YASUKO KONDO,t DALI YIN,t GENE H. BARNE11,*. RAMI KAAKAJI,*. JOHN W. PETERSON,t. TATSUO MORIMURA,t. HIROAKI KUBO,t JUJI TAKEUCHI,t. AND.
Involvement apoptosis endothelial
of mterleukin-
1 3-converting
of bFGF-deprived cells
enzyme
murme
in
aortic
SEUI KONDO,5” YASUKO KONDO,t DALI YIN,t GENE H. BARNE11,* RAMI KAAKAJI,* JOHN W. PETERSON,t TATSUO MORIMURA,t HIROAKI KUBO,t JUJI TAKEUCHI,t AND BARBARA P. BARNA* Departments of *Neurosurget.y and tNeurosciences The Cleveland Clinic Foundation, USA; and Department of Neurosurgery, National Utano Hospital, Kyoto, Japan
Apoptosis (programmed cell death) is an essential physiological process that is genetically regulated and contributes to the balance between cell growth, differentiation, and the maintenance of normal cells. Recent studies show that deprivation of growth factor induces apoptosis in endothelial cells. However, the molecular mechanisms regulating apoptosis remain unclear. In this study, we demonstrate that deprivation of basic lIbroblast growth factor (bFGF) increased the expression of interleukin-1J-converting enzyme (ICE) protein, and subsequently induced apoptosis in murine aortic endothelial (MAE) cells. In contrast, the proteins of the tumor suppressor p53 and c-myc were undetected during apoptosis. This apoptosis was suppressed by the tetrapeptide ICE inhibitor, Ac-YVAD-CMK. Overexpression of murine ICE, in addition, induced apoptosis in MAE cells using gene transfer techniques. These results strongly suggest that ICE may mediate apoptosis in bFGF-deprived endothelial cells, and the suppression of ICE function could represent a novel approach for the protection of endothelial cells from damages.-Kondo, S., Kondo, Y., Yin, D., Barnett, G. H., Kaakaji, R., Peterson, J. W., Morimura, T., Kubo, H., Takeuchi, J., Barna, B. P. Involvement of interleukin1 converting enzyme in apoptosis of bFGF-deprived murine aortic endothelial cells. FASEBJ. 10, 1192-1197 (1996) ABSTRACT
Key Words: ICE
.
MAE cell
cell viability
.
apoptosis
bFGF
deprivation
CEREBROVASCULAR DISORDER, CORONARYartery disease, pulmonary venous thromboembolism, and systemic and pulmonary hypertension account for one-third of the national annual mortality (1). A common feature of many forms of these vascular diseases is that endothelial function is impaired. Metabolic, mechanical, and immunologic injury to the endothelium may disturb the homeostatic balance within the vasculature by inducing endothelial dysfunction. Clarifying the molecular mecha-
1192
Cleveland,
Ohio 44195,
nisms inducing endothelial cell damage may offer insight into new modes of treatment. Recently, we and others have reported that deprivation of growth factor induces apoptosis (programmed cell death) in cultured endothelial cells (2, 3). Apoptosis is an essential physiologic process in the normal development of multicellular organisms. It is characterized by chromatin condensation and DNA fragmentation (4, 5). A variety of stimuli are known to induce or suppress apoptosis (6-12). However, the precise molecular mechanisms regulating apoptosis are unknown, The tumor suppressor gene p53 and the cellular protooncogene c-myc encode ubiquitously express nuclear phosphoproteins that function as transcriptional regulators controlling cell proliferation, differentiation, and apoptosis (13-16). Moreover, interleukin1 p-converting enzyme (ICE)2 gene, a mammalian homolog of the Caenorhabditis elegans cell death gene ced-3 (17), has been identified as the regulator of apoptosis in several cells (18, 19). Therefore, we wished to determine whether the above apoptosis-related genes p53, c-myc, and ICE were involved in the apoptosis of bFGF-deprived murine aortic endothelial (MAE) cells.
MATERIALS Endotheial
AND
METHODS
cells
MAE cells were obtained as previously described cultured in MCDB 107 (Kyokuto Pharmaceutical supplemented with 20% heat-inactivated fetal calf Laboratories, Grand Island, N.Y.), 2 ng/ml bFGF Takeda Chemical Industries, Ltd. (Osaka, Japan), penicillin and streptomycin.
1To whom correspondence Neurosurgery/S80, Clinic
Brain
Foundation,
fibroblast tetrazolium (benzimide)
growth
Euclid
9500
2Abbreviations:
ICE, factor;
bromide;
be addressed,
Center/Cancer
Ave., Cleveland,
interleukin-1-converting MIT,
Hoechst
trihydro-chloride;
fetal calf serum;
should
Tumor
(3). The cells were Ind., Tokyo, Japan) serum (FCS: GIBCO kindly supplied by and the antibiotics
at: Department
Center,
The
of
Cleveland
OH 44195, USA. enzyme; bFGF, basic
3-(4,5-dimethyl-2.thiazolyl)-2,5-diphenyl
33258, MAE,
PBS, phosphate-buffered
the DNA binding murine saline;
0892.6638/96/0010-1
aortic
fluorochrome endothelial;
bis FCS,
m, murine.
192/$01.50
© FASEB
Cell viability Cell viability was assayed by the ability of cells to convert soluble MIT into an insoluble, dark blue formazan reaction product (Cell Proliferation kit I, Boehringer Manheim Biochemicals, Indianapolis, Ind.) as described previously (9). MAE cells were seeded at i04 cells/well (0.1 ml) in 96-well, flat-bottomed plates (Coming, Corning, N.Y.) and incubated overnight at 37#{176}C. The bFGF-containing medium was then replaced with bFGF-free medium as previously described (3). To demonstrate the specificity of bFGF in MAE cells, anti-bFGF immunoneutralizing antibody (50 ng/mI) was added to the bFGF-containing medium in some experiments as previously described (3). After incubation for 1 to 4 days, MIT assay was performed. The statistical significance of findings was assessed using the paired Students t test.
Apoptotie
features
of MAE cells by deprivation
of bFGF
To detertnine whether MAE cells deprived of bFGF displayed an apoptotic morphology, cells were stained with Hoechst 33258 as described previously (20). Five hundred cells were counted and scored for the incidence of apoptotic chromatin changes under fluorescence microscopy. Furthermore, free 3’-OH ends generated by endonuclease cleavage of gerlomic DNA during apoptosis were labeled with a comtnercial kit (ApopTag; Oncor, Gaithersburg, McI.) based on a method similar to that of Cavrieli et al. (21), but using digoxigenin-11-dUTP as label.
Immunoblotting
assay
MAE cells deprived of bFGF were rinsed three times with phosphatebuffered saline (PBS), scraped off with a rubber policeman, pelleted at 3000 x g for 5 mm, and lysed in 300 j.il of freshly prepared extraction buffer (10 mM Tris-HCI pH 7, 140 mM sodium chloride, 3 mM magnesium chloride, 0.5% NP-40, 2 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 5 tnM dithiothreitol) for 20 mm on ice as described previously (20). Equal amounts of protein estimated by the BioRad protein assay (BioRad, Richmond, Calif.) were separated by electrophoresis on a 10 or 8% polyacrylamide gel in SDS and thereafter subjected to electrotransfer onto nitrocellulose. The membranes were subjected to iramunoblotting using enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, Ill.) detection system according to the manufacturer’s instructions. The monoclonal antibodies used were anti-p53, anti-c.myc (Oncogene Science, Uniondale, N.Y.), and anti-ICE (Santa Cruz, Calif.). The intensity of each band was quantitated by densitometry.
ICE inhibition
assay
Because the results of Immunoblotting analysis showed the increased expression of ICE protein during apoptosis, we determined whether a specific
Torrance, deprivation 1 day
ICE
tetrapeptide
inhibitor,
Ac-YVAD-CMK (BACHEM,
Inc..
Calif.),
affected the viability and apoptosis of MAE cells after of bFGF. This inhibitor was added (5, 10, or 50 f.tM) to cells
before
the initiation
of deprivation.
ICE transfection To determine whether overexpression of ICE induced apoptosis in MAE cells, the ICE-lacZ fusion gene (pactM10Z containing the intact murinc mn] ICE eDNA fused to the Escherichia coli lacZ gene, kindly supplied by J. Yuan) was used (18). The day before ICE transfection, MAE cells were seeded at i05 cells/mi in Lab-Tek chamber slides. MAE cells were transfected with the ICE-lacZ or control gene (pactgal’) construct by lipofectamine-mediated gene transfer (GIBCO BRL). The cells were incubated for 5 h in OPTI-MEM medium (GIBCO BRL) containing 2 1g of DNA and 5 .tl of lipofectamine, then an equal volume of culture medium containing 40% FCS and 4 ng/ml bFGF was added without
removing
the
transfer
mixture.
To
detect
the
expression
of
chimeric gene in transfected MAE cells, cells were fixed with 1% formaldehyde and 0.2% glutaraldehyde for 5 mm, rinsed three times with PBS, and stained in X-Gal buffer (0.4 mg/ml 5-bromo-4-chloro-3indoxyl -galactoside, 4 mM K3Fe(CN)6, 4 mM KtFe(CN)#{243}-3H2O,and 2 mM MgCl2 in 0.1 M sodium phosphate buffer [pH 7.5]) at 37#{176}C for 3
EFFECT OF ICE ON APOPTOSIS
IN ENDOTHELIAL
CELLS
h. MAE cells were then stained apoptotic morphology.
with Hoechst 33258
(8 .tg/ml)
to detect
RESULTS Effect of bFGF deprivation apoptosis in MAE cells
on viability
and
As shown in Fig. 1A, MAE cell death was first apparent 1 day after deprivation of bFGF. By day 4, only 20% of the initial MAE cell population had survived. The administration of bFGF alone retained the viability of MAE cells, but about 15% of the cumulative cell number decreased compared to controls containing complete medium supplemented with FCS and bFGF (data not shown). The administration of anti-bFGF MoAb induced cell death in MAE cells cultured in bFGF-containing medium to almost the same extent as did deprivation of bFGF. In FCS alone, the viability of MAE cells decreased to almost same extent as viability of cells deprived of both FCS and bFGF. As shown in Fig. lB. 70 or 78% of MAE cells stained with Hoechst 33258 showed clear nuclear condensation with the typical apoptotic profile 4 days after deprivation of either bFGF alone or bFGF and FCS. 90% of MAE cells retained normal nuclear shape after deprivation of FCS alone (Fig. 1B). To confirm that DNA fragmentation occurred in MAE cells undergoing chromatin condensation by deprivation of bFGF, we used an in situ end labeling assay to detect DNA breakage. Nearly all cells containing condensed chromatin were also positive for DNA breakage (data not shown). The degree of DNA fragmentation as well as apoptotic cells in MAE cells increased in a time-dependent manner (Fig. 1B, C). These results demonstrated that bFGF deprivation induced apoptotic cell death in MAE cells. Expression of p53, c-Myc, bFGF-deprived MAE cells
and ICE proteins
in
To determine whether deprivation of bFGF affected the levels of the apoptosis-related proteins p53, c-Myc, and ICE in MAE cells, immunoblotting assay was performed. Neither p53 nor c-Myc protein was detected in untreated and bFGF-deprived MAE cells (Fig. 2), although we cannot mle out the possibility that levels were below the sensitivity of the antibodies used. MAE cells cultured with FCS and bFGF expressed low levels of ICE protein, but its expression was remarkably increased fivefold 2 days
after
deprivation
of bFGF.
These
results
suggested
that accumulated ICE protein might be related to the induction of apoptosis in MAE cells by bFGF deprivation. Effect of ICE inhibitor on apoptosis bFGF-deprived MAE cells
in
To further examine the role of ICE protein in MAE cells undergoing apoptosis due to bFGF deprivation, we used a specific inhibitor of ICE. As shown in Fig. 3A, B, the administration of the peptide Ac-YVAD-CMK partially
1193
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Figure 1. Apoptotic cell death of MAE cells by deprivation of bFGF. A) Viability of MAE cells after deprivation of bFGF. MAE cells were seeded at a density of iO cells/well (0.1 ml) in 96-well, flat-bottomed plates and incubated overnight. Percentage of cell viability was determined by MIT assay. Values represent the means ± SD of results of three separate experiments. B) The effect of bFGF deprivation on the percentage of MAE cells exhibiting morphological changes of apoptosis. A total of 500 cells stained with Hoechst 33258 were counted. Values represent the means ± so of results of three separate experiments. C) Percentage of DNA fragmentation in individual cells was determined by in situ end labeling assay (Apoptag kit). A total of 500 cells stained were counted. Values represent the means ± SD of results of three separate experiments.
80 0
160
0
0
0 0.
a.
C)
-
20
40
20
-
0
rrT
0
I
1
2
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3
4
5
0
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Days 3.
Figure
Effect
MAE
results of three
separate
4
5
MIT
assay) and apoptosis
of deprivation
(B,
assay. Values
Hoechst 33258 staining) of represent the means ± SD of
experiments.
The p53 protein is a negative regulator of cell proliferation that arrests cells in G1 and induces apoptosis (13, 14), and the c-Myc protein is a positive regulator of cell cycle and may also induce apoptosis (15). These proteins function closely in the induction of apoptosis in growth factor-deprived several cells (16). In the present study, however, the protein levels of p53 and c-myc were undetected in MAE cells undergoing apoptosis.
X-Ga!
3
Days
of the tetrapeptide ICE inhibitor (YVAD-CMK) on cell viability (A, cells. YVAD-CMK was added to aliquots 1 day before the initiation
bFGF-deprived
2
Hoechst
33258
bFGF is a potent endogenous mitogen for endothelial cells (41). The intracellular signaling mechanisms that mediate bFGF-induced angiogenesis, however, have not been fully identified. Recent studies have found that the effect of bFGF on endothelial cells is mediated through activation of the intracellular enzyme protein kinase C (PKC) (42, 43). Activation of PKC has been suggested as a mechanism for cytokine-mediated protection against apoptosis (44, 45). It is possible that ICE-mediated apoptosis in endothelial cells may be ameliorated by PKC activation, but the relationship between ICE and PKC in these cells remains to be explored. We thank pactgal’
p3actM1OZ(mICE
search Fund
expression)
Dr. Junying
Yuan for kind
gifts of the pactM1OZ and in part by the CCF-Re-
plasmids. This study was supported
(5514) and in part by the John Gagliarducci
Fund.
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Figure
4.
mICE.
MAE
control Hoechst from
1196
Induction cells
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were
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with
by overexpression the mICE-lacZ
of
pactJgal’ vector; 24 h later, they were fixed and stained with 33258 after X-Gal staining (x400). The left and right panels are
the same
fields
and at the same
Vol.lOAugustl996
magnification,
2.
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Received for publication November Accepted for publication March
27. 29,
1995. 1996.
1197
