© 1999 American Heart Association, Inc.
Molecular Medicine |
A Role for Reactive Oxygen Species in Endothelial Cell Anoikis
From the Cardiology Branch (A.E.L., H.I., I.I.R., K-S.K., T.F.) and Pathology Section (K.T., Z-Y.Y., V.J.F.), National Heart, Lung, and Blood Institute, NIH, Bethesda, Md.
Correspondence to Toren Finkel, MD, PhD, Cardiology Branch, NHLBI, NIH, 10 Center Dr, MSC 1650, Building 10, Room 7B-15, Bethesda, MD 20892-1650. E-mail finkelt{at}gwgate.nhlbi.nih.gov
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Abstract |
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Abstract—When adherent cells, such as epithelial or endothelial cells, are detached and continuously maintained in suspension, they undergo a form of programmed cell death termed anoikis. We demonstrate that coincident with endothelial cell detachment, there is a dramatic rise in the intracellular level of reactive oxygen species (ROS). Reattachment to a solid surface rapidly attenuates the level of ROS. The mitochondria appear to be the major source of the detachment-induced rise in ROS. The change in the intracellular redox state appears to contribute to endothelial anoikis, because treatment with either the cell-permeant antioxidant N-acetylcysteine or the flavin protein inhibitor diphenylene iodonium is demonstrated to reduce oxidant levels and protect against subsequent cell death. Similarly, the endogenous intracellular level of ROS is shown to correlate with the extent of cell death. Finally, we demonstrate that the activities of both caspases and of the c-Jun N-terminal kinases are modulated by the rise in intracellular ROS levels. These results suggest that oxidants serve as signaling molecules and regulators of anoikis.
Key Words: JNK • caspase • hydrogen peroxide • mitochondria
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Disruption of cell attachment to extracellular matrix results in activation of programmed cell death by a process termed anoikis. Many types of cells exhibit this property (reviewed in Reference 11 ), which may have important implications in both physiological and pathological processes. In particular, the loss of anoikis is thought to contribute to the anchorage-independent growth of tumor cells and, thus, to play a role in metastatic disease. In addition, anoikis appears critical to cavity formation in the embryo2 and glandular development in the adult.3
Previous studies have demonstrated that endothelial
cells undergo anoikis.4 This process can be inhibited in
both endothelial cells and other cell types by
selective engagement of integrin receptors or activation of
integrin-dependent cellular signaling pathways.1 4 5
Similarly, treatment of endothelial cells with an
vß3 integrin
antagonist induces endothelial cell
apoptosis and regression of tumor
neovascularization,6 which suggests that regulation of
endothelial anoikis may have important clinical
benefits.
The signaling pathway that is initiated by cell detachment and leads to cell death is not completely understood. Early studies demonstrated that expression of oncogenic forms of ras or src could rescue cells from anoikis.7 Some recent studies have suggested that the stress-regulated, c-Jun amino-terminal kinase (JNK) pathway, which appears to be activated by detachment, is critical for anoikis.8 9 Nonetheless, in other studies, it was suggested that although JNK is activated in suspended cells, its activation does not correlate with survival.10 Alternatively, the phosphatidylinositol-3 kinase/Akt pathway has been implicated as an important determinant in anoikis.11 Finally, activation of the caspase family of cysteine proteases appears to be critical in anoikis, as bcl-2, CrmA and the peptide caspase inhibitor zVAD-fmk all appear to protect cells.8 9 10 In some studies, caspase activation has been linked to JNK activation in a postulated positive feedback loop,8 9 whereas other studies have disputed this association.10
In certain instances, reactive oxygen species (ROS) have been implicated as important downstream mediators of apoptosis.12 13 14 Nonetheless, apoptosis can occur in the presence of hypoxic or even anoxic conditions,15 16 a situation in which ROS levels should be low or absent. The role of ROS in anoikis has not been studied. Recent evidence suggests that disruption of integrin contact in fibroblasts can lead to cell detachment that is preceded by a rise in intracellular ROS levels.17 In this report, we demonstrate that detachment of endothelial cells results in a dramatic rise in ROS levels and, furthermore, that this rise in ROS contributes to anoikis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells
Human umbilical vein endothelial cells were obtained from Clonetics and were grown in endothelial growth medium (EGM; Clonetics) supplemented with 20% FCS and plated on dishes coated with 0.1% gelatin. Cells from passages <10 were routinely used.
Measurements of ROS
For measurement of intracellular ROS, cells were loaded with 5
µg/mL of 2',7'-dihydrodichlorofluorescin diacetate (Molecular Probes)
for 5 minutes. Cells were subsequently imaged, and the
fluorescence of dichlorofluorescin (DCF) was quantified on an
arbitrary gray scale (0–256) using a Leica laser scanning confocal
fluorescent microscope with false color imaging, as previously
described.18 Attached cells routinely had levels of DCF
fluorescence of 
20 units, whereas the fluorescent
intensity of cells undergoing detachment varied between 100 and 200
units. In certain cases, cells were treated with the mitochondrial
electron transport inhibitor rotenone (1 µmol/L) for
1 hour before assessment with DCF. Direct visualization of
mitochondrial ROS was made using dihydrorhodamine (DHR123). Previous
studies have demonstrated that this compound selectively accumulates in
the mitochondria, where it is oxidized by mitochondrial ROS to the
fluorescent rhodamine derivative.19 Cells were
incubated with DHR123 (1 µmol/L) for 1 hour before visualization
by confocal microscopy. Measurement of DCF and DHR123
fluorescence by confocal microscopy was made 30 seconds after
trypsin or other methods of detachment were initiated.
For cell sorting experiments, cells were trypsinized and loaded with 5 µg/mL DCF for 15 minutes before measurement of intracellular ROS by a Coulter fluorescence-activated cell sorter (FACS). A bell-shaped distribution curve of the cellular fluorescence was routinely observed, and the 10% of cells producing the lowest and highest levels of ROS were sorted into 2 separate tubes and used for subsequent studies.
Anoikis Assays
Confluent cells in 35-mm dishes were trypsinized, and
5x105 cells were suspended in 2.0 mL of medium,
placed in microcentrifuge tubes, and set at slow rotation at
37°C for the indicated times. Cell death was assessed by the
following 3 methods: trypan blue exclusion, a cell death–detection
ELISA (Boehringer Mannheim) that quantifies the level of
cytoplasmic histone-associated DNA fragments composed of mono- or
oligonucleosomes, and DNA laddering. All experiments for viability and
DNA fragmentation were performed in triplicate, and the results shown
are from 1 of at least 3 similar experiments (mean±SD). Where
indicated, cells were preincubated in N-acetylcysteine (NAC;
30 mmol/L) for 24 hours or with diphenylene iodonium (DPI) (5
µmol/L) for 2 hours before detachment. Alternatively, where
indicated, cells were treated while suspended with zVAD-fmk (100
µmol/L). Unless stated otherwise, trypan blue exclusion was assessed
after 24 hours in suspension. A time course of DNA fragmentation
demonstrated that peak activity was noted after 12 hours of suspension
(data not shown). Therefore, unless stated otherwise, DNA fragmentation
assays were performed after 12 hours in suspension using triplicate
samples, as previously described.20
Kinase Assays
Confluent cells were trypsinized and suspended via slow rotation
in microcentrifuge tubes for the indicated times, after which
cells were harvested and 125 µg of extract was immunoprecipitated
with a JNK1 antibody (Santa Cruz Biotechnology). Analysis of
JNK activity was as previously described using a truncated form of
activating transcription factor-2 (amino acids 1–96) as a
substrate.21
Caspase Assays
Caspase assays were carried out using the ApoAlert
fluorescent assay kit (Clontech) modified for detection of
caspase-3 activity. Confluent cells (1x107) were
harvested and lysed in 180 µL of the included cell lysis buffer, and
protein concentrations were equalized for each condition. Subsequently,
60 µg of cell lysate was combined with an equal amount of substrate
reaction buffer. This reaction mixture was incubated with a caspase-3
fluorescent substrate (acetyl-Asp-Glu-Val-Asp-MCA
[4-methyl-coumaryl-7-amide; Peptides International, Louisville,
Ky]) at a final concentration of 50 µmol/L for 30
minutes at 37°C, and then fluorescence was quantified on a
Millipore Cytofluor 2350 fluorescent plate reader. The data
presented are from 1 experiment (mean±SD) performed in
triplicate and are representative of 3 similar
experiments.
Statistics
Statistical comparison between groups was made by a Student
2-tailed t test (in figures, asterisk indicates
P<0.05). When >2 groups are compared, the data were first
analyzed by an ANOVA followed by a 2-tailed t
test.
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Results |
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As previously observed in both epithelial7 and endothelial4 cells, maintaining endothelial cells in suspension resulted in a loss in cell viability. As demonstrated in Figure 1A
, cell viability decreased over a
24-hour period in suspension with a 
75% loss of cell viability over
that time frame. Consistent with other studies,1
cell death occurred via apoptosis, as assessed both by
morphological criteria (data not shown) and by DNA fragmentation assays
(Figure 1B) and DNA laddering (Figure 1C).
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A variety of evidence has implicated ROS as mediators of cell
death. We therefore sought to understand whether ROS had a role in
endothelial anoikis. Intracellular levels of ROS were
measured by using the peroxide-sensitive fluorophore DCF. As
demonstrated in Figure 2A, levels of ROS
were low in plated, confluent endothelial cells.
Detachment of cells using EDTA alone, mechanical disruption (data not
shown), or trypsinization (Figure 2B) all produced a rapid rise
in ROS. After detachment, levels of DCF fluorescence increased
5- to 10-fold (Figure 2C). Although cells maintained in
suspension continued to show high levels of ROS, reattachment to the
plate resulted in a rapid decline in DCF fluorescence (Figure 2D).
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To further characterize the source of the detachment-induced rise in
ROS, we directly imaged mitochondrial ROS. Endothelial
cells were loaded with DHR123, a nonfluorescent compound that
is selectively concentrated in the mitochondria, where its rate of
conversion to a fluorescent rhodamine species is determined by
mitochondrial ROS levels.19 As demonstrated in Figure 3A, detachment led to a 3-fold
increase in DHR123 fluorescence. Similarly, treatment with
rotenone, a specific inhibitor of mitochondrial electron
transport, resulted in a significant inhibition in the rise of DCF
fluorescence after detachment (Figure 3B).
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To understand whether the change in the intracellular redox state was
an important determinant of cell death, we attempted to correlate the
level of ROS with the extent of anoikis. The level of DCF
fluorescence in suspended cells followed a bell-shaped
distribution curve (Figure 4A). Using a
FACS, we sorted suspended cells on the basis of level of
fluorescence. Analysis of cells exhibiting the upper
and lower 10% of DCF fluorescence demonstrated that the
intracellular levels of ROS correlated with the extent of cell death
(Figure 4B and 4C).
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To further strengthen the correction between the redox state and the
degree of anoikis, we treated endothelial cells with
the peroxide-scavenging cell-permeant antioxidant NAC or with the
flavin protein inhibitor DPI. As shown in Figure 5A, treatment with NAC or DPI reduced the
level of intracellular H2O2
after cell detachment. Similarly, assessment of cell viability after 24
hours in suspension demonstrated that both NAC and DPI protected cells
from anoikis (Figure 5B).
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We next sought to understand the mechanism by which ROS participate in
modulating cell death. Some studies have suggested that the activation
of the JNK is essential for anoikis.8 9 As demonstrated in
Figure 6, JNK activity was substantially
increased the longer cells were maintained in suspension.
Consistent with the protective effects observed with NAC,
treatment with this antioxidant inhibited the rise in JNK activity. A
similar inhibition was observed with DPI treatment (data not
shown).
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Similarly, as shown in Figure 7A, caspase
activity in untreated cells increased as a function of time in
suspension. At each point, treatment with NAC or DPI reduced the level
of caspase activity. Similarly, sorting cells on the basis of
intracellular level of DCF fluorescence demonstrated a
correlation between intracellular
H2O2 levels and caspase
activity (Figure 7B). The higher the level of DCF
fluorescence, the higher the level of caspase activity and the
higher the subsequent level of cell death (see also Figure 4
).
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Although these results suggest that the intracellular redox state
modulates both JNK and caspase activity, these pathways may not be
completely independent. Indeed, recent evidence suggests that the
activation of JNK during anoikis may be dependent on the cleavage of
mitogen-activated protein/extracellular signal–regulated
kinase kinase (MEKK) by caspases.9
Consistent with a role for caspases in the activation of JNK,
the peptide caspase inhibitor zVAD significantly inhibited
suspension-induced JNK activation (Figure 8A). Treatment of suspended cells with
zVAD also rescued them from anoikis as assessed qualitatively by DNA
laddering (Figure 8B) or quantitatively by trypan blue exclusion
(Figure 8C).
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Discussion |
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Our results demonstrate that endothelial cell detachment leads to a rapid rise in the level of ROS. The source of the ROS appears to be the mitochondria, given that the increase in cytosolic ROS can be inhibited by pretreatment with rotenone, a specific inhibitor of mitochondrial electron transport. In addition, direct visualization by DHR123 demonstrated an abrupt rise in mitochondrial ROS levels after cell detachment. Although superoxide anions are the most likely species of ROS generated by the mitochondria, further studies, perhaps using spin-trapping techniques, are required to validate this assumption. The importance of the change in the ROS for anoikis is demonstrated by the observations that the intracellular level of ROS correlates with the extent of cell death and that decreasing the levels of ROS protects cells from anoikis. Finally, the rise in ROS appears to modulate the activity of both JNK and intracellular caspases.
It is presently unclear how cell detachment signals a rapid
increase in intracellular ROS. A recent study noted that in
fibroblasts, disruption of the actin cytoskeleton by an antibody to
5ß1 integrin led to a
change in cell shape, the activation of rac1, and the subsequent
generation of ROS.17 It is possible that similar events
may take place with other methods of cell detachment. A growing body of
evidence suggests that in addition to their role in phagocytic oxidant
generation, rac proteins may regulate ROS levels in nonphagocytic
cells.17 21 22 23 24 Consistent with this, we have
noted that expression of a dominant negative rac1 gene (N17rac1)
reduces the rise in ROS seen with endothelial cell
detachment (A.E.L., T.F., unpublished observations, 1998). It
should be noted, however, that all of our results were obtained with
cultured cells plated on gelatin. The influence of long-term culturing
of cells and the contribution of various components of the
extracellular matrix on the extent of anoikis requires further
study.
One possible mediator of the rise in ROS occurring during anoikis is ceramide. Production of ceramide is triggered by a variety of stressful and apoptotic stimuli.25 26 A recent report suggests that levels of ceramide also rise during anoikis.27 In addition, several recent studies suggest considerable cross-talk and cross-regulation between rac1 and ceramide signaling.28 29 Similarly, treatment of cells or isolated mitochondria with exogenous ceramide causes an increase in the mitochondrial release of ROS, which could be blocked by rotenone.30
Finally, our results suggest that both JNK and caspase activity are sensitive to the redox state of the cell. The role of JNK in anoikis is controversial, with some studies suggesting that the kinase is activated by caspases and required for cell death.8 9 In contrast, other studies suggest that in anoikis, JNK is activated in a caspase-independent fashion and is superfluous in the cell death pathway.10 Our results in endothelial cells suggest that JNK activity is regulated by caspase activity, given that zVAD-fmk blocks JNK activation. As such, the ability of antioxidants to modulate both JNK and caspase-3 activity may reflect the ability to modulate an upstream component common to both pathways.
One upstream activator of JNK that has recently been
described to be redox sensitive is apoptosis signal–regulating
kinase-1 (ASK1). This kinase is at the level of a
mitogen-activated protein kinase kinase kinase (MAPKKK). Yeast
2 hybrid analysis has demonstrated that ASK1 binds directly to
thioredoxin, an antioxidant protein.31 Changes in the
redox state alter ASK1-thioredoxin interactions leading to ASK1
dimerization and activation.31 32 Although it is
presently unknown whether ASK1 regulates caspase activity in
addition to JNK activity, expression of a dominant negative ASK1 can
block tumor necrosis factor-–induced
apoptosis.33
In summary, we demonstrate that endothelial cell detachment results in a rapid and dramatic rise in intracellular ROS levels. The rise in ROS appears to be important in modulating the subsequently observed cell death and therefore suggests that in endothelial cells, anoikis occurs through a redox-sensitive pathway.
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Acknowledgments |
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We thank Judith Taylor for expert preparation of the manuscript. A.E.L. was supported by the NIH Clinical Research Training Program.
Received December 9, 1998; accepted June 11, 1999.
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K. Aoshiba, K. Yasuda, S. Yasui, J. Tamaoki, and A. Nagai Serine proteases increase oxidative stress in lung cells Am J Physiol Lung Cell Mol Physiol, September 1, 2001; 281(3): L556 - L564. [Abstract] [Full Text] [PDF]
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V. J. Thannickal and B. L. Fanburg Reactive oxygen species in cell signaling Am J Physiol Lung Cell Mol Physiol, December 1, 2000; 279(6): L1005 - L1028. [Abstract] [Full Text] [PDF]
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 K. Irani Oxidant Signaling in Vascular Cell Growth, Death, and Survival : A Review of the Roles of Reactive Oxygen Species in Smooth Muscle and Endothelial Cell Mitogenic and Apoptotic Signaling Circ. Res., August 4, 2000; 87(3): 179 - 183. [Abstract] [Full Text] [PDF]
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S. W. Ballinger, C. Patterson, C.-N. Yan, R. Doan, D. L. Burow, C. G. Young, F. M. Yakes, B. Van Houten, C. A. Ballinger, B. A. Freeman, et al. Hydrogen Peroxide- and Peroxynitrite-Induced Mitochondrial DNA Damage and Dysfunction in Vascular Endothelial and Smooth Muscle Cells Circ. Res., May 12, 2000; 86(9): 960 - 966. [Abstract] [Full Text] [PDF]
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C. Cahilly, C. M. Ballantyne, D.-S. Lim, A. Gotto, and A. J. Marian A Variant of p22phox, Involved in Generation of Reactive Oxygen Species in the Vessel Wall, Is Associated With Progression of Coronary Atherosclerosis Circ. Res., March 3, 2000; 86(4): 391 - 395. [Abstract] [Full Text] [PDF]
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 C. A. Knight-Lozano, C. G. Young, D. L. Burow, Z. Y. Hu, D. Uyeminami, K. E. Pinkerton, H. Ischiropoulos, and S. W. Ballinger Cigarette Smoke Exposure and Hypercholesterolemia Increase Mitochondrial Damage in Cardiovascular Tissues Circulation, February 19, 2002; 105(7): 849 - 854. [Abstract] [Full Text] [PDF]
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