Mast cell chymase limits the cardiac efficacy of Ang I-converting enzyme inhibitor therapy in rodents.
ABSTRACT Ang I-converting enzyme (ACE) inhibitors are widely believed to suppress the deleterious cardiac effects of Ang II by inhibiting locally generated Ang II. However, the recent demonstration that chymase, an Ang II-forming enzyme stored in mast cell granules, is present in the heart has added uncertainty to this view. As discussed here, using microdialysis probes tethered to the heart of conscious mice, we have shown that chronic ACE inhibitor treatment did not suppress Ang II levels in the LV interstitial fluid (ISF) despite marked inhibition of ACE. However, chronic ACE inhibition caused a marked bradykinin/B2 receptor-mediated increase in LV ISF chymase activity that was not observed in mast cell-deficient KitW/KitW-v mice. In chronic ACE inhibitor-treated mast cell-sufficient littermates, chymase inhibition decreased LV ISF Ang II levels substantially, indicating the importance of mast cell chymase in regulating cardiac Ang II levels. Chymase-dependent processing of other regulatory peptides also promotes inflammation and tissue remodeling. We found that combined chymase and ACE inhibition, relative to ACE inhibition alone, improved LV function, decreased adverse cardiac remodeling, and improved survival after myocardial infarction in hamsters. These results suggest that chymase inhibitors could be a useful addition to ACE inhibitor therapy in the treatment of heart failure.
- SourceAvailable from: Sebastien Fuchs[show abstract] [hide abstract]
ABSTRACT: There is uncertainty about the contribution of angiotensin-converting enzyme (ACE) to angiotensin II formation, with recent studies suggesting that non-ACE enzymes may be the predominant pathway of angiotensin II formation in kidney, heart, and lung. To investigate the role of ACE in angiotensin II formation, we measured angiotensin I and II levels in blood, kidney, and heart of 2 mouse genetic models (ACE.1 and ACE.4) of reduced somatic ACE gene expression and in blood, kidney, heart, lung, adrenal, and brain of mice administered the ACE inhibitor lisinopril. We also measured the levels of bradykinin (1-9) and its ACE metabolite bradykinin (1-7). Reduced ACE gene expression and ACE inhibition had similar effects on angiotensin and bradykinin peptide levels. Angiotensin II levels were reduced by 70% to 97% in blood, 92% to 99% in kidney, 93% to 99% in heart, 97% in lung, and 85% in adrenal and brain. The marked reductions in angiotensin II/angiotensin I ratio indicated that ACE was responsible for at least 90% of angiotensin I conversion to angiotensin II in blood, kidney, heart, lung, and brain, and at least 77% in adrenal. Blood bradykinin (1-9) levels were increased 6.4-fold to 8.4-fold. Heart bradykinin (1-9) levels were increased in ACE.4 mice and the bradykinin (1-7)/bradykinin (1-9) ratio was reduced in kidney and heart of ACE.4 mice and heart of lisinopril-treated mice. These studies demonstrate that ACE is the predominant pathway of angiotensin II formation in blood and tissues of mice and plays a major role in bradykinin (1-9) metabolism in blood and, to a lesser extent, in kidney and heart.Hypertension 05/2004; 43(4):854-9. · 6.87 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Angiotensin-converting enzyme inhibitors have beneficial effects that are presumably mediated by decreased angiotensin II (ANG II) production. In this study, we measure for the first time ANG I and ANG II levels in the interstitial fluid (ISF) space of the heart. ISF and aortic plasma ANG I and II levels were obtained at baseline, during intravenous infusion of ANG I (5 microM, 0.1 ml/min, 60 min), and during ANG I + the angiotensin-converting enzyme inhibitor captopril (cap) (2.5 mM, 0.1 ml/min, 60 min) in six anesthetized open-chested dogs. ISF samples were obtained using microdialysis probes inserted into the left ventricular myocardium (3-4 probes/dog). ANG I increased mean arterial pressure from 102+/-3 (SEM) to 124+/-3 mmHg (P < 0.01); addition of cap decreased MAP to 95+/-3 mmHg (P < 0.01). ANG I infusion increased aortic plasma ANG I and ANG II (pg/ml) (ANG I = 101+/-129 to 370+/-158 pg/ml, P < 0.01; and ANG II = 22+/-40 to 466+/-49, P < 0.01); addition of cap further increased ANG I (1,790+/-158, P < 0.01) and decreased ANG II (33+/-49, P < 0.01). ISF ANG I and ANG II levels (pg/ml) were > 100-fold higher than plasma levels, and did not change from baseline (8,122+/-528 and 6,333+/-677), during ANG I (8,269+/-502 and 6, 139+/-695) or ANG I + cap (8,753+/-502 and 5,884+/-695). The finding of very high ANG I and ANG II levels in the ISF vs. intravascular space that are not affected by IV ANG I or cap suggests that ANG II production and/or degradation in the heart is compartmentalized and mediated by different enzymatic mechanisms in the interstitial and intravascular spaces.Journal of Clinical Investigation 08/1997; 100(2):253-8. · 12.81 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Here we propose a molecular model for bradykinin receptor-operated and second messenger (inositol-1,3,4,5-tetrakisphosphate)-evoked Ca2+ influx and its potentiation by oncogenic Ras, which is not store-depletion-induced, so-called capacitative, Ca2+ influx. The principal idea for this hypothesis stems from observation that two bradykinin B2 receptor-activated signal pathways, protein tyrosine phosphorylation and formation of inositol tetrakisphosphate, merge during the Ca2+ influx process and that GTPase activating-protein 1 (GAP 1) is inositol tetrakisphosphate binding protein.Immunopharmacology 12/1999; 45(1-3):7-11.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
Mast cell chymase limits the cardiac efficacy
of Ang I–converting enzyme inhibitor
therapy in rodents
Chih-Chang Wei,1,2 Naoki Hase,3 Yukiko Inoue,4 Eddie W. Bradley,1 Eiji Yahiro,5 Ming Li,5
Nawazish Naqvi,5 Pamela C. Powell,1,2 Ke Shi,1 Yoshimasa Takahashi,3 Keijiro Saku,6
Hidenori Urata,4 Louis J. Dell’Italia,1,2,7 and Ahsan Husain5
1Department of Medicine, University of Alabama at Birmingham. 2VA Medical Center, Birmingham, Alabama. 3Teijin Institute for Bio-medical Research,
Teijin Pharma Ltd., Tokyo, Japan. 4Department of Cardiovascular Diseases, Fukuoka University Chikushi Hospital, Fukuoka, Japan. 5Division of Cardiology,
Department of Medicine, Emory University, Atlanta, Georgia. 6Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan.
7Department of Physiology and Biophysics, University of Alabama at Birmingham.
Ang I–converting enzyme (ACE), a membrane-bound zinc metallo-
peptidase, converts the prohormone Ang I to Ang II and inactivates
bradykinin (1). Many large, prospective, randomized clinical trials
over the last 20 years have shown the usefulness of ACE inhibitors
in reducing overall mortality in patients with myocardial infarction
(MI) and various degrees of LV systolic dysfunction (2–4). Although
the mechanisms underlying these beneficial effects are not fully
understood, suppression of Ang II in the heart and an improved
hemodynamic state are thought to be important. The identifica-
tion of an ACE-independent mast cell (MC) pathway for Ang II gen-
eration in the human heart raised the possibility that chronic ACE
inhibitor therapy may not completely suppress Ang II (5–7), which
may in turn cause adverse LV remodeling by activating Ang II recep-
tor subtypes 1 (AT1 receptor) and 2 (AT2 receptor) (8, 9).
Chymase, an efficient Ang II–forming serine protease (6), is
mainly found in MCs. In the human heart, it is also found in the
cardiac interstitial space and in some cardiac ECs (10). Chymases
have also been reported in cultured neonatal rat ventricular car-
diomyocytes (11) and rat VSMCs (12). EM-immunohistochemical
studies using human heart tissue indicate that the positively
charged chymase molecule is associated with the matrix within the
cardiac interstitial fluid (ISF) space (10). This localization suggests
a role for chymase in interstitial Ang II formation, as does the find-
ing that, in anesthetized dogs, Ang II levels in the cardiac ISF are
not suppressed by acute ACE inhibitor administration (13). These
studies also indicate the presence of a functional chymase-depen-
dent Ang II–forming pathway in the heart. However, studies with
conscious baboons questioned this notion. For example, using
direct coronary artery infusions of [Pro11,DAla12]Ang I, a substrate
that is converted to Ang II by chymase but not ACE, Hoit et al. (14)
were unable to demonstrate a change in cardiac function, despite
the fact that the non-ACE–dependent Ang II–forming activity is
higher than ACE-dependent Ang II–forming activity in baboon
heart homogenates. Because chymase is activated and stored in
secretory granules, the possibility exists that chymase activity in
tissue homogenates does not reflect extracellular chymase activ-
ity in the hearts of conscious animals, which could be minimal.
Its interstitial localization in histological tissue sections may be
exaggerated because nonfailing human hearts used to study its
localization were obtained from victims of accidents, who were
subjected to a number of drugs that could lead to chymase release,
including anesthetics. Moreover, protease inhibitors present in ISF
obtained from skin blisters have been shown to inhibit chymase
activity (15). If these inhibitors occur in the cardiac interstitium,
they could ensure that chymase remains constitutively inactivated.
In addition, the identification of distinct enzymes from other cell
types, such as cathepsin G from neutrophils (16), which can also
form Ang II, makes the importance of MC-mediated Ang II forma-
tion in the heart uncertain.
Chronic ACE inhibitor treatment influences plasma Ang II levels
in a biphasic manner (17, 18). The immediate response is a marked
fall in plasma Ang II levels. But over time, plasma Ang II levels
Authorship?note: Chih-Chang Wei, Naoki Hase, and Yukiko Inoue contributed
equally to this work.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J Clin Invest. 2010;120(4):1229–1239. doi:10.1172/JCI39345.
Related Commentary, page 1028
1230?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
return to near normal levels despite substantial ACE inhibition.
Because ACE is also a kininase, tissue and plasma bradykinin levels
are markedly elevated during chronic ACE inhibitor treatment (1).
Here we report that cardiac ACE inhibition produces a bradykinin-
dependent release of chymase from MCs in conscious mice, which
maintains cardiac ISF Ang II levels. These studies not only demon-
strate the in vivo functionality of the cardiac non-ACE pathway but
also show that it originates from MCs. Our findings challenge the
notion that the cardiac efficacy of ACE inhibitors requires Ang II
suppression in the heart. We also show that, in animals treated
with an ACE inhibitor, chymase inhibition improves LV function
and decreases adverse cardiac remodeling after MI.
MCs are the major source of the non-ACE Ang II–forming activity in LV
homogenates. We observed similar levels of ACE (ACE inhibitor–sen-
sitive Ang II–forming activity) and non-ACE (ACE inhibitor–resis-
tant Ang II–forming activity) Ang II–forming activities in mouse LV
homogenates (Figure 1A). The mouse heart, thus, has the potential
to regulate Ang II locally through these 2 pathways. One source of
the non-ACE activity is the MC, which elaborates chymase (16). To
evaluate this, we studied the effect of MC deficiency on non-ACE
activity in LV homogenates. LV non-ACE activity was reduced by
more than 95% (P < 0.001) in MC-deficient KitW/KitW-v (W/Wv) mice
(19), relative to MC-sufficient WT littermates (Figure 1A), implicat-
ing MCs as the major source of LV non-ACE activity. Interestingly,
total LV homogenate Ang II–forming activity in W/Wv mice was not
different from that in WT mice because of a 3-fold increase in W/Wv
LV ACE activity (Figure 1A). Quantitative immunofluorescence
showed a 2.6-fold higher ACE immunoreactivity in W/Wv LV tissue
sections relative to WT (23 ± 7.6 versus 59 ± 4.4 AU/μm2 in WT and
W/Wv LVs, respectively; n = 5/group; P < 0.01) (Figure 1, B and C);
in W/Wv mice, ACE immunoreactivity was mainly associated with
cardiomyocytes (Figure 1C). However, ACE mRNA levels in isolated
W/Wv cardiomyocytes were similar to those in WT cardiomyocytes
(0.066 ± 0.009 versus 0.0512 ± 0.003 ACE/GAPDH mRNA tran-
script ratio in WT and W/Wv, respectively; n = 6–7 cardiomyocyte
isolations/group). It is tempting to speculate that decreased shed-
ding of cell surface ACE, which may be regulated by ACE secretase
(20), rather than increased transcription, produced higher ACE lev-
els in W/Wv LVs; however, this increase is unlikely to be due to sup-
pressed chymase-dependent Ang II formation because, in WT mice,
chronic inhibition of mouse MC protease-4 (MMCP4), the major
Ang II–forming chymase in the LV (see below), did not increase LV
ACE immunoreactivity (data not shown).
ACE and non-ACE Ang II–forming activities and ACE immunoreactiv-
ity in WT and W/Wv LV homogenates (A). Values are mean ± SEM. In
each group, n = 6. All measurements were made in 7-week-old mice.
Ang II–forming activity: total, which was determined in the absence of
any inhibitors; ACE, that level of the total that was inhibited by the ACE
inhibitor lisinopril (10 μM); and non-ACE, the total minus the ACE activ-
ity. ***P < 0.001. Photomicrographs showing ACE immunoreactivity in
(B) WT and (C) W/Wv LV tissue sections. ACE (red); CD31, which iden-
tifies ECs (green); and DAPI, which identifies nuclei (blue). Note ACE
immunoreactivity in clusters of cardiomyocytes in the W/Wv LV tissue
section (C) but not in WT LV section (B). ACE immunoreactivity was
only weakly associated with CD31-positive ECs. Scale bar: 20 μm.
Basal Ang II (A), Ang I (B), ACE activity (C), and chymase activity
(D) in the LV ISF of conscious WT and W/Wv mice. In vivo microdialy-
sis was used to collect samples for these measurements. In addition,
using this procedure, [Pro10]Ang I and [Pro11,DAla12]Ang I conversion
to Ang II in the LV ISF was used to measure ACE- and chymase-like
activities, respectively. Values are mean ± SEM. In each group, values
in parentheses represent n. **P < 0.01.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
In the basal state, cardiac, chymase-like activity is not in the ISF sub-
compartment. Steady-state ISF Ang I/Ang II ratios in the LV of
conscious mice, as measured by in vivo microdialysis, were greater
than 1 (approximately 3.6) (Figure 2, A and B), suggesting that
the rate-limiting step in Ang II generation in the LV ISF is Ang I
activation. Next, we estimated ACE- and chymase-like Ang II–
forming activities in the LV ISF of conscious mice. We used
[Pro10]Ang I and [Pro11,DAla12]Ang I conversion to Ang II in the
LV ISF as a measure of ACE- and chymase-like activities, respec-
tively. While [Pro10]Ang I is converted to Ang II by ACE, but not
chymase (21), [Pro11,DAla12]Ang I is converted to Ang II by chy-
mase, but not ACE (14, 22). These selective substrates were deliv-
ered in the lumen of the microdialysis probe in vivo. Increase in
Ang II over baseline in the microdialysis probe retentate (which
involves the diffusion of Ang II, generated from these substrates
by ISF enzymes, from the ISF to the lumen of the microdialysis
probe) during the infusion of [Pro10]Ang I or [Pro11,DAla12]Ang I
was taken to represent in vivo LV interstitial ACE- and chymase-
like activities, respectively. In conscious WT mice, LV ISF ACE-
and chymase-like activities were 0.6 and 0.08 pg Ang II formed/h,
respectively (Figure 2, C and D), which indicates that ACE is the
dominant Ang II–forming activity in the LV ISF. Moreover, LV
ISF chymase-like activity in conscious WT mice was similar to
that in W/Wv mice (Figure 2D), which, because they are deficient
in cardiac chymase-like activity (Figure 1A), suggests that under
basal conditions, chymase in the LV is not located extracellularly.
But in W/Wv mice, LV ISF Ang II levels were 1.8-fold higher than
in WT mice (P < 0.01; Figure 2A), which was associated with a
3-fold increase in LV tissue ACE activity (P < 0.001; Figure 1A)
and a trend toward an increase in LV ISF ACE activity (Figure
2C). This association and low chymase-like activity in the LV ISF
of conscious WT and W/Wv mice suggest a dominant role for
local ACE in regulating LV ISF Ang II levels.
Chronic ACE inhibition causes chymase release into the LV ISF from
MCs. To directly test the importance of ACE in regulating intersti-
tial Ang II, we examined the effect of chronic (2 weeks) oral ACE
inhibitor treatment (150 mg captopril/kg/d) on LV ISF Ang II.
We took measurements of Ang II that enters the microdialysis
probe per hour to directly reflect the level of Ang II in the LV ISF
of the conscious mouse, realizing, of course, that factors such
as peptide transfer rates in vivo are difficult to model based on
idealized transfer rates determined in in vitro experiments with
microdialysis probes. Chronic oral ACE inhibition with captopril
produced an 85% decrease in LV ISF ACE activity (P < 0.05) in WT
mice (Figure 3A), but it did not significantly lower ISF Ang II com-
pared with vehicle controls (Figure 3B). In contrast, plasma Ang II
levels decreased by approximately 2-fold (P < 0.05) after chronic
ACE inhibition (Figure 3C). This was despite the fact that LV ISF
and plasma Ang I levels increased by about 2-fold (P < 0.01) in both
compartments (Figure 3, D and E). These findings could be inter-
preted in a number of ways. First, in the setting of chronic ACE
inhibition, chymase, rather than ACE, is the major Ang II–forming
enzyme in the LV ISF. Second, the rate of uptake of Ang II from the
plasma, rather than the plasma Ang II concentration, is the chief
determinant of LV ISF Ang II.
In the absence of a known mechanism for actively importing
Ang II to the LV ISF, we considered the role of chymase and MCs
in regulating LV ISF Ang II. Chronic ACE inhibition produced
a 14-fold increase in LV ISF chymase-like activity (Figure 3F)
(P < 0.01) and increased LV MMCP4 mRNA levels by approximately
10-fold (0.0034 ± 0.00023 versus 0.036 ± 0.008 MMCP4/GAPDH
mRNA transcript ratio in vehicle- and captopril-treated WT mice,
respectively; n = 5/group; P < 0.05). A time-course analysis indicated
that LV ISF chymase activity increased as early as 24 hours after
captopril administration, but at no time point were LV ISF Ang II
levels suppressed by ACE inhibition (Supplemental Table 1; sup-
Effect of chronic ACE inhibitor treat-
ment on angiotensins and Ang II–form-
ing enzyme activities in conscious mice.
Effect of chronic captopril treatment
(150 mg captopril/kg/d; for 2 weeks) on
(A) LV ISF ACE activity, (B) LV ISF, (C)
plasma Ang II, (E) plasma Ang I, and
(G) MC density in WT mice. Effect of
chronic captopril treatment on (D) LV
ISF Ang I and (F) LV ISF chymase
activity in WT and W/Wv mice. Values
are mean ± SEM. In each group, values
in parentheses represent n. *P < 0.05;
**P < 0.01; ***P < 0.001. Veh, vehicle;
1232?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
plemental material available online with this article; doi:10.1172/
JCI39345DS1). We also found that basal LV ISF chymase activity
in MC-deficient W/Wv mice was low and similar to that observed in
MC-sufficient WT mice, but the chronic ACE inhibitor–dependent
14-fold increase (P < 0.01) in this activity seen in WT mice was not
observed in W/Wv mice (Figure 3F). This suggests that MCs are the
main source of the chymase-like activity that is released into the
LV ISF during chronic ACE inhibition.
Chronic ACE inhibition increased LV ISF Ang I levels in WT mice
(Figure 3D). Because cardiac interstitial MCs elaborate renin (23),
it is possible that this increase is produced by MC renin release.
We tested this by comparing LV ISF Ang I levels, basal and after
chronic ACE inhibition, in WT and W/Wv mice. We found that
both basal and ACE inhibitor–induced LV ISF Ang I levels were
not different between WT and W/Wv mice (Figure 3D), which does
not support a role for MC renin in regulating LV ISF Ang I in the
setting of ACE inhibition.
Bradykinin mediates chymase release into the LV ISF during chronic ACE
inhibition. ACE inhibition prevents bradykinin degradation, and in
rats, it increases LV ISF bradykinin levels (24). Because bradykinin
elicits an inflammatory response (25), we evaluated the role of bra-
dykinin/kinin B2 receptor activation on MC chymase release. We
found that kinin B2 receptors are localized on MMCP4-positive
MCs (Figure 4) — MMCP4 is the major Ang II–forming chymase
in the LV (see below) — and the kinin B2 receptor antagonist Hoe-
140 prevented the 14-fold increase LV ISF chymase-like activity in
conscious WT mice caused by chronic ACE inhibition (Figure 5A).
This indicates that chymase release into the LV ISF produced by
ACE inhibition requires kinin B2 receptor activation and implies
that bradykinin degranulates MCs.
We studied the in vivo effect of bradykinin on LV MC degranula-
tion. MC numbers in LV sections were quantified using Giemsa
(30–40 ×20 LV fields/mouse), which specifically stains MC gran-
ules. When MCs fully degranulate, Giemsa cannot visualize them.
This and the difficulty with recognizing a partially degranulated
MC have led some investigators to measure apparent MC loss as
an estimate of degranulation (26). 24-hour microdialysis-based
infusion of 5 ng/ml bradykinin decreased the number of iden-
tifiable LV MCs by 35% (0.38 ± 0.05 and 0.59 ± 0.08 MCs/mm2
in the bradykinin and vehicle groups, respectively; n = 5/group;
P < 0.05); 7-day pretreatment with 0.5 mg/kg/d Hoe-140 prevented
this decrease (0.67 ± 0.06 MCs/mm2, n = 5). Together with kinin
B2 receptor localization on MCs (Figure 4, A–D), our findings
suggest the possibility that bradykinin/kinin B2 receptors medi-
ate MC degranulation in the LV of the conscious mouse. A 10-fold
increase in MMCP4 mRNA levels in the LV (see above) coupled
with a decrease in the number of identifiable MCs in WT LV after
chronic ACE inhibitor treatment support this notion, although
this latter change failed to reach significance (Figure 3G). We then
tested to determine whether LV ISF Ang II formation in ACE-inhib-
ited mice also requires kinin B2 receptor activation and found that
Bradykinin B2 receptors in LV MMCP4+ MCs. Pho-
tomicrographs of a LV section from an 8-week-
old WT mouse stained for (A) MMCP4 to identify
MC, (B) DAPI to detect nuclei, (C) and the B2
kinin receptor as well as the (D) composite image.
Arrows show the location of the MMCP4+ MC.
Scale bar: 20 μm.
Effect of chronic (2 weeks) ACE inhibition (Captopril, 150 mg/kg/d) on
(A) chymase activity and (B) Ang II in the LV ISF on conscious WT
mice concurrently treated with vehicle, chymase inhibitor (CI-A, 200
mg/kg, b.i.d.) (CI), or bradykinin B2 receptor antagonist (Hoe-140, 0.5
mg/kg/d). Values are mean ± SEM. In each group, values in parenthe-
ses represent n. *P < 0.05; ***P < 0.001.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
Hoe-140 produced a greater than 10-fold decrease in LV ISF Ang II
in chronic ACE-inhibited mice (Figure 5B) (P < 0.05).
MMCP4 regulates Ang II in the LV ISF. The mouse genome has mul-
tiple chymase genes: MMCP1, -2, -4, -5, -9, -10, and -L (27). Quanti-
tative RT-PCR shows that MMCP4/5 gene expression in the LV was
higher than MMCP1/2 gene expression (Figure 6). Other chymase
mRNA transcripts could not be detected in the LV. In W/Wv LVs,
all chymase mRNA transcripts were undetectable (Figure 6), which
suggests that chymases are chiefly elaborated by MCs in the LV.
Purified skin MMCP4 has Ang II–forming activity, but it also
degrades Ang II (Supplemental Figure 1, A and B), whereas purified
heart MMCP5 does not metabolize Ang I or Ang II (Supplemental
Figure 2, A and B) because it is an elastase (Supplemental Figure 2C)
(28, 29). Because (a) MMCP4 is a more than 100-fold more efficient
Ang II–forming enzyme than MMCP1 or -2 (22) and (b) LV MMCP1
and 2 mRNA transcripts are low abundance (Figure 6), the dominant
Ang II–forming chymase in the mouse LV is likely to be MMCP4.
To evaluate this, we examined several human chymase inhibitors
and found that 4-[1-(4-methyl-benzo[b]thiophen-3-yl methyl)-1H-
benzimidazol-2-yl sulfanyl]-butyric acid (CI-A) inhibited purified
MMCP4 with high affinity (KI = 39.7 ± 1.7 nM; n = 4). Chronic ACE
inhibition produced a 14-fold increase in LV ISF chymase activity
(P < 0.01) (Figure 3F). In these ACE inhibitor–treated WT mice, CI-A
treatment (200 mg/kg, b.i.d.) caused a 5-fold (P < 0.05) inhibition
of LV ISF chymase activity and decreased LV ISF Ang II by 16-fold
(P < 0.05) (Figure 5, A and B). Moreover, chronic ACE inhibition
decreased LV ISF Ang II by 69% ± 27% in MC-deficient W/Wv mice
(n = 22; P < 0.001), but not in WT mice (Figure 3B). Taken together,
these findings provide evidence that MC MMCP4 release regulates
LV ISF Ang II during chronic ACE inhibition.
Chymase limits the efficacy of ACE inhibitor therapy in MI. Here we
show that ACE inhibition causes LV MC chymase release, which
then activates Ang I. Previous in vitro studies suggest that chymase
also activates/inactivates proteins, which could be important in
the pathogenesis of cardiac diseases if they occur in vivo (7). Given
this, we tested to determine whether chymase limits ACE inhibitor
efficacy in the post-MI heart. The adult mouse heart has chymase
activity, but a direct Ang II effect on cardiomyocytes in vivo is not
easily demonstrable (30, 31). We chose the hamster as a model
system because Ang II–forming chymase activity and chymase-to-
ACE activity ratio in hamster heart homogenates is similar to that
observed in humans (32) and, as in the human heart (33), Ang II
produces a positive inotropic effect in the hamster heart (21, 34).
We tested to determine whether the beneficial effects of ACE
inhibitor therapy are limited by chymase in hamsters with MI. Post-
MI LV function was analyzed by echocardiography. In hamsters, LV
ejection fraction (EF) and fractional shortening (FS) were depressed
by 36% (P < 0.001) and 50% (P < 0.001), respectively, by MI (Figure
7, A and B). In the myocardium, distal to the site of infarction, aver-
age cardiomyocyte diameters (Figure 7C) and fibrosis (Figure 7D)
were increased by 31% (P < 0.001) and 36% (P < 0.01), respectively,
35 days after MI. ACE inhibitor therapy (10 mg temocapril /kg/d)
started 24 hours after MI and continued for 34 days, improved LV
EF and FS by 20% (P < 0.01) and 26% (P < 0.01), respectively, and
decreased infarct size by 26% (P < 0.001) (Figure 7, A, B, and E).
But MI-induced increase in cardiomyocyte diameter and fibrosis in
the noninfarcted LV was not altered by ACE inhibition (Figure 7,
C and D). To determine whether potential ACE inhibitor–induced
chymase release limits the beneficial effects of ACE inhibitor treat-
ment, we examined the difference in the effect of chymase inhibi-
tion in the untreated versus the ACE inhibitor–treated post-MI
heart. The hamster has 2 known chymases. Hamster chymase-1,
which converts Ang I to Ang II (35), is a homolog of MMCP4 (27),
and hamster chymase-2, which does not, is a homolog of MMCP5
(36). We tested and found that 4-[1-(naphthalen-1-yl methyl)-1H-
benzimidazol-2-yl sulfanyl]-butyric acid (CI-B) inhibits purified
hamster chymase-1 with a KI of 30.6 ± 3.75 nM (n = 4). Compared
with vehicle treatment, CI-B (100 mg/kg/d; started 24 hours after
MI for 34 days) therapy produced an 18% (P < 0.001) decrease in
average cardiomyocyte diameter in the noninfarcted LV (Figure
7C) and reduced infarct size by 24% (P < 0.001) (Figure 7E). There
was a small improvement in LV function, but this change did not
reach significance. In contrast, when compared with ACE inhibitor
monotherapy, combination therapy (10 mg temocapril/kg/d plus
100 mg CI-B/kg/d; started 24 hours after MI for 34 days) improved
LV EF and FS by 16% (P < 0.05) and 19% (P < 0.05), respectively, and
reduced infarct area by 51% (P < 0.01) and LV end-diastolic dimen-
sion by 24% (P < 0.05) (Figure 7, A, B, E, and F). In the distal myocar-
dium, combination therapy, relative to ACE inhibitor monotherapy,
reduced MI-induced interstitial fibrosis by 50% (P < 0.0002) (Figure
7D) and cardiomyocyte diameters by 18% (P < 0.001) (Figure 7C).
These beneficial effects were unlikely to be due to differences in
hemodynamic effects of ACE/chymase inhibition because mean
arterial blood pressures or heart rates in hamsters with MI were
unaffected by drug treatments (Supplemental Table 2).
Using Kaplan-Meier survival analysis, we found that post-MI
survival was greater in hamsters treated with an ACE and chymase
inhibitor combination, relative to vehicle-treated hamsters, than
with ACE inhibitor alone (Figure 7G). Remarkably, in the combi-
nation therapy group, survival was no different from that observed
in sham-operated controls. Together, these findings suggest that
ACE inhibitor treatment causes chymase to become an important
contributor to cardiac dysfunction and adverse remodeling in the
post-MI hamster heart.
Here we show that ACE is the key Ang II–forming enzyme in the LV
ISF of conscious mice. However, although chronic ACE inhibitor
β-actin–normalized MMCP1, MMCP2, MMCP4, and MMCP5 mRNA
levels in LV tissues from WT and W/Wv mice. Values are mean ± SEM;
n = 7 in each group. **P < 0.01; ***P < 0.001.
1234?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
therapy inhibits ISF ACE activity, it does not lower ISF Ang II lev-
els. This apparent paradox is due to the fact that the chronic ACE
inhibitor treatment changes the normal balance of Ang II–forming
activity in the LV ISF from being predominantly ACE dependent to
being predominantly chymase dependent. The discovery of an ACE
inhibitor–induced, chymase-dependent Ang II formation mecha-
nism in the LV raises questions regarding our clinical assumptions
about the rationale for using ACE inhibitors in treating cardiac
diseases, namely, to suppress Ang II in the heart.
Based on (a) chymase gene expression profiling in the mouse
LV, (b) enzyme kinetics with mouse chymases whose genes are
highly expressed in the LV, and (c) in vivo conscious mouse
studies with a selective chymase substrate ([Pro11,DAla12]Ang I)
(14) and a specific chymase inhibitor targeted toward the only
LV-expressed chymase that has efficient Ang II–forming activity
(i.e., MMCP4), we propose that MMCP4 is the enzyme respon-
sible for the non-ACE Ang II–forming activity in the mouse LV.
Our immunohistochemical study shows occurrence of MMCP4-
positive MCs in the LV. In addition, we show that in MC-deficient
W/Wv mice, MMCP4 mRNA is undetectable, non-ACE Ang II–
forming activity is undetectable, and ACE inhibitor–dependent
increase in LV ISF chymase activity does not occur. Based on these
findings, we propose that the MMCP4-positive MC in the mouse
LV is the likely source of the non-ACE Ang II–forming activity in
the LV ISF. While more extensive adoptive transfer experiments
might conceivably strengthen this conclusion, technical limita-
tions make this approach difficult or impossible. Thus, it is possi-
ble that non-MC, c-kit–expressing cells in the LV could potentially
account for the chymase activity that we detect.
ACE inhibition also decreases bradykinin degradation, which
increases ISF bradykinin (24). It is possible that the release of MC
chymase in vivo is a direct effect of bradykinin on MCs. This is sug-
gested by our identification of kinin B2 receptors on LV MMCP4-
positive MCs as well as the well-established reports that kinin B2
Effect of ACE inhibitor, chymase inhibitor, or combination therapy on LV function, cardio-
myocyte size, interstitial fibrosis, infarct size, and survival in hamsters after MI. EF (A), FS
(B), average cardiomyocyte diameter (C), LV interstitial fibrosis (D), infarct size (E), and
LV end-diastolic dimension (F) in hamsters with MI treated with vehicle (MI) ACE inhibitor
(temocapril, 10 mg/kg/d) (ACEI), chymase inhibitor (CI-B, 100 mg/kg/d) (CI), or combina-
tion therapy (10 mg temocapril/kg/d + 100 mg CI-B/kg/d) (ACEI + CI) started 24 hours
after MI for 34 days or after a sham operation. (G) Actuarial survival after MI in hamsters
following treatment with vehicle, ACE inhibitor, chymase inhibitor, or combination therapy
or after a sham operation. Values are mean ± SEM. In each group, values in parentheses
represent n. *P < 0.05; **P < 0.01; ***P < 0.001.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
receptor activation mobilizes inositol 1,4,5-trisphosphate–evoked
Ca2+ in several cell types (37) and that increases in intracellular
Ca2+ or inositol 1,4,5-trisphosphate cause MC degranulation (38).
The finding that kinin B2 receptor antagonism prevents ACE
inhibitor–dependent release of chymase into the LV ISF supports
a role for bradykinin in this process; however, it does not exclude
the possibility that the effect is indirect.
The notion that enzymes other than ACE are involved in tis-
sue Ang II generation in vivo has been keenly debated. The basis
for this debate revolves around the effect of ACE inhibition on
circulating and tissue Ang II levels. On the one hand, it has been
shown that tissue Ang II levels are almost completely suppressed
by acute ACE inhibition in rodents (39), and plasma Ang II levels
are markedly suppressed in hypertensive patients after acute ACE
inhibitor therapy (17). On the other hand, chronic ACE inhibitor
therapy does not suppress plasma Ang II in hypertensive patients
(17). This phenomenon, also known as “ACE inhibitor escape,” has
been attributed to incomplete tissue ACE inhibition in the face
of elevated plasma Ang I levels (40, 41) or, by some, to a method-
ological artifact (39). Lack of a mechanistic understanding of this
phenomenon and the fact that tissue Ang II is a complex measure-
ment that simultaneously determines Ang II in multiple tissue
compartments (e.g., interstitial, cell associated, and intravascular)
has impeded the successful resolution of this debate. We show that
chymase, although abundant in tissue homogenates, is relatively
low in the LV interstitium of conscious mice. Importantly, we also
show that chronic ACE inhibition causes a bradykinin-mediated
release of MC chymase into the LV ISF. Although ACE inhibition
rapidly increases bradykinin levels, its effect on LV interstitial chy-
mase levels may be gradual. MC degranulation with the sequestra-
tion of the positively charged chymase by the negatively charged
intracellular matrix (10, 42) may be required to elevate interstitial
chymase, which then helps to maintain steady-state LV ISF Ang II
at pre-ACE inhibition levels — our data suggest that after ACE
inhibition is initiated, the increase in ISF chymase levels in the
mouse LV may take as little as 24 hours.
Could the benefits of chronic ACE inhibitor use in the treat-
ment of cardiac diseases be limited because of ACE inhibitor–
dependent chymase release into the cardiac ISF? Chymase release
not only sustains Ang II levels in the heart, but also processes a
number of substrates including endothelin-1, pro–MMP-9, and
pro–MMP-3 that promote tissue remodeling (Figure 8) (7, 43).
We used an orally active chymase inhibitor to directly determine
whether the potential benefits of ACE inhibitor use in MI are lim-
ited by chymase. Chronic ACE inhibition improved LV function
after MI. Because the beneficial effect of chymase inhibition was
greater when combined with ACE inhibition, we conclude that
ACE inhibition in the heart causes the release of chymase. LV ISF
Ang II and chymase activity could not be measured directly due
to difficulties of microdialysis probe insertion in the infarcted
heart. But one possibility is that this mechanism limits ACE
inhibitor–dependent reduction in LV ISF Ang II by now main-
taining LV ISF Ang II at high levels via chymase-dependent Ang II
generation. Another possibility is that the effects of chymase on
other regulatory proteins (Figure 8) contribute to adverse post-
MI LV remodeling and to deterioration of LV function that lim-
its post-MI survival. Thus, for example, chymase inhibition may
suppress pro-MMP activation caused by the enzymatic actions of
ACE inhibitor–dependent chymase release from MCs. This could
explain the more compact and smaller area of scar seen with com-
bination therapy relative to ACE inhibitor therapy alone. Drugs
were given 24 hours after left anterior descending artery occlu-
sion. This timing was based on the reported maximal increase in
LV free wall ACE and chymase activities that occurs 3 days after
MI in hamsters (44). If the initial infarct size was the same but
scar formation was improved by chymase inhibitor addition to
ACE inhibitor therapy, LV function could improve because of a
decrease in LV end-diastolic dimension. We propose that the car-
diac benefits of ACE inhibition may be better realized by combin-
ing it with simultaneous chymase inhibition. Cardiac diseases,
including hypertensive heart disease, viral myocarditis, and ath-
erosclerotic coronary artery disease, the prelude to MI, which are
treated with ACE inhibitors, have an inflammatory component
that is associated with MC accumulation in the heart (7). This
suggests a role for chymase inhibition in improving outcomes for
heart disease patients on ACE inhibitor therapy.
Study limitations. In studying the cellular pathway(s) responsible for
chymase release, we used the mouse model. Its choice was primarily
dictated by the availability of mice with genetic MC deficiency (19)
and from analyses that showed equivalent levels of ACE and non-
ACE activity in LV homogenates from WT controls of MC-deficient
W/Wv mice (Figure 1). We show that this WT mouse contains a chy-
mase (MMCP4) in the heart that is a net Ang II–forming enzyme.
Although there are many similarities in the substrate recognition
profiles of human chymase and MMCP4, human chymase differs
from MMCP4 in that the latter, but not the former, has apprecia-
ACE-dependent bradykinin degradation limits baseline secretion of MC
chymase in the LV. Chronic inhibition of ACE reduces its direct effects
on Ang I to Ang II conversion, but it increases bradykinin/B2 recep-
tor–dependent chymase release from MCs, which counteracts the
direct effect of ACE inhibition on Ang II formation through the chymase
pathway of Ang II formation. In this scheme, we suggest that the effect
of bradykinin on MC chymase release is direct; however, it is also
possible that the bradykinin/B2 receptor mechanism indirectly causes
chymase release. Chymase release also has a myriad of effects on
factors that promote the inflammatory response and tissue remodeling.
ET-1, endothelin-1; TIMP-1, tissue inhibitor-1 of metalloproteinase.
1236?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
ble angiotensinase activity, which allows human chymase to more
robustly generate Ang II (6, 28). Thus, studies in the mouse may
underestimate the overall contribution of the human MC non-ACE
pathway in local cardiac Ang II formation.
The mouse model has a second limitation in that its cardiomy-
ocytes in vivo from adult animals are relatively unresponsive to
Ang II (30, 31). This led us to choose the hamster for functional
studies. In the adult hamster, as with humans, cardiomyocytes are
responsive to Ang II (21, 33, 34, 45). Species variability in chymases
is striking. Realizing this, we first validated the use of the chymase
inhibition strategy in hamsters. We purified hamster chymase 1, a
homolog of MMCP4, showed that it is Ang II forming, and identi-
fied an orally active high-affinity inhibitor (CI-B) of this enzyme.
The effect(s) of species differences in AT2 receptor in the heart,
which has been shown in some studies to be beneficial (46) and
in others to be detrimental (9), could also be a limitation with
respect to the implied importance of chymase-based Ang II gen-
eration in humans with MI. But the importance of the AT2 recep-
tor is difficult to “factor in” because its role in the human heart
is as yet undefined. Based on these limitations, we realize that
the significance of ACE inhibitor–dependent chymase release as
a therapeutic target in MI necessarily requires clinical evidence.
Nevertheless, the importance of our studies is that they provide
an evidence-based hypothesis for a study of the role of cardiac MC
chymase in patients that are currently treated with ACE inhibitors.
Because ACE inhibitors are some of the most effective and widely
used drugs in the treatment of heart diseases, minimizing adverse
effects, such as chymase release, has the potential to be highly use-
ful in the treatment of heart failure, let alone other cardiovascular
disorders that have an inflammation component.
Mice. Ten-week-old MC-deficient W/Wv mice (background, C57BL/6) (19)
and their MC-sufficient WT littermates were purchased from the Jackson
Laboratory. All animal studies were approved by the University of Alabama
at Birmingham IACUC guidelines for animal care and treatment.
Measurement of ACE and non-ACE Ang II–forming activities in the mouse LV
tissue homogenates. LV tissue from WT and W/Wv mice was homogenized
in 2 ml of 50 mM NaH2PO4 buffer, pH 7.4, using a Polytron homogenizer
at 9,000 rpm for 15 seconds at 4°C, then centrifuged at 30,000 g for 20
minutes at 4°C and the pellet retained. This procedure was repeated
twice. Resulting supernatant fractions, S-1 and S-2, were kept. The pellet
was resuspended in 0.5 ml 50 mM NaH2PO4 buffer, pH 7.4, containing
100 mM NaCl and 10 mM MgCl2, and 5 μl of this was added to 35 μl of
assay buffer (20 mM Tris-HCl, pH 8.0, containing 0.5 M KCl and 0.01%
Triton X-100) containing either no further additive or 10 μM lisinopril
and preincubated for 30 minutes at 0°C. Ten microliters of 1 mM Ang I
was added to 40 μl of the preincubated aortic extract, and incubations
were for 40 minutes at 37°C. Reactions were terminated by the addition of
300 μl ethanol. Precipitated proteins were removed by centrifugation, and
the supernatant containing angiotensins was dried. The residue, resus-
pended in 125 μl of distilled water, was applied to a C18 HPLC column
(Vydac); angiotensins were separated as described (22, 27). Total and ACE-
independent Ang II–forming activities were determined in duplicate from
assays containing either buffer or lisinopril, respectively. ACE-dependent
Ang II–forming activity was taken as total minus ACE-independent activ-
ity. Assays were optimized to ensure that Ang II generation was linear with
time. With crude membrane preparations, positively charged chymases
were quantitatively retained in the pellet, as was ACE, which was mem-
brane bound. Ang II–forming activity in S-1 and S-2 was negligible.
Preparation and implantation of microdialysis probes. Microdialysis probes
were inserted into the LV myocardium of 10-week-old male W/Wv and age-
matched WT mice using a modification of a previously described procedure
(24). Briefly, each microdialysis probe consisted of a semipermeable mem-
brane fiber (Hospal) with a molecular weight cutoff of 35 kDa (internal
diameter = 250 μm) with Pebax tubing (outer diameter = 200 μm) inserted
within it and sealed in place at each end of the dialysis fiber such that 4 mm
of semipermeable membrane fiber remained exposed at the center of the
assembly; this exposed region resided within the LV myocardium free wall.
A BV-1 tapered needle with 1-cm length of 7-0 Prolene suture was secured
in one end of the probe and a 2-cm length of polyethylene PE-50 tubing
was secured to the other end. With animals under general anesthesia (iso-
flurane, 2%–3%) and using mechanical ventilation, the microdialysis probe
was implanted in the LV. An incision was made between the sixth and sev-
enth ribs on the left side, intercostal muscles were cut, and the ribs spread.
The heart was thus exposed and the microdialysis probe inserted caudal to
rostral in the LV myocardium. The microdialysis probe was fixed in the myo-
cardium such that the dialysis fiber would not work itself out of the LV wall.
The Pebax tubing exited the thoracic cavity one intercostal space above and
one below the incision. The lungs then were fully inflated, and the sixth and
seventh ribs were sutured together. The ends of the probe were transferred
subcutaneously to the base of the neck and exteriorized and secured with
silicone sealant (Kwik-Cast; World Precision Instruments). After implanta-
tion, the probe was perfused with 0.9% saline using a precision infusion
syringe pump (BAS) at a flow rate of 0.5 μl/min. The mice were then allowed
to recover for 1 day before ISF collections were implemented. At the end of
the ISF collection, Evans blue dye was introduced into the probe to visu-
ally determine whether the probe was positioned correctly in the LV. The
dialysate was collected from the outflow tube in 200 μl Eppendorf tubes
containing 10 μl 1 M acetic acid at 0°C. At the end of the collection period,
the Eppendorf tubes with their contents were frozen at –80°C.
Mouse experimental protocols. In the first protocol, mice, divided into 3
groups, each group subdivided by the genotypes WT and W/Wv, were used
for measurement of ISF and plasma Ang I and Ang II concentrations. At
day 1, randomized WT and W/Wv mice started with vehicle or oral ACE
inhibitor–captopril (150 mg/kg/d) treatment. At day 8, microdialysis
probe was implanted inside LV myocardium in both genotypes. At day 9,
after 24 hours recovery from surgery, ISF dialysate was collected from
the microdialysis probes implanted in conscious, nonsedated mice over
a 24-hour collection period by infusion of 0.9% saline at a flow rate of
0.5 μl/min; this was used for the measurement of baseline LV ISF Ang II.
At day 10, ISF infusion protocol was implemented in WT and W/Wv mice
for the infusion of ACE-selective ([Pro10]Ang I) or chymase-selective Ang I
([Pro11,DAla12]Ang I) substrates or 0.9% saline at a flow rate of 0.5 μl/min.
This protocol allowed the collection of dialysates for (a) baseline ISF Ang I
measurement following ISF infusion of 0.9% saline after oral vehicle or
captopril treatment for 10 days; (b) ISF Ang II measurement following
ISF infusion (0.5 μl/min) of 500 nM [Pro11,DAla12]Ang I after vehicle or
oral captopril treatment for 10 days; or (c) ISF Ang II measurement follow-
ing ISF infusion (0.5 μl/min) of 500 nM [Pro10]Ang I after vehicle or oral
captopril treatment for 10 days. These protocols allowed the determina-
tion of baseline LV ISF Ang II and Ang I as well as chymase-dependent
Ang II–forming activity ([Pro11,DAla12]Ang I–dependent Ang II formation
– baseline Ang II in the LV ISF) and ACE-dependent Ang II–forming activ-
ity ([Pro10]Ang I–dependent Ang II formation – baseline Ang II in the LV
ISF) after 10 days of vehicle treatment or 10 days of ACE inhibitor treat-
ment. Pooled blood samples for measurement of angiotensins in plasma
were collected by heart puncture at the time of sacrifice.
In the second protocol, at day 1, WT mice were given either (a) oral capto-
pril treatment (150 mg/kg/d) (mouse group A); (b) oral captopril treatment
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
(150 mg/kg/d; Sigma-Aldrich) supplemented with the B2 receptor antago-
nist Hoe-140 (0.5 mg/kg/d; Sigma-Aldrich) (mouse group B); or (c) oral
captopril treatment (150 mg/kg/d) supplemented with the chymase inhibi-
tor CI-A (200 mg/kg, b.i.d.; Teijin Pharma Limited) (group C). At day 8,
microdialysis probe was implanted inside LV myocardium. At day 9, after
24 hours recovery from surgery, ISF dialysate collection was implemented
from the implanted microdialysis probes in conscious, nonsedated mice in
groups A–C over a 24-hour collection period during which 0.9% saline was
infused at a flow rate of 0.5 μl/min. This was used for the measurement
of baseline LV ISF Ang II during either chronic ACE inhibitor treatment,
chronic ACE inhibitor treatment plus B2 receptor blockade, or chronic
ACE inhibitor treatment plus chymase inhibitor treatment. Immediately
following this, 500 nM [Pro11,DAla12]Ang I was infused (0.5 μl/min) into
the microdialysis probe and dialysate was collected over the next 24 hours
for the measurement of Ang II in order to calculate LV ISF chymase activity.
In these experiments, captopril was prepared in drinking water, CI-A was
orally administered by gavage, and Hoe-140 (American Peptide Company)
was administered using osmotic minipumps. From the measurement of
[Pro11,DAla12]Ang I–dependent Ang II formation – baseline Ang II in the
LV ISF in each group, we calculated the effects of chronic ACE inhibition
(group A), chronic ACE inhibition plus B2 receptor blockade (group B),
and chronic ACE inhibition plus chronic chymase inhibition (group C) on
LV ISF chymase activity.
Measurements of angiotensins in blood plasma, LV ISF. Angiotensin peptide concen-
trations were determined using solid-phase extraction, HPLC, and RIA (24).
Immunohistochemistry. Mouse hearts were immersion fixed in 4% para-
formaldehyde and stored in 70% ethanol until paraffin embedding and
sectioning. Sections (5 μm) were mounted on slides, deparaffinized
in xylene, and placed in ethanol. Tissue sections were treated with the
Mouse on Mouse Immunodetection Kit Fluorescein (FMK-2201; Vector
Labs) in conjunction with mouse B2 bradykinin receptor mAb (610452,
1:50; BD Transduction Laboratory). Sections were blocked with 5% goat
serum in 1% bovine serum for 1 hour at 22°C. Rabbit polyclonal Ab was
made against the MMCP4 sequence ETPSVNVIPLPRPSD; it recognizes
a peptide sequence unique to this isoform. The MMCP4 polyclonal Ab
(1:100) or ACE polyclonal Ab (1:100; sc20891; Santa Cruz Biotechnol-
ogy Inc.) in 5% goat serum was applied to sections for 12 hours at 4°C.
The sections were incubated with Alexa Fluor 488 (green) or 594 (red)
goat anti-mouse or rabbit to visualize the specific stains. Secondary anti-
bodies were from Molecular Probes. Sections were counterstained with
DAPI (blue) (Molecular Probes) to reveal nuclei. Image acquisition was
performed on a Leica DM6000B epifluorescence microscope (Leica Micro-
systems) with a Hamamatsu ORCA ER cooled CCD camera and SimpleP-
CI software (Compix Inc.). Images were adjusted appropriately to remove
Determination of mRNA expression in the mouse LV. For mRNA transcript
measurements, mRNA was either extracted from freshly dissected LVs or
cardiomyocytes isolated from individual adult mouse hearts (47). Total
RNA was extracted with RNAgents (Invitrogen). mRNA was reverse tran-
scribed using Transcriptor reverse transcriptase (Roche) according to
the manufacturer’s protocols. Real-time PCR reactions were carried out
using iCycler and iQ SYBR Green Supermix (Bio-Rad). The primer sets for
β-actin, GAPDH, and MMCP1, -2, -4, -5, -9, -10, and -L were as previously
reported (22, 47). Forward and reverse mouse ACE PCR primers were
5′-GGGGGCCAAGCTCAAGCAGG-3′ and 5′-GCACTGCCCGGTCCAG-
GTTC-3′, respectively. Real-time PCR conditions for these primers were
optimized as 3 mM Mg2+, 5 pmol primer, and 50 ng cDNA in a total reac-
tion volume of 25 μl. The amplifying conditions were determined empir-
ically and were 95°C for 30 seconds, 62°C for 30 seconds (or 59°C for
30 seconds for GAPDH), and 72°C for 60 seconds for 45 cycles. All PCR
products appeared as single bands of the expected molecular size by 1.5%
agarose gel fractionation; these PCR products were sequenced to identify
target cDNAs. In addition to confirming that all PCR products appeared
as a single band of the expected molecular size, we also verified that each
PCR product consisted of only a single species, as determined by melting
curve analysis. To determine copy number, standard curves for each target
transcript were established using purified PCR products. Standard curves
were linear and the correlation coefficients were greater than 0.99.
Hamsters. Syrian hamsters, 8 weeks old, were obtained from SLC Japan.
The animals were housed in individual cages under controlled conditions
of constant temperature and humidity and exposed to a 12-hour dark/
12-hour light cycle. The hamsters had free access to drinking water and either
a standard chow diet or diet chow containing 0.1% CI-B. All animal studies
were approved by the Internal Review Committee of Fukuoka University.
Induction of MI in hamsters. MI was induced by permanent ligation of
the left anterior descending coronary artery as previously described (48).
Briefly, under pentobarbital (50 mg/kg i.p.) anesthesia, hamsters were intu-
bated and artificially ventilated. After left thoracotomy was performed at
the fifth intercostal space, the pericardium was incised and the heart was
exteriorized. A 6-0 silk suture was placed around the left coronary artery
at the most proximal position. In sham-operated hamsters, the suture was
placed beside the coronary artery. The muscle and skin layers were sutured
and the thoracic cavity was closed.
Hamster experimental protocol. CI-B (Teijin Pharma Ltd.) was used in studies
with hamsters. Its KI value against hamster chymase-1 was estimated to be
approximately 30 nM. In a preliminary experiment, 4 hamsters were given
0.1% CI-B in food for 5 days and blood samples were collected to measure
the serum concentration of CI-B. The CI-B concentration was measured
by reverse-phase HPLC using a linear gradient (from 0.1% acetic acid con-
taining 0% acetonitrile to 0.1% acetic acid containing 60% acetonitrile). The
serum concentration of CI-B was 7.9 ± 2.1 μM, which was approximately
250-fold higher than its KI value against hamster chymase. The ACE inhibi-
tor temocapril was provided by Sankyo Co. The dose of temocapril used
(10 mg temocapril /kg/d, oral) was based on previously published data.
For hamster studies, a single experienced investigator performed all MI
surgeries. Once MI or sham surgery was performed, the hamsters with
MI were randomized. The hamster groups were treated according to the
following protocol: group 1, sham-operated; group 2, MI without drug
intervention; group 3, MI with temocapril (10 mg/kg/d); group 4, MI with
0.1% CI-B in food (100 mg/kg/d); group 5, MI with temocapril (10 mg/
kg/d) and 0.1% CI-B in food. Cardiac echocardiography procedures used
to obtain the EF, FS, and LV end-diastolic dimension were performed as
previously described by us (48). The survival rates for each treatment group
were evaluated over 5 weeks of treatment. At the end of the treatment peri-
od, hamsters were sacrificed to collect hearts.
Pathological examination of hamster hearts. The hearts were rinsed in ice-cold
saline, and the atria and ventricles were separated to measure ventricular
weight. They were then cut at the level of the papillary muscles through the
short axis. Tissue blocks were fixed in 4% formaldehyde for 48 hours. After
dehydration, the sections were embedded in paraffin, and 4-μm–thick
sections were cut. Deparaffinized sections were stained with H&E and
Masson’s trichrome. After dehydration in 96% ethanol, the sections were
mounted. The epicardial circumferential lengths of the infarcted and non-
infarcted LV were determined by computerized morphometry. The infarct
zone of the LV was measured and presented as a percentage of the entire
epicardial circumferential length of the LV. Average myocytes diameter
measurements were performed on myocytes in 10 ×400 fields. To avoid
individual myocyte cross sections that were grossly ellipsoid in shape, the
ratio of the long and short axis diameters was measured. Measurements
in which this ratio exceeded 1.5 were excluded. Average value from each
1238? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 4 April 2010
hamster heart was based on the data from 130 myocytes. Myocyte diameter
was determined on an iMac computer using the public domain NIH Image
Program. In order to determine fibrosis area, 6 ×200 fields were analyzed in
the midwall area of the noninfarcted interventricular septum. Fibrosis area
was expressed as the percentage of aniline blue–positive areas in the fields
analyzed relative to the total area of the fields analyzed. Measurements
were restricted to the interstitial fibrosis; perivascular and endocardial
fibrosis were excluded from the measurements.
Purification of mouse and hamster chymases. MMCP4 and MMCP5 were puri-
fied from mouse skin and heart, respectively, according to the methods
described previously (49, 50). Hamster chymase-1 was purified from the
tongue using procedures described previously (6).
Determination of chymase substrate specificities. All synthetic angiotensins
were from Bachem California Inc. 30 mM of Ang I, Ang II, [Val8]Ang I,
and [Val8]Ang II were incubated with the purified MMCP4 and MMCP5 in
20 mM Tris-HCl buffer, pH 7.5, containing 0.5 M NaCl, in a total volume
of 50 μl at 37°C for 30 minutes (6). Chymase concentrations were adjusted
so that less than 30% of the substrate was consumed during the assay. The
reactions were terminated by addition of 50 μl ice-cold 0.25% trifluoroace-
tic acid. 20 μl of the resulting reactions was applied to a BEH C18 reverse-
phase HPLC column (100 mm × 1.7 mm i.d.; Waters Corp.), which was
developed using 20-minute linear gradients with acetonitrile containing
0.1% trifluoroacetic acid. The elution position of Ang I, Ang II, [Val8]Ang I,
[Val8]Ang II, and Ang I–(5–10) was determined using pure synthetic angio-
tensin peptides as standards.
Determination of KI. KI values for chymase inhibition were determined using
Suc-Ala-His-Pro-Phe-pNA or MeOsuc-Ala-Ala-Pro-Val-pNA (Bachem).
Enzyme activities were evaluated by measuring p-nitroaniline release from
the synthetic substrates. KI values were determined from plots of enzyme
activity versus inhibitor concentration in which inhibitor concentration
was corrected by using the Km and concentration of the substrate (S) used
for the competition (KI = IC50/(1 + ([S]/Km))).
Statistics. All data are presented as mean ± SEM. In some cases, an
unpaired Student’s t test was used for statistical comparisons between the
WT and W/Wv groups. Groups were compared statistically using ANOVA,
and the significance of differences was determined by the Bonferroni’s test.
P values of less than 0.05 were considered to indicate statistical signifi-
cance. For differences in mortality, a Kaplan-Meier survival analysis was
performed and differences between the groups were tested by a log-rank
analysis using the Cox-Mantel test. Hamsters that died within 24 hours
after the operation were excluded from the survival-rate analysis.
We thank Robert M. Graham for his helpful comments. This study
was supported by grants R01HL60707 and R01HL54816 (to L.J.
Dell’Italia); R01HL79040 (to A. Husain); a Specialized Centers of
Clinically Oriented Research grant in Cardiac Dysfunction from the
NIH (P50HL077100); and a Scientist Development grant from the
National American Heart Association (0130306N to C.-C. Wei).
Received for publication March 26, 2009, and accepted in revised
form January 20, 2010.
Address correspondence to: Hidenori Urata, Department of
Cardiovascular Diseases, Fukuoka University Chikushi Hospi-
tal, 1-1-1 Zokumyoin, Chikushino 818-8502 Fukuoka, Japan.
Phone: 81.92.801.1011; Fax: 81.92.865.2692; E-mail: uratah@
fukuoka-u.ac.jp. Or to: Louis J. Dell’Italia, Department of Medi-
cine, Center for Heart Failure Research, University of Alabama at
Birmingham, 434 BMR2, 901 19th St. S, Birmingham, AL 35294.
Phone: 205.789.0212; Fax: 205.996.2586; E-mail: dell’Italia@
physiology.uab.edu. Or to: Ahsan Husain, Division of Cardiology,
Emory University, 101 Woodruff Circle, 319 Woodruff Memorial
Research Building, Atlanta, GA 30322. Phone: 404.727.8125; Fax:
404.727.3572; E-mail: firstname.lastname@example.org.
Ming Li’s present address is: Victor Chang Cardiac Research Insti-
tute, Darlinghurst, New South Wales, Australia.
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