Moderate Caveolin-1 Downregulation Prevents NADPH Oxidase

Transcription

Moderate Caveolin-1 Downregulation Prevents NADPH OxidaseDependent
Endothelial Nitric Oxide Synthase Uncoupling by Angiotensin II in Endothelial
Cells
Irina Lobysheva, Géraldine Rath, Belaïd Sekkali, Caroline Bouzin, Olivier Feron,
Bernard Gallez, Chantal Dessy and Jean-Luc Balligand
Arterioscler Thromb Vasc Biol published online Jun 9, 2011;
DOI: 10.1161/ATVBAHA.111.230623
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association.
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Moderate Caveolin-1 Downregulation Prevents NADPH
Oxidase–Dependent Endothelial Nitric Oxide Synthase
Uncoupling by Angiotensin II in Endothelial Cells
Irina Lobysheva, Géraldine Rath, Belaïd Sekkali, Caroline Bouzin, Olivier Feron, Bernard Gallez,
Chantal Dessy, Jean-Luc Balligand
Objective—We analyzed the role of caveolin-1 (Cav-1) in the cross-talk between NADPH oxidase and endothelial nitric
oxide synthase (eNOS) signaling in endothelial caveolae.
Methods and Results—In intact endothelial cells, angiotensin II (AII) concurrently increased NO and O2⫺䡠 production (to
158⫾12% and 209⫾5% of control). NO production was sensitive to inhibition of NADPH oxidase and small interfering
RNA downregulation of nonreceptor tyrosine kinase cAbl. Reciprocally, L-NAME, a NOS inhibitor, partly inhibited
O2⫺䡠 stimulated by AII (by 47⫾11%), indicating eNOS uncoupling, as confirmed by increased eNOS monomer/dimer
ratio (by 35%). In endothelial cell fractions separated by isopycnic ultracentrifugation, AII promoted colocalization of
cAbl and the NADPH oxidase subunit p47phox with eNOS to Cav-1-enriched fractions, as confirmed by proximity
ligation assay. Downregulation of Cav-1 by small interfering RNA (to 50%), although it preserved eNOS confinement,
inhibited AII-stimulated p47phox translocation and NADPH oxidase activity in Cav-1-enriched fractions and reversed
eNOS uncoupling. AII infusion produced hypertension and decreased blood Hb-NO in Cav-1⫹/⫹ mice but not in
heterozygote Cav-1⫹/⫺ mice with similar Cav-1 reduction.
Conclusion—Cav-1 critically regulates reactive oxygen species– dependent eNOS activation but also eNOS uncoupling in
response to AII, underlining the possibility to treat endothelial dysfunction by modulating Cav-1 abundance. (Arterioscler
Thromb Vasc Biol. 2011;31:00-00.)
Key Words: angiotensin II 䡲 endothelium 䡲 free radicals/free-radical scavengers 䡲 nitric oxide 䡲 superoxide
R
educed bioavailability of endothelium-derived nitric oxide (NO), a key regulator of vascular homeostasis, is a
hallmark of endothelial dysfunction, a recognized risk factor
for cardiovascular diseases. Many of these diseases (eg,
hypertension and diabetes) are associated with activation of
the renin-angiotensin system.1 Its effector, angiotensin II
(AII), activates vascular NADPH oxidase, one of the main
cellular sources of superoxide anion radicals (O2⫺䡠) that
perpetuates a second cascade of signaling events triggered by
reactive oxygen species (ROS) formation.2,3 In the course of
these events, impairment of endothelial NO bioavailability is
caused by scavenger reactions with O2⫺䡠 or inhibition of the
endothelial NO synthase (eNOS) (eg, by ROS-activated
PYK24). Moreover, the activity of eNOS can shift from NO
to O2⫺䡠 production (ie, eNOS “uncoupling”) in case of
shortage of its substrate, L-arginine; oxidation of the cofactor
(6R)-5,6,7,8-tetrahydrobiopterin or of eNOS itself; changes
of the eNOS phosphorylation pattern; or disruption of the
protein-protein interaction (eg, with hsp905–7), all of which
may be caused by ROS production.8,9 On the other hand,
increasing evidence suggests a signaling role for ROS in the
vasculature beyond NO scavenging. For example, short-time
exposure of endothelial cells (ECs) to AII (30 minutes)
increases eNOS activity (and NO production) via AT1 receptor/NADPH oxidase; in this setting, hydrogen peroxide resulting from superoxide dismutation has been proposed as a
mediator of eNOS activation.10
Spatial compartmentation may be one explanation for this
apparent paradox, but it has been very little explored in the
context of the cross-talk between NO and ROS in ECs. eNOS
is a well-known “resident” of endothelial caveolae, where its
activity is tightly regulated by its interaction with the structural caveolar protein, caveolin-1 (Cav-1).12,13 The mechanism(s) of NADPH oxidase activation by AII has been more
intensively studied in vascular smooth muscle. The assembly
of a functional enzyme requires at least 2 main membrane
components: one of the NOX homologs (such as gp91phox,
or NOX2; NOX1; and NOX4, all identified in the endothe-
Received on: August 9, 2010; final version accepted on: May 23, 2011.
From the Unit of Pharmacology and Therapeutics, Institute of Clinical and Experimental Research (I.R., G.R., B.S., C.B., O.F., C.D., J.-L.B.) and the
Biomedical Magnetic Resonance Unit, Louvain Drug Research Institute (B.G.), Université catholique de Louvain, Brussels, Belgium.
Correspondence to Jean-Luc Balligand, Unit of Pharmacology and Therapeutics (FATH 5349), Institute of Clinical and Experimental Research, UCL,
52 Avenue E. Mounier, B-1200 Brussels, Belgium (E-mail [email protected]); or Irina Lobysheva, Unit of Pharmacology and Therapeutics
(FATH 5349), Institute of Clinical and Experimental Research, UCL, 52 Avenue E. Mounier, B-1200 Brussels, Belgium (E-mail
[email protected]).
© 2011 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.111.230623
Downloaded from atvb.ahajournals.org at University
of Pennsylvania on June 30, 2011
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Arterioscler Thromb Vasc Biol
September 2011
lium) and p22phox, a stabilizer. Activation of NOX2/NOX1
requires the additional association of the cytosolic p47phox,
an organizer of its functional assembly, and of the activator
p67phox, which were shown to translocate to the membrane
after stimulation.14 Recent evidence suggests that this multisubunit assembly might take place in caveolin-enriched
membrane fractions, ie, endothelial rafts/caveolae.15 However, a role for Cav-1 in orchestrating the interaction between
NO and ROS from eNOS and NADPH oxidase in rafts/
caveolae following activation by AII was never investigated.
This is important because (1) Cav-1 abundance is known to
be regulated by disease states, and in particular, we showed
that its increase in ECs in hypercholesterolemic states causes
NO-dependent endothelial dysfunction16; and (2) moderate
downregulation of Cav-1 (as obtained with therapeutic concentrations of statins) potentiates eNOS activation.17,18 Therefore, we hypothesized that Cav-1 abundance may critically
control the assembly of caveolar signalosomes containing
both eNOS and NADPH oxidase on AII stimulation of ECs
and that its downregulation may decrease ROS production
while maintaining NOS activity, thereby tilting the balance
toward preserved NO production and bioavailability.
Materials and Methods
Materials and Cell Cultures
All chemicals of ultrahigh grade were purchased from Sigma-Aldrich Chemical Inc or Alexis Biochemical Inc. Primary cultures of
human umbilical vein ECs (HUVECs) and bovine aortic ECs
(BAECs) were purchased from Clonetics (Lonza, Switzerland).
Murine ECs were isolated from aortas of mice haploinsufficient for
Cav-1 (Cav-1⫹/⫺) and wild-type littermates of the same C57Bl6
background (Cav-1⫹/⫹). For details, see the Supplemental Data,
available online at http://atvb.ahajournals.org.
Animals
Male 16-week-old mice, Cav-1⫹/⫺ and Cav-1⫹/⫹, were anesthetized
and implanted with miniaturized telemetry devices (Datascience
Corp) and osmotic minipumps (model 2002, Alzet) for AII delivery
(1.1 mg/kg per day) or saline as described previously19 and in the
Supplemental Data. Mice were injected with heparin and anesthetized, and venous blood was obtained by a puncture of the right
ventricle, immediately frozen in calibrated tubes (0.2 mL) at 77 K,
and processed for EPR measurements. Experiments conformed with
the Guide for the Care and Use of Laboratory Animals and with the
recommendations of the local ethics committee.
Small Interfering RNA Transfection
RNA oligonucleotides complementary to Cav-1 and cAbl target
sequences (Qiagen) were used to silence respective gene expression.
A detailed description of the construction, transfection, and control
small interfering RNA (siRNA) is available in the Supplemental
Data.
Purification of Caveolae-Enriched
Membrane Fractions
Caveolin-enriched membranes were isolated by isopycnic ultracentrifugation of lysates of ECs against sucrose gradient (35% to 5%) as
described previously.20 The fractions were collected (0.5 mL each),
tested for NADPH oxidase activity by EPR spin trapping with
5.5-dimythyl-1-pyrolline-N-oxide (DMPO), and concentrated for
immunoblotting. A detailed description of the protocols for cell
fractionation is available in the Supplemental Data.
Immunoprecipitation and Immunoblotting
Cells were lysed with cold lysis buffer, homogenized, and analyzed
with appropriate antibodies as described previously.21 For eNOS
monomer/dimer assay, nondenatured cell lysates in ice-cold buffer
were separated by low-temperature SDS-PAGE for immunoblotting.
Signals were quantified by densitometry, normalized to loading
control, and expressed as ratio of monomeric/monomeric⫹dimeric
eNOS. A detailed description of these assays is available in the
Supplemental Data.
Immunofluorescence Microscopy and Proximity
Ligation Assay In Situ
Confluent HUVECs were preincubated with low-serum medium and
stimulated or not with AII before fixation. Then cells were permeabilized, washed, incubated with 5% of bovine serum albumin, and
then sequentially incubated with the appropriate antibodies. Speciesspecific secondary antibodies (Alexa and DyLight IgG) for immunocytochemistry or Duolink proximity ligation assay (PLA) probes
(Olink Bioscience, Sweden) were added according to the manufacturers’ protocols. Colocalized p47phox and Cav-1 bound to the
specific antibodies were detected after reporter DNA circularization
with a size-limiting linker oligonucleotide, ligation, and rolling circle
amplification. Amplified reporter DNA was detected using complementary fluorescent probes. After nuclear staining with Hoechst
33342 and cytoskeleton with phalloidin, images were acquired with
a ZeissImager.Z1 fluorescence microscope equipped with an ApoTome device using ⫻20, ⫻40, or ⫻63 oil-immersion objective
lenses and analyzed with AxioVision and Duolink BlobFinder
software. A detailed description of the protocols is available in the
Supplemental Data.
Measurements of NO and Superoxide Anions
by EPR
Bioavailable NO was assayed by EPR spin trapping in ECs as
previously reported.21,22 Briefly, serum-starved cells (treated with
AII with/without inhibitors or the respective vehicle) were incubated
with a colloid solution of the NO spin trap, diethyldithiocarbamateiron complex in culture dishes in a CO2 incubator. After medium
removal, cells were scraped on ice, and extracts were frozen in
calibrated tubes in liquid nitrogen. The formed paramagnetic NOadduct, accumulated at plasma cell membranes, was assayed by a
Bruker EMX100 spectrometer.
The level of circulating Hb-NO was assayed in whole blood of
mice from the EPR signal of 5-coordinate-␣-Hb-NO as described
previously.19 The EPR spectra of whole blood were recorded by a
Bruker EMX100 spectrometer. The level of Hb-NO was quantified
from hfs of the signal after subtraction of EPR signal of free radicals
using Microcal Origin software.
Extracellular O2⫺䡠 formation was assayed by EPR spin trapping
using DMPO (high purity, Alexis after charcoal filtration) as
described previously.23 Cells were preincubated in Krebs-DTPAHepes buffer with pharmacological reagents or vehicle and stimulated (or not) with AII in the presence of DMPO (60 mmol/L) at
37°C in a CO2 incubator. The extracellular medium was transferred
in a capillary for immediate measurement of formed paramagnetic
adducts by a Bruker EMX100.
The details of the protocols, EPR parameters and spectrum
characteristics are available in the Supplemental Data.
Statistical Analysis
Data are presented as mean values⫾standard error. Statistical significance was assessed by group comparison using 1-way or 2-way
ANOVA, where appropriate (Microcal Origin). Results were considered significant at P⬍0.05.
Results
AII Activates the Concurrent Production of NO
and O2ⴚ䡠 in ECs With Ensuing eNOS Uncoupling
We first analyzed NO formation in the membranes of intact
ECs by EPR spectroscopy using colloidal spin trapping
Lobysheva et al
Figure 1. AII stimulation of ECs induces NO and O2⫺䡠 production and promotes eNOS uncoupling. Production of NO at the
membrane (A) and extracellular production of O2⫺䡠 (D) were
measured by EPR spin trapping from intact BAECs with or without L-NAME (2 mmol/L, 30 minutes) or CuZn-SOD (150 U/mL)
and stimulation with AII (2 ␮mol/L, 30 minutes; n⫽3 to 10). Cntl
indicates control. B, Typical EPR spectra (with [Fe(II)(DETC)2] of
untreated (control) and AII-treated BAECs, with or without MnTBAP (100 ␮mol/L), or L-NAME (2 mmol/L). C, Effect of PEG-SOD
(70 U/mL, 30 minutes) on AII-stimulated NO production in
BAECs (measured as in A). n⫽3 to 10. *P⬍0.05 between conditions. E, Typical accumulated EPR spectra (with DMPO) of
untreated (control) and AII-treated BAECs, with or without
CuZn-SOD (150 U/mL). F, Representative eNOS immunoblotting
signals from nondenatured EC lysates with or without AII (as in
A) or H2O2 (5 mmol/L, 15 minutes); densitometric analysis of
eNOS monomer/(dimer⫹monomer) ratio, quantified from 6 independent preparations is shown below. *P⬍0.05 between conditions analyzed by 1-tailed Wilcoxon test.
agents. NO production, assayed by EPR as accumulated
paramagnetic complex of nitrosylated [Fe(II)(DETC)2], was
increased to 158⫾12% of basal level after 30 minutes of
stimulation of ECs with AII (2 ␮mol/L). This increase was
abrogated on incubation with the NOS inhibitor, L-NAME
(Figure 1A). Typical EPR spectra obtained after stimulation
of ECs (⬇2⫻106) by AII are shown on Figure 1B. The NO
signal is increased on incubation of ECs with superoxide
dismutase (SOD), suggesting scavenging of the NO radical
by O2⫺䡠 concurrently produced at the membrane. Indeed,
AII-stimulated NO production was potentiated to 314⫾14%
of baseline after 30 minutes of preincubation of ECs with
polyethylene glycol-conjugated SOD (PEG-SOD) (Figure
1C). This time was shown to be sufficient for the association
of PEG-SOD to the plasma membrane.24 A similar potentiation of AII-stimulated NO formation was observed in cells
pretreated 30 minutes with the SOD mimetic MnTBAP
(Supplemental Figure IA and Figure 1B).
We next applied EPR spin trapping using DMPO to
monitor extracellular superoxide anion formation from intact
ECs. Stimulation with AII (30 minutes) also increased O2⫺䡠
production to 209⫾5% of basal level in the cell supernatant
(Figure 1D). Incubation with SOD strongly suppressed the
EPR signal, confirming that the observed paramagnetic
DMPO-OH radicals mostly resulted from the reaction with
O2⫺䡠 Accumulated EPR spectra obtained after stimulation of
ECs (⬇1⫻106) are shown on Figure 1E. Notably, L-NAME
(which inhibited NO production, Figure 1A) also partly
Caveolin Regulates eNOS/NOX Cross-Talk
3
diminished the O2⫺䡠 signal in AII-stimulated ECs (Figure 1D).
This is contrary to the expectation if NO scavenged only O2⫺䡠;
instead, it suggested O2⫺䡠 production directly from the NOS
electron chain in uncoupled eNOS, which is known to be
inhibited by L-NAME,23 as observed here under conditions of
concurrent NO and O2⫺䡠 production after AII short-term
stimulation.
To confirm eNOS uncoupling, we assayed the monomer/
dimer ratio of eNOS proteins by Western blotting in nonreducing conditions. We found that the ratio of monomer/
(dimer⫹monomer) proteins increased by 35% over control
(n⫽6; P⬍0,05) after 30 minutes of cell stimulation with AII
(Figure 1F).
eNOS uncoupling was previously described to be associated with specific alterations in phosphorylation patterns.
Accordingly, we observed combined eNOS dephosphorylation at Thr495 and Ser1177, despite clearly activated upstream kinases for the latter (ie, phosphatidylinositol 3-kinase/Akt; Supplemental Figure IIA to IID), a pattern
previously associated with eNOS uncoupling.25 It also
suggested the involvement of phosphatases (to dephosphorylate Ser1177/1179), as supported from the effect of
okadaic acid, an inhibitor of protein phosphatases 1 and
2A,26 which restored AII-stimulated phosphorylation of
eNOS at Ser1177 (Supplemental Figure IID). Altogether,
the phosphorylation pattern, shift to the monomeric form
of eNOS, and L-NAME sensitivity of O2⫺䡠 production
strongly indicate eNOS uncoupling.
Redox-Sensitive Pathway of eNOS Activation by
AII: Critical Role of cAbl
Although oxidant species resulting from the simultaneous
formation of NO and O2⫺䡠 can uncouple eNOS and decrease
NO bioavailability at the membrane, as illustrated above, they
may also activate intracellular signaling through redoxsensitive kinases implicated in eNOS activation by ROS.4
Indeed, in our ECs, preincubation with a water-soluble
cell-permeating antioxidant, Trolox, abrogated the AIIstimulated NO signal (Supplemental Figure IA), in contrast to
the effects of SOD shown above (Figure 1C). Conversely, on
stimulation of ECs with the Ca2⫹ ionophore A23187, all
antioxidants consistently and exclusively increased the NO
signal (Supplemental Figure IB), indicating specific ROSdependent signaling downstream AII receptor activation.
To study redox-sensitive signaling elements for eNOS
activation by AII, ECs were treated with an inhibitor of
phosphatidylinositol 3-kinases, LY294002, and apocynin, an
inhibitor of NADPH oxidase assembly. Both agents decreased AII-stimulated extracellular O2⫺䡠 and, notably, also
NO production in ECs (Figure 2A and 2B). We then tested
the involvement of the redox-sensitive c-Src and cAbl tyrosine kinases using an inhibitor of nonreceptor c-Src kinase,
PP2, and siRNA targeting cAbl, respectively. PP2 fully
suppressed NO formation stimulated by AII (Figure 2C).
Similarly, AII-stimulated NO production was strongly inhibited in ECs (Figure 2D) after siRNA downregulation of cAbl
(by more than 60%, Figure 2E) but not with control siRNA
(Figure 2D).
4
September 2011
IIIB). Likewise, with AII, we observed a strong redistribution
to the same light, caveolin-rich fractions of the tyrosine
kinase, cAbl (Supplemental Figure IVA and IVB), which we
showed to be critical for eNOS activation (see above).
Cav-1 Downregulation Inhibits p47phox
Recruitment and NADPH Oxidase Activation in
LD, Caveolin-Rich Membrane Fractions While
Preserving eNOS Confinement
Figure 2. AII-stimulated NO and O2⫺䡠 production in ECs are
dependent on redox-sensitive phosphatidylinositol 3-kinase,
cSrc, and cAbl kinases. EPR spin trapping analysis of O2⫺䡠 (A)
and NO production (B and C) from BAECs with or without
LY294002 (Ly, 10 ␮mol/L), apocynin (Apo, 200 ␮mol/L), or PP2
(10 ␮mol/L, all for 30 minutes), and stimulation with AII
(2 ␮mol/L, 30 minutes). Cntl indicates control. D, NO production
in HUVECs pretreated with/without AII (as in A), with PEG-SOD
(70 U/mL; 30 minutes) after transfection with cAbl siRNA or control siRNA (C siRNA). E, Representative cAbl (and ␤-actin) immunoblotting signals and densitometric analysis from HUVECs
treated with cAbl or control siRNA. n⫽3 to 10. *P⬍0.05 between
conditions.
AII Induces Colocalization of eNOS, cAbl, and
p47phox and Assembly of NADPH Oxidase in
Low-Density, Caveolin-Rich Membrane Fractions
We next examined whether such redox signaling was influenced by compartmentation of the 2 enzymatic systems.
To analyze the colocalization of Cav-1 and p47phox, a
critical organizer of NADPH-oxidase assembly, in intact EC,
we used a PLA combined with fluorescence microscopy,
where fluorescent dots quantitatively reflect physical proximity of 2 different immunodetected proteins. In resting cells,
a low PLA signal indicated some colocalization between the
2 proteins; however, the signal doubled after 15 minutes of
AII stimulation (Figure 3A and 3B; n⫽3; P⬍0.05).
To further analyze spatial compartmentation of eNOS and
NADPH oxidase, total homogenates of ECs, stimulated or not
with AII, were separated on a multiple-step discontinuous
sucrose density gradient and each fraction analyzed for
NADPH oxidase activity (Figure 3D) and by immunoblotting
(Figure 3F). NADPH oxidase activity was measured in all
fractions from unstimulated cells and in fractions from
AII-stimulated cells. A typical result is presented in Figure
3D, and mean values normalized to protein content in
low-density (LD) and high-density (HD) fractions are shown
in Figure 3E. On AII stimulation, a peak of NADPH oxidase
activity was observed in LD fractions, corresponding to
caveoline-1-enriched fractions, as identified by immunoblotting (Figure 3F, left). This indicated the assembly of a
functional NADPH oxidase on AII stimulation in caveolinrich membrane fractions where eNOS is colocalized (Figure
3F, left). Consistent with the PLA observations, p47phox and
NOX2, a main membrane component of NADPH oxidase,
were translocated to Cav-1-enriched fractions on AII stimulation (Figure 3F, left, and Supplemental Figure IIIA and
Next, we analyzed the effect of moderate Cav-1 downregulation on the distribution of the above proteins in ECs
stimulated with AII. Transfection with anti-Cav-1 siRNA (to
reduce Cav-1 abundance by ⬇50%, see Figure 4C) abrogated
the peak of NADPH oxidase activity in LD fractions of cells
stimulated by AII, whereas it was unaffected in cells transfected with control siRNA (Figure 3E). Notably, Cav-1
downregulation altered the translocation of p47phox after
AII, which decreased in the caveolin-rich fractions (Figure
3F, right, and 3G). This was confirmed by a separate analysis
of the membrane distribution of p47phox, which decreased in
cells transfected with Cav-1 siRNA (Supplemental Figure
IVC), and by PLA in intact cells, where the colocalization
signal was abrogated after Cav-1 downregulation (Figure 3C;
for higher magnification, see Supplemental Figure VA). This
indicates that the recruitment of p47phox and assembly of
functional NADPH oxidase in caveolin-rich membrane fractions is critically modulated by Cav-1 abundance. Conversely, Cav-1 downregulation did not alter the level of eNOS
in LD fractions in AII-stimulated cells (Figure 3F).
Cav-1 Downregulation Inhibits O2ⴚ䡠 and Reverses
eNOS Uncoupling at the Membrane of
AII-Treated ECs
We next examined the impact of Cav-1 downregulation on
O2⫺䡠 and NO bioavailability at the membrane of intact cells.
Consistent with measurements in cell fractions (Figure 3E),
transfection with anti-Cav-1 siRNA inhibited AII-stimulated
O2⫺䡠 production (Figure 4A, right); it also reduced (but did
not abrogate) NO production stimulated by AII (Figure 4B),
as expected from its O2⫺䡠 dependence (see Figure 2B and
Supplemental Figure IA). Importantly, it also abolished the
effect of PEG-SOD preincubation on AII-stimulated NO
production. This suggests that the NO signal at the membrane
was less sensitive to O2⫺䡠 after Cav-1 downregulation, consistent with reduced assembly of NADPH oxidase as observed above. To assess the impact on eNOS uncoupling, we
measured the L-NAME-sensitive production of O2⫺䡠. As
previously illustrated in Figure 1D, in control cells, the
AII-stimulated O2⫺䡠 production was inhibited by L-NAME,
indicating functional eNOS uncoupling (Figure 4A, left).
This L-NAME-sensitive production of O2⫺䡠 was abrogated
after Cav-1 downregulation; instead, L-NAME treatment
increased the O2⫺䡠 signal after AII stimulation (Figure 4A,
right), as expected from inhibition of NO production from a
functional eNOS.
Cav-1 Downregulation Prevents AII-Induced
Hypertension and Preserves Circulating Hb-NO
Levels In Vivo
To extend the functional relevance of these observations in
vivo, we examined the impact of a similar moderate down-
Lobysheva et al
5
Figure 3. AII-stimulated p47phox and Cav-1 colocalization in ECs. PLA for p47phox and Cav-1 (A and C) in HUVECs stimulated or not
by AII (2 ␮mol/L). Positive interactions are visualized as red dots, nuclei are stained in blue, and phalloidin staining is green. Representative images using a ⫻20 (A, top, and C) and ⫻63 (A, bottom) oil-immersion objective lenses are shown for resting (A and C, left) and
AII-stimulated cells (A and C, right). See also Supplemental Figure V for a higher magnification of C. B, Quantitative analysis, expressed
as PLA signal per cell, from randomly selected ⬎11 images for each condition from 3 independent experiments. *P⬍0.05 analyzed by
1-tailed Wilcoxon test. Cntl indicates control. D, NADPH oxidase activity measured by EPR spin trapping (DMPO) in fractions from
isopycnic ultracentrifugation of ECs lysates after stimulation (F) or not (䡺) by AII. Spectra were acquired after 10 minutes of incubation
with DMPO (60 mmol/L) and NADPH (0.1 mmol/L) at 37°C. E. Mean values of NADPH oxidase activity, normalized by protein content,
in LD or HD fractions of HUVECs transfected with control or Cav-1 siRNA with/without AII stimulation (30 minutes). F, Representative
immunoblotting signals from fractions obtained as in D from HUVECs transfected with control (left) or Cav-1 (right) siRNA. G, Densitometric analysis of p47phox abundance in LD fraction normalized by total signal (LD⫹HD), n⫽3. *P⬍0.05 compared with control.
regulation of Cav-1 using Cav-1⫹/⫺ haploinsufficient mice.
First, we verified that Cav-1 was downregulated in aortic
extracts of Cav-1⫹/⫺ to 53⫾7% of the level in Cav-1⫹/⫹
(Figure 5C). Then, we analyzed the colocalization of Cav-1
and p47phox in murine aortic ECs by PLA and observed that
the PLA signal was suppressed in Cav-1⫹/⫺ ECs (Figure 5A
and 5B, left) and, contrary to control cells, was not increased
under AII stimulation (Figure 5A and 5B, right, and 5D).
Furthermore, we assayed circulating NO bioavailability and
systolic blood pressure regulation in heterozygote Cav-1⫹/⫺
mice and their Cav-1⫹/⫹ littermates after 1 week of AII (or
vehicle) administration by osmotic mimipumps. In Cav-1⫹/⫹
mice, systolic blood pressure measured by implanted telemetry before and after 1 week of AII infusion significantly
increased (from 115.3⫾1.5 to 134.6⫾3.5 mm Hg); in parallel, blood levels of the 5-coordinate-␣-Hb-NO complex,
measured by EPR as an index of vascular NO bioavailability,
decreased to 55⫾15% of control (Figure 5E and 5F, left).
Figure 4. Moderate Cav-1 downregulation reverses eNOS uncoupling in vitro. A, Extracellular O2⫺䡠 production (EPR spin trapping) in
intact HUVECs, transfected with control or Cav-1 targeted siRNA and pretreated with L-NAME (2 mmol/L) or vehicle (30 minutes) before
stimulation by AII (2 ␮mol/L, 30 minutes; n⫽6 to 7; *P⬍0.05 between different conditions). Cntl indicates control. B, NO production
(EPR spin trapping) in intact BAECs transfected with control or Cav-1 targeted siRNA and pretreated with PEG-SOD (30 minutes) or
vehicle before stimulation by AII (2 ␮mol/L, 30 minutes; n⫽3 to 5). C, Representative immunoblotting signals for Cav-1 (and ␤-actin)
and densitometric analysis from ECs lysates transfected or not with Cav-1 siRNA. (n⫽7). *P⬍0.05 between conditions.
6
September 2011
Figure 5. Moderate Cav-1 downregulation prevents AII-induced
p47phox and Cav-1 interaction in mouse ECs in vitro, preserves
NO bioavailability, and prevents hypertension development in
haploinsufficient Cav-1⫹/⫺ mice in vivo. A and B, PLA signal (red
dots) of Cav-1 and p47phox interaction in aortic ECs isolated
from haploinsufficient Cav-1⫹/⫺ mice (B) and WT littermate (A)
and treated with/without AII (DAPI for nuclei staining in blue;
cytoskeleton in green; nuclear dots represent non specific binding of trapped antibodies). Images were obtained using a ⫻40
objective lens. C, Representative immunoblotting signals for
Cav-1 (and ␤-actin) from aortic extracts of Cav-1⫹/⫹ and Cav1⫹/⫺ mice and densitometric analysis (n⫽4). D, Quantitation of
PLA signals from n⫽3 to 6 independent experiments as illustrated in A and B. *P⬍0.05 between conditions. E, Systolic
blood pressure measured by implanted telemetry in conscious
Cav-1⫹/⫺ and Cav-1⫹/⫹ mice before and after 1 week of AII
infusion (1.1 mg/kg per day) or saline (n⫽7 mice each). F, Level
of Hb-NO (as 5-coordinate-␣-HbNO complex, by EPR) in
venous blood of Cav-1⫹/⫺ and Cav-1⫹/⫹ mice after 1 week of
AII infusion as in E (n⫽5 mice each). *P⬍0.05 between conditions. Cntl indicates control.
Conversely, the increase in systolic blood pressure was
significantly lower in Cav-1⫹/⫺ mice under AII infusion
(8.9⫾3.1 compared with 19.3⫾3.4 mm Hg in Cav⫹/⫹ mice),
and NO bioavailability measured as Hb-NO level was unchanged (Figure 5E and 5F, right).
Discussion
Our data show that (1) AII activates both NO and ROS
production in EC, and the latter both reduces NO bioavailability at the membrane but mediates AII signaling to eNOS
through ROS-sensitive Src and cAbl tyrosine kinases; (2)
Cav-1 mediates the recruitment of cAbl and p47phox and the
assembly of a functional NADPH oxidase by AII in caveolinrich membrane fractions, where eNOS is colocalized; (3)
colocalization of AII-activated eNOS and NADPH oxidase
promotes eNOS uncoupling; and (4) moderate downregula-
Figure 6. Diagram summarizing the effect of moderate Cav-1
downregulation on AII signaling to eNOS and NADPH oxidase in
ECs. Left: Normal Cav-1 expression levels (as in Cav-1⫹/⫹
mice). Top: At resting state, eNOS activity is low because of
inhibitory interaction with Cav-1 in caveolae. Bottom left: On AII
stimulation of AT1 receptors (AT1R), Cav-1 recruits p47phox for
the assembly of functional NOX2-containing NADPH oxidase in
caveolae; this promotes O2⫺䡠 production that (1) activates eNOS
activity (through ROS-sensitive intracellular signaling; see Figure
2) but also (2) produces eNOS uncoupling, thereby shifting
some eNOS to monomeric form producing more O2⫺䡠, resulting
in endothelial dysfunction. Right: Moderate Cav-1 downregulation (as in haploinsufficient Cav-1⫹/⫺ mice). Top right: The abundance of Cav-1 is decreased, but eNOS confinement is
unchanged in caveolae (see Figure 3). Bottom right: On AII stimulation, less p47phox is recruited, resulting in lower NADPH oxidase activation and less O2⫺䡠 production in caveolae (see Figure
3); this (1) lowers ROS-dependent activation of eNOS but (2)
prevents eNOS uncoupling, resulting in preserved NO bioavailability and endothelial function.
tion of Cav-1 prevents all of the above while maintaining NO
bioavailability in vitro and in vivo.
Our model, as summarized in Figure 6, shows that although oxidant species resulting from the simultaneous formation of NO and O2⫺䡠 can uncouple eNOS and decrease NO
bioavailability at the membrane, they may also activate
intracellular signaling through redox-sensitive kinases implicated in eNOS activation by ROS.4,26 These results are
consistent with previous demonstrations of eNOS activation
by NADPH oxidase-derived oxidants, such as H2O2,10 and
more recently by overexpression of NOX5 in ECs.27 In the
latter study, as in ours (Figure 1C), the ROS-mediated
production of NO was potentiated on incubation with extracellular SOD, suggesting a dual effect of ROS to decrease
bioavailability of NO at the cell surface but activating eNOS
through intracellular ROS signaling. Our study adds the
identification of cAbl as critical for AII-mediated activation
of eNOS, which may have implications for the mechanistic
understanding of vascular side effects of new anticancer
drugs targeting this and other tyrosine kinases.28
Lobysheva et al
Previous studies, including ours, demonstrated basal interaction of eNOS and Cav-116,17 in endothelial caveolae;
however, the role of Cav-1 in NADPH-oxidase assembly and
activation in ECs remained poorly defined. We now demonstrate that the recruitment of p47phox and assembly of
functional NADPH oxidase in caveolin-rich membrane fractions is critically modulated by Cav-1 abundance. Conversely, Cav-1 downregulation did not alter the confinement
of eNOS in LD fractions in AII-stimulated cells (Figure 3F),
consistent with the predominant role of eNOS prenylation for
membrane anchoring.29
Our data further demonstrate that colocalization of
NADPH oxidase with eNOS in Cav-1-rich rafts/caveolae
both sustains ROS-mediated activation of eNOS by AII and
simultaneously promotes eNOS uncoupling (see Figure 6,
lower left). The latter can be reversed by downregulating of
Cav-1 while maintaining a functional eNOS at the membrane
(Figure 6, lower right).
Partial downregulation of endothelial Cav-1 may then
confer the double advantage of increasing NO output with
vasodilators and reducing eNOS uncoupling through attenuation of NADPH oxidase assembly and activation in response
to AII (and possibly other prooxidant agonists). We previously showed that similar moderate reductions of Cav-1
expression are associated with a potentiation of agonistinduced activation of eNOS in vitro and in vivo, through the
enzyme’s release from its allosteric inhibitory interaction
with caveolin.30 Importantly, this may not be accompanied
with further nitrosative stress, as produced with complete
abrogation of Cav-1 expression,31 because the residual caveolin expression maintains a physiological activation of eNOS
(see Figure 4A), as opposed to deregulated NO production.
Therapeutically, this can be achieved with HMG-CoA reductase inhibition using statins, which we showed to produce a
similar 40% to 50% downregulation of endothelial Cav-1 in
vitro and improvement in endothelial function in vivo.17,18
Downregulation of Cav-1 may also decrease ROS production
in vascular smooth muscle cells, in addition to eNOS regulation in endothelium.
In conclusion, the present study revealed the importance of
Cav-1 for NADPH oxidase activation in response to AII in
ECs, as well as of the spatial confinement of O2⫺䡠 production
both for eNOS activation and its uncoupling. It also demonstrates the possibility of preventing eNOS uncoupling by
moderate Cav-1 downregulation, opening the possibility to
therapeutically modulate the adverse effects of the renin-angiotensin system in vascular disease.
Acknowledgments
The authors thank Hrag Esfahani and Delphine DeMulder for expert
technical assistance.
Sources of Funding
This work was funded by the Politique Scientifique Federale (IAP
P6-30), the Communauté Française de Belgique (ARC06/11-339),
the Fondation Jean Leducq, the European Commission (FP6-IP
“EUGeneHeart”), and the Fonds National de la Recherche Scientifique. Drs Dessy and Feron are FNRS Senior Research Associate and
Research Director, respectively.
7
Disclosures
None.
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1
Lobysheva I.I. et al. – Supplemental Data to Caveolin regulates eNOS/NOX cross-talk.
Moderate Caveolin-1 Downregulation Prevents NADPH-oxidase Dependent eNOS
Uncoupling by Angiotensin II in Endothelial Cells
Irina Lobysheva*1, Géraldine Rath1, Belaïd Sekkali1, Caroline Bouzin1, Olivier Feron1,
Bernard Gallez2, Chantal Dessy1 and Jean-Luc Balligand*1
SUPPLEMENTAL DATA
MATERIALS AND METHODS
Materials and cell cultures: Antibodies (Ab) for eNOS, Cav-1, NOX2 and β-actin detection
were purchased from BD Biosciences, Abcam and Sigma respectively. Anti-phospho-eNOS
(phospho-Ser1177), Akt (phospho-Ser473) and cAbl Abs were purchased from Cell Signaling
Technology Inc. (Beverly, USA); anti-phosphorylated eNOS (Thr495) and p47phox Abs from
Upstate. Primary cultures of human umbilical vein endothelial cells (HUVECs), bovine aortic
endothelial cells (BAECs) were purchased from Clonetics (Lonza Group Ltd, Switzerland)
and maintained according to recommended protocol in a CO2 incubator. All chemicals of ultra
high grade were purchased from Sigma-Aldrich Chemical Inc. or Alexis Biochemical Inc.,
Benelux. Murine aortic endothelial cells were isolated from aortas of adult mouse (8-12
weeks old), haploinsufficient for Cav-1 (Cav-1+/-) and wild-type littermate of the same
C57Bl6 background (Cav-1+/+), as described previously1.
Briefly, after anesthetizing of
mice, aortas were perfused with 2 ml sterile solution (Dulbecco’s Modified Eagle Medium,
1% PS, 10% FCS purchased from Invitrogen, Gibco). Then thoracic aortas were dissected,
cleaned from connective tissues and fat, and cut into small rings. Rings were placed into 12well plate coated with fibronectin, 0.1% (Sigma), and maintained in EGM-2 (Lonza Group
Ltd, Switzerland). Rings were elevated after 3-4 days, when endothelial cells formed the
colonies inside the rings. The cells were maintained in plastic dishes with EGM-2 in a 37°C
incubator, and the media was replaced after 1 day. Cells were positively characterized to
2
eNOS and VE-cadherin expression by flow cytometry and for expression of von Willebrand
factor. Cells were used for immunocytochemistry and PLA after second passage.
Animal experiments: Experiments conformed with the Guide of the Care and Use of
Laboratory Animal and local ethics committee. Male, 16 weeks old heterozygote Cav-1+/- and
wild-type littermate of the same C57Bl6 background (Cav-1+/+) were implanted with
miniaturized telemetry devices (Datascience Corp., USA) as described previously2 and after
recovery, long-term (24 hours) online recordings were acquired (baseline) and digitized.
Then, osmotic mini-pumps (Alzet, Model 2002, Durect Corporation Cupertino, USA) for
delivery of AII (1.1 mg/kg per day) or saline were surgically inserted in a subcutaneous pouch
and online recordings repeated after 7 days. Further, mice were injected with heparin (IP: 100
units /25g), anesthetized, and venous blood was obtained by a puncture of the right ventricle,
immediately frozen in calibrated tubes (0.2 ml each) at 77K and processed for EPR
measurements.
siRNA transfection: For Cav-1 and cAbl gene expression silencing, cells were transfected
with a Lipofectamine kit (Qiagen Science Inc. Benelux) according to the manufacturer’s
protocol with siRNA duplexes against Cav-1 (5´-AACGAGAAGCAAGTGTACGAC-3´ for
HUVECs and 5´-AAGATGTGATTGCAGAACCAG-3´ for BAECs), and human cAbl (5´AAAGGTGAAAAGCTCCGGGTC-3´) or universal control oligonucleotides (AllStars),
Purification of caveolae-enriched micro-domains and cytosolic/membrane fractions
separation: Caveolin-enriched membranes were isolated by isopycnic ultracentrifugation as
described previously3. Briefly, lysates of ECs were collected with a cold solution of Na2CO3
containing protease and phosphatase inhibitor cocktail (PIC), homogenized and separated by
ultracentrifugation (100,000 g, 18 hours, 4°C) against sucrose gradient (35% to 5%). The
fractions were collected (0.5 ml each), tested for NADPH oxidase activity by EPR spin
3
trapping with DMPO and concentrated by centrifugation after precipitation with ammonium
sulfate (35%) for immunoblotting.
Membrane and cytosolic fractions were separated by ultracentrifugation (100,000 g for 1
hour), the supernatant (cytosolic fraction)
and precipitate (membranes fraction) were
collected and resuspended in lysis buffer for immunoblotting.
Immunoprecipitation and Immunoblotting : Cells were lysed with cold lysis buffer,
homogenized, and analyzed directly as described previously 4. Samples in SDS-PAGE buffer
were heated
and separated by electrophoresis. For eNOS monomer/dimer assay, non-
denatured cell lysates in ice-cold buffer (composition in mmol/liter: Tris-HCl, 50, pH 8; NaCl,
180; EDTA, 0.5; NP40, 0.2%; phenylmethylsulfonyl fluoride, 100; DTT, 1; PIC) were
separated with 2xSDS sample buffer by low temperature SDS-PAGE at 30 mA. After transfer
to nitrocellulose or PVD membranes, proteins were immunoblotted with appropriate
antibodies and signals quantified by densitometry (using ImageJ). Results were expressed as
ratios of monomeric/monomeric+dimeric eNOS, phosphorylated/unphosphorylated protein or
normalized to β-actin as loading control, where appropriate.
Immunofluorescence Microscopy and Proximity Ligation Assay (PLA) in situ: HUVECs
were grown to 90% confluence on gelatinized glass cover-slips, incubated with medium,
containing 0.2% serum for 4 h. and stimulated or not with AII (2 μM) before fixation (4%
paraformaldehyde, PBS). Then cells were permeabilized with Saponin (0.1%), washed with
PBS, incubated with 5% of BSA, then sequentially with a goat anti-Cav-1 and rabbit antip47phox antibodies with intermittent washing (PBS, 1% BSA). Species-specific secondary
antibodies (Alexa Fluor568 donkey anti-goat IgG, and Jackson IR Laboratories Inc. DyLight
488 donkey anti-rabbit IgG) for immunocytochemistry or Duolink PLA probes (anti-Goat
Minus and anti-Rabbit Plus antibodies linked to their “reporter” oligonucleotides, Olink
Bioscience, Sweden) were added according to the manufacturer’s protocol. Co-localized
4
p47phox and Cav-1 bound to the specific antibodies were detected after “reporter” DNA
circularization with a size-limiting linker oligonucleotide, ligation and rolling circle
amplification with a polymerase. Amplified “reporter” DNA was then detected using
complementary fluorescent probes. After nuclear staining with Hoechst 33342 and
cytoskeleton with Phalloidin-FITC (2μg/mL), images were acquired with a ZeissImager.Z1
fluorescence microscope, equipped with an ApoTome device, using 20x, 40x or 63x oil
immersion objective lenses and analyzed with AxioVision software completed with Duolink
BlobFinder for fluorescent dots quantification.
Measurements of NO: Bioavailable NO was assayed by EPR spin trapping in ECs as
previously reported
4, 5
. Briefly, serum-starved cells (pretreated with AII with/without
inhibitors or their respective solvents, when applicable) were incubated with a colloid solution
of the NO spin trap, diethyldithiocarbamate-iron complex in culture dishes in a CO2
incubator. After culture medium removal, cells were scraped on ice, extracts frozen in
calibrated tubes in liquid nitrogen and processed for EPR. The formed paramagnetic NOadduct, accumulated at plasma cell membranes, was assayed by a Bruker EMX100
spectrometer (X-band, microwave frequency 9.35 GHz, modulation frequency, 100 kHz) with
setting: microwave power (MP), 20 mW; modulation amplitude (MA), 0.5 mT; 10 scans,
77K. The amplitude of the third hyperfine (hf) component of the EPR signal (g1 = 2.035; Ahfs
= 1.3 mT) was used for analysis of the NO production using WINEPR software.
The level of circulating Hb-NO was assayed in whole blood of mice from the EPR signal of 5coordinate- -Hb-NO as described by us previously 2. The EPR spectra of whole blood were
recorded by a Bruker EMX100 spectrometer with setting: MP, 20 mW; MA, 0.7 mT; time
constant (TC), 163 ms; 10 scans, 77K. The level of 5-coordinate- -Hb-NO was quantified
from hf signal after subtraction of EPR signal of free radicals using Microcal Origin software.
5
Superoxide anions measurements by EPR: Extracellular O2-. formation was assayed by EPR
spin trapping using 5.5-dimythyl-1-pyrolline-N-oxide (DMPO, high purity, Alexis
Biochemical Inc. after charcoal filtration) as described previously 6. Cells were preincubated
in KREBS-DTPA-Hepes buffer (0.1 mmol/liter DTPA, 10 mmol/liter HEPES, pH 7.5) with
pharmacological reagents or vehicle, and stimulated (or not) with AII in presence of DMPO
(60 mmol/liter) at 37°C in a CO2 incubator. The extracellular medium was transferred in a
capillary for immediate measurement of formed paramagnetic adducts by a Bruker EMX100
with setting: MP, 20 mW; MA, 0.1 mT; TC, 163 ms.; 5 scans. The amplitude of the second hf
component of the DMPO-OH EPR signal (g = 2.006, aN = 1.49 mT and aßH = 1.49 mT) was
used for analysis.
References to Supplemental Methods
1.
Sonveaux P, Martinive P, DeWever J, Batova Z, Daneau G, Pelat M, Ghisdal
P, Grégoire V, Dessy C, Balligand JL, Feron O. Caveolin-1 expression is critical for vascular
endothelial growth factor-induced ischemic hindlimb collateralization and nitric oxidemediated angiogenesis. Cardiovasc Res. 2004;95:154-61.
2.
Desjardins F, Lobysheva I, Pelat M, Gallez B, Feron O, Dessy C, Balligand JL.
Control of blood pressure variability in caveolin-1-deficient mice: role of nitric oxide
identified in vivo through spectral analysis. Cardiovasc Res. 2008;79:527-36.
3.
Saliez J, Bouzin C, Rath G, Ghisdal P, Desjardins F, Rezzani R, Rodella LF,
Vriens J, Nilius B, Feron O, Balligand JL, Dessy C. Role of caveolar compartmentation in
endothelium-derived hyperpolarizing factor-mediated relaxation: Ca2+ signals and gap
junction function are regulated by caveolin in endothelial cells. Circulation. 2008;117:106574.
4.
Dessy C, Saliez J, Ghisdal P, Daneau G, Lobysheva II, Frerart F, Belge C,
Jnaoui K, Noirhomme P, Feron O, Balligand JL. Endothelial beta3-adrenoreceptors mediate
nitric oxide-dependent vasorelaxation of coronary microvessels in response to the thirdgeneration beta-blocker nebivolol. Circulation. 2005;112:1198-205.
5.
Kleschyov AL, Munzel T. Advanced spin trapping of vascular nitric oxide
using colloid iron diethyldithiocarbamate. Methods Enzymol. 2002;359:42-51.
6
6.
Xia Y, Tsai AL, Berka V, Zweier JL. Superoxide generation from endothelial
nitric-oxide synthase. A Ca2+/calmodulin-dependent and tetrahydrobiopterin
regulatory process. J Biol Chem. 1998;273:25804-8.
SUPPLEMENTAL FIGURES
Figure I. Differential modulation of NO production by ROS scavengers in
endothelial cells stimulated by Angiotensin II. (A). Nitric oxide production stimulated by
Angiotensin II (2 μmol/liter) in cultured BAECs, and assayed by EPR as accumulated
paramagnetic NO complex in presence of the spin trapping agent [Fe(II)(DETC)2] (0.4 mmol/L,
7
30 minutes), was potentiated in cells pretreated 30 minutes with the superoxide dismutase
mimetic, MnTBAP (50 μmol/liter). By contrast, a water-soluble cell-permeating antioxidant,
Trolox (100 μmol/liter), decreased the AII-stimulated NO signal to basal level. (B) These
antipathetic effects were not observed in BAECs stimulated by a receptor-independent
agonist, Ca2+-ionophore A23187 (1 μmol/liter). n= 3-5; * P<0.05, between different conditions;
ns - difference with A23187-stimulated NO production was nonsignificant.
8
Figure II. Time-dependent changes in phosphorylation of eNOS and Akt by
Angiotensin II in BAECs. Phosphorylation of eNOS was analyzed at Ser-1177 and Thr-495
residues after AII stimulation of endothelial cells. AII decreased Thr-495 phosphorylation of
eNOS at 30 minutes (A and C) without significant change of phosphorylation at Ser-1177 (B
and D, left). Okadaic acid (OA, 100 nM), a potent inhibitor of protein phosphatases 1 and 2A,
increased basal phosphorylation and significantly augmented AII-induced phosphorylation of
eNOS at Ser-1177 (D, right). Akt phosphorylation at Ser-473 was significantly increased from
baseline levels in BAECs after 10 minutes of AII stimulation, but dephosphorylated at 30
minutes (E,F). Densitometric analyses from 3 to 4 independent preparations (C, D, F). *
P<0.05, between different conditions.
Figure III. Translocation of NOX2 to caveolin-1-enriched fractions after AII
stimulation of endothelial cells. A. Representative immunoblotting signals (NOX2) from the
lysate of HUVECs stimulated or not by AII, and separated on a multiple-step discontinuous
sucrose density gradient as described in Material and Methods. Fractions were collected from
9
the top to the bottom of the tubes and total lysate, light-density (LD, 4-6), and heavy-density
(HD, 7-10) fractions were analyzed by WB. We observed a redistribution of NOX2 from HD
to LD (caveolin-rich) fractions upon AII stimulation (A, from left to right). Densitometric
analyses of light-density fractions (LD) normalized to total density (LD+HD) for NOX2
presented in (B). n = 3 independent preparations, *, P<0.05.
Figure IV. Translocation of cAbl to caveolin-1-enriched fractions after AII
stimulation of endothelial cells. To analyze spatial compartmentation of the proteins
10
potentially involved in AII signaling, total homogenates of ECs stimulated or not by AII, were
separated on a multiple-step discontinuous sucrose density gradient as described in Material
and Methods. Fractions were collected from the top to the bottom of the tubes and analyzed
separately. We observed a redistribution of cAbl from heavy to light, caveolin-rich fractions
upon AII stimulation (A-B). Representative Western blot analysis of subcellular fractions
obtained from lysates of BAECs non-stimulated (upper panel) and stimulated by AII (2
μmol/liter for 30 minutes, bottom panel) and probed with antibodies for Cav-1 and cAbl (A).
Densitometric analyses of light-density fractions (2-6) normalized to total density (2-10) for
Cav-1 and cAbl (B). This experiment was repeated twice with similar results. C. Abundance
of p47phox, an organizer of NADPH oxidase assembly, in the membrane fraction of
endothelial cells was decreased by Cav-1 downregulation. Representative Western blots
analysis of lysates obtained from BAECs transfected with siRNA targeting Cav-1 or control
and separated to membrane and cytosolic fractions as described in Materials and Methods.
Immunoblots were probed with antibodies for Cav-1, p47phox and β-actin. D. Densitometric
analyses normalized to β-actin abundance (from 2 independent experiments).
11
Figure V. Effect of Cav-1 downregulation on co-localization of Cav-1 and p47phox
in HUVECs treated or not by Angiotensin II. Proximity ligation assay (PLA) for p47phox
12
and Cav-1 in HUVECs transfected with Cav-1 siRNA (A) or control siRNA (B) and
stimulated or not by AII (2 μM, 15 min.). Positive interactions are visualized as red dots,
nuclei are stained in blue, cytoskeleton staining is green. HUVECs were transfected and
stimulated by AII as described in Supplemental Material and Methods, before fixation.
Representative images are shown for resting (A, B, left) and AII-stimulated cells (A, B, right).
C. Quantitative analysis, expressed as PLA signal per cell, from randomly selected images
(more than 200 cells were analyzed) for each condition. n = 4-7 independent preparations, *,
P<0.05.

Moderate Caveolin-1 Downregulation Prevents NADPH Oxidase

Transcription

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