Chymase-Dependent Angiotensin II Formation in
Human Vascular Tissue
Shinji Takai, PhD; Denan Jin, MD; Masato Sakaguchi, MSc; Mizuo Miyazaki, MD, PhD
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Background—Some reports have suggested that, in vitro, human heart chymase in homogenates contributes little to
angiotensin (Ang) II formation in the presence of natural protease inhibitors such as a-antitrypsin. We studied whether
chymase bound to heparin, resembling an in vivo form, could contribute to Ang II formation in the presence of natural
protease inhibitors.
Methods and Results—The Ang II formation was increased time-dependently after incubation in an extract (1 mg of
protein/mL) of human vascular tissues containing Ang I. The concentration of Ang II in the extract after incubation for
30 minutes was 1.6760.06 nmol/mL, and we regarded this quantity of Ang II as 100%. The Ang II formation was
inhibited 10%, 95%, and 96% by 1 mmol/L lisinopril, 100 mmol/L chymostatin, and 0.1 g/L a-antitrypsin, respectively.
The extract was applied to a heparin affinity column. After the column was washed with PBS, the eluted PBS contained
a weak Ang II-forming activity, which was completely inhibited by lisinopril. The eluted PBS, to which .0.8 mol/L
NaCl had been added, showed a strong Ang II-forming activity which was inhibited by chymostatin and a-antitrypsin.
After the application of the extract, the column was washed with PBS and then an Ang I solution in PBS was applied
to the column. The Ang II formation in the PBS eluted from the incubated column was increased time-dependently. The
concentration of Ang II in the PBS (1 mL) eluted from the column after incubation for 30 minutes was 2.5660.28
nmol/mL, and we regarded this quantity of Ang II as 100%. To study the effects of inhibitors, the extract (1 mg of
protein/mL) was applied to a heparin affinity column (1 mL) which was preequilibrated with PBS (3 mL); 100 mmol/L
chymostatin or 0.1 g/L a-antitrypsin in PBS (1 mL) was then applied to the column. After the column was washed with
PBS (3 mL), Ang I solution (1 mg/mL) in PBS was applied to the column, and the column was incubated for 30 minutes.
The Ang II formation in the PBS eluted from the column was suppressed up to 5% by application of chymostatin,
although this was not affected by application of a-antitrypsin.
Conclusions—These findings suggest that human chymase bound to heparin plays a functional role in Ang II formation
in the presence of natural protease inhibitors such as a-antitrypsin. (Circulation. 1999;100:654-658.)
Key Words: chymase n angiotensin n heparin n vascular tissue
C
presence of these inhibitors, chymase-dependent Ang II formation was not detected. They therefore concluded that the
chymase-dependent Ang II-forming pathway is emphasized in
the absence of these inhibitors in human heart homogenate and
in the membrane extract but that chymase in human heart may
only play a small role in Ang II formation in vivo. Zisman et al16
reported that an ACE inhibitor reduced Ang II formation by 89%
after intracoronary infusion of Ang I, and they also concluded
that the ACE-dependent Ang II pathway was the predominant
pathway for Ang II formation in human heart. However, we
demonstrated that 30% of Ang I-dependent vasocontractile
responses in isolated human gastroepiploic arteries were suppressed by captopril, an ACE inhibitor; the remainder was
blocked by chymostatin, a chymase inhibitor.17 These findings
suggested that this chymase-like enzyme has a functional role in
Ang II formation in human vascular tissue. Recently, we isolated
and identified this enzyme as chymase.12
hymase is a chymotrypsin-like serine protease contained
in the secretory granules of mast cells. Chymases have
been isolated and their enzymatic characteristics have been
studied in rats,1–3 dogs,4,5 hamsters,6,7 monkeys,8 and humans. 9 –12 In general, these chymases hydrolyze the
C-terminal side of proteins after aromatic amino acids such as
Phe, Tyr, and Trp. Using the substrate angiotensin (Ang) I,
hamster,6 monkey,8 and human9 –12 chymases cleave the
Phe8-His9 bond of Ang I to yield Ang II, whereas rat
chymase10 cleaves the Tyr4-Ile5 bond to form inactive
fragments. Chymase is also involved in processing various
hormonal peptides,13,14 but it is unclear whether chymase
plays a major role in physiological and pathophysiological
functions in vivo.
Recently, Kokkonen et al15 demonstrated that chymase was
responsible for most Ang II formation in human heart homogenate in the absence of natural protease inhibitors, whereas in the
Received October 16, 1998; revision received April 14, 1999; accepted April 15, 1999.
From the Department of Pharmacology, Osaka Medical College, Takatsuki City, Osaka, Japan.
Correspondence to Shinji Takai, PhD, Department of Pharmacology, Osaka Medical College, Takatsuki City, Osaka 569-8686, Japan. E-mail
[email protected]
© 1999 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
654
Takai et al
Human chymase is stored in a macromolecular complex
with heparin proteoglycan in the cytoplasmic secretory granules in mast cells, and the complexed enzyme has maximal
activity immediately on release into the extracellular matrix
in vascular tissues after degranulation.18,19 The complexed
enzyme is more resistant to inhibition by macromolecular
protease inhibitors such as a-antitrypsin than the purified
enzyme in vitro.20
In the present study, we investigated whether or not
chymase bound to heparin has an Ang II-forming activity in
the presence of natural protease inhibitors such as
a-antitrypsin in the extract of human vascular tissue.
Methods
Materials
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Ang I, Ang II, and chymostatin were obtained from the Peptide
Institute (Minoh, Japan). Aprotinin and a-antitrypsin were purchased
from Sigma Chemical Co (St. Louis, Mo). The heparin affinity
column (1 mL) was purchased from Amersham Pharmacia Biotech
Ltd (Uppsala, Sweden). Dulbecco’s phosphate buffered saline (PBS)
was obtained from Gibco (Rockville, Md).
Human Vascular Tissues
Human gastroepiploic arteries were obtained from the surgically
resected stomachs of 8 patients (3 men and 2 women, mean age of
48.869 years) who underwent total gastrectomy because of gastric
cancer. All patients had no apparent vascular complications (eg,
hypertension, atherosclerosis, and diabetic vasculopathy). Their
arteries were transported in ice-cooled Tyrode’s solution consisting
of 137 mmol/L NaCl, 2.7 mmol/L KCl, 1.8 mmol/L CaCl2,
1.1 mmol/L MgCl2, 0.42 mmol/L NaH2PO4, 12 mmol/L NaHCO3,
and 5.7 mmol/L glucose, pH 7.4, and were stored at 4°C before use
in experiments.
Preparation of Extract from Human
Vascular Tissue
The arteries were minced and homogenized in 10 volumes (wt/vol)
of Tyrode’s solution consisting of 137 mmol/L NaCl, 2.7 mmol/L
KCl, 1.8 mmol/L CaCl2, 1.1 mmol/L MgCl2, 0.42 mmol/L NaH2PO4,
12 mmol/L NaHCO3, and 5.7 mmol/L glucose, pH 7.4. The homogenate was centrifuged at 20 000g for 30 minutes at 4°C. The
supernatant was discarded, and the pellets were resuspended and
homogenized in 10 volumes (wt/vol) of 10 mmol/L phosphate
buffer, pH 7.4, containing 2 mol/L potassium chloride and 0.1%
(v/v) Nonidet P-40. The homogenate was stored overnight at 4°C and
centrifuged at 20 000g for 30 minutes at 4°C. The supernatant was
called the extract.
Ang I Conversion in Extract
Aliquots of the extract (1 mg of protein/mL) were incubated for 0 to
60 minutes at 37°C with 1 mg/mL of Ang I in PBS (final incubation
volume of 0.1 mL). For the studies of inhibitor effects, 1 mmol/L
lisinopril, 100 mmol/L chymostatin, 1 mmol/L aprotinin, or 0.1 g/L
a-antitrypsin was added and preincubation was conducted for 10
minutes at 37°C, followed by incubation for 30 minutes at 37°C with
1 mg/mL of Ang I in PBS (final incubation volume of 0.1 mL). The
reaction was terminated by addition of 0.15 mL of 150 g/L
trichloroacetic acid, followed by centrifuging at 20 000g for 10
minutes. The supernatant was collected for the measurement of the
Ang II formation.
Heparin Column
The extract (0.1 mg of protein/mL) was applied to a heparin affinity
column (1 mL) which was preequilibrated with PBS. The column
was eluted with PBS (1 mL), to which was added 0, 0.2, 0.4, 0.6, 0.8,
1.0, 1.2, 1.4, 1.6, or 1.8 mol/L NaCl. Each of these media eluting
Chymase-Dependent Ang II Formation
655
from the column was collected for measurement of the Ang II
formation. To study the effects of enzyme inhibitors on the Ang II
formation, aliquots of the media eluted from the column were
preincubated for 10 minutes at 37°C with 1 mmol/L lisinopril,
100 mmol/L chymostatin, or 0.1 g/L a-antitrypsin, followed by
incubation for 30 minutes at 37°C with 1 mg/mL Ang I in PBS (final
incubation volume of 0.1 mL).
The extract (1 mg of protein/mL) was applied to a heparin affinity
column (1 mL) which was preincubated at 37°C and was preequilibrated with PBS. Ang I solution (1 mg/mL) in PBS was applied to
the column, and the column was incubated at 37°C for 0, 5, 10, 30,
or 60 minutes. After the incubation, the column was eluted with PBS
(1 mL), and the medium was collected for measurement of the Ang
II formation. To study the effects of inhibitors, the extract (1 mg of
protein/mL) was applied to a heparin affinity column (1 mL) which
was preequilibrated with PBS (3 mL); 100 mmol/L chymostatin or
0.1 g/L a-antitrypsin in PBS (1 mL) was then applied to the column.
After the column was washed with PBS (3 mL), Ang I solution (1
mg/mL) in PBS was applied to the column, and the column was
incubated at 37°C for 30 minutes. After the incubation, the column
was eluted by PBS (1 mL), and the PBS eluted from the column was
collected for measurement of the Ang II formation.
Determination of Ang I Fragments and
Protein Concentration
The media from the homogenate, the extract, or the slices were
applied to a TSKgel ODS-80 TM column (4.6 mm3250 mm ID,
Tosoh), which was eluted with a linear gradient of methanol (10% to
90%) in 10 mmol/L phosphoric acid, pH 3.8, at a flow rate of 0.5
mL/min.8,12 The protein concentration of the extract was measured
by bicinchoninic acid protein assay reagent (Pierce Chemical) using
bovine serum albumin as a standard.
Statistical Analysis
All values are expressed as mean6SEM. Two-way ANOVA and
Student’s t test were used for statistical comparisons. P,0.05 was
considered statistically significant.
Results
Ang I Conversion Extract from Human
Vascular Tissue
Ang II formation was observed after incubation of the extract
(1 mg of protein/mL) from human vascular tissues with Ang
I (1 mg/mL) and Ang II was found to be linearly generated up
to 60 minutes.20a The effects of the enzyme inhibitors were
investigated with Ang I conversion for 30 minutes during the
phase of linear formation of this product. The concentration
of Ang II in the extract after incubation for 30 minutes was
1.6760.06 nmol/mL, and we regarded this quantity of Ang II
as 100%. The Ang II formation was not inhibited by aprotinin
(1 mmol/L). The Ang II formation was inhibited 10% by
lisinopril (1 mmol/L) and was inhibited 95% by chymostatin
(100 mmol/L) and 96% by a-antitrypsin (0.1 g/L) (Figure 1).
Binding Affinity of Chymase to Heparin
The Ang II formation in PBS (1 mL) eluted from the column
after incubation with Ang I (1 mg/mL) for 30 minutes was
0.2560.01 nmol/mL, although it was not detectable in the
eluted PBS that contained up to 0.6 mol/L NaCl (Figure 2). In
the eluted PBS, to which had been added 0.8, 1.0, and 1.2
mol/L NaCl, the Ang II formation was 0.0860.02,
1.0260.09, and 0.1260.02 nmol/mL, respectively (Figure 2).
Ang II was not detected in the buffer containing over 1.4
mol/L NaCl (Figure 2). The Ang II formation in the PBS
656
Circulation
August 10, 1999
Figure 1. Effects of protease inhibitors on Ang II formation after
incubation of the extract from human gastroepiploic arteries for
30 minutes with 1 mg/mL of Ang I (n58). Formation of Ang II in
the absence of protease inhibitors was regarded as 100%. Inhibitory effects were determined using 1 mmol/L lisinopril,
100 mmol/L chymostatin, or 0.1 g/L a-antitrypsin. Data are presented as mean6SEM. *P,0.05; **P,0.01 vs control.
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
eluted from the column was completely inhibited by lisinopril
(1 mmol/L) but not by chymostatin (100 mmol/L) or
a-antitrypsin (0.1 g/L), whereas that in the eluted PBS to
which had been added between 0.8 and 1.2 mol/L NaCl was
completely suppressed by chymostatin (100 mmol/L) or
a-antitrypsin (0.1 g/L), but not by lisinopril (data not shown).
Effects of Inhibitors on Chymase Bound
to Heparin
After application of the extract (1 mg of protein/mL), Ang I
(1 mg/mL) was applied to the column and then the column
was incubated for 0, 5, 10, 30, or 60 minutes. The concentrations of Ang II in the PBS (1 mL) eluted from the column
were 0, 0.3460.03, 0.7860.08, 2.5660.28, and 5.1260.67
nmol/mL, respectively (Figure 3). We regarded the quantity
of Ang II after incubation for 30 minutes as 100% and studied
the effects of inhibitors on this Ang II formation. After
application of the extract (1 mg of protein/mL), chymostatin
(100 mmol/L) and a-antitrypsin (0.1 g/L) were applied the
column; Ang I was then applied to the column. The Ang II
formation in the PBS eluted from the column was suppressed
Figure 2. Ang II formation after incubation of medium eluted
from application of extract from human gastroepiploic arteries
by PBS (1 mL), to which had been added 0, 0.2, 0.4, 0.6, 0.8,
1.0, 1.2, 1.4, 1.6, or 1.8 mol/L NaCl, for 30 minutes with 1
mg/mL of Ang I (n58). Data are presented as mean6SEM.
Figure 3. Time course of Ang II formation in medium from the
column, which was incubated after application of the extract
from human gastroepiploic arteries and 1 mg/mL of Ang I (n58).
Data are presented as mean6SEM.
up to 5% by application of chymostatin, although this was not
affected by application of a-antitrypsin (Figure 4).
Discussion
The study results showed that Ang I was converted to Ang II
time-dependently in the extract of human vascular tissue. The
Ang II formation was suppressed by 90%, 5%, and 4% with
lisinopril, chymostatin, and a-antitrypsin, respectively. These
results suggested that chymase rather than ACE predominantly converted Ang I to Ang II in the extract of human
vascular tissue. Results similar to these were reported by
Urata et al11 in the extract of human heart membrane. In
contrast, Zisman et al16 showed that Ang II formation after
intracoronary administration of Ang I in the human heart was
reduced by 89% with an ACE inhibitor. These authors
suggested that the predominant pathway for Ang II formation
by chymase in human heart tissue extract, as reported by
Urata et al,11 could be observed only in the absence of internal
inhibitors. Kokkonen et al15 suggested that most Ang II
formation in the heart homogenate was a chymase-dependent
pathway. However, this pathway is inhibited by human
interstitial fluid containing a-antitrypsin, and these authors
concluded that human chymase was not responsible for Ang
Figure 4. Effects of protease inhibitors on Ang II formation in
medium from column, which was incubated for 30 minutes after
application of extract from human gastroepiploic arteries and 1
mg/mL of Ang I (n58). Formation of Ang II in absence of protease inhibitors was regarded as 100%. Inhibitory effects were
determined using 100 mmol/L chymostatin or 0.1 g/L
a-antitrypsin. Data are presented as mean6SEM. **P,0.01 vs
control.
Takai et al
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
II formation in vivo. In this study, we also observed that the
Ang II formation in the extract from human vascular tissue in
the absence of the inhibitors was predominantly a chymasedependent pathway, but Ang II formation was not detected in
the presence of a-antitrypsin. Therefore, chymase-dependent
Ang II formation in the extract was suppressed in the
presence of the natural protease inhibitors.
In vivo, chymase is stored in a macromolecular complex
with heparin proteoglycan within mast cell granules; the
enzyme remains complexed after degranulation.21 The complexed enzyme is more resistant to inhibition by macromolecular protease inhibitors, such as a-antitrypsin, than the
purified enzyme.20 In our study, the Ang II formation in the
PBS eluted from the heparin column after application of the
extract from human vascular tissues was inhibited by lisinopril only, suggesting that the Ang II formation was dependent
on ACE. On the other hand, in the eluted PBS containing 0.8,
1.0, and 1.2 mol/L NaCl, all of the Ang II formation was
inhibited by chymostatin or a-antitrypsin but not by lisinopril, suggesting that the Ang II formation was dependent on
chymase. These findings clearly showed that chymase, but
not ACE, was bound to the heparin column in PBS. Furthermore, we demonstrated, for the first time, that the chymase
bound to the heparin gel converted Ang I to Ang II and the
conversion of Ang I to Ang II was completely suppressed by
chymostatin but not by a-antitrypsin. The difference in the
inhibitory effects of chymostatin (M r 5604) 22 and
a-antitrypsin (Mr550 000)23 may be due to the different sizes
of the molecules. Therefore, natural serine protease inhibitors
such as a-antitrypsin hardly inhibit the chymase bound to
heparin as in the in vivo condition.
The chymase substrate in vivo has not yet been elucidated.
For hormonal peptides, human chymase converts Ang I to
Ang II but hardly hydrolyzes other peptides.11,12 Ang II is
known to play a role in maintaining blood pressure. The
chymase-dependent Ang II formation may be irrelevant to
blood pressure because ACE inhibitors are clinically effective
for various types of hypertension. Recent reports suggest that
Ang II plays crucial roles in the migration and proliferation of
vascular tissues.24,25 For example, an ACE inhibitor was
effective in preventing the proliferation of vascular tissue
after balloon injury of vessels in rats.26 Rat vascular tissues
contain only ACE as the Ang II-forming enzyme,17 and these
results suggest that vascular Ang II formation plays a crucial
role in tissue proliferation. On the basis of these reports, we
asked whether an ACE inhibitor suppresses human vascular
restenosis after percutaneous transluminal coronary angioplasty in humans: the result was negative.27,28 Such species’
differences in the effects of ACE inhibitors on neointimal
formation after injury may depend on whether or not a given
species possesses Ang II-forming chymase in vascular tissue.
Previously, we reported that dogs had a chymase-like Ang
II-forming enzyme in vascular tissues,29 and chymase-like
activities in the injured vessels were significantly increased in
comparison with those in noninjured vessels.30 In this model,
an Ang II receptor antagonist was effective in preventing
neointimal formation after balloon injury of vessels in dogs
but an ACE inhibitor was ineffective.31 Therefore, the
chymase-dependent Ang II formation in vascular tissue may
Chymase-Dependent Ang II Formation
657
be closely related to promoting growth. However, this role of
chymase in vivo has yet to be demonstrated by antiproliferative effects of a chymase inhibitor in this model.
In conclusion, natural internal inhibitors such as a-antitrypsin
strongly inhibited the chymase-dependent Ang II formation in
the extract, but chymase bound to heparin, as in the in vivo
condition, converted Ang I to Ang II in the presence of natural
protease inhibitors in plasma and vascular tissues. These findings suggest that chymase plays a functional role in Ang II
formation in human vascular tissues in vivo.
Acknowledgments
This study was supported in part by Grant-in-Aid for Encouragement
of Young Scientists 10770047 from the Ministry of Education,
Science, Sports, and Culture, Japan, and by a research grant (1998)
from Jinsenkai, the Alumni of Osaka Medical College.
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Chymase-Dependent Angiotensin II Formation in Human Vascular Tissue
Shinji Takai, Denan Jin, Masato Sakaguchi and Mizuo Miyazaki
Circulation. 1999;100:654-658
doi: 10.1161/01.CIR.100.6.654
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