytic
reserve
F
Glycolytic function
120
Basal OCR
0.000
E
ECAR (mpH/min/RFU)
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**
Glycolytic
capacity
90
0.015
0.000
Glycolysis
60
Time (min)
D
*** ***
Basal
ECAR
2-Deoxy-D
glucose
siNeg
siPFKFB3 A
siPFKFB3 B
siNeg
siPFKFB3 1
siPFKFB3 2
0.006
Oligomycin
0.006
0.000
siPFKFB3 siPFKFB3
1
2
0.002
Glucose
OCR (pMoles/min/RFU)
siNeg
FK
FB
si
N
FK
***
***
ECAR (mpH/min/RFU)
55
2
Tubulin
eg
60
50
0.008
ECAR (mpH/min/RFU)
PFKFB3
si
P
PFKFB3 mRNA
expression (% siNeg)
100
0
C
Glycolytic function
0.008
150
0.003
0.002
0.001
0.000
KLF2
-
-
+
+
KLF2
-
-
+
+
KLF2
-
-
+
+
PFKFB3
-
+
-
+
PFKFB3
-
+
-
+
PFKFB3
-
+
-
+
Figure 5. 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3) is required for endothelial glycolysis and partially reverses
Krüppel-like factor 2 (KLF2)–mediated repression of endothelial glycolytic function. A, PFKFB3 mRNA levels (top) were measured by
quantitative real-time polymerase chain reaction in human umbilical vein endothelial cells (HUVECs) at 48 hours after transfection with
control siRNA (siNeg) or 2 different siRNA directed against PFKFB3 (siPFKFB3; 1 and 2; n=3). Representative Western blot analysis
­(bottom) of total cell protein lysates from HUVECs after 48 hours transfection with siNeg or siPFKFB3; 1 and 2, probed against antibodies
for PFKFB3 and α-tubulin, which served as a loading control. B, Extracellular acidification rate (ECAR) profile showing glycolytic function in HUVECs transfected with siNeg or siPFKFB3 at 48 hours after transfection. Vertical lines indicate the time of addition of glucose
(10 mmol/L), oligomycin (3 μmol/L) and 2-deoxy-D glucose (100 mmol/L; n≥3). C, Quantification of glycolytic function parameters from B
(n≥3). D, Basal Oxygen consumption rate (OCR) levels, all values normalized to DNA content measured by Seahorse flux analysis (n≥3).
E, Representative ECAR profile showing glycolytic function in HUVECs transduced with control, PFKFB3, KLF2, or KLF2 and PFKFB3.
Vertical lines indicate the time of addition of glucose (10 mmol/L), oligomycin (3 μmol/L), and 2-deoxy-D glucose (100 mmol/L). F–J,
Quantification of glycolytic function parameters (F) basal ECAR, (G) glycolysis, (H) maximum glycolytic capacity, (I) glycolytic reserve,
and (J) Basal OCR levels normalized to DNA content measured by Seahorse flux analysis (n=7). In all graphs, data represent mean±SEM
*P<0.05, **P<0.01, ***P<0.001.
144 Arterioscler Thromb Vasc Biol January 2015
Cumulative sprout length (µm)
Sprouting assay
1500
KLF2
KLF2
PFKFB3
Mock
PFKFB3
KLF2
KLF2
PFKFB3
***
*
500
-
B
+
+
-
+
+
Tube formation
15000
***
10000
*
5000
0
KLF2
PFKFB3
C
-
-
-
+
+
-
+
+
DMSO
Aortic ring outgrowth assay
3-PO
ns
6000
*
WT
Cumulative tube length (µm)
PFKFB3
1000
0
KLF2
PFKFB3
D
Mock
4000
2000
0
EC-KO
Discussion
Laminar blood flow induces a quiescent endothelial cell phenotype. It has recently been shown that endothelial cell metabolism,
and in particular glycolysis, is a key determinant of endothelial
cell phenotype.7 Here, we show that fluid shear stress induces
endothelial cell metabolic quiescence, via induction of the
key transcriptional regulator KLF2. Laminar flow reduces the
expression of several genes involved in glucose metabolism and,
in particular, PFKFB3 via KLF2-mediated repression of its promoter activity. These results provide a mechanism by which a
biomechanical stimulus reduces endothelial cell metabolic activity, thereby inducing a quiescent phalanx cell-like phenotype.
It has been suggested that factors that induce endothelial
tip cell phenotype, such as hypoxia-inducible factor 1-alpha
activation and vascular endothelial growth factor or fibroblast
growth factor stimulation induce glycolysis in endothelial
cells, dependent on PFKFB3.9,16 Because KLF2 is known to
inhibit hypoxia-inducible factor 1-alpha15 and vascular endothelial growth factor signaling,17 these mechanisms may contribute to the effects of KLF2 on glycolysis. Furthermore,
KLF2 heterozygous mice, which would putatively display
increased PFKFB3 expression, show an increase in capillary
density15 that is perhaps driven by an increase in glycolysis.
It has recently been shown that PFKFB3-induced glycolysis is required for proper angiogenic behavior in vitro and
in vivo,9,11 and that transient chemical inhibition of PFKFB3
blocks pathological angiogenesis.10 How PFKFB3 expression
is physiologically regulated in endothelial cells has not been
described before. Furthermore, shear stress and KLF2 also
regulate the expression of several other proteins involved in
glycolysis, including hexokinase-2 and PFK1. The inhibition
of expression of these proteins could additionally contribute
to the metabolically quiescent phenotype elicited by shear
stress or KLF2 overexpression in endothelial cells. In fact,
PFKFB3 overexpression alone does induce endothelial sprouting and tube formation activity but does not fully alleviate
A
Cumulative outgrowth length (µm)
Downloaded from http://atvb.ahajournals.org/ by guest on August 3, 2017
quiescence,15 and PFKFB3 is known to be important for angiogenesis.9 We, therefore, determined whether KLF2 reduces
endothelial angiogenic function via reduction of PFKFB3. An
endothelial spheroid sprouting assay, which allows endothelial
cell outgrowth into a 3-dimensional (3D) matrix, showed that
KLF2 indeed reduces endothelial cell sprouting (Figure 6A).
Interestingly, PFKFB3 overexpression significantly induced
sprouting of KLF2 overexpressing endothelial cells. These
results were confirmed in a second angiogenesis assay, where
endothelial cells form a 2D vessel network (Figure 6B). Here,
KLF2 likewise reduced network formation, whereas PFKFB3
overexpression significantly reversed KLF2-mediated inhibition of endothelial network formation. Conversely, endothelial
cell–specific deletion of KLF2 induces sprouting angiogenesis in an aortic ring outgrowth assay, which is dependent on
PFKFB3, as shown by treatment with the PFKFB3 inhibitor 3-PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one;
Figure 6C). Together, these experiments show that KLF2
represses endothelial cell metabolism, in part, through transcriptional inhibition of PFKFB3, thereby inducing a metabolic quiescent endothelial cell phenotype (Figure 6D).
DMSO 3-PO DMSO 3-PO
WT
EC-KO
Glucose
Shear stress
Glucose
G6P
KLF2
PFKFB3
PFKFB3
F6P
F-2,6BP +
Glycolysis
F-1,6BP
PEP
Oxidative
metabolism
Pyruvate
Lactate
EC quiescence
Figure 6. 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase-3
(PFKFB3) partially reverses Krüppel-like factor 2 (KLF2)–mediated
repression of angiogenic activity. A, Spheroids were allowed to
sprout in a 3-dimensional (3D) matrix for 24 hours from cells transduced with mock control, PFKFB3, KLF2, or KLF2 and PFKFB3.
Cumulative sprout length was quantified from 10 sprouts per
condition. Representative images are shown here (n=3). B, Tube
formation network in a 2D matrix was allowed to form for 24 hours
from cells transduced with mock control, PFKFB3, KLF2, or KLF2
and PFKFB3. Cumulative tube length was quantified from 5 random microscopic fields per condition. Representative images are
shown here (n=4). C, Endothelial sprouts from aorta of wild-type
(WT; KLF2 fl/fl) and EC-knockout (KO; Cdh5-CreERT2;KLF2fl/fl)
mice treated with or without 50 μmol/L 3-PO (3-(3-pyridinyl)-1(4-pyridinyl)-2-propen-1-one) were allowed to develop >7 days
and stained with isolectin B4. Cumulative outgrowth length was
quantified from 3 aortic rings per condition. Representative images
are shown here (n=6). D, Schematic representation of the effect
of shear stress on KLF2 upregulation in endothelial cells, which
leads to a direct repression of PFKFB3 transcription and, in turn,
reduces glycolysis and mitochondrial respiration in cells thus
inducing a quiescent phenotype to the endothelium. In all graphs,
data represent mean±SEM *P<0.05, ***P<0.001.
Doddaballapur et al Shear Stress Inhibits Endothelial Cell Metabolism 145
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the KLF2-mediated inhibition of endothelial cell activation
(Figures 5 and 6). Induction of hexokinase-2 and PFK1 is probably not required to overcome the quiescent state induced by
KLF2 fully because these 2 proteins do not seem to be required
for endothelial glycolysis (Figure V in the online-only Data
Supplement). The role of PFKFB3 in shear stress–induced
endothelial phenotype in vivo also remains to be established.
Next to repression of glycolytic activity, KLF2 and shear
stress also reduce mitochondrial content and activity. Even
though endothelial cells do not rely on oxidative phosphorylation for generating ATP,9 mitochondria are known to induce
oxidative stress, regulate calcium signaling, and control
apoptosis.18 Interestingly, KLF2 and shear stress are known
to reduce oxidative stress in endothelial cells19 and inhibit
apoptosis (Figure 3). The reduction of mitochondrial content
and increase in membrane potential by KLF2 could contribute
to these antiapoptotic and atheroprotective cellular effects of
shear stress. Even though KLF2 and shear stress both repress
mitochondrial metabolism, the shear stress–mediated repression seems to be independent of KLF2 (data not shown),
indicating that other shear stress–induced signaling pathways
repress mitochondrial content and activity.
The function of the endothelium is highly dynamic. On
hypoxia, endothelial cells need to migrate and proliferate to form
new blood vessels rapidly, but under homeostatic resting conditions the endothelium needs to maintain a quiescent state. In this
resting state, endothelial cells need to facilitate proper transfer
of nutrients and oxygen to the underlying tissues. Indeed, KLF2
reduces metabolic activity of endothelial cells, which results in
increased levels of glucose underneath the endothelial cells in
our transwell experiments (Figure 2C). It is tempting to speculate
that the shear stress–mediated reduction in endothelial cell glucose consumption ensures a maximal delivery of nutrients and
oxygen to the adjacent tissues. Furthermore, the present data provide a molecular mechanism for the well-known and important
switch of endothelial cell activation and angiogenesis back to a
quiescent functional endothelium once blood flow is established.
Acknowledgments
We thank Dr Auwerx (EPFL [École polytechnique Fédérale de
Lausanne], Switzerland) for discussions and advice and Denise
Berghäuser and Ariane Fischer for technical support.
Sources of Funding
The study was supported by the German Center for Cardiovascular
Research DZHK (BMBF) to S. Dimmeler and W. Chen, the LOEWE
Center for Cell and Gene Therapy (State of Hessen) to A. Doddaballapur,
R.A. Boon, and S. Dimmeler , a VENI grant (91613050) from NWO/
ZonMw to R.H.H., the European Research Council (Advanced grant
Angiomirs) to S. Dimmeler and the Deutsche Forschungsgemeinschaft
(SFB834/B9) to R.A. Boon.
Disclosures
None.
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Significance
Laminar shear stress regulates endothelial cell phenotype by inducing quiescence, but whether metabolic changes contribute to this process
was not known. This study show that laminar shear stress affects endothelial cell glycolytic metabolism, which controls angiogenic behavior
of endothelial cells. These findings provide mechanistic insight into how the switch between the resting and the angiogenic state of endothelial cells is regulated by physiological stimuli.
Downloaded from http://atvb.ahajournals.org/ by guest on August 3, 2017
Laminar Shear Stress Inhibits Endothelial Cell Metabolism via KLF2-Mediated
Repression of PFKFB3
Anuradha Doddaballapur, Katharina M. Michalik, Yosif Manavski, Tina Lucas, Riekelt H.
Houtkooper, Xintian You, Wei Chen, Andreas M. Zeiher, Michael Potente, Stefanie Dimmeler
and Reinier A. Boon
Arterioscler Thromb Vasc Biol. 2015;35:137-145; originally published online October 30, 2014;
doi: 10.1161/ATVBAHA.114.304277
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2014 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/35/1/137
Data Supplement (unedited) at:
http://atvb.ahajournals.org/content/suppl/2014/10/30/ATVBAHA.114.304277.DC1
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Supplementary Material
Supplementary figure I
Shear direction
A
o
90
75
60
*
30
o
90
15
0
1
2
3
2-NBDG Signal
KLF2 levels in the heart
4
C
1.5
ECAR/OCR ratio
D
0.3
1.0
0.5
0.0
5
***
WT
EC-KO
0.2
0.1
0.0
Apoptosis (Annexin V)
6
% Apoptotic cells
KLF2 mRNA (fold change)
Angle to shear direction
45
0
B
o
0
ECAR/OCR
Angle to shear direction
90
Mock
KLF2
p=0.19
4
2
0
Mock
KLF2
Supplementary figure I.
(A) 2-NBD-Glucose uptake by HUVECs on a μ-slide Y-shaped (Ibidi) exposed to 20dyn/cm2 shear stress for 72
hours was analyzed by flow cytometry. 10 images each from high shear and low shear region were analyzed for
2-NBD-Glucose fluorescence and cell alignment. *p<0.05 by Pearson’s correlation. (B) Total RNA from hearts of
WT and EC-KO mice were isolated and analyzed for KLF2 mRNA expression normalized to RPLP0 mRNA. (n≥4)
(C) Ratio of basal ECAR and basal respiration OCR as measured by Seahorse XFe analyzer in mock- and KLF2transduced HUVECs. (n=3). (D) Apoptosis as measured by Annexin V staining by flow cytometry in mock- and
KLF2 transduced HUVECs. (n=5). In all graphs, data represent mean ± S.E.M *p<0.05, ***p<0.001 (See also
figure 1 and 2)
Supplementary figure II
A
B
Phospho-kinase proteome profile
p-AMPKα1 (T174) levels
Ratio Lenti-KLF2/Mock (log2)
0
1
2
E
Lenti-Mock
Lenti-KLF2
C
2-NBDG (MFI)
***
50
***
0.002
1000
H
0.04
0.5
ECAR (mpH/min)
0.03
0.02
0.01
si
AM
PK
α1
si
1
AM
PK
α1
2
*
10
5
-
-
-
+
+
-
Basal OCR
75
**
15
0
KLF2
L-NAME
g
Ne
si
I
Basal ECAR
20
g
Ne
si
1.0
Glycolytic
reserve
OCR (pMoles/min)
Basal OCR
si
AM
PK
α1
si
1
AM
PK
α1
2
OCR (pMoles/min/RFU)
2000
1.5
0.0
g
Ne
si
3000
Mitochondrial DNA content
0.000
0.00
4000
si
AM
PK
α1
si
1
AM
PK
α1
2
F
0.004
Glycolytic
capacity
KLF2
Glucose uptake
0
g
Ne
si
siAMPKα1 1
siAMPKα1 2
Glycolysis
Mock
si
AM
PK
α1
si
1
AM
PK
α1
2
AMPKα1 mRNA
expression (% siNeg)
100
siNeg
G
1000
5000
150
0
*
2000
D
AMPKα1 knockdown
Glycolytic function
Basal
ECAR
3000
0
0.006
ECAR (mpH/min/RFU)
4000
3
p-AMPKα1 (pg/ml)
-1
Relative DNA content
(mtDNA vs. nDNA)
-2
AMPKa1 (T174)
HSP60
B-Catenin
c-Jun (S63)
JNK pan (T183/Y185 T221/Y223)
STAT6 (Y641)
AMPKa2 (T172)
STAT5a (Y694)
STAT2 (Y689)
Fgr (Y412)
p38a (T180/Y182)
Chk-2 (T68)
Lyn (Y397)
Lck (Y394)
FAK (Y397)
STAT5a/b (Y694/Y699)
HSP27 (S78/S82)
p53 (S392)
STAT5b (Y699)
GSK-3a/b (S21/S9)
MSK1/2 (S376/S360)
Akt (S473)
CREB (S133)
p53 (S46)
Src (Y419)
PRAS40 (T246)
p53 (S15)
TOR (S2448)
Yes (Y426)
PDGF Rb (Y751)
EGF R (Y1086)
Hck (Y411)
Fyn (Y420)
Akt (T308)
STAT3 (Y705)
RSK1/2/3 (S380/S386/S377)
p70 S6 Kinase (T421/S424)
PLC-g1 (Y783)
p27 (T198)
WNK1 (T60)
p70 S6 Kinase (T389)
ERK1/2 (T202/Y204 T185/Y187)
PYK2 (Y402)
STAT3 (S727)
eNOS (S1177)
+
+
50
25
0
KLF2
L-NAME
-
-
-
+
+
-
+
+
Supplementary figure II.
(A) Phospho-kinase array profile showing phosphorylation levels of proteins in KLF2-transduced HUVEC
versus mock-transduced controls. Code in parenthesis indicates site of phosphorylation and those highlighted
in red represent proteins with significant changes in levels as analyzed by two way ANOVA. (n=4)
(B) Phosphorylated-AMPKa1 (174 levels) measured in mock- and KLF2 transduced cell lysates. Final sample
concentration was calculated using corrected absorbance values against standard curve concentration. (n=3).
(C) AMPKa1 mRNA levels were measured by real time qPCR in HUVECs at 48 hours after transfection with
control siRNA (siNeg) or two different siRNA directed against AMPKa1 (siAMPKa1; 1 and 2), expression
normalized to RPLP0 mRNA (n=3). 48 hours after transfection with siNeg or siAMPKa1, HUVECs were
analyzed for (D) Glucose uptake by flow cytometry analysis of 2-NBD-Glucose fluorescence (E) ECAR levels
normalized to DNA content to quantify glycolytic function (F) Mitochondrial DNA content measured by
quantitative real time PCR of mitochondrial encoded gene ND1 and nuclear encoded RPLP0. Relative levels
were normalized to median values (G) Basal OCR levels normalized to DNA content measured by Seahorse
flux analysis. (n=3). Mock-and KLF2-transduced HUVECs treated with 1mM L-NAME (Nω-nitro-L-argininemethyl ester hydrochloride) for 4 hours were analyzed for (H) Basal ECAR levels and (I) basal OCR by
Seahorse XF flux analysis (n=4). In all graphs, data represent mean ± S.E.M *p<0.05, **p<0.01, ***p<0.001
Supplementary figure III
A
B
C
PFKFB3 (WT):
GAATGCGGCCCGCCCCGAGGCT
PFKFB3 (Mutant):
GAATGCGGAATTAACCGAGGCT
Supplementary figure III.
(A) Graphical representation showing evolutionary conserved regions in PFKFB3 genome (adapted from rVISTA
2.0, http://rvista.dcode.org/) (B) Enlarged region from (A) within red box showing conserved Sp1/KLF binding
sites on PFKFB3 promoter (B) Partial sequence of PFKFB3 promoter constructs wildtype (WT) Sp1/KLF binding
site and mutated (mut) binding site (highlighted in red) cloned into firefly luciferase reporter plasmids. (See also
figure 4)
Supplementary figure IV
A
mRNA (fold change)
20
KLF2
15
PFKFB3
***
***
10
***
***
5
0
KLF2
-
+
-
+
PFKFB3
-
-
+
+
Supplementary figure IV.
(A) Total cell mRNA was analyzed for expression of KLF2 and PFKFB3 by real time qPCR in HUVECs transduced with mock, KLF2, PFKFB3 or KLF2 and PFKFB3 lentivirus. All expression levels were normalized to
RPLP0 mRNA (n=6). In all graphs, data represent mean ± S.E.M ***p<0.001 (See also figure 5)
Supplementary figure V
B
50
***
***
1
2
K2
H
si
K2
H
si
C
0.002
0.000
si
H
K2
eg
N
i
s
0.004
si
N
eg
0
0.006
2
100
Basal OCR
1
150
OCR (pMoles/min/RFU)
HK2 mRNA expression
(% siNeg)
HK2 knockdown
si
H
K2
A
Glycolytic function
ECAR (mpH/min/RFU)
0.003
siNeg
siHK2 1
siHK2 2
0.002
0.001
0.000
F
0.004
0.002
3
K1
2
Glycolytic function
0.003
ECAR (mpH/min/RFU)
si
PF
K1
si
PF
si
PF
K1
1
0.000
3
2
0.006
K1
si
PF
si
PF
K1
1
0
***
0.008
si
PF
50
Basal OCR
K1
100
si
N
eg
PFK1 mRNA expression
(% siNeg)
150
***
Glycolytic
reserve
E
PFK1 knockdown
***
Glycolytic
capacity
si
N
eg
D
Glycolysis
OCR (pMoles/min/RFU)
Basal
ECAR
siNeg
siPFK1 1
siPFK1 2
siPFK1 3
0.002
0.001
0.000
Basal
ECAR
Glycolysis
Glycolytic
capacity
Glycolytic
reserve
Supplementary figure V.
(A) HK2 mRNA levels were measured by real time qPCR in HUVECs at 48 hours after transfection with
control siRNA (siNeg) or two different siRNA directed against HK2 (siHK2; 1 and 2), expression
normalized to RPLP0 mRNA (n=3). 48 hours after transfection with siNeg or siHK2, HUVECs were
analyzed for (B) basal OCR and (C) ECAR levels to quantify glycolytic function as measured by Seahorse
flux analysis, both normalized to DNA content (n=3).
(D) PFK1 mRNA levels were measured by real time qPCR in HUVECs at 48 hours after transfection with
control siRNA (siNeg) or three different siRNA directed against PFK1 (siPFK1; 1, 2 and 3), expression
normalized to RPLP0 mRNA (n=3). 48 hours after transfection with siNeg or siPFK1, HUVECs were
analyzed for (B) basal OCR and (C) ECAR levels to quantify glycolytic function as measured by Seahorse
flux analysis, both normalized to DNA content (n=3). In all graphs, data represent mean ± S.E.M *p<0.05,
**p<0.01, ***p<0.001.
Material and methods
Cell culture, transfection and lentivirus
Pooled Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from Lonza
(Verviers, Belgium) and cultured in EBM (Lonza) supplemented with 10%FBS (Invitrogen) and
EGM-SingleQuots (Lonza). Cells were grown at 37°C and 5% CO2 and cell number was
determined by NucleoCounter (ChemoMetec A/S). Lipofectamine RNAiMax (Life
Technologies) was used to transfect 60-70% confluent HUVECs with 70nM siRNA (Sigma)
against PFKFB3, AMPKα1, HK2 and PFK1 according to manufacturer’s instructions. Universal
negative control siRNA (Sigma) was used as control. Cells were treated Nω-nitro-L-argininemethyl ester hydrochloride (L-NAME, 1mM, Sigma). Long term overexpression of KLF2 1 and
shRNA mediated silencing of KLF2 was done as previously described 2. PFKFB3 cDNA was
obtained in pENTR221 (Source Bioscience) and shuttled into plenti4 vector (Invitrogen).
Lentiviral particles were generated as previously described 3.
Shear stress
1x105 HUVECs were plated overnight on µ -Slides I 0.4 Luer (Ibidi) and exposed to laminar flow
at a shear stress of 20 dynes/cm2 for 72 hours controlled by an Ibidi perfusion system. HUVECs
seeded on µ-Slides I (Ibidi) were used as static controls.
Cone plate viscometer setup
500,000 HUVECs were seeded overnight on 6cm dishes and were subjected to a fluid shear
stress of 12 dyn/cm2 for 48 hours in a cone plate viscometer as previously described4. HUVECs
maintained under static conditions served as controls.
In-vitro Glucose up-take
HUVECs were incubated with 100µM of 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2Deoxyglucose (2-NBDG, Life Technologies) for 1hour at 37°C and 2-NBDG fluorescence was
measured in the FITC channel (FL-1) using a FACS Canto II device (BD Biosciences).
Glucose uptake imaging
1x105 HUVECs were plated overnight on μ-slide y shaped (Ibidi) and exposed to laminar flow at
a shear stress of 20 dynes/cm2 for 72 hours controlled by an Ibidi perfusion system. Slides were
incubated with 1mM of 2-NBDG (Life Technologies) for 30 min at 37°C and confocal
micrographs were imaged on LSM 780, Axio Observer (Carl Zeiss) with a LD LCI PlanApochromat 25x/0.8 Imm Korr DIC M27 objective. 10 images each were analyzed from straight
channel high shear region and branched low shear regions for 2-NBDG mean fluorescence and
cell alignment using NIH ImageJ digital image analysis software.
Glucose transwell assay
Mock and KLF2 transduced cells were seeded on fibronectin coated 24-well ThinCert transwell
PET inserts (Greiner bio-one). Cells were washed twice in Hank’s BSS (GE healthcare) and
incubated with 100μM 2-NBDG (Life Technologies) and 10μM 20kDa -Dextran (TMR-Dextran,
Sigma), which served as an internal control, in Hank’s BSS for 1 hour at 37°C. TMR-dextran
fluorescence (Ex 525nm and Em 580-640nm) and 2-NBDG fluorescence (Ex 490nm and Em
510-570nm) were measured in duplicates from Hank’s BSS in the compartment underneath the
cells using a GloMax®-Multi+ Microplate Multimode Reader with Instinct®
RNA isolation and Real Time Quantitative Polymerase Chain Reaction
Total RNA was isolated from cultured cells using miRNeasy kits (Qiagen) according to the
manufacturer’s instruction. cDNA was synthesized from 0.5-1µg RNA using MuLV reverse
transcriptase (Life Technologies) and random hexamers (Thermo Scientific). Real time
quantitative PCR was carried out using Fast SYBR Green (Applied Biosystems) in a
StepOnePlus machine (Applied Biosystems). Gene expression was normalized to RPLP0 or
GAPDH. Primer sequences are listed in table 1.
Mitochondrial DNA content measurement
Total DNA was isolated from cells using DNeasy Blood and Tissue kits (Qiagen) as per
manufacturer’s instruction. 30ng DNA was analyzed by real time qPCR for mitochondrial
encoded gene ND1 and nuclear encoded gene RPLP0. The ratio of the relative levels of
mitochondrial ND1 DNA and nuclear RPLP0 DNA was used to express mitochondrial DNA
content.
Mice experiments
All mice experiments were carried out in accordance with the principles of laboratory animal
care as well as according to the German national laws. The studies have been approved by the
local ethical committee (Regierungspräsidium Darmstadt, Hessen). Cdh5-CreERT2 mice 5 and
KLF2 flox/flox 6 were described previously and kindly provided by Dr. Adams (Münster,
Germany) and Dr. Sebzda (Vanderbilt University, TN), resp. Animals were administered with
seven injections of 2 mg tamoxifen base each (Sigma) intraperitoneally over a period of two
weeks and sacrificed in the third week.
Ex-vivo glucose up-take in mice hearts
Mice hearts were subjected to Langendorff mediated perfusion and digestion as previously
described 7 along with 50µM 2-NBDG. The digest was incubated with 200µM 2-NBDG and 5ul
CD31-APC antibody (BD Pharmingen) for 30 min at 37°C and analyzed for CD31+ endothelial
cell population and 2-NBDG fluorescence using a FACS Canto II device
Measurement of cellular metabolism
HUVECs were seeded overnight at 6x104 cells per well on fibronectin (Sigma) coated Seahorse
XF96 polystyrene tissue culture plates (Seahorse Bioscience). The plate was incubated in
unbuffered DMEM assay medium (Sigma) for 1 hour in a non-CO2 incubator at 37°C before
measuring in an XFe 96 extracellular flux analyzer (Seahorse Bioscience). Both OCR (oxygen
consumption rate) and ECAR (extracellular acidification rate) were measured over 4 min periods
with a mixing of 2 min in each cycle, with five cycles in total. Inhibitors and activators were
used at the following concentrations: Glucose (10mM), Oligomycin (3µM), 2-DG (100mM),
FCCP (1µM), Antimycin A (1.5µM) and Rotenone (3µM). Cellular DNA content using DAPI
(Roche) was measured on a microplate reader (TECAN) and the data is represented as OCR or
ECAR normalized to DNA content (RFU). Each measurement was averaged from triplicate
wells.
Mitochondrial activity measurement
Cells were incubated with 0.5mg/ml of MTT (Dimethylthiazol-zyl-diphenyltetrazoliumbromide)
for 4 hours at 37°C. After a wash with PBS, cells were lysed 30 min at room temperature and
absorbance of the cleared lysate was photo metrically measured at 550nm.
ATP assay
Cell titer Glo luminescent cell viability assay kit (Promega) was used to quantify ATP levels in
cells. 3x104 cells in 100µl culture media were incubated with 100µl Cell-Titer-Glo Reagent
(Promega) for 10 min at room temperature. Luminescence signal was measured with FlourChem
M system (Proteinsimple) and signal normalized to background media control. All conditions
were assayed in triplicates.
Mitochondrial membrane potential measurement
Mitochondrial membrane potential in HUVECs was assessed using MitoPT® TMRM Assay Kit
(Immunochemistry Technologies LLC). Cells were incubated with 100nM TMRM for 20 min at
37°C and the orange red fluorescence (FL2) in cells was analyzed using FACS Canto II device
(BD Biosciences).
Proliferation assay
Cells were incubated with 10mM BrdU for 45 min and subsequent staining was performed using
BrdU Flow Kit (BD Pharmingen) as per manufacturer’s instruction. Cells were stained with
2.5µl of anti-BrdU-V450 for 20min and 10µl of 7-AAD for 10min, both at room temperature and
analyzed using FACS Canto II device (BD Biosciences)
Caspase 3/7 activity
Apo-ONE Homogeneous Caspase-3/7 assay kit (Promega) was used as per manufacturer’s
instruction and Caspase 3/7 activity was analyzed by measuring fluorescence at with an
excitation of 490nm and emission of 510-570nm using a GloMax®-Multi+ Microplate
Multimode Reader with Instinct® (Promega). Mock transduced cells treated with 200nM of
staurosporine for 4 hours were used as a positive control.
Apoptosis measurement
Mock- and KLF2 transduced cells were stained with Annexin V V450 (BD Biosciences) and 7AAD (BD Biosciences) in binding buffer (BD Biosciences) for 15 min at room temperature as
per manufacturer’s instructions. Apoptosis was quantified by flow cytometry analysis on a FACS
Canto II device (BD Biosciences)
Senescence staining
Acidic β-gal staining mixture was added onto cells for 24 hours as per manufacturer’s instruction
(Cell signaling). Cells were imaged on a bright field microscope (Axiovert 100, Zeiss) and 5
image fields were analyzed per condition for positive β-gal staining using digital image analysis
software (AxioVision Rel. 4.8, Carl Zeiss).
Phospho kinase proteome profiling
5x106 mock- and KLF2- transduced cells were processed using Human Phospho-Kinase Array
Kits (Proteome ProfilerTM R&D Systems, Minneapolis, MN) according to manufacturer’s
instructions. The phosphorylation level of proteins was analyzed by quantification of membrane
profile panels by NIH ImageJ digital image analysis software with a Dot Blot Analyzer macro
plugin. The signal values were subtracted from negative control values (PBS) and further
normalized to whole membrane signal intensity.
AMPKα1 phosphorylation measurement
2x107 mock- and KLF2- transduced cells were analyzed using DuoSet IC Human Phospho
AMPKα1 (T174) elisa kits (R&D Systems) according to manufacturer’s instruction. Absorbance
was measured at 450nm using a GloMax®-Multi+ Microplate Multimode Reader with Instinct®
(Promega) and wavelength correction was set to 560nm. Concentration was determined from a
standard curve performed along with the samples.
RNA sequencing
For RNA sequencing, 0.5μg total RNA isolated from static HUVEC and shear stress exposed
HUVEC were used. Sequencing libraries were prepared as described previously8. The deep
sequencing data was mapped against the reference genome GRCH37 by applying tophat2.
Afterwards the transcript abundance was calculated by Cufflinks v2.2 based on the Ensemble
annotation. The sequence data have been deposited in the NCBI GEO database under accession
number GSE54384.
Western blotting
HUVECs were lysed in RIPA buffer (Sigma) supplemented with protease and phosphatase
inhibitors (Roche) for 20min on ice. Western blot analysis was performed as described
previously by using antibodies against PFKFB3, HK2, PFKP (1:1000, Cell signaling), KLF2 9
and Tubulin (1:2000, Thermo Scientific). All protein levels were normalized to tubulin signal.
Luciferase assay
Luciferase promoter reporter experiments were performed as described 10. Specifically, the
region bearing the putative KLF2 binding site in PFKFB3 promoter or a mutated version of the
KLF2 binding site (see Figure S3) were cloned into firefly luciferase reporter plasmid pGL4.10
according to manufacturer’s instructions (Promega). Cells were co-transfected with luciferase
plasmid and pGL4 Renilla plasmid (Promega) as control for transfection efficiency by
electroporation using Neon transfection system (Invitrogen). The activity of Luciferase and
Renilla was assessed after 24 hours with the Dual Luciferase reporter assay system (Promega)
Tube formation assay
HUVECs were cultured at a density of 1x105 on Matrigel basement membrane matrix (BD) and
tube formation was analyzed after 24 hours. Cumulative tube length was quantified from
microscopic images taken from 5 random fields for every condition. Quantification was carried
out by digital image analysis software (AxioVision Rel. 4.8, Carl Zeiss)
Spheroid based angiogenic assay
Endothelial cell spheroids were generated as previously described 11. Cumulative length of
sprouts was quantified from 10 spheroids for every condition. Quantification was carried out by
digital image analysis software (AxioVision Rel. 4.8, Carl Zeiss)
Aortic Ring Outgrowth Assay
Aortae were isolated from WT (KLF2 fl/fl) and EC-KO (Cdh5-CreERT2;KLF2fl/fl) mice and
cultured as previously described12. Briefly, cleaned 1 mm long aortic tissue was embedded in rat
tail collagen type I gel (1mg/ml, Millipore) in a 96 well plate and cultured in DMEM/F-12
medium (Life technologies) containing 2.5% FBS and treated with or without 3-PO (50µM,
Merck Millipore). Endothelial sprouts were allowed to develop over 7 days, which were fixed
with 4% PFA and stained with biotin Isolectin B4 (Vector laboratories) and streptavidin
AlexaFluor 488 (Molecular Probes). Photomicrographs of sprouts from aortic rings were taken
with Axio Observer.Z1 microscope (Zeiss) and cumulative outgrowth length from 3 rings per
condition was quantified using NIH Image J digital image analysis software
Statistical analysis
Data are expressed as mean ± S.E.M. GraphPad Prism 5 software was used to assess statistical
significance by student t-test or Mann-Whitney U test when comparing two groups or analysis of
variance (ANOVA) followed by Bonferroni’s correction or Kruskal-Wallis test with Dunns
correction for multiple comparisons. Statistical significance was defined as follows: *p<0.05,
**p<0.01, ***p<0.001
Table 1. Oligonucleotide primer sequence list
Target
gene
KLF2
RPLP0
GAPDH
HK2
PFKFB3
PFK1
AMPKα1
mt.ND1
p21
KLF2
RPLP0
Species
Forward primer
Reverse primer
Human
Human
Human
Human
Human
Human
Human
Human
Human
Mouse
Mouse
CGGCAAGACCTACACCAAGAG
TCGACAATGGCAGCATCTAC
ATGGAAATCCCATCACCATCTT
GCTTGGAGCCACCACTCACCC
GGAGGCTGTGAAGCAGTACA
ACTTGGAAGAGATCGCCACA
AGGCTCCACGAAGGAGCTGGAT
CCCTAAAACCCGCCACATCT
CAGCATGACAGATTTCTACC
GGCGCATCTGCGTACACA
GCGTCCTGGCATTGTCTGT
CTGTGTGCGTGCGCAGAT
ATCCGTCTCCACAGACAAGG
CGCCCCACTTGATTTTGG
AGCCAGGAACTCTCCGTGTTCTGT
CAGCTAAGGCACATTGCTTC
CCCAGGTAGGCCTCGAATC
ATGGACCACCATATGCCTGTGACAA
GAGCGATGGTGAGAGCTAAGGT
CAGGGTATGTACATGAGGAG
GCATCCTTCCCAGTTGCAAT
GAAGGCCTTGACCTTTTCAGTAAG
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