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-induced endothelial cell apoptosis: dual regulation by reactive oxygen species
Division of Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
1Correspondence: Division of Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Ross 1023, 720 Rutland Ave., Baltimore MD 21205, USA. E-mail: kirani{at}mail.jhmi.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B), increased
activity of caspase-3, and markedly augmented endothelial cell
susceptibility to TNF-induced apoptosis. Direct inhibition of NF-B
through adenoviral expression of the super repressor form of inhibitor
of B
(I-B S32/36A) also increased susceptibility of HUVECs to
TNF-induced apoptosis. Rotenone, a mitochondrial electron transport
chain inhibitor, suppressed TNF-induced mitochondrial ROS production,
proteolytic cleavage of procaspase-3, and apoptosis. These findings
show that Rac1 is an important regulator of TNF-induced ROS production
in endothelial cells. Moreover, they suggest that Rac1-dependent ROS,
directly or indirecly, lead to protection against TNF-induced death,
whereas mitochondrial-derived ROS promote TNF-induced
apoptosis.—Deshpande, S. S., Angkeow, P., Huang, J., Ozaki,
M., Irani, K. Rac1 inhibits TNF--induced endothelial cell apoptosis:
dual regulation by reactive oxygen species.
Key Words: Apoptosis • ROS • Rac1 • nuclear factor-kappa B • caspase-3
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The vascular endothelial
monolayer serves as a nonthrombogenic barrier between the bloodstream
and the vascular wall, and endothelial cell dysfunction and loss are
characteristic of atherosclerotic lesions (2)
. Since
apoptotic cell death is a common feature of human atheromas
(3)
, it is hypothesized that endothelial cell apoptosis
induced by cytokines such as tumor necrosis factor-alpha (TNF) or other
cytotoxic agents is important in the pathogenesis of atherosclerotic
vascular disease. This hypothesis is supported by evidence that
apoptotic vascular endothelial cells become procoagulant, and many of
the agents associated with atherosclerosis induce apoptosis in
endothelial cells (4
, 5)
.
The TNF-induced signal transduction pathway involves the intracellular
production of reactive oxygen species (ROS). There is conflicting
evidence regarding the source(s) and roles of these ROS in modulating
TNF-induced apoptosis (6)
. It is clear that in addition to
initiating pathways leading to cell death, TNF also activates
anti-apoptotic signaling pathways, including but not limited to
induction of the transcription factor nuclear factor-kappa B (NF-B)
(7
8
9)
. NF-B-regulated genes can then inhibit cell
death by modulating a number of enzymes that are involved in apoptosis,
including the cysteine proteases, caspases (10
, 11)
. Since
NF-B activation is redox-sensitive (12)
, it is tempting
to speculate that one role of the intracellular ROS produced by TNF may
be to serve as second messengers in this anti-apoptotic signaling
pathway.
Stimuli, such as TNF, which act via the TNF receptor superfamily lead
to the activation of the Rho family of small GTP binding proteins
(13
, 14)
. Rac1, a member of this family, has been shown to
regulate the activity of NF-B via the production of ROS
(12)
. This is believed to occur through a Rac1-dependent
plasma membrane NAD(P)H oxidase present in many nonphagocytic cells
(15
, 16)
, including cells comprising the vascular wall
(17)
, that is functionally similar to the phagocyte NADPH
oxidase (18)
. However, the functional significance of such
an oxidase in the context of endothelial cell apoptosis is not known.
This study examines the role of a Rac1-dependent oxidase in regulating
TNF-induced ROS production, NF-B activation, and caspase processing
and activity in vascular endothelial cells. Furthermore, it also asks
how ROS, dependent and independent of Rac1 regulation, affect
TNF-induced endothelial cell apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adenoviruses
The adenoviruses encoding the myc epitope-tagged cDNA of the
dominant negative and constitutively active forms of rac1 (AdRac1N17
and AdRac1V12) were constructed in our laboratory and have previously
been described (12
, 19)
. The adenovirus Adßgal, encoding
the Escherichia coli lacZ gene, was used as a control
(19)
. Identical results were obtained using Addl312, a
control adenovirus lacking a transgene (data not shown). AdSOD encoding
human Cu-Zn superoxide dismutase, AdCat encoding human erythrocyte
catalase, and AdIB(S32/36A) encoding the hemagglutinin-tagged super
repressor mutant of the NF-B inhibitor IB- have been described
previously (20
21
22)
. Increased intracellular SOD and
catalase activities using these viruses have been reported (20
, 21)
. Adenoviral stocks were prepared in HEK 293 cells, purified
on a double cesium gradient, and titered using a standard plaque assay.
Infections were carried out at a multiplicity of infection (MOI) of 50
or 200 for 16 h. Protein expression and biochemical or functional
assays described below were carried out 48 h after infection.
Expression of the epitope-tagged proteins was assessed in 20 µg of
whole cell lysates using a myc-specific or anti-hemagglutinin antibody
(9E10, Santa Cruz, Santa Cruz, Calif.; 0.2 µg/ml). In some cases,
protein loading was determined by immunoblotting with an antibody to
tubulin (Sigma, St. Louis, Mo.).
ROS measurements
HUVECs infected with the indicated adenoviruses were stimulated
with TNF, where indicated, for the specified time and intracellular ROS
were detected by fluorescence of 2'-7'-dichlorodihydrofluorescein
diacetate (DCF-DA) using a Zeiss confocal laser scanning microscope as
described previously (12)
. Absolute fluorescence was
quantified on a scale of 0–255 with MetaMorph software. Mitochondrial
ROS were detected in a similar fashion using dihydrorhodamine 123 (DHR)
as described previously (23)
. Basal DHR fluorescence was
higher than DCF fluorescence. Results shown are from a representative
experiment and are the mean ± SE of the absolute DCF
or DHR fluorescence of 40 random cells. Where indicated, cells were
treated with rotenone (1 µM) for 30 min prior to imaging.
Apoptosis assay
Quantitative measurements of apoptosis were performed using a
colorimetric enzyme-linked immunoassay (ELISA) that quantifies DNA
fragmentation by measuring cytoplasmic histone-DNA fragments (Cell
Death ELISA, Boehringer Mannheim, Mannheim, Germany). HUVECs infected
with adenoviruses were stimulated with TNF (R&D; Abingdon, Oxon, U.K.
20 ng/ml) and apoptosis was assessed 24 h later according to the
manufacturer’s recommendations. Where indicated, cells were treated
with rotenone for 30 min before addition of TNF. The measured optical
densities (O.D.405 at 20 min after incubation
with substrate) obtained from the ELISA were expressed as fold change
in apoptosis compared to Adßgal 50 MOI. Each condition was done in
triplicate and values represent the mean ± SE.
Electrophoretic mobility shift assay
HUVECs infected with the indicated adenovirus were stimulated
with TNF for 30 min before collecting nuclear extracts as described
previously (12)
. 10 µg of extract was incubated with
105 cpm of a 32P-labeled
B binding consensus oligonucleotide
(5'-AGTTGAGGGGACTTTCCCAGGC-3') or a mutant B oligonucleotide
(5'-AGTTGAGGCGACTTTCCCAGGC-3') for 15 min in binding buffer (10 mM Tris
pH 7.4, 80 mM KCl, 5% glycerol, 1 mM DTT, 0.25 µg dIdC) at room
temperature. Incubation mixtures were run out on a 6% polyacrylamide
gel and autoradiographed.
NF-B reporter assay
2 x 105 HUVECs were transfected with
2 µg of a 3x B chloramphenicol acetyltransferase reporter
construct (pMHC-CAT) and 0.2 µg of a constitutive firefly luciferase
plasmid (pRSV-Luc, Promega, Madison, Wis.) in 12 µl of Lipofectamine
(Gibco-BRL, Grand Island, N.Y.). Five hours after transfection, the
cells were infected at 200 MOI with the indicated adenovirus. Where
indicated, cells were stimulated with TNF (20 ng/ml) 24 h after
infection and whole cell lysates collected 16 h later. CAT assay
was performed on lysates as described previously (12)
.
Luciferase signal was measured using the luciferase assay system
(Promega) according to the manufacturer’s recommendations.
14C counts were normalized to luciferase light
units and are expressed as relative NF-B activity compared to
Adßgal 200 MOI. Values represent the mean ± SE.
Procaspase-3 levels and caspase-3 activity
Cell lysate (30 µg) was separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membrane. The membrane was immunoblotted with an
antibody to full-length procaspase-3 (Santa Cruz, E-8) or an antibody
that detects both the full-length (115 kDa) and the 85–89 kDa cleaved
fragment of PARP (Santa Cruz, H-250). Immunoblotting with an antibody
to -tubulin (Sigma) was done to confirm equal protein loading.
The membranes were developed by ECL (Amersham). Band intensities were
quantified by densitometry and normalized to -tubulin where
indicated. Representative blots from experiments that were repeated at
least once are shown.
Statistical analyses
A paired Student’s t test was used for statistical
comparison between experimental groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In addition, infection with AdIB(S32/36A) resulted in
high levels of expression of this super repressor mutant of IB, as
judged by Western blotting (Fig. 1B
).
|
Rac1 is partly responsible for the TNF-induced oxidative burst
We then examined the effect of TNF stimulation on
intracellular ROS production. TNF stimulation of HUVECs infected with
Adßgal resulted in a burst of ROS peaking at 15 min (Fig. 2A
), as measured by DCF fluorescence, a fluorophore that has
been extensively used as a general marker of overall intracellular ROS
production. When compared to Adßgal-infected cells (Fig. 2B
, 2c
), cells infected with AdRac1N17 showed a
significant decrease in the peak TNF-induced oxidative burst (Fig. 2B
, 2d
). The basal DCF fluorescence in both the
Adßgal and AdRac1N17-infected cells was similar (Fig. 2B
, 2a
and b
). Adenoviral-mediated overexpression of
either superoxide dismutase or catalase markedly decreased the
TNF-induced oxidative burst, suggesting that both superoxide and
hydrogen peroxide were being generated (Fig. 2C
).
Quantification of the DCF fluorescence revealed that infection with
AdRac1N17 diminished the TNF-induced peak to 63% of that seen in cells
infected with Adßgal (Fig. 2C
), suggesting that
Rac1-dependent pathways were only partially responsible for the
TNF-induced oxidative burst. In addition, infection of HUVECs with
AdRac1V12 also resulted in an increase in DCF fluorescence that was
lower in magnitude than that seen with TNF stimulation, again
implicating Rac1-independent source(s) as contributing to TNF-induced
ROS production. Rotenone, a specific inhibitor of site I of the
mitochondrial electron transport chain, partially suppressed the
TNF-induced oxidative burst, whereas adenoviral expression of catalase
or superoxide dismutase, two nonspecific antioxidant enzymes, led to
complete inhibition (Fig. 2C
).
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Rac1 does not regulate TNF-induced mitochondrial ROS generation
We then determined whether Rac1-independent sources of ROS
were contributing to the TNF-induced oxidative burst. Based on the
partial effect of rotenone in suppressing overall TNF-induced oxidant
production, we looked at the contribution of mitochondrial oxidases to
this phenomenon. DHR, a fluorophore that has been shown to be a
specific marker of mitochondrial ROS production (23
, 24)
,
was used for this purpose. As shown in Fig. 2D
, TNF addition
to Adßgal-infected cells resulted in an increase in the fluorescence
of DHR. Infection with AdRac1N17 did not result in suppression of the
TNF-induced increase in DHR fluorescence, indicating that Rac1 does not
regulate mitochondrial ROS production. This was corroborated in cells
infected with AdRac1V12, which demonstrated no increase in DHR
fluorescence over control Adßgal-infected cells (not shown). Finally,
treatment with rotenone completely abolished the TNF-induced increase
in DHR fluorescence.
Rac1-regulated ROS production is responsible for TNF-induced
NF-B activation
Rac1 is known to regulate the transcriptional activity of NF-B
(12)
. We therefore examined the role of Rac1 and
Rac1-regulated and independent ROS in TNF-stimulated NF-B activation
in HUVECs. Electrophoretic mobility shift assays (EMSA) were performed
to determine NF-B DNA binding activity in HUVECs (Fig. 3A
). HUVECs infected with Adßgal had basal constitutive
NF-B DNA binding activity. This activity was not different from that
in uninfected cells (not shown). Stimulation of Adßgal-infected cells
with TNF resulted in an increase in NF-B DNA binding activity that
was suppressible by treatment with the chemical, cell-permeable
antioxidant N-acetyl-L-cysteine (NAC). The use of antibodies against
the p65 and p50 subunits of NF-B determined that at least part of
this TNF-induced activity was due to the p50/65 heterodimer. Infection
with AdRac1N17 resulted in a decrease in basal NF-B DNA binding
activity as well as abrogation of the TNF-induced activity. Infection
with AdIB(S32/36A) achieved the same result. In contrast, treatment
with rotenone had little effect on TNF-induced NF-B DNA binding
activity. Finally, infection with AdRac1V12 simulated TNF in inducing
NF-B DNA binding activity, which was inhibited by pretreatment with
NAC.
|
The anti-apoptotic effect of NF-B is due to its transcriptional
activity (7
, 9)
. Having shown that Rac1 regulates
TNF-induced DNA binding activity of the transcriptionally active p50/65
heterodimer of NF-B, we next examined NF-B
trans-activation activity using a B chloramphenicol
acetyl transferase reporter (Fig. 3B
). TNF led to an
induction of NF-B trans-activation activity in
Adßgal-infected cells. Infection with AdRac1N17 substantially reduced
this activity, as did AdIB(S32/36A). Treatment with rotenone had
little effect on trans-activation by NF-B. Therefore, the
TNF-induced NF-B DNA binding and trans-activation
activities corresponded with each other and were primarily dependent on
Rac1-regulated ROS production, not ROS derived from mitochondria.
Rac1-regulated ROS production and NF-B activation protect
against TNF-induced apoptosis
Having demonstrated a role for Rac1-regulated ROS production in
NF-B activation in HUVECs, we investigated the functional
significance of this activation with regard to its role in modulating
endothelial cell apoptosis. Apoptosis was quantified with an ELISA that
measures cytoplasmic histone-DNA
fragments(Fig. 4
). TNF addition resulted in no significant increase in apoptosis in
cells infected with Adßgal, regardless of the MOI used. In contrast,
TNF addition to AdRac1N17-infected HUVECs led to a marked induction of
apoptosis. This increase was dependent on the MOI used, and therefore
the expression of Rac1N17. A similar effect was achieved with the
AdIB(S32/36A). It is noteworthy that in addition to making the cells
susceptible to TNF-induced apoptosis, expression of IB(S32/36A) also
led to an increase in basal apoptosis. To determine the role of
mitochondrial ROS production in the induction of apoptosis, we also
treated AdRac1N17-infected cells with rotenone. Rotenone abrogated the
susceptibility of HUVECs expressing Rac1N17 to TNF-induced apoptotic
death.
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Rac1-regulated and mitochondrial ROS production differentially
modulate procaspase-3 cleavage and caspase-3 activity
Caspases are cysteine proteases that have a central role in
apoptosis (25)
. Proteolytic cleavage of procaspases
results in the generation of active caspases. The anti-apoptotic effect
of some oxidant species, such as nitric oxide, has been attributed to
their effect on inhibiting the processing and activation of certain
procaspases, including the executioner procaspase-3 (26)
.
We therefore investigated the role of Rac1-regulated ROS and
mitochondrial ROS in the processing of procaspase-3 and activity of
caspase-3. As shown in Fig. 5A
, TNF stimulation resulted in a similar decrease in
procaspase-3 levels in both Adßgal and AdRac1N17-infected cells,
indicating that Rac1N17 has no effect on proteolytic processing of
procaspase-3. Treatment with rotenone, however, inhibited the
TNF-induced reduction in procaspase-3 levels, suggesting that
mitochondrial ROS promote the proteolytic cleavage of procaspase-3.
This provides a mechanism for the anti-apoptotic effect of rotenone in
our cell system. We next examined the levels and processing of poly
ADP-ribose polymerase (PARP), an important substrate for caspase-3
(27)
. Infection of HUVECs with AdRac1N17 resulted in a
marked potentiation of TNF-induced PARP cleavage, as compared to
Adßgal-infected cells (Fig. 5B
). Thus, inhibition of
Rac1-regulated pathway(s) did not alter TNF-induced processing of
procaspase-3, but did substantially increase the proteolytic activity
of caspase-3, as measured by cleavage of PARP. Addition of the NO donor
S-nitroso-acetyl-penicillamine (SNAP) did not decrease the degree of
TNF-induced PARP cleavage in cells expressing Rac1N17.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 24
, 28)
.
Indeed, many of the changes associated with apoptosis occur at the
mitochondria and some anti-apoptotic proteins are known to act as
antioxidants at mitochondrial sites (29)
. Our findings do
identify a TNF-induced mitochondrial source of ROS in endothelial
cells, but distinguish it from a Rac1-regulated source of ROS. This
distinction is critical since we also suggest that ROS, depending on
their source of origin, regulate TNF-induced apoptosis in diametrically
opposite ways. Such apparently paradoxical effects have been shown for
other second messengers, most notably nitric oxide (30)
,
and may well be achieved through subcellular compartmentation of the
ROS. Compartmentation could expose the same ROS to quite different
redox milieus, thereby leading to the development of structurally and
functionally very different second-generation species. In addition,
localized production of ROS may be critical in determining their access
to key cellular targets.
TNF is known to generate superoxide in various cell types
(6)
. Our finding that complete suppression of the
TNF-induced oxidative burst by SOD in HUVECs suggests that superoxide
is the primary species of oxidant generated. In addition, inhibition of
the TNF-induced increase in DCF fluorescence by catalase shows that
hydrogen peroxide, a dismutation product of superoxide, is also being
produced. It is worthwhile noting that TNF addition resulted in a
three- to fourfold increase in total cellular ROS production (DCF
fluorescence) but only a 30% increase in mitochondrial ROS (DHR
fluorescence). This modest, but significant, increase in
mitochondrial-derived ROS is consistent with previous reports examining
TNF-induced redox changes in the mitochondria (28)
, and
suggests that strict compartmentalization of the ROS generated may be
more important than absolute increases in ROS levels in determining the
effect induced by ROS. The difference in fold induction of total
cellular and mitochondrial ROS also suggests that the mitochondria are
not the only source of Rac1-independent ROS production. Additional
TNF-activated enzymatic sources may include xanthine oxidase
(31)
and arachidonate metabolism (32
, 33)
.
Whatever the nature of the enzymatic systems involved in TNF-induced
ROS generation, the fact that rotenone offered complete protection
against TNF-induced apoptosis shows that mitochondrial-derived ROS are
chiefly responsible for promoting apoptotic death in endothelial
cells.
In dissecting the mechanism of action of Rac1-regulated and
mitochondrial ROS we observed that the TNF-induced cleavage of the
precursor of the critical downstream executioner caspase, caspase-3,
was not affected by Rac1N17. Since Rac1 and Rac1-regulated ROS were
essential for NF-B activation, this suggests that NF-B activation
does not inhibit TNF-induced procaspase-3 cleavage. Other studies have
similarly reported the lack of effect of NF-B on TNF-induced
proteolysis of procaspase-3 (11)
. The fact that TNF
induced only partial processing of procaspase-3 may reflect the
relative resistance of HUVECs to this form of cell death.
The effect of rotenone on TNF-induced procaspase-3 cleavage implicates
mitochondrial ROS in caspase-3 processing. Rotenone-sensitive ROS
production has been shown to result in sequential dysregulation of
mitochondrial functions, leading to caspase-3 activation
(34)
. Notably, Rac1N17 did not affect the processing of
procaspase-3 into the active caspase, but did markedly potentiate the
TNF-induced activity of caspase-3, as evidenced by cleavage of PARP.
This suggests that Rac1-mediated mechanisms, directly or indirectly,
suppress caspase-3 activity once activated by mitochondrial ROS. In
this context, it should be noted that suppression of caspase-3 activity
by NO protects against TNF/actinomycin D-induced apoptosis in
hepatocytes. (26)
. Moreover, inducible NO synthase is
regulated by NF-B (35)
. However, in our model, addition
of the NO donor SNAP did not suppress caspase-3 activity in cells
expressing Rac1N17, showing that solely increasing intracellular NO
levels is not sufficient to counteract the effect of Rac1N17. This
implicates other B-regulated anti-apoptotic proteins such as c-IAP1
and c-IAP2, which are known to inhibit caspase-3 activity
(10)
in the Rac1-dependent survival pathway.
Alternatively, Rac1-regulated ROS could directly participate in the
suppression of caspase-3 activity.
Note that HUVECs in culture display basal NF-B activity that was
further induced by TNF. Such basal activity has also been observed in
other vascular cell types and has been shown to be necessary for their
proliferation (36)
. Expression of Rac1N17 and
IB(S32/36A) both led to a decrease in this activity, whereas
expression of Rac1V12 stimulated this DNA binding activity.
Corresponding with these observations, we noted that expression of
Rac1N17 suppressed HUVEC growth and expression of Rac1V12 stimulated
early cell growth (not shown). Therefore, this Rac1-regulated,
redox-sensitive basal NF-B activity may be necessary for endothelial
cell proliferation.
IB(S32/36A) was more effective in blocking NF-B-mediated
trans-activation than Rac1N17, and this correlated with a
higher degree of both basal and TNF-induced apoptosis in HUVECs
infected with AdIB(S32/36A). This difference in the effectiveness of
IB(S32/36A) and Rac1N17 in inhibiting NF-B activation, combined
with the inability of Rac1N17 to completely suppress NF-B-mediated
trans-activation, suggests the concomitant involvement of
Rac1-independent pathways in TNF-induced NF-B activation in HUVECs.
Nonetheless, the effect of Rac1N17 on DNA binding,
trans-activation, and apoptosis, taken together, strongly
implicate Rac1 and Rac1-controlled ROS production as important in the
activation of the transcriptionally active, anti-apoptotic p50/65
NF-B heterodimer. These findings are consistent with observations
that gene transcription is important in TNF-induced anti-apoptotic
signaling in endothelial cells (37)
.
Our data also show that in contrast to Rac1-regulated ROS,
mitochondrial-derived ROS do not mediate TNF-induced NF-B
activation. This is in contrast to another report suggesting that
mitochondrial ROS production is responsible for ceramide-induced
NF-B activation (38)
. This difference in observations
may be due to the different cell types and the nature of the stimuli
used in the two studies.
Although we have specifically examined NF-B induction and inhibition
of the activity of the executioner caspase-3 as possible mechanisms for
the anti-apoptotic effect of Rac1, other proteins that may cross talk
with NF-B may also contribute to the resistance of endothelial cells
to cytokine-induced apoptosis. Rac1 may regulate the activation of
cytokine-stimulated anti-apoptotic signaling proteins such as A1, A20,
c-Jun N-terminal kinases (JNKs), PI 3-kinase, and Akt, some of which
are sensitive to the redox state of the cell (39
, 40)
, and
participate in NF-B activation (41
42
43)
.
In summary, previous studies have shown that primary human endothelial
cells are protected from apoptotic pathways initiated by engagement of
Fas (APO1) and the TNF receptor 1 (44
, 45)
. Our study
confirms this observation and also proposes a hitherto unrecognized
Rac1 and ROS-regulated survival pathway for this phenomenon. Such a
pathway complements the demonstrated role of Rac1-regulated ROS in
cellular proliferation (46)
. It also presents an
alternative to the characterization of ROS and NF-B as contributing
to endothelial dysfunction (47)
and therefore being
pro-atherogenic and/or pro-thrombotic. Our data imply that ROS by
simultaneously functioning as both pro- and anti-apoptotic messengers
in response to a single stimulus can have both a protective and
deleterious role in endothelial cells. Thus, strategies aimed at
inhibiting the production of ROS or activation of NF-B in
endothelial cells with the goal of preventing endothelial damage or
dysfunction may have the untoward effect of leading to endothelial cell
loss and therefore promote vascular disease. A similar protective
function of ROS against pro-apoptotic stimuli has been shown in other
models of cellular injury and death (48
, 49)
. Therefore,
the concept of the duality of ROS produced in response to a single
stimulus as being both pro- and anti-apoptotic may also have relevance
outside of endothelial cell biology. In this regard, identification of
the Rac1 GTPase as a modulator of survival in cytokine-induced
apoptosis in tumor cells may have important implications for cancer
immunotherapy.
|
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ACKNOWLEDGMENTS |
|---|
B(S32/36A), A. Baldwin for the
pMHC-CAT, T. Reilly for help with confocal microscopy, P. J.
Goldschmidt-Clermont, C. J. Lowenstein, T. Finkel, R. R.
Ratan, and S. Mattagajasingh for their constructive criticisms of the
manuscript, K. Baughman, E. Marban, and S. Dirks for their
encouragement, and P. Emig for secretarial assistance. This work was
supported by the Johns Hopkins University Clinician Scientist Award
(K.I.), a grant from the W. W. Smith Charitable Trust (K.I.), the
CardioFellows Foundation (P.A.), the Bernard Foundation, and an
endowment from Mr. and Mrs. Abraham Weiss.
Received for publication October 18, 1999.
Revision received March 1, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B trans-activation activity as measured by a
