BIOCHEMICAL
Vol. 166, No. 3, 1990 May 16, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1194-1200
APOPTOSIS OF VASCULAR ENDOTHELIAL CELLS BY FIBROBLAST GROWTH FACTOR DEPRIVATION Satohiko
Araki’.
IDepartment of Nagoya University, 2National
3,
l,* Kazuhiko
Institute Tokyo
Kaji’
School of Science, Nagoya 464-01, Japan
of Radiological Chi ba-shi , Japan
Metropolitan Itabashi-ku.
April
Shimada’ Hayashi
Molecular Biology, Chikusa-ku, Institute Anagawa,
3Tokyo Received
Yoshiya and Hiroshi
of 173,
Sciences,
Gerontology, Japan
1990
Survival and proliferation of many types of vascular endotielial cells are influenced by fibroblast growth factor (FCF) . Removal of FGF from the medium of human umbilical vein endothelial cells (HUVECl in culture resulted in death of the cells. Here we show that the death caused by deprivation of FGF is active death or apoptosis, and the process of apoptosis can be inhibited by cycloheximide. an inhibitor of protein synthesis. The present study shows apoptosis occurs in endothelial cells in The process of active death of vascular endothelial culture. cells is inhibited by growth factor. This mechanism may be important for the regulation of vascular organization through the degeneration of vessels. OiwoAcademicc-ress, I~C.
Vascular
endothelial vascular
clotting,
In cancer
ability
to
blood
vessels
and
body.
system
Recent suggest
cognate
regulatory
the
systembm8.
*To
whoa
ooo6-291x/90 Copyright All rights
reprint
addition,
form
the
role
control
cells
blood
are
5
on of
factors We analyzed
requests
the
for the
should
many
equally of of
types
of
death
of
be
addressed.
1194
growth The
control
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
important
and
are
in
cells
vascular
blood-
vascular
also
establishment
versatile
roles
of
vessels3’4.
degeneration
studies the
important
embryogenesis
new
their
many
and
development,
their
the
play
contraction,
permeabiIity2. healing,
cells
wound
because
of
formation
of
important
to
the cell
in
immune death involved
endothelial
by in
Vol.
BIOCHEMICAL
168, No. 3, 1990
cells
from
the
positive
role
culture
of
human
endothelial
in
point
from
is
of
umbilical
however.
after
the
cells
had
grown
confluently.
study
how
cells
die
after
removal
culture
medium. MATERIALS
the
the
of
of
to
play
for
availability
Removal
decided
from
also
culture
a
A primary
suitable
veins,
FGF
may
degeneration.
cells
because
RESEARCH COMMUNICATIONS
a death
vascular
cells
difficulty.
such
vascular
cells
even
that
controlling
umbilical
Endothelial
death
view
AND BIOPHYSICAL
the
study tissue.
present medium
some
resulted
Thus, of
of
FGF
in
we
from
the
AND METHODS
Reagents. FGF was extracted from bovine brains by the method of Lobb’o. MCDB-104 was purchased from Kyokuto pharmaceutical industry, Tokyo, Japan. Fetal bovine serum (FBS) was purchased from GIBCO, Grand island, NY. Cycloheximide, proteinase K, and ethidium bromide were purchased from Sigma Chemical Company, St.Louis. MO. w cultures. Human umbilical vein endyt4helial cells (HLJVEC) were obtained by the method of Jaffe . The cells were cultured on collagen-coated plastic dish in MCDB-104 supplemented with 10% FBS and 70ng/ml FGF at 37C in 5% CO2 and 95% air. The cultures were stained by a rabbit anti-human Factor VIII antibody to confirm that they were endothelial cells. For experiments, HLJVEC were grown until they become confluent. After several days at confluent, The medium was then replaced one lacking FGF. The number of floating or trypsinized cells were determined by a coulter counter. Throughout the experiments, we used cells with a population doubling level of 10 to 20.
by
Analysis of DNA fragmentation. Cells were incubated in digestion buffer containing 0.2 mg/ml proteinase K at 50C for 12hours. The cellular DNA was extracted twice with a 1:l phenol/chloroform mixture and once with chloroform. DNA precipitation was performed using ethanol. The samples were electrophoresed on a 1.5% agarose gel in Tris-athetate buffer. Following electrophoresis, the gel was stained with ethidium bromide and photographed on an UV transilluminator.
RESULT Two round medium 1). neither
hours up
after
and
The
floating attached
deprivation
eventually
leaving
AND DISCUSSION of
detached
many
holes cells
onto
on
the
could a new
dish
FGF,
some
from
the
sheet
of
be
dish
1195
started and
proliferated
with
to
floated
in
cells
(Fig.
confluent
recovered nor
cells
the (cl
medium, cell
the
and in
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
1196
RESEARCH COMMUNICATIONS
Vol.
168, No. 3, 1990
several
BIOCHEMICAL
thousands)
concluded Using
even
that this
the
in
cells
criterion,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
presence
that we
of
floated
studied
FGF.
into
the
Thus,
the
time
medium
course
of
we
were
dead.
FGF-deficient
death. About
0.1
conditions other
% of
used words,
FGF was into
about
removed
the
to
indicates
this
study
while
maintaining
symmetrical
process
of
order
be
observed6’7’g-11.
then
floating
effect
of
in
degraded but
is
culture On
living
removal
(Fig.
2a). very
and
likely
apoptosis,
the
cells.
showed
characteristic
in
death
not
the
protein (Fig.
2b).
we and
living of
cell
that
cells. DNA should
had
been
a characteristic no
DNA in the
quickly
do
delay
dying
pattern
fragmentation
cells
lysate fragmentation
was
killed was
added
observation
death,
a dying
contrary,
if
rather
fragmentation
DNA in
The
FGF was
involves the
DNA in
to
addition
The
When
floating
started
This
inhibited
dish,
slightly. no
FGF
in
hour.
cells
medium
level.
the
per
If
the
FGF-deficient
The the
into
death,
over
4 h later.
background
death
from
for
about
characterize
fragmentation.
6 hr,
2 h,
analyzed
FGF-deficient
for
of
cycloheximide
to
If
thawing
number
kinetics
since
observed
the
cells
electrophoretically
of
medium, constant
experimental
confluence;
turned
FGF-deficient
synthesis
the
cells
original the
under
the
a maximum
the
that
detached
% of the
of
exhibit
In
hour
remained
number
returned
every
0.1
reaching the
died
from
medium
increase, back,
in
cells
by
incubated was
freeze-andat
observed
37 C (Fig.
Fig. 1. FGF-deficient death of HUVEC observed under the microscope. al Confluent culture of HUVEC. The medium contained MCDB-104 (basal medium), 10 % fetal bovine serum, 70 nglml FGF, and 100 uglml heparin. bl Confluent culture 3 h after FGF deprivation. Some cells rounded up but still remained at the spots. cl “Worm-eat en” structure of otherwise confluent cell-sheet, observed away into
6 h after FGF deprivation. the medium leave holes
in
1197
the
Cells which sheet.
have
floated
Vol.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
a
RESEARCH COMMUNICATIONS
b
3 f ; 2 a 1
1 0
0
2
1- 0
6
4
Time(h)
4
Timi(days)
Fig. 2. The time course of cell death. a) The rate of cell death after FGF deprivation was measured by determining the number of cells detached from the dish. Several sets of plates containing confluent cultures of HUVEC were washed by fresh medium free of FGF at time 0. Every hour, the medium was removed and replaced by new medium. The number of detached cells contained in the older medium was counted, and plotted against time. The numbers represent the population of cells that had died during previous hour. At the arrow. FGF was added back to the replacing medium for one set of plates (0). but not the others (0 1. b) Inhibition of cell death by cycloheximide. A longer time course than a) was obtained after the removal of FGF. The number of cells remained on the dish was counted. The symbols are; (0 ), control; (0 ). -FGF; (A), -FGF +cycloheximide. The concentration of cycloheximide was 1 ug/ml; inhibition was partial, but higher concentrations caused cytotoxicity.
These
3). is
results
show
that
the
th
dea
caused
by
FGF-deprivation
apoptosis. Apoptosis
is
HUVEC
undergoes
bovine
serum
likely
to
in
vascular
the
cells
vessel The
instead be
also
cell
mechanism to
isolate
apoptosis
from
Thus
a
dead
not
an
since
cells
fashion.
1198
the of
several
unique
of
fetal
factors
is
Apoptosis characteristic cells
similar
conditions
for
the the
living
example,
medium
apoptosis.
because from
For
endothelial
suited
apoptosis,
set of
under
especially of
withdrawal
is
cord
is
process.
suppression
umbilical
system
complex
FGF12. for
undergoes
present
by of
required
from
one
rather
apoptosis
endothelial
molecular allows
likely
of from 12 .
study
of
detachment cells
lung
the process
in
a
single
Vol. 168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
4.0 2.0 ‘1.0 0.5
Fig. 3. Degradation of DNA in dead cells. DNA was extracted from living cells (attached to dish), dead cells (floating in the medium), and cells killed by repeated freezing and thawing. The DNA was then analyzed by agarose gel electrophoresis. Lane 1, DNA from living cells; lane 2, DNA from killed cells, subsequently incubated at 37 C, for 6 h; lane 3, DNA from dead cells collected from the medium 6 h after FGF deprivation; lane 4, molecular weight markers (lambda/Hind III digest). Molecular weights are expressed in terms of kilo basepairs.
ACKNOWLEDGMENTS We
thank
Thanks
are
Harumi
Ohyama
Robert also
Macnab due
for
to their
for
critical
Mitsuyoshi helpful
reading Matsuo,
Takeshi
of
the
manuscript.
Yamada,
and
discussions.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9.
Gospodarowicz, D.. Brown, K.D., Bridwell, C.R.,and B.R. J.Cell Biol. (1978) 77, 774-788. Jaffe, E.A. (1984) Biology of endothelial cells, Martinus Nijhoff Publishers, Boston. Foikman, J.,and Haudenschild, C.C. (1980) Nature 556. R.N..and Sherline, P. (1984) Ann.N.Y.Acad.Sci Mascardo, 451-453. O’Shea, J.D., Nightingale, M.G..and Charnley. W.A. Biol.Reprod. 17.162-177. Williams, G.T., Smith, C.A., Spooncer, E., Dexter, Taylor, D.R. (1990) Nature 343, 76-79. Smith, C.A., Williams, G.T.. Kingston, R., Jenkinson. E.J. ,and Owen, J.J.T. (1989) Nature 337. 181-184. Res. Duke, R.C.,and Cohen, J.J. (1986) Lymphokine Kerr, J.F.R.. Wyllie. A.H..and Currie, A.R. (1972) Br.J.Cancer 26. 239-257.
1199
Zetter, pp.l-456, 288,
551435,
(1977 T.M.
5,
, and
289-299.
Vol.
10. 11. 12. 13. 14.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
Wyllie, A.H. (1980) Nature 284, 555-557. Wyllie, A.H., Morris, R.G., Smith, A.L.,and J.Pathol. 142, 67-77. Araki, S. et al. unpublished observations. Jaffe. E.A., Nachman, R.L., Becker, C.G.,and (1973) J.Clin.Invest. 52, 2745-2756. Biochemistry Lobb, R.R.,and Fett, J.W. (1984)
1200
RESEARCH COMMUNICATIONS
Dunlop, Minlck, 23,
D.
(1984)
R.C. 6295-6296.
Vol. 166, No. 3, 1990 May 16, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1194-1200
APOPTOSIS OF VASCULAR ENDOTHELIAL CELLS BY FIBROBLAST GROWTH FACTOR DEPRIVATION Satohiko
Araki’.
IDepartment of Nagoya University, 2National
3,
l,* Kazuhiko
Institute Tokyo
Kaji’
School of Science, Nagoya 464-01, Japan
of Radiological Chi ba-shi , Japan
Metropolitan Itabashi-ku.
April
Shimada’ Hayashi
Molecular Biology, Chikusa-ku, Institute Anagawa,
3Tokyo Received
Yoshiya and Hiroshi
of 173,
Sciences,
Gerontology, Japan
1990
Survival and proliferation of many types of vascular endotielial cells are influenced by fibroblast growth factor (FCF) . Removal of FGF from the medium of human umbilical vein endothelial cells (HUVECl in culture resulted in death of the cells. Here we show that the death caused by deprivation of FGF is active death or apoptosis, and the process of apoptosis can be inhibited by cycloheximide. an inhibitor of protein synthesis. The present study shows apoptosis occurs in endothelial cells in The process of active death of vascular endothelial culture. cells is inhibited by growth factor. This mechanism may be important for the regulation of vascular organization through the degeneration of vessels. OiwoAcademicc-ress, I~C.
Vascular
endothelial vascular
clotting,
In cancer
ability
to
blood
vessels
and
body.
system
Recent suggest
cognate
regulatory
the
systembm8.
*To
whoa
ooo6-291x/90 Copyright All rights
reprint
addition,
form
the
role
control
cells
blood
are
5
on of
factors We analyzed
requests
the
for the
should
many
equally of of
types
of
death
of
be
addressed.
1194
growth The
control
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
important
and
are
in
cells
vascular
blood-
vascular
also
establishment
versatile
roles
of
vessels3’4.
degeneration
studies the
important
embryogenesis
new
their
many
and
development,
their
the
play
contraction,
permeabiIity2. healing,
cells
wound
because
of
formation
of
important
to
the cell
in
immune death involved
endothelial
by in
Vol.
BIOCHEMICAL
168, No. 3, 1990
cells
from
the
positive
role
culture
of
human
endothelial
in
point
from
is
of
umbilical
however.
after
the
cells
had
grown
confluently.
study
how
cells
die
after
removal
culture
medium. MATERIALS
the
the
of
of
to
play
for
availability
Removal
decided
from
also
culture
a
A primary
suitable
veins,
FGF
may
degeneration.
cells
because
RESEARCH COMMUNICATIONS
a death
vascular
cells
difficulty.
such
vascular
cells
even
that
controlling
umbilical
Endothelial
death
view
AND BIOPHYSICAL
the
study tissue.
present medium
some
resulted
Thus, of
of
FGF
in
we
from
the
AND METHODS
Reagents. FGF was extracted from bovine brains by the method of Lobb’o. MCDB-104 was purchased from Kyokuto pharmaceutical industry, Tokyo, Japan. Fetal bovine serum (FBS) was purchased from GIBCO, Grand island, NY. Cycloheximide, proteinase K, and ethidium bromide were purchased from Sigma Chemical Company, St.Louis. MO. w cultures. Human umbilical vein endyt4helial cells (HLJVEC) were obtained by the method of Jaffe . The cells were cultured on collagen-coated plastic dish in MCDB-104 supplemented with 10% FBS and 70ng/ml FGF at 37C in 5% CO2 and 95% air. The cultures were stained by a rabbit anti-human Factor VIII antibody to confirm that they were endothelial cells. For experiments, HLJVEC were grown until they become confluent. After several days at confluent, The medium was then replaced one lacking FGF. The number of floating or trypsinized cells were determined by a coulter counter. Throughout the experiments, we used cells with a population doubling level of 10 to 20.
by
Analysis of DNA fragmentation. Cells were incubated in digestion buffer containing 0.2 mg/ml proteinase K at 50C for 12hours. The cellular DNA was extracted twice with a 1:l phenol/chloroform mixture and once with chloroform. DNA precipitation was performed using ethanol. The samples were electrophoresed on a 1.5% agarose gel in Tris-athetate buffer. Following electrophoresis, the gel was stained with ethidium bromide and photographed on an UV transilluminator.
RESULT Two round medium 1). neither
hours up
after
and
The
floating attached
deprivation
eventually
leaving
AND DISCUSSION of
detached
many
holes cells
onto
on
the
could a new
dish
FGF,
some
from
the
sheet
of
be
dish
1195
started and
proliferated
with
to
floated
in
cells
(Fig.
confluent
recovered nor
cells
the (cl
medium, cell
the
and in
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
1196
RESEARCH COMMUNICATIONS
Vol.
168, No. 3, 1990
several
BIOCHEMICAL
thousands)
concluded Using
even
that this
the
in
cells
criterion,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
presence
that we
of
floated
studied
FGF.
into
the
Thus,
the
time
medium
course
of
we
were
dead.
FGF-deficient
death. About
0.1
conditions other
% of
used words,
FGF was into
about
removed
the
to
indicates
this
study
while
maintaining
symmetrical
process
of
order
be
observed6’7’g-11.
then
floating
effect
of
in
degraded but
is
culture On
living
removal
(Fig.
2a). very
and
likely
apoptosis,
the
cells.
showed
characteristic
in
death
not
the
protein (Fig.
2b).
we and
living of
cell
that
cells. DNA should
had
been
a characteristic no
DNA in the
quickly
do
delay
dying
pattern
fragmentation
cells
lysate fragmentation
was
killed was
added
observation
death,
a dying
contrary,
if
rather
fragmentation
DNA in
The
FGF was
involves the
DNA in
to
addition
The
When
floating
started
This
inhibited
dish,
slightly. no
FGF
in
hour.
cells
medium
level.
the
per
If
the
FGF-deficient
The the
into
death,
over
4 h later.
background
death
from
for
about
characterize
fragmentation.
6 hr,
2 h,
analyzed
FGF-deficient
for
of
cycloheximide
to
If
thawing
number
kinetics
since
observed
the
cells
electrophoretically
of
medium, constant
experimental
confluence;
turned
FGF-deficient
synthesis
the
cells
original the
under
the
a maximum
the
that
detached
% of the
of
exhibit
In
hour
remained
number
returned
every
0.1
reaching the
died
from
medium
increase, back,
in
cells
by
incubated was
freeze-andat
observed
37 C (Fig.
Fig. 1. FGF-deficient death of HUVEC observed under the microscope. al Confluent culture of HUVEC. The medium contained MCDB-104 (basal medium), 10 % fetal bovine serum, 70 nglml FGF, and 100 uglml heparin. bl Confluent culture 3 h after FGF deprivation. Some cells rounded up but still remained at the spots. cl “Worm-eat en” structure of otherwise confluent cell-sheet, observed away into
6 h after FGF deprivation. the medium leave holes
in
1197
the
Cells which sheet.
have
floated
Vol.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
a
RESEARCH COMMUNICATIONS
b
3 f ; 2 a 1
1 0
0
2
1- 0
6
4
Time(h)
4
Timi(days)
Fig. 2. The time course of cell death. a) The rate of cell death after FGF deprivation was measured by determining the number of cells detached from the dish. Several sets of plates containing confluent cultures of HUVEC were washed by fresh medium free of FGF at time 0. Every hour, the medium was removed and replaced by new medium. The number of detached cells contained in the older medium was counted, and plotted against time. The numbers represent the population of cells that had died during previous hour. At the arrow. FGF was added back to the replacing medium for one set of plates (0). but not the others (0 1. b) Inhibition of cell death by cycloheximide. A longer time course than a) was obtained after the removal of FGF. The number of cells remained on the dish was counted. The symbols are; (0 ), control; (0 ). -FGF; (A), -FGF +cycloheximide. The concentration of cycloheximide was 1 ug/ml; inhibition was partial, but higher concentrations caused cytotoxicity.
These
3). is
results
show
that
the
th
dea
caused
by
FGF-deprivation
apoptosis. Apoptosis
is
HUVEC
undergoes
bovine
serum
likely
to
in
vascular
the
cells
vessel The
instead be
also
cell
mechanism to
isolate
apoptosis
from
Thus
a
dead
not
an
since
cells
fashion.
1198
the of
several
unique
of
fetal
factors
is
Apoptosis characteristic cells
similar
conditions
for
the the
living
example,
medium
apoptosis.
because from
For
endothelial
suited
apoptosis,
set of
under
especially of
withdrawal
is
cord
is
process.
suppression
umbilical
system
complex
FGF12. for
undergoes
present
by of
required
from
one
rather
apoptosis
endothelial
molecular allows
likely
of from 12 .
study
of
detachment cells
lung
the process
in
a
single
Vol. 168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
4.0 2.0 ‘1.0 0.5
Fig. 3. Degradation of DNA in dead cells. DNA was extracted from living cells (attached to dish), dead cells (floating in the medium), and cells killed by repeated freezing and thawing. The DNA was then analyzed by agarose gel electrophoresis. Lane 1, DNA from living cells; lane 2, DNA from killed cells, subsequently incubated at 37 C, for 6 h; lane 3, DNA from dead cells collected from the medium 6 h after FGF deprivation; lane 4, molecular weight markers (lambda/Hind III digest). Molecular weights are expressed in terms of kilo basepairs.
ACKNOWLEDGMENTS We
thank
Thanks
are
Harumi
Ohyama
Robert also
Macnab due
for
to their
for
critical
Mitsuyoshi helpful
reading Matsuo,
Takeshi
of
the
manuscript.
Yamada,
and
discussions.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9.
Gospodarowicz, D.. Brown, K.D., Bridwell, C.R.,and B.R. J.Cell Biol. (1978) 77, 774-788. Jaffe, E.A. (1984) Biology of endothelial cells, Martinus Nijhoff Publishers, Boston. Foikman, J.,and Haudenschild, C.C. (1980) Nature 556. R.N..and Sherline, P. (1984) Ann.N.Y.Acad.Sci Mascardo, 451-453. O’Shea, J.D., Nightingale, M.G..and Charnley. W.A. Biol.Reprod. 17.162-177. Williams, G.T., Smith, C.A., Spooncer, E., Dexter, Taylor, D.R. (1990) Nature 343, 76-79. Smith, C.A., Williams, G.T.. Kingston, R., Jenkinson. E.J. ,and Owen, J.J.T. (1989) Nature 337. 181-184. Res. Duke, R.C.,and Cohen, J.J. (1986) Lymphokine Kerr, J.F.R.. Wyllie. A.H..and Currie, A.R. (1972) Br.J.Cancer 26. 239-257.
1199
Zetter, pp.l-456, 288,
551435,
(1977 T.M.
5,
, and
289-299.
Vol.
10. 11. 12. 13. 14.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
Wyllie, A.H. (1980) Nature 284, 555-557. Wyllie, A.H., Morris, R.G., Smith, A.L.,and J.Pathol. 142, 67-77. Araki, S. et al. unpublished observations. Jaffe. E.A., Nachman, R.L., Becker, C.G.,and (1973) J.Clin.Invest. 52, 2745-2756. Biochemistry Lobb, R.R.,and Fett, J.W. (1984)
1200
RESEARCH COMMUNICATIONS
Dunlop, Minlck, 23,
D.
(1984)
R.C. 6295-6296.
