- Email: [email protected]
Induction by fibroblast growth factor of angiotensin converting enzyme in vascular endothelial cells invitro
Vol. 145, No. 3, 1987 June 30, 1987
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1211-1216
INDUCTION BY FIBROBLAST GROWTH FACTOR OF ANGIOTENSIN CONVERTING ENZYME IN VASCULAR ENDOTHELIAL CELLS --IN VITRO Tetsuro
Okabe,
The Third University
Received
Kazuo Hiroshi
Yamagata, Hidaka,
Michio Fujisawa, Fumimaro and Yoshimi Umezawa
Department of Internal Medicine, Faculty of Tokyo, Tokyo 113, Japan, and Department Juntendo University, Tokyo 113, Japan
May 15,
Takaku,
of Medicine, of Pathology,
1987
SUMMARY: Induction of vascular endothelial cells with pituitary fibroblast growth factor (FGF) provoked an increase in angiotensin converting The stimulatory effect of FGF on ACE activity was enzyme activity. dose-dependent (EDso = 1.0 rig/ml). Our results suggest a possible role for pituitary FGF in regulation of ACE production in vascular 0 1987 Academic Press, Inc. endothelial cells.
The by two
production
of angiotensin renin
enzymes;
controlling
release
regulation
functions.
peptidase
located
degrades
bradykinin
of
pressure
isolating chose the shown the growth
and
endothelial cultured
to
of
increase
effects factor
of
largely
ignored.
culture
exhibit
a wide
assigned
cells
have cells
ACE production. ACE production various
on ACE activity
peptide
of
range
vascular
important
roles
fluid
exchange.
established
as a convenient
(2). growth
in endothelial
1211
but
of
to
a carboxydicells,
angiotensin in
the
factors
II.
regulation
Techniques (1).
system
this
the
degradative
report, including
for
We therefore to
investigate
glucocorticoids In
factors
endothelial
I
Recently,
The
enzyme,
angiotensin
been
regulated
studied,
converting
transvascular
endothelial
regulation
has been
surface
is
enzyme.
intensively
converts
been
substrate
been
luminal
and
have
have
renin
converting
Angiotensin
on the
peptides
blood
in
from
angiotensin
renin
cells
synthetic
These
of
of ACE activity
Endothelial and
and
II
have we
been
examined
fibroblast
cells. 0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. Ail rights of reproduction in any ,form reserved.
Vol. 145, No. 3, 1987
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALSANDMETHODS Materials: Fibroblast growth factor (FGF), Epidermal growth factor stimulating activity (MSA), insulin, Triiodo(EW t Multiplication thyronine, and dexamethasone were purchased from Collaborative Research Inc. Heparin, 12-O-tetradecanoylphorbol 13-acetate (TPA), progesterone, estradiol, testosterone, and cholera toxin were obtained from Sigma Chemical Co. Cell culture and medium: Endothelial cells were collected from a fresh porcine aorta using collagenase digestion as described by Booyse et Cloning of the endothelial cells was performed by single cell al (3). platings as described originally by Puck et al (4). The endothelial cells were cultured in plastic dishes (Falcon 3002) in Ham's F-10 nutrient mixture supplemented with 10% fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 us/ml). Subcultures were performed using 0.25% trypsin and 0.02% EDTA solution (Gibco) when the cells reached confluency. There was no evidence of transformation or loss of endothelial monolayer under these conditions. Media, serum, and antibiotics were obtained from Flow Laboratories Inc. Conversion of angiotensin I to angiotensin II by endothelial cells: ACE activity was measured by adding angiotensin I to the cultured endothelial cells and measuring generated angiotensin II by radioimmunoassay as described previously (5). After confluency, the culture medium was removed, and the cells were washed twice with F-10 medium, and then incubated in serum-free F-10 medium in the presence of angiotensin I (final concentration = 12 rig/ml). After 5 hr of incubations at 37°C in a 5% CO%chamber, the medium was harvested and stored at -20°C for later angio ensin II analysis. Simultaneously, as a control, the medium without cells was incubated and harvested. Angiotensins were purchased from Protein Research Foundation, Osaka, Japan. Radioimmunoassay for angiotensin II: The generated angiotensin II was measured by direct radioinununoassay performed according to the Briefly, 700 ul of 0.1 M Tris buffer (pH 7.4) method described (5). containing & mM EDTA and 0.025% bovine serum albumin, and 100 ul of 0.005 uCi I-angiotensin II (approximately 10,000 cpm; New England Nuclear Co.) were mixed with 100 ul angiotensin II antiserum )l : 100,000 titer). The sample (100 ul) was added and the reaction mixture was incubated for 16-20 hr at 4°C. Antibody-bound and free angiotensin II were separated by the addition of a dextran-coated charcol suspension. Bound radioactivity was counted with a [email protected] counter. Without addition of the test sample, 75% of I-angiotensin II was bound to antiserum at a final titer of 1 : 100,000. There was less than 0.5% cross-reactivity to angiotensin I.
RESULTSAND DISCUSSION When the
endothelial
angiotensin
II
(Fig.
Angiotensin
1).
of vascular
cells
were incubated
with
angiotensin
I,
was generated in the medium in a time-dependent manner II
was not generated in the incubation
smooth muscle cells
or without
the endothelial
not shown) that
I 10 nM dexamethasone could
our endothelial
cells
Conversion of angiotensin
in
confirmation I to angiotensin 1212
of II
cells
induce ACE activity the
earlier
report
medium (data in (2).
was increased by incubat-
Vol. 145, No. 3, 1987
BIOCHEMICAL
0
AND BIOPHYSICAL
1
4
8
Incubation Figure
ing
the
cells
with factor(s)
dexamethasone which
fibroblast
growth factor
in the endothelial
cells
in
may be
we examined several
activity,
a dose-related involved
peptide
(FGF)(6).
2.
factors
in
manner. the
regulation
In search of
ACE
and hormones including
FGF was found to induce ACE activity
in a dose dependent manner (Fig.
Dexamethasone (nM) Figure
Time (hrf
Conversion of angiotensin I to angiotensin II in serum-free cultures of vascular endothelial cells. At time = 0, culture medium containing no fetal bovine serum was placed over confluent cultures of endothelial cells in Falcon 60-mm dishes. The generated angiotensin II was measured by direct radioimmunoassay after 1, 4, and 8 hours. Each point represents mean value from three dishes. Bars, S.D.
1.
of peptide
RESEARCH COMMUNICATIONS
2).
The
FGF (rig/ml)
Effect of fibroblast growth factor on ACE activity in endothelial cells. Confluent cultures of endothelfal cells were incubated in serum-free F-10 medium containing various concentrations of pituitary fibroblast growth factor. Conversion of angiotensin I to angiotensin II was examined as described in Fig. 1. The generated angiotensin II was measured after 5 hr of incubations. Each point represents mean value from three dishes. Bars, S.D.
1213
Vol. 145, No. 3, 1987
Figure
other
3.
8lOCHEMlCAL
factors,
cant effects
such as insulin,
epidermal growth factor
stimulating
on the ACE activity.
were strongly
inhibited
(data not shown). 95% by captopril
by treating
factor
has been shown to stimulate
cells.
However, the physiological elucidated.
However, endothelial confluency
(7).
about
was achieved
at
peptide
factors
in the endothelial this
pituitary
angiotensin
2.5-fold
was
not
peptide II.
role of this
Several
3).
glands (6).
The
of mesoderm-derived pituitary
peptide has
FGF has been shown to stimulate
cells
in sparsely populated cultures.
cultures,
within
In
(Fig. may play
FGF was found to induce
24 hr.
Half-maximal
the FGF-treated
significantly
including
cells
for ACE (Fig.
have been shown to cease DNAsynthesis after
1.0 rig/ml.
cells
97% by EDTA (1 mM) and
a wide variety
In the confluent
ACE activities
endothelial
cells
with cycloheximide
from pituitary
Pituitary
the growth of vascular endothelial
of the endothelial
the cells
inhibitor
isolated
(EGF), growth
(MSA) shows no signifi-
was inhibited
(10 uM), a specific
not been fully
activity
The ACE activity
The activity
FGF is a basic protein
Other
RESEARCH COMMUNICATIONS
Effect of EDTA and captopril (SQ 14225) on the ACE activity. The endothelial cells were incubated in the presence of EDTA (1 rnM) or captopril ( 1 uM). Conversion of angiotensin I to The angiotensin II was examined as described in Fig. 1. generated angiotensin II was measured after 3 hr of incubations. Each column represents mean value from three dishes. Bars, S.D.
hormone, or multiplication
cells
AND BIOPHYSICAL
increased
insulin 4).
1214
(data
These observations
lines
number of not
shown).
did not induce ACE activity
an important
converging
cultures,
stimulation
of
role
in
suggest that production
of
evidences suggest that
Vol. 145, No. 3, 1987
Figure
BIOCHEMICAL
AND BIOPHYSICAL
Effect of various factors and in endothelial cells. Confluent
4.
were incubated in factors or hormones to angiotensin II
serum-free for 24 hr. was examined
RESEARCH COMMUNlCATlONS
hormones
on the
ACE activity
cultures of endothelial F-10 medium containing
Conversion as
cells various
of angiotensin
described
in
Fig.
I
1.
The
generated angiotensin II was measured after 5 hr of incubations. Each column represents mean value from three dishes. Bars, S.D.
an action the
of
angiotensin
development
angiotensin given
of arterial system
directly
into
has
been
via
demonstrated
ADH secretion, nervous
the
and
II
produce
has
been
Our
analysis in
the
also
of
of brain,
rise
that
the
cells
pituitary will
physiological thereby
intake.
1215
controlling
angiotensin
arterial
pressure water
in
the
intake, central
FGF increases be the
role
of
ventricles
control
functions
when
types
cerebral in
to
physiological
endothelial
or
renin-
pressure
administration
considered
observations
blood
various
supply
contributes of the
with
a sustained
of
by
lower
animals
blood
system
Antagonists
to
Chronic
to
nervous
(8).
of
cerebral
production
peptide water
(9).
(11).
central
reported
brain
a variety
experimental
endocrine or/and
II
system
angiotensin of
the
either
Angiotensin
(10).
been
hypertesion brain
on the hypertension
have
into
experimental II
II
of
beginning this
blood
neuropressure
Vol. 145, No. 3, 1987
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Okabe, T., and Takaku, F. (1986) Biochem. Biophys. Res. Commun. l34, 344-3.50. Mendelsohn, F.A.O., Lloyd, C.J., Kachel, C., and Funder, J.W. (1982) J. Clin. Invest. 70, 684-692. Booyse, F.M., Sedlak, B.J., and Rafelson, M.E. (1975) Thromb. Diathes. Haemorrh. (stuttg.) 34, 3722-3726. Puck, T.T., Marcus, P.I., and Cieciura, S.J. (1956). J. Exp. Med. 103, 273-289. Okabe, T., Yamagata, K., Fujisawa, M., Watanabe, J., Takaku, F., Lanzillo, J.J., and Fanburg, B.L. (1985) J. Clin. Invest. fi, 911914. Bohlen, P., Baird, A., Esch, F., Ling, F., and Gospodarowicz, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 8l, 5364-5368. Schwartz, S,M. (1978) --In vitro l4, 966-980. Severs, W.B., and Danniels-Severs, A.N. (1973) Pharmcol. Rev. 25, 415-449. Reid, I.A. (1977) Circ. Res. 4l, 147-153. Fukiyama, K., McCubbin, J.W., and Page, I.H. (1971) Clin. Sci. (London) 40, 283-291. Fitzsimons, J.T. (1972) Physiol. Rev. 52, 468-561.
1216
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1211-1216
INDUCTION BY FIBROBLAST GROWTH FACTOR OF ANGIOTENSIN CONVERTING ENZYME IN VASCULAR ENDOTHELIAL CELLS --IN VITRO Tetsuro
Okabe,
The Third University
Received
Kazuo Hiroshi
Yamagata, Hidaka,
Michio Fujisawa, Fumimaro and Yoshimi Umezawa
Department of Internal Medicine, Faculty of Tokyo, Tokyo 113, Japan, and Department Juntendo University, Tokyo 113, Japan
May 15,
Takaku,
of Medicine, of Pathology,
1987
SUMMARY: Induction of vascular endothelial cells with pituitary fibroblast growth factor (FGF) provoked an increase in angiotensin converting The stimulatory effect of FGF on ACE activity was enzyme activity. dose-dependent (EDso = 1.0 rig/ml). Our results suggest a possible role for pituitary FGF in regulation of ACE production in vascular 0 1987 Academic Press, Inc. endothelial cells.
The by two
production
of angiotensin renin
enzymes;
controlling
release
regulation
functions.
peptidase
located
degrades
bradykinin
of
pressure
isolating chose the shown the growth
and
endothelial cultured
to
of
increase
effects factor
of
largely
ignored.
culture
exhibit
a wide
assigned
cells
have cells
ACE production. ACE production various
on ACE activity
peptide
of
range
vascular
important
roles
fluid
exchange.
established
as a convenient
(2). growth
in endothelial
1211
but
of
to
a carboxydicells,
angiotensin in
the
factors
II.
regulation
Techniques (1).
system
this
the
degradative
report, including
for
We therefore to
investigate
glucocorticoids In
factors
endothelial
I
Recently,
The
enzyme,
angiotensin
been
regulated
studied,
converting
transvascular
endothelial
regulation
has been
surface
is
enzyme.
intensively
converts
been
substrate
been
luminal
and
have
have
renin
converting
Angiotensin
on the
peptides
blood
in
from
angiotensin
renin
cells
synthetic
These
of
of ACE activity
Endothelial and
and
II
have we
been
examined
fibroblast
cells. 0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. Ail rights of reproduction in any ,form reserved.
Vol. 145, No. 3, 1987
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALSANDMETHODS Materials: Fibroblast growth factor (FGF), Epidermal growth factor stimulating activity (MSA), insulin, Triiodo(EW t Multiplication thyronine, and dexamethasone were purchased from Collaborative Research Inc. Heparin, 12-O-tetradecanoylphorbol 13-acetate (TPA), progesterone, estradiol, testosterone, and cholera toxin were obtained from Sigma Chemical Co. Cell culture and medium: Endothelial cells were collected from a fresh porcine aorta using collagenase digestion as described by Booyse et Cloning of the endothelial cells was performed by single cell al (3). platings as described originally by Puck et al (4). The endothelial cells were cultured in plastic dishes (Falcon 3002) in Ham's F-10 nutrient mixture supplemented with 10% fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 us/ml). Subcultures were performed using 0.25% trypsin and 0.02% EDTA solution (Gibco) when the cells reached confluency. There was no evidence of transformation or loss of endothelial monolayer under these conditions. Media, serum, and antibiotics were obtained from Flow Laboratories Inc. Conversion of angiotensin I to angiotensin II by endothelial cells: ACE activity was measured by adding angiotensin I to the cultured endothelial cells and measuring generated angiotensin II by radioimmunoassay as described previously (5). After confluency, the culture medium was removed, and the cells were washed twice with F-10 medium, and then incubated in serum-free F-10 medium in the presence of angiotensin I (final concentration = 12 rig/ml). After 5 hr of incubations at 37°C in a 5% CO%chamber, the medium was harvested and stored at -20°C for later angio ensin II analysis. Simultaneously, as a control, the medium without cells was incubated and harvested. Angiotensins were purchased from Protein Research Foundation, Osaka, Japan. Radioimmunoassay for angiotensin II: The generated angiotensin II was measured by direct radioinununoassay performed according to the Briefly, 700 ul of 0.1 M Tris buffer (pH 7.4) method described (5). containing & mM EDTA and 0.025% bovine serum albumin, and 100 ul of 0.005 uCi I-angiotensin II (approximately 10,000 cpm; New England Nuclear Co.) were mixed with 100 ul angiotensin II antiserum )l : 100,000 titer). The sample (100 ul) was added and the reaction mixture was incubated for 16-20 hr at 4°C. Antibody-bound and free angiotensin II were separated by the addition of a dextran-coated charcol suspension. Bound radioactivity was counted with a [email protected] counter. Without addition of the test sample, 75% of I-angiotensin II was bound to antiserum at a final titer of 1 : 100,000. There was less than 0.5% cross-reactivity to angiotensin I.
RESULTSAND DISCUSSION When the
endothelial
angiotensin
II
(Fig.
Angiotensin
1).
of vascular
cells
were incubated
with
angiotensin
I,
was generated in the medium in a time-dependent manner II
was not generated in the incubation
smooth muscle cells
or without
the endothelial
not shown) that
I 10 nM dexamethasone could
our endothelial
cells
Conversion of angiotensin
in
confirmation I to angiotensin 1212
of II
cells
induce ACE activity the
earlier
report
medium (data in (2).
was increased by incubat-
Vol. 145, No. 3, 1987
BIOCHEMICAL
0
AND BIOPHYSICAL
1
4
8
Incubation Figure
ing
the
cells
with factor(s)
dexamethasone which
fibroblast
growth factor
in the endothelial
cells
in
may be
we examined several
activity,
a dose-related involved
peptide
(FGF)(6).
2.
factors
in
manner. the
regulation
In search of
ACE
and hormones including
FGF was found to induce ACE activity
in a dose dependent manner (Fig.
Dexamethasone (nM) Figure
Time (hrf
Conversion of angiotensin I to angiotensin II in serum-free cultures of vascular endothelial cells. At time = 0, culture medium containing no fetal bovine serum was placed over confluent cultures of endothelial cells in Falcon 60-mm dishes. The generated angiotensin II was measured by direct radioimmunoassay after 1, 4, and 8 hours. Each point represents mean value from three dishes. Bars, S.D.
1.
of peptide
RESEARCH COMMUNICATIONS
2).
The
FGF (rig/ml)
Effect of fibroblast growth factor on ACE activity in endothelial cells. Confluent cultures of endothelfal cells were incubated in serum-free F-10 medium containing various concentrations of pituitary fibroblast growth factor. Conversion of angiotensin I to angiotensin II was examined as described in Fig. 1. The generated angiotensin II was measured after 5 hr of incubations. Each point represents mean value from three dishes. Bars, S.D.
1213
Vol. 145, No. 3, 1987
Figure
other
3.
8lOCHEMlCAL
factors,
cant effects
such as insulin,
epidermal growth factor
stimulating
on the ACE activity.
were strongly
inhibited
(data not shown). 95% by captopril
by treating
factor
has been shown to stimulate
cells.
However, the physiological elucidated.
However, endothelial confluency
(7).
about
was achieved
at
peptide
factors
in the endothelial this
pituitary
angiotensin
2.5-fold
was
not
peptide II.
role of this
Several
3).
glands (6).
The
of mesoderm-derived pituitary
peptide has
FGF has been shown to stimulate
cells
in sparsely populated cultures.
cultures,
within
In
(Fig. may play
FGF was found to induce
24 hr.
Half-maximal
the FGF-treated
significantly
including
cells
for ACE (Fig.
have been shown to cease DNAsynthesis after
1.0 rig/ml.
cells
97% by EDTA (1 mM) and
a wide variety
In the confluent
ACE activities
endothelial
cells
with cycloheximide
from pituitary
Pituitary
the growth of vascular endothelial
of the endothelial
the cells
inhibitor
isolated
(EGF), growth
(MSA) shows no signifi-
was inhibited
(10 uM), a specific
not been fully
activity
The ACE activity
The activity
FGF is a basic protein
Other
RESEARCH COMMUNICATIONS
Effect of EDTA and captopril (SQ 14225) on the ACE activity. The endothelial cells were incubated in the presence of EDTA (1 rnM) or captopril ( 1 uM). Conversion of angiotensin I to The angiotensin II was examined as described in Fig. 1. generated angiotensin II was measured after 3 hr of incubations. Each column represents mean value from three dishes. Bars, S.D.
hormone, or multiplication
cells
AND BIOPHYSICAL
increased
insulin 4).
1214
(data
These observations
lines
number of not
shown).
did not induce ACE activity
an important
converging
cultures,
stimulation
of
role
in
suggest that production
of
evidences suggest that
Vol. 145, No. 3, 1987
Figure
BIOCHEMICAL
AND BIOPHYSICAL
Effect of various factors and in endothelial cells. Confluent
4.
were incubated in factors or hormones to angiotensin II
serum-free for 24 hr. was examined
RESEARCH COMMUNlCATlONS
hormones
on the
ACE activity
cultures of endothelial F-10 medium containing
Conversion as
cells various
of angiotensin
described
in
Fig.
I
1.
The
generated angiotensin II was measured after 5 hr of incubations. Each column represents mean value from three dishes. Bars, S.D.
an action the
of
angiotensin
development
angiotensin given
of arterial system
directly
into
has
been
via
demonstrated
ADH secretion, nervous
the
and
II
produce
has
been
Our
analysis in
the
also
of
of brain,
rise
that
the
cells
pituitary will
physiological thereby
intake.
1215
controlling
angiotensin
arterial
pressure water
in
the
intake, central
FGF increases be the
role
of
ventricles
control
functions
when
types
cerebral in
to
physiological
endothelial
or
renin-
pressure
administration
considered
observations
blood
various
supply
contributes of the
with
a sustained
of
by
lower
animals
blood
system
Antagonists
to
Chronic
to
nervous
(8).
of
cerebral
production
peptide water
(9).
(11).
central
reported
brain
a variety
experimental
endocrine or/and
II
system
angiotensin of
the
either
Angiotensin
(10).
been
hypertesion brain
on the hypertension
have
into
experimental II
II
of
beginning this
blood
neuropressure
Vol. 145, No. 3, 1987
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Okabe, T., and Takaku, F. (1986) Biochem. Biophys. Res. Commun. l34, 344-3.50. Mendelsohn, F.A.O., Lloyd, C.J., Kachel, C., and Funder, J.W. (1982) J. Clin. Invest. 70, 684-692. Booyse, F.M., Sedlak, B.J., and Rafelson, M.E. (1975) Thromb. Diathes. Haemorrh. (stuttg.) 34, 3722-3726. Puck, T.T., Marcus, P.I., and Cieciura, S.J. (1956). J. Exp. Med. 103, 273-289. Okabe, T., Yamagata, K., Fujisawa, M., Watanabe, J., Takaku, F., Lanzillo, J.J., and Fanburg, B.L. (1985) J. Clin. Invest. fi, 911914. Bohlen, P., Baird, A., Esch, F., Ling, F., and Gospodarowicz, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 8l, 5364-5368. Schwartz, S,M. (1978) --In vitro l4, 966-980. Severs, W.B., and Danniels-Severs, A.N. (1973) Pharmcol. Rev. 25, 415-449. Reid, I.A. (1977) Circ. Res. 4l, 147-153. Fukiyama, K., McCubbin, J.W., and Page, I.H. (1971) Clin. Sci. (London) 40, 283-291. Fitzsimons, J.T. (1972) Physiol. Rev. 52, 468-561.
1216