Download Increased Connective Tissue Growth Factor Relative

Increased Connective Tissue Growth Factor Relative to
Brain Natriuretic Peptide as a Determinant of
Myocardial Fibrosis
Norimichi Koitabashi, Masashi Arai, Shinya Kogure, Kazuo Niwano, Atai Watanabe, Yasuhiro Aoki,
Toshitaka Maeno, Takashi Nishida, Satoshi Kubota, Masaharu Takigawa, Masahiko Kurabayashi
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Abstract—Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was to
investigate the role of connective tissue growth factor (CCN2/CTGF), a profibrotic cytokine of the CCN (Cyr61, CTGF,
and Nov) family, and its functional interactions with brain natriuretic peptide (BNP), an antifibrotic peptide, in the
development of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsy
samples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiac
myocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. In
a rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportion
to one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP was
positively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression.
Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cells
demonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, G
protein– coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereas
aldosterone and transforming growth factor-␤ preferentially induced expression of the CTGF gene. Finally, exogenous
BNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase in
CTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis and
diastolic heart failure. (Hypertension. 2007;49:1120-1127.)
Key Words: extracellular matrix 䡲 hypertrophy 䡲 cardiac function 䡲 connective tissue growth factor
䡲 natriuretic peptide
E
pidemiological studies have established that 40% to 50%
of patients with heart failure have normal or minimally
impaired left ventricular (LV) ejection fraction, a clinical
syndrome that is commonly referred to as diastolic heart
failure (DHF). These patients typically have cardiac hypertrophy that is induced by long-standing hypertension or by
primary hypertrophic cardiomyopathy, as well as increased
passive LV stiffness.1 Among various molecular mechanisms
that regulate LV stiffness,2 abnormalities in the transcriptional or posttranscriptional regulation of the collagen gene
can result in the disproportionate accumulation of fibrous
tissue and elevation of stiffness in the hypertrophied heart.2,3
Recent studies have shown that, in addition to mechanical
load, autocrine, paracrine, and endocrine factors, such as
angiotensin II, aldosterone (Aldo), endothelin-1 (ET1), natriuretic peptides, osteopontin, and transforming growth
factor-␤1 (TGF-␤), play important roles in the development
of myocardial hypertrophy and fibrosis.4,5 However, the precise
molecular mechanisms that initiate and promote myocardial
fibrosis and increases in ventricular stiffness remain largely
unknown.
Connective tissue growth factor (CCN2/CTGF) belongs to
the CCN (Cyr61, CTGF, and Nov) family of immediate early
genes, which are highly conserved among species.6 This
cysteine-rich secreted protein may contribute to progressive
fibrosis and excessive scarring in various systemic and local
fibrotic diseases.6 Further, CTGF expression is increased in
the hypertrophied and failing myocardium of experimental
animal models.7,8 CTGF is also an essential mediator for the
biological actions of TGF-␤6 and its downstream signal transduction elements.9 However, a recent in vitro study demonstrated that CTGF is 1 of the earliest growth factors
transcriptionally induced by hypertrophic stimuli in cardiac
myocytes (CMs).10
Received July 24, 2006; first decision August 13, 2006; revision accepted February 25, 2007.
From the Department of Medicine and Biological Science (N.K., M.A., S.K., K.N., A.W., Y.A., T.M., M.K.), Gunma University Graduate School of
Medicine, Gunma, Japan; and the Department of Biochemistry and Molecular Dentistry (T.N., S.K., M.T.), Okayama University Graduate School of
Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
Correspondence to Masashi Arai, Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-22
Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail [email protected]
© 2007 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/HYPERTENSIONAHA.106.077537
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CTGF vs BNP and Myocardial Fibrosis
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Figure 1. Myocardial fibrosis and CTGF
protein in endomyocardial biopsy samples. A through D, Main panels show highpower fields (⫻400). Left small panels
show low-power fields (⫻100). All scale
bars are 50 ␮m. A and C, Masson’s
trichrome staining; B and D, immunostaining for CTGF; A and B, NF group: A
77-year– old man with mild LV hypertrophy
and chronic hypertension, without previous history of pulmonary congestion. His
LV ejection fraction was 45%. Plasma
BNP concentration was 76.2 pg/mL. C
and D, DHF group: A 74-year– old man
with mild LV hypertrophy, chronic atrial
fibrillation, and hypertension. His LV ejection fraction was 75%. Plasma BNP concentration was 135 pg/mL. E, Comparison
between NF (n⫽15) and DHF (n⫽31) in
MFA estimated by Masson’s trichrome
staining in NF (n⫽15) and DHF (n⫽31). F,
Comparison between NF (n⫽15) and DHF
(n⫽31) in positive-stained area with CTGF
antibody. G, Correlation between MFA and
CTGF-stained area among all of the
patients enrolled (n⫽46). 䡬, NF; ●, DHF.
In this study, to confirm the involvement of CTGF in the
myocardial fibrosis, we first investigated CTGF protein production in myocardial biopsy samples of patients with DHF.
Secondly, by using the pressure overload rat model with a
suprarenal abdominal aortic constriction (AC), which mimics a
model of DHF,5 we determined and compared the temporal
changes of CTGF, TGF-␤, and an antifibrotic peptide, brain
natriuretic peptide (BNP).11 Because the collagen accumulation
level is reflected by the balance of profibrotic factors and
antifibrotic factors,12 we investigated their functional interactions, especially between CTGF and BNP, in the development of
myocardial fibrosis and DHF.
Methods
An expanded Methods section is available online at http://
hyper.ahajournals.org.
Forty-six consecutive patients with normal or minimally impaired
LV ejection fraction (⬎40%), estimated by echocardiography, who
underwent endomyocardial biopsy of the LV-free wall in Gunma
University Hospital were enrolled in this study (Table S1). All of the
patients were clinically stable when the biopsy was performed. Of
these patients, 31 patients who had a previous history of overt heart
failure within the preceding year in the absence of impaired systolic
function as estimated by echocardiography were designated as the
DHF group. Another 15 patients without a previous history of heart
failure were designated as the nonfailing (NF) group. The clinical
diagnosis and the exclusion criteria are described in the expanded
Methods section.
AC was established with a 21G silver clip13 in male Wistar rats
(Charles River, Japan) weighing 250 to 300 g. Cell culture, histochemical analysis and immunostaining, hemodynamic measurements
in AC rats, RNA isolation and Northern blot analysis, Western
blotting, and statistical analysis are described in the expanded Methods
section online.
Results
Elevated Levels of CTGF Protein in CM
Correlates With Myocardial Interstitial Fibrosis in
Patients With Preserved Ejection Factor
Clinical characteristics of the NF and DHF groups are
summarized in Table S1. There were no significant differences in age, sex, clinical diagnosis, and frequency of
complicated disease, except for atrial fibrillation, when comparing the 2 groups. Sixty-one percent of DHF patients had
been given previous medication, including angiotensinconverting enzyme inhibitors and/or ␤-adrenoceptor blockers, because of their previous history of congestive heart
failure. Pulmonary artery wedge pressure and LV end-diastolic pressure were not different when comparing the 2
groups. Furthermore, there were no significant differences in
echocardiographic parameters when comparing the 2 groups
except for left atrial dimension. Plasma BNP concentration
was significantly elevated in the DHF group. Representative
LV biopsy samples taken from a patient from the NF group
with hypertension and a patient from the DHF group are
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Figure 2. Gene expressions in hypertrophied rat hearts. A, Temporal expression
patterns of mRNAs normalized to 18S
rRNA. S-0, rat euthanized immediately
after sham operation; AC-1, 4, 7, 14, and
28, rats euthanized on designated day
after AC or sham operation; *P⬍0.05 vs
sham-operated rats at same postoperative
periods. B and C, Correlation between
CTGF and BNP mRNA levels in AC rats on
days 1, 4, and 7 (B) and on days 14 and
28 (C). D and E, Representative Northern
blot analysis of CTGF, BNP, TGF-␤, and
COL1A1 (procollagen type 1␣1) mRNAs
and 28S and 18S rRNAs on day 4 (D) and
day 28 (E).
illustrated in Figure 1. Biopsies from the NF patient showed
mild hypertrophic myocytes but no interstitial fibrosis by
Masson’s trichrome staining (Figure 1A). CTGF immunostaining of serial sections showed a small amount of CTGF
protein in the myocytes (Figure 1B). By contrast, biopsies
from the DHF patient showed interstitial fibrosis (Figure 1C)
and an abundance of CTGF protein in CM (Figure 1D).
Quantitative analysis revealed that myocardial fibrosis area
(MFA) and CTGF-stained area were significantly elevated in
DHF patients (Figure 1E and 1F). Interestingly, the CTGFstained area correlated with MFA (r⫽0.638; P⬍0.001;
Figure 1G).
CTGF and BNP Gene Expression Are
Coordinately Induced Early in the Development of
Cardiac Hypertrophy and Fibrosis
To investigate the role of CTGF for the development of
cardiac fibrosis, we created a rat pressure overload cardiac
hypertrophy model by constricting the abdominal aorta. In
accordance with the increase of systolic blood pressure, on
day 4 after AC operation and until day 28, LV weight/body
weight ratio, a parameter of LV hypertrophy, significantly
increased (Table S2). Furthermore, MFA was significantly
increased on day 14 after AC.
Quantitative Northern blot analysis revealed that CTGF
mRNA levels peaked on day 1, whereas TGF-␤ mRNA levels
increased gradually and peaked on day 7 (Figure 2A).
Furthermore, procollagen type 1␣1 (COL1A1) mRNA levels
were significantly increased on day 7 and continued to
increase until day 28. Interestingly, the temporal course of
changes in BNP mRNA was similar to that of CTGF mRNA,
particularly from day 1 to day 7 (N⫽22; r⫽0.836; P⬍0.001;
Figure 2B and 2D).
High CTGF/BNP Expression Ratio Is Associated
With Myocardial Fibrosis and Ventricular
Stiffness at a Later Stage of Cardiac Hypertrophy
Although a correlation between CTGF and BNP mRNA
expression was observed during the entire experimental
period (N⫽69; r⫽0.804; P⬍0.001; Figure not shown), the
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Figure 3. LV diastolic function and myocardial fibrosis in rats with different ratios
of CTGF and BNP mRNAs. Representative
pressure–volume loops (A) and echocardiograms (B). Left, central and right panels
show sham, an AC rat with comparable
mRNA levels of CTGF and BNP, and an AC
rat with disproportionate increase of CTGF
against BNP, respectively. A, End-diastolic
relationships are depicted in broken lines.
B, Differences in cardiac parameters
between upper and lower 50th percentile
groups of CTGF/BNP expression ratio in
day 28 AC rats. In each subset, 䡺 represents the lower group (n⫽7), and f represents the higher group (n⫽7). EDPVR,
slope of end-diastolic pressure volume
relationship; ␶, monoexponential time constant of relaxation; LVW/BW, ratio of LV
weight to body weight at sacrifice; Sarcomeric ␣-actin, the amount of sarcomeric
␣-actin in the heart extracts estimated by
Western blot.
correlation was weaker between day 14 and day 28 (N⫽19;
r⫽0.645; P⬍0.001; Figure 2C and 2E) when compared with
the early stage of cardiac hypertrophy (day 1 to day 7;
r⫽0.836; Figure 2B). As shown in Figure 2C, some rats
expressed disproportionately abundant CTGF mRNA. Furthermore, those rats with high CTGF mRNA levels relative to
BNP mRNA levels also showed marked upregulation of
COL1A1 mRNA (Figure 2E, lanes 4 and 6). By contrast, rats
with proportional increases in both CTGF and BNP mRNA
levels showed only mild upregulation of COL1A1 mRNA
(Figure 2E, lanes 3 and 5). Finally, rats with low CTGF
mRNA levels relative to BNP mRNA levels showed low
levels of COL1A1 mRNA (Figure 2E, lane 7).
Hemodynamic analysis was performed in rats on day 28.
LV contractility indices, calculated using the pressure-volume
relationship, were comparable when comparing shamoperated and AC rats (Table S3). Diastolic indexes, that is,
time constant of relaxation (␶) and the slope of end-diastolic
pressure-volume relationship (EDPVR slope), were significantly higher in the AC rats than in the sham-operated rats.
Representative hemodynamic data and histochemical
staining of CTGF in a sham-operated and 2 AC rats with
comparable or disproportionate mRNA levels for CTGF and
BNP are illustrated in Figure 3A and Figure S1. A rat with
high CTGF levels related to BNP mRNA levels showed a
steeper slope of EDPVR (Figure 3A), a high E/A ratio in
Doppler echocardiography (Figure S1A), severe interstitial
fibrosis, and positive immunostaining against CTGF in CM
(Figure S1B) when compared with a sham-operated rat and a
rat with comparable mRNA levels of CTGF and BNP.
To further characterize hearts with high CTGF mRNA
levels relative to BNP mRNA levels, AC rats were classified
according to the upper or lower 50th percentile groups of the
CTGF/BNP expression ratio on day 28. The mean ratio of the
CTGF/BNP mRNA level was 1.2 (Figure 3B). AC rats with
a higher CTGF/BNP mRNA ratio (n⫽7) showed elevated
EDPVR slope, higher E/A ratio, and increased MFA (Figure
3B) relative to AC rats with the lower CTGF/BNP ratio
(n⫽7). By contrast, LV relaxation (␶), contractility (ejection
factor), or LV hypertrophy (LV weight/body weight ratio)
was similar when comparing the 2 groups (Figure 3B). The
protein content of sarcomeric ␣-actin was also similar when
comparing the 2 groups (Figure 3B). Interestingly, the CTGF/
BNP expression ratio correlated with EDPVR slope (r⫽0.720;
P⬍0.001; Figure S2) and with COL1A1 mRNA expression
(r⫽0.458; P⬍0.001; Figure S2). The ratio also significantly
correlated with the E/A ratio, expression levels of procollagen
type 3␣1 (COL3A1), and MFA (Table S4). On the other hand,
LV contractility indexes, ␶, and mRNA expressions of TGF␤
and sarcoplasmic reticulum Ca2⫹ ATPase (SERCA) 2a were not
correlated with the ratio (Table S4). Finally, plasma concentration of Aldo was significantly elevated in AC rats with a higher
CTGF/BNP ratio (Figure S3), whereas plasma TGF-␤ and ET-1
concentration was not significantly different when comparing
the 2 groups.
CTGF Is Secreted From CM
The molecular basis of the production of CTGF in the heart
and the functional interaction with other neurohumoral factors was investigated using rat neonatal primary CM and
cardiac fibroblasts (CFBs). Immunofluorescent study with
anti-CTGF antibody revealed production of CTGF in cultured
CMs (Figure 4A) and CFBs (vimentin-positive cells; Figure
S4). Administration of recombinant CTGF resulted in a
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Figure 4. CTGF expression in cultured cardiac myocytes (CM) and its paracrine effect on COL1A1 expression in cardiac fibroblasts
(CFB). A, Immunofluorescent imaging of CTGF (detected by Cy3) and actinin (detected by FITC) protein. Actinin is a sarcomeric protein,
which indicates CM. Bar, 20 ␮m. B, COL1A1 mRNA levels in cultured cardiac fibroblasts (CFB) treated with recombinant human CTFG
in designated concentrations for 24 h. The result was confirmed by triplicate experiments. C, COL1A1 mRNA levels in CFB 24 h after
the addition of conditioned medium from CM cultured in the presence or absence of TGF-␤ (10 ng/mL), ET1 (0.1 ␮mol/L), or Aldo (1
␮mol/L) for 24 h. Upper and middle panels: Western blot showing the amount of CTGF protein in cell lysates and in the culture media
of CM, respectively. Lower panel: Northern blot showing COL1A1 mRNA levels in CFB simulated by conditioned medium. Experiments
were performed in triplicate. D, The effect of an anti-CTGF neutralizing antibody on COL1A1 mRNA levels in CFB. Experimental conditions were identical to those in panel C. Veh-medium, medium with the solvent of TGF-␤; ␣CTGF, anti-CTGF neutralizing antibody (␮g/
mL); ␣TGF␤, anti-TGF-␤neutralizing antibody (␮m/mL); IgG; normal goat IgG, used as a control for the anti-CTGF antibody. The bar
graphs show mean mRNA levels based on 4 independent experiments. *P⬍0.05 versus Veh-medium with normal IgG; †P⬍0.05 versus
TGF␤-treated-medium with normal IgG.
dose-dependent increase in COL1A1 mRNA levels in cultured CFB (Figure 4B). Profibrotic stimulation with TGF-␤,
ET1, and Aldo resulted in increased CTGF production and
release into the culture medium from the myocytes (Figure 4C).
Furthermore, conditioned medium from these CMs enhanced
COL1A1 mRNA levels in CFBs (Figure 4C), suggesting that
CMs may regulate collagen production in CFBs. When the
TGF-␤–treated medium was preincubated with anti-CTGF antibody, COL1A1 mRNA induction was abolished in CFBs
(Figure 4D). Pretreatment of the CM-cultured medium with both
anti-CTGF and anti–TGF-␤ antibodies further suppressed the
COL1A1 mRNA, suggesting that the induction of the COL1A1
gene by TGF-␤ and even the basal expression level of the
COL1A1 gene in CFBs are mediated through TGF-␤– dependent
and CTGF-dependent pathways.
Common and Uncommon Stimuli Triggering
CTGF and BNP Gene Transcription in CMs
To further characterize the correlation between CTGF and
BNP mRNA induction in AC rats, the role of mechanohmoral
and neurohumoral stimuli on CTGF and BNP induction was
investigated. Cyclic stretch induced a rapid increase in CTGF
and BNP mRNA levels (Figure 5A). Furthermore, G protein–
coupled receptor ligands, such as ET1 (Figure 5B), norepinephrine, and angiotensin II (Figure S5A), increased CTGF
and BNP levels in a dose-dependent manner. By contrast,
Aldo and TGF-␤ stimulation resulted in increases in CTGF
mRNA levels and decreases or no effect on BNP mRNA
levels (Figure 5B). The differential effect of TGF-␤ and Aldo
on CTGF and BNP mRNA levels was also confirmed by
comparing the temporal induction pattern of these genes by
TGF-␤ and Aldo with that induced by ET1 (Figure 5C).
BNP Suppresses CTGF Expression in CMs
Because BNP and TGF-␤ have opposing biological effects,14
the effect of BNP on CTGF expression was investigated.
CTGF mRNA levels decreased 2 hours after administration
of synthetic BNP (Figure S5B). Synthetic BNP-mediated
inhibition of CTGF expression was completely blocked by
the protein kinase G inhibitor KT5823, suggesting that the
Koitabashi et al
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Figure 5. Effects of various hypertrophyassociated stimuli on the CTGF and BNP
expression in CMs. A and B, Northern blot
showing the effect of cyclic cell stretching
(A) and various humoral factors (4 hours;
B) on CTGF and BNP mRNA levels in
CMs. Experiments were performed at least
in triplicate. C, Temporal patterns of CTGF
and BNP mRNA levels in response to
TGF-␤ (10 ng/mL), ET1 (0.1 ␮mol/L), or
Aldo (1 ␮mol/L) stimulation. Each dot indicates the mean intensity of 4 independent
experiments relative to the value in the
vehicle-treated sample in each time point.
D, Western blot showing the effect of
TGF-␤, ET1, and Aldo and the inhibitory
effect of BNP on CTGF protein levels. Synthetic BNP (sBNP) was added with TGF-␤,
ET1, or Aldo, and cells were incubated for
24 hours. Bar graphs show mean CTGF
protein levels based on 4 independent
experiments. *P⬍0.01 vs vehicle, †P⬍0.05
vs maximal level of CTGF protein induced
by these stimuli.
BNP– cGMP–protein kinase G pathway plays a critical role in
regulating CTGF expression in CM (Figure S5C). Furthermore, the effect of BNP was also evident in the context of
enhanced production of the CTGF protein in response to
profibrotic stimuli, such as TGF-␤, ET1, and Aldo (Figure 5D).
fibrosis, through an increase of CTGF, significantly contributes to the development of DHF. Based on the staining
pattern with CTGF antibody, strong staining was mainly
observed in CMs rather than in the interstitium (Figure 1D),
suggesting that CMs are largely responsible for the production of CTGF in the DHF heart.
Discussion
DHF, Fibrosis, and CTGF
In the present study, patients with DHF had greater amounts
of interstitial fibrosis when compared with patients without a
previous history of congestive heart failure. Excessive collagen deposition contributes to abnormal passive diastolic
ventricular stiffness3 and leads to pulmonary edema.1 Importantly, MFA, the degree of the interstitial fibrosis, significantly correlated with the abundance of CTGF-positive CMs
(Figure 1G). By contrast, neither MFA nor the percentage of
CTGF-positive CMs was correlated with LV ejection fraction, an index of systolic function, in our study subjects (data
not shown). Endomyocardial biopsy can merely disclose
histological changes of a limited portion of whole heart, and
this immunohistochemical analysis is not a quantitative measurement of CTGF protein. The amount of biopsy samples
was not enough to isolate protein for Western blotting.
However, multiple samplings from different portions of the
same heart and the staining of serial section with normal IgG
as a reference of nonspecific staining minimized sampling
and technical variations. Our study suggests that excess
CMs Produce CTGF
CTGF is overexpressed in numerous fibrotic diseases, and the
degree of overexpression correlates with the severity of
disease.6 Collagen is mainly produced by fibroblasts in many
organs. However, various other cells, including fibroblasts,
secrete humoral factors to initiate collagen production in
fibroblasts.4,5 Although previous reports mainly focused on
CFBs as CTGF-producing cells in the heart,15 the present
study demonstrated that a significant amount of CTGF is
produced by CMs in the hypertrophied rat heart and in the
hearts of patients with DHF.
Interestingly, cultured CFB had higher basal levels of
CTGF mRNA than CM (Figure S4A). However, unlike the
significant induction in CM, CTGF mRNA levels in CFBs
were only minimally affected by extrinsic stimuli (CFBs,
Figure S4B; CMs, Figure 5B), which is consistent with
observations by Kemp et al.10 The inducibility of CTGF in
CMs, along with the fact that BNP, an antifibrotic factor, is
also produced by CMs, raises the possibility that CTGF
produced in CMs regulates collagen production in CFBs.
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Indeed, the present study demonstrated that CTGF was
secreted into the cultured medium of CMs and that this
conditioned medium induced increases in COL1A1 mRNA
levels in CFBs (Figure 4C). In addition, neutralization of
conditioned medium with CTGF antibody sufficiently
blunted the fibrotic signal from CMs to CFBs (Figure 4D).
These data suggest that there is molecular communication in
a paracrine manner between CMs and CFBs and that this
process regulates production of collagen.
CTGF/BNP Balance Regulates Cardiac Fibrosis
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Because we could not obtain a well-working antibody for
BNP immunostaining, we were unable to estimate the BNP
protein level in myocardial biopsy samples. However, the
percentage of CTGF-positive staining cells in biopsy samples
correlated with the plasma BNP level (r⫽0.41; P⬍0.05;
Figure not shown). A close correlation between CTGF and
BNP mRNA induction was also seen in the pressure overload
rat heart and in cultured CMs under cell stretch or stimulation
with G protein– coupled receptor ligands. Although the precise mechanisms for this induction were not investigated in
this study, preliminary studies demonstrated that the ET1induced increase in CTGF and BNP mRNA levels was
blocked by inhibitors of mitogen-activating protein kinases,
protein kinase C, and protein kinase A (data not shown).
These data suggest that coordinated expression of the CTGF
and BNP genes may be mediated by these signaling
pathways.
Most importantly, the CTGF/BNP ratio in CM significantly
correlated with indices of fibrosis and diastolic function, such
as the slope of EDPVR, E/A ratio, COL1A1 mRNA levels,
and MFA (Figure 3 and Table S4). Furthermore, AC rats with
comparable levels of CTGF mRNA and BNP mRNA expression showed mild production of CTGF protein and sparse
fibrosis in the myocardium. Sarcomeric ␣-actin content was
not different between the high CTGF/BNP ratio group and
the comparable ratio group, which suggests that myocyte loss
is not responsible for the change of the ratio (Figure 3B).
SERCA2a is a principal protein responsible for the initiation
of the diastolic phase through its ability to remove cytoplasmic Ca2⫹.2 However, SERCA2a mRNA level was not correlated with the CTGF/BNP ratio (Table S4). These data
suggest that the CTGF/BNP ratio does not associate with LV
diastolic function, which is related to Ca2⫹ removal from
cytoplasm.
The identity of upstream factors responsible for the disproportionate expression of CTGF and BNP in CMs remains
unclear. AC causes severe hypertension and increases in the
levels of various neurohumoral factors, such as renin and
angiotensin II.16 Interestingly, rats with a higher CTGF/BNP
ratio had a higher plasma Aldo concentration and a tendency
toward higher plasma TGF-␤ and ET1 concentrations than
the rats with a lower CTGF/BNP ratio (Figure S3). In
addition, in vitro study demonstrated that Aldo and TGF-␤
induced increases in CTGF mRNA but not in BNP mRNA in
contrast to the response to cell stretch or G protein– coupled
receptor ligands (Figure 5A through 5C). Therefore, at least
in the present model, Aldo may be an upstream factor
responsible for disproportionate CTGF expression.
Figure 6. A scheme illustrating that an abundance of CTGF relative to BNP in CMs promotes pathogenic collagen production in
CFBs.
In addition to the effect on body fluid homeostasis and
blood pressure control, BNP can exert antihypertrophic and
antifibrotic effects in the stressed myocardium.11 The present
study demonstrated that BNP suppressed basal CTGF expression level in CMs via its effects on protein kinase G (Figure
S4C). The effect of BNP on CTGF expression was also
observed under various profibrotic stimuli, such as ET1,
Aldo, and TGF-␤ (Figure 5D). Thus, the increase of CTGF
and/or decrease of BNP in CMs may play a central role in the
induction of excessive myocardial fibrosis and abnormal
diastolic function (Figure 6).
To dissect the role of CTGF in the development of DHF,
we used a rat pressure-overloaded model as a preserved
systolic but impaired diastolic function model. Given that
collagen accumulation is regulated by a balance of its synthesis
and degradation, the pressure-overloaded model may be
characterized as a “synthesis”-dominant model.12 On the
other hand, myocardial infarction is a “accelerated synthesis
and accelerated degradation” model with respect to collagen
turnover.17 Myocardial infarction is another leading cause to
provoke cardiac fibrosis. Therefore, our hypothesis should be
also tested in the ischemic heart model, as well as the
pressure-overloaded cardiac hypertrophy model.
Perspectives
CTGF is a secreted protein, and plasma CTGF concentration
correlates with the severity of several systemic fibrotic
disorders.18 Measurement of plasma CTGF concentrations is
easier and less invasive than assessment of CTGF levels in
biopsy samples. Furthermore, the present data suggest that
plasma concentration of CTGF or the ratio of plasma concentration of CTGF:BNP may be a diagnostic marker for
myocardial fibrosis. In addition, our data showing the inducibility of CTGF in CMs and the myocardial responsiveness to
Koitabashi et al
exogenously administered CTGF suggest that CTGF plays an
active role in cardiac progressive fibrosis and, thus, becomes
a good candidate molecule as a target of antifibrotic therapy.
Conclusions
The present study demonstrated the following: (1) production
of CTGF from CMs is associated with the myocardial
interstitial fibrosis and DHF; (2) increased CTGF expression
relative to BNP expression triggers excessive cardiac fibrosis
via BNP-mediated suppression of CTGF expression; and (3)
Aldo and TGF-␤ induce a disproportionate induction of
CTGF and BNP expression, whereas a mechanical stretch of
CM and G protein– coupled receptor ligands induces proportionate CTGF and BNP expression. These data suggest that
CTGF is a key molecule in the process of cardiac fibrosis and
that it may serve as a diagnostic marker and therapeutic target
for cardiac fibrosis and DHF.
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Acknowledgments
We are grateful to Miki Yamazaki for her technical assistance with
the cardiac myocytes culture and Yoshiko Nonaka for her excellent
preparation of histological samples.
Sources of Funding
This work was supported in part by a Grant-in-Aid for Scientific
Research (KAKENHI B-17390224 and S-15109010) from the Japan
Society for the Promotion of Science.
Disclosures
None.
References
1. Zile MR, Baicu CF, Gaasch WH. Diastolic heart failure–abnormalities in
active relaxation and passive stiffness of the left ventricle. N Engl
J Med. 2004;350:1953–1959.
2. Kass DA, Bronzwaer JG, Paulus WJ. What mechanisms underlie diastolic
dysfunction in heart failure? Circ Res. 2004;94:1533–1542.
3. Mundhenke M, Schwartzkopff B, Strauer BE. Structural analysis of
arteriolar and myocardial remodelling in the subendocardial region of
patients with hypertensive heart disease and hypertrophic cardiomyopathy. Virchows Arch. 1997;431:265–273.
4. Manabe I, Shindo T, Nagai R. Gene expression in fibroblasts and fibrosis:
involvement in cardiac hypertrophy. Circ Res. 2002;91:1103–1113.
CTGF vs BNP and Myocardial Fibrosis
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5. Kai H, Kuwahara F, Tokuda K, Imaizumi T. Diastolic dysfunction in
hypertensive hearts: roles of perivascular inflammation and reactive myocardial fibrosis. Hypertens Res. 2005;28:483– 490.
6. Blom IE, Goldschmeding R, Leask A. Gene regulation of connective
tissue growth factor: new targets for antifibrotic therapy? Matrix Biol.
2002;21:473– 482.
7. Ohnishi H, Oka T, Kusachi S, Nakanishi T, Takeda K, Nakahama M, Doi
M, Murakami T, Ninomiya Y, Takigawa M, Tsuji T. Increased expression
of connective tissue growth factor in the infarct zone of experimentally
induced myocardial infarction in rats. J Mol Cell Cardiol. 1998;30:
2411–2422.
8. Matsui Y, Sadoshima J. Rapid upregulation of CTGF in cardiac myocytes
by hypertrophic stimuli: implication for cardiac fibrosis and hypertrophy.
J Mol Cell Cardiol. 2004;37:477– 481.
9. Abreu JG, Ketpura NI, Reversade B, De Robertis EM. Connective-tissue
growth factor (CTGF) modulates cell signalling by BMP and TGF-beta.
Nat Cell Biol. 2002;4:599 – 604.
10. Kemp TJ, Aggeli IK, Sugden PH, Clerk A. Phenylephrine and
endothelin-1 upregulate connective tissue growth factor in neonatal rat
cardiac myocytes. J Mol Cell Cardiol. 2004;37:603– 606.
11. Cameron VA, Ellmers LJ. Minireview: natriuretic peptides during development of the fetal heart and circulation. Endocrinology. 2003;144:
2191–2194.
12. Diez J, Gonzalez A, Lopez B, Querejeta R. Mechanisms of disease:
pathologic structural remodeling is more than adaptive hypertrophy in
hypertensive heart disease. Nat Clin Pract Cardiovasc Med. 2005;2:
209 –216.
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Transcription of the SERCA2 gene is decreased in pressure-overloaded
hearts: A study using in vivo direct gene transfer into living myocardium.
J Mol Cell Cardiol. 1999;31:2167–2174.
14. Kapoun AM, Liang F, O’Young G, Damm DL, Quon D, White RT,
Munson K, Lam A, Schreiner GF, Protter AA. B-type natriuretic peptide
exerts broad functional opposition to transforming growth factor-beta in
primary human cardiac fibroblasts: fibrosis, myofibroblast conversion,
proliferation, and inflammation. Circ Res. 2004;94:453– 461.
15. Ahmed MS, Oie E, Vinge LE, Yndestad A, Oystein Andersen G,
Andersson Y, Attramadal T, Attramadal H. Connective tissue growth
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activation in heart failure in rats. J Mol Cell Cardiol. 2004;36:393– 404.
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Anderson GH Jr. Preoperative and postoperative renin levels in coarctation of the aorta. Circulation. 1982;66:513–514.
17. Jugdutt BI. Ventricular remodeling after infarction and the extracellular
collagen matrix: when is enough enough? Circulation. 2003;108:
1395–1403.
18. Dziadzio M, Usinger W, Leask A, Abraham D, Black CM, Denton C,
Stratton R. N-terminal connective tissue growth factor is a marker of the
fibrotic phenotype in scleroderma. Qjm. 2005;98:485– 492.
Increased Connective Tissue Growth Factor Relative to Brain Natriuretic Peptide as a
Determinant of Myocardial Fibrosis
Norimichi Koitabashi, Masashi Arai, Shinya Kogure, Kazuo Niwano, Atai Watanabe, Yasuhiro
Aoki, Toshitaka Maeno, Takashi Nishida, Satoshi Kubota, Masaharu Takigawa and Masahiko
Kurabayashi
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Hypertension. 2007;49:1120-1127; originally published online March 19, 2007;
doi: 10.1161/HYPERTENSIONAHA.106.077537
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2007 American Heart Association, Inc. All rights reserved.
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Increased connective tissue growth factor relative to brain natriuretic peptide
as a determinant of myocardial fibrosis
Norimichi Koitabashi, Masashi Arai, Shinya Kogure, Kazuo Niwano, Atai Watanabe,
Yasuhiro Aoki, Toshitaka Maeno, Takashi Nishida, Satoshi Kubota, Masaharu Takigawa,
Masahiko Kurabayashi
ONLINE DATA SUPPLEMENT
Address correspondence to:
Masashi Arai MD, PhD
Department of Medicine and Biological Science
Gunma University Graduate School of Medicine
3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
E-mail: [email protected]
TEL: (+81) 27-220-8142
FAX: (+81) 27-220-8158
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
Materials and Methods
Patients
The study was approved by the local ethics committee and conforms to the ethical
guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained
from all patients.
Forty-six consecutive patients with normal or minimally impaired left ventricular (LV)
ejection fraction (>40%) estimated by echocardiography who underwent endomyocardial
biopsy of the LV free wall in Gunma University Hospital were enrolled in this study.
Clinical diagnosis of these patients included hypertrophic cardiomyopathy (n=13),
hypertensive
heart
disease
(n=15),
dilated
cardiomyopathy
(n=7),
alcoholic
cardiomyopathy (n=3), sick sinus syndrome (n=1), hyperthyroidism (n=1), idiopathic
ventricular tachycardia (n=1), and other diseases (n=5). Of these patients, 31 patients who
had previous history of overt heart failure within the preceding year (i.e. dyspnea and rales
due to pulmonary congestion, as confirmed by chest radiography) in the absence of
impaired systolic function as estimated by echocardiography were designated as the
diastolic heart failure (DHF) group. Heart failure was clinically diagnosed according to the
criteria used in the Framingham Heart Study project
2
1
and the elevation of plasma BNP
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
concentration was confirmed. All patients in the DHF group showed any of
echocardiographic criteria of DHF, i.e. impaired relaxation, pseudonormal, and restrictive
patterns. Another 15 patients without a previous history of heart failure were designated as
the non-failing (NF) group. Patients with significant coronary stenosis in angiography,
moderate or severe valvular disease, secondary hypertension, renal failure (serum creatinine
concentration >2.0mg/dl), myocarditis, epicarditis, or uncontrolled decompensated
congestive heart failure were excluded. Patients with cardiac sarcoidosis pathologically
diagnosed by their endomyocardial biopsy (i.e., lymphocytic infiltration) were also
excluded from the present analysis. At least two endomyocardial samples were obtained
from the LV free wall in each patient, and hemodynamic parameters were measured with an
LV and Swan-Ganz catheters. Two-dimensional, M-mode, and Doppler ultrasound
recordings were performed in each patient using transthoracic echocardiography, and left
ventricular ejection fraction and mass index was calculated from the echocardiogram.
Peripheral blood samples were obtained within a week before or after cardiac
catheterization for determination of plasma brain natriuretic peptide (BNP) concentration.
BNP levels were measured using immunoradiometric assay.
Histochemical analysis and immunostaining
3
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
Endomyocardial biopsy samples were immediately fixed in 10% buffered formalin,
embedded in paraffin. Masson’s trichrome staining was performed for detection of collagen,
and five high-power field (X200) color images were randomly selected in each sample.
Myocardial fibrosis area (MFA) was determined by blue staining and quantified by an
automated image analysis system (MacScope)2.
Immunostaining with a human connective tissue growth factor (CTGF) antibody (Santa
Cruz Biotechnology, Inc.) and with a normal goat IgG1 (R&D systems) as a negative
control for non-specific staining was performed in the same serial sections as that used in
the MFA study. Variation of control IgG1 staining among samples was minimized, and
sections that demonstrated significantly higher staining intensity with CTGF antibody than
with control IgG1 were selected for densitometry 3. Average data of the percentage of
positively stained area relative to the sample area in 5 different positions in each sample
was used to determine the “CTGF-stained area”.
Animal models
Constriction of the suprarenal abdominal aorta was established with a 21G silver clip 4 in
male Wister rats (Charles River, Japan) weighing 250-300 g after intraperitoneal (IP)
injection of pentobarbital. After hemodynamic measurement on the experimental day, the
4
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
heart was excised and weighed. The LV was divided into three pieces for histological
analysis, RNA isolation and protein extraction. All animal experiments were performed
according to the Guide for the Care and Use of Laboratory Animals published by the US
National Institutes of Health and were approved by the Animal Research Committee of the
Gunma University Graduate School of Medicine.
Hemodynamic measurements in rats
Before constriction of the suprarenal abdominal aorta, blood pressure was measured by the
tail-cuff method.
On Day 28, hemodynamic parameters were measured using a pressure-volume (PV)
catheter. Rats were anesthetized with 2% isoflurane, and tracheostomy was performed to
allow mechanical ventilation. The LV apex was exposed under sternotomy, and a microtip
PV catheter (SPR-838, Millar Instruments) was advanced through the apex along the
longitudinal axis. Absolute volume was calibrated, and PV data were measured at a steady
state and during transient reduction of venous return, as described previously 5. Blood
pressure was measured in rats before sacrifice on Days 1, 4, 7 and 14 using a fluid-filled
manometer via the carotid artery under isoflurane anesthesia 4
To assess ventricular function and hypertrophy, transthoracic echocardiography was
5
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
performed with a 10-MHz transducer (EUB-6000, HITACHI) in all rats on Days 0, 1, 4, 7,
14 and 28. Rats were sedated with ketamine (40 mg/kg IP) and xylazine (10 mg/kg IP) to
maintain blood pressures equivalent to the awake condition. M-mode tracings and
transmitral pulse wave Doppler spectra were measured as described previously 6.
RNA isolation and Northern blot analysis
Total cellular RNA was isolated using the ISOGEN reagent (Nippongene) in accordance
with the manufacturer’s instruction. Probes for Northern blots were as follows: 1) rat CTGF
(nucleotide +1201~1795 bp; Acc. No. NM_022266)
7
isolated using RT-PCR; 2) rat
procollagen type 1α1 (COL1A1) (nucleotide +5096~5669 bp; Acc. No. Z78279) isolated
using RT-PCR; 3) rat procollagen type 3α1 (COL3A1) (nucleotide +2046~2350 bp; Acc.
No. XM_216813) isolated using RT-PCR; 4) a 628-bp fragment of the rat BNP cDNA 8;
and 5) a 490-bp fragment of the rat transforming growth factor (TGF) β1 cDNA 9; 6) rat
sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) (nucleotide +3557-3865 bp; Acc. No.
J04023) isolated usinf RT-PCR. Messenger RNA levels were quantified using scanning
autoradiographs and computerized optical densitometry and were normalized with 28S
rRNA.
6
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
Plasma analysis in rats
Blood sample was obtained via the carotid artery before sacrifice. Plasma TGF-β
concentration was examined by enzyme-linked immunosorbent assay. Plasma endothelin
(ET)1 and aldosterone (Aldo) concentration were examined by radioimmunoassay.
Cell culture
Neonatal rat cardiac myocytes were isolated from 1- to 3-day-old Wistar rats, as previously
described 10, and were seeded on gelatin-coated tissue culture plates or FlexWell plates
(Flexcell International) 10. Cardiac myocytes were cultured for 24 h in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine serum and 0.1 mmol/L
bromodeoxyuridine and then switched to DMEM containing 0.1%
insulin/transferring/selenium (Gibco) before being stimulated with various agents 24 h later.
For cell stretch experiments, cardiac myocytes were stretched biaxially (15%, 0.5 Hz) using
a FlexCell Strain Unit (FX-4000; FlexCell International). Control myocytes were cultured
on FlexWell plates without mechanical stretch.
Neonatal rat cardiac fibroblasts were prepared as described previously
10
. After the
second passage, cells were plated (3×105 cells) in 60 mm-culture dishes and grown in
DMEM containing 10% FBS. Just before reaching confluence, the medium was replaced
7
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
with serum-free DMEM or with conditioned medium from cardiac myocytes culture.
Cardiac myocyte-conditioned medium was prepared as a supernatant of cultured media
after 24 h stimulation of cardiac myocytes by TGF-β, ET-1 or Aldo. The medium for
neonatal cardiac fibroblasts was replaced with the cardiac myocyte-conditioned medium,
and fibroblasts were harvested 24 hrs after the replacement. In neutralizing antibody
experiments, antibodies for CTGF and TGF-β (R&D systems) and normal IgG (R&D
systems) were supplemented at the time of medium replacement.
Immunofluorescent microscopic analysis
Immunofluorescent microscopic analysis was performed with CTGF antibody and
Cy3-conjugated anti-goat IgG antibody (Sigma) in methanol-fixed cultured cells. Mouse
monoclonal sarcomeric actinin antibody (Sigma) and FITC-conjugated anti-mouse IgG
antibody (Sigma) were used for detection of cardiac myocytes. Mouse monoclonal
vimentin antibody (Sigma) was used for detection of cardiac fibroblasts.
Western blotting
The protein extracts of in vivo experiments were homogenized with buffer containing 10
mmol/L imidazole, 300 mmol/L sucrose, and protease inhibitors. Cultured cells were lysed
8
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
by adding ice-cold radioimmunoprecipitation buffer. Protein concentration was determined
by the Bradford dye-binding method (BioRad). The cell lysates or culture media were
subjected to electrophoresis on a SDS-13% polyacrylamide gel and transferred to
nitrocellulose membranes. Membranes were then blocked in TBS (10 mmol/L Tris, pH 7.6,
and 150 mmol/L NaCl) containing 5% skim milk, followed by overnight incubation with
anti-CTGF antibody (Santa Cruz). Chemiluminescent detection was performed with the
enhanced chemiluminescence protocol (ECL; Amersham Bioscience). After CTGF
detection, the membranes were stripped and reprobed with anti sarcomeric α-actin (Sigma)
as an internal control.
Reagents
Synthetic rat BNP and recombinant human ET-1 were obtained from the Peptide Institute.
Norepinephrine, Ang II, Aldo, and human recombinant TGF-β were obtained from Sigma,
Bachem, Acros Organics, and Roche, respectively. Recombinant human CTGF was purified
as previously described 11. KT5823 was obtained from Calbiochem.
Statistical analysis
Data are expressed as means±SD. Overall differences within groups were determined by
9
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
one-way analysis of variance. When this test indicated that differences existed, individual
experimental groups were compared by Bonferroni’s test. Categorical variables were
analyzed by the χ2 test or Fisher’s exact probability test when necessary. Bivariate
correlations between variables were assessed by simple least-squares linear regression
analysis. A probability value <0.05 was considered statistically significant.
10
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
References for Supplemental Methods
1.
McKee PA, Castelli WP, McNamara PM, Kannel WB. The natural history of
congestive heart failure: the Framingham study. N Engl J Med.
1971;285:1441-1446.
2.
Querejeta R, Varo N, Lopez B, Larman M, Artinano E, Etayo JC, Martinez Ubago
JL, Gutierrez-Stampa M, Emparanza JI, Gil MJ, Monreal I, Mindan JP, Diez J.
Serum carboxy-terminal propeptide of procollagen type I is a marker of myocardial
fibrosis in hypertensive heart disease. Circulation. 2000;101:1729-1735.
3.
Wallace CK, Stetson SJ, Kucuker SA, Becker KA, Farmer JA, McRee SC, Koerner
MM, Noon GP, Torre-Amione G. Simvastatin decreases myocardial tumor necrosis
factor alpha content in heart transplant recipients. J Heart Lung Transplant.
2005;24:46-51.
4.
Takizawa T, Arai M, Yoguchi A, Tomaru K, Kurabayashi M, Nagai R. Transcription
of the SERCA2 gene is decreased in pressure-overloaded hearts: A study using in
vivo direct gene transfer into living myocardium. J Mol Cell Cardiol.
1999;31:2167-2174.
11
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
5.
Pacher P, Mabley JG, Liaudet L, Evgenov OV, Marton A, Hasko G, Kollai M, Szabo
C. Left ventricular pressure-volume relationship in a rat model of advanced
aging-associated heart failure. Am J Physiol Heart Circ Physiol.
2004;287:H2132-2137.
6.
Masuyama T, Yamamoto K, Sakata Y, Doi R, Nishikawa N, Kondo H, Ono K,
Kuzuya T, Sugawara M, Hori M. Evolving changes in Doppler mitral flow velocity
pattern in rats with hypertensive hypertrophy. J Am Coll Cardiol.
2000;36:2333-2338.
7.
Yokoi H, Mukoyama M, Sugawara A, Mori K, Nagae T, Makino H, Suganami T,
Yahata K, Fujinaga Y, Tanaka I, Nakao K. Role of connective tissue growth factor in
fibronectin expression and tubulointerstitial fibrosis. Am J Physiol Renal Physiol.
2002;282:F933-942.
8.
Kojima M, Minamino N, Kangawa K, Matsuo H. Cloning and sequence analysis of
cDNA encoding a precursor for rat brain natriuretic peptide. Biochem Biophys Res
Commun. 1989;159:1420-1426.
9.
Tsuji T, Okada F, Yamaguchi K, Nakamura T. Molecular cloning of the large subunit
of transforming growth factor type beta masking protein and expression of the
mRNA in various rat tissues. Proc Natl Acad Sci U S A. 1990;87:8835-8839.
12
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
10.
Yokoyama T, Sekiguchi K, Tanaka T, Tomaru K, Arai M, Suzuki T, Nagai R.
Angiotensin II and mechanical stretch induce production of tumor necrosis factor in
cardiac fibroblasts. Am J Physiol. 1999;276:H1968-1976.
11.
Nishida T, Nakanishi T, Shimo T, Asano M, Hattori T, Tamatani T, Tezuka K,
Takigawa M. Demonstration of receptors specific for connective tissue growth
factor on a human chondrocytic cell line (HCS-2/8). Biochem Biophys Res Commun.
1998;247:905-909.
13
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
Figure legends for supplemental figures
Supplemental Figure I
LV diastolic function and myocardial fibrosis in rats with different ratios of CTGF and BNP
mRNAs. Representative echocardiograms (A), and histological analysis (B); In each row,
left, central and right panels show sham, a AC rat with comparable mRNA levels of CTGF
and BNP, and a AC rat with disproportionate increase of CTGF against BNP, respectively;
(A) M-mode echocardiography (left) and transmitral Doppler flow pattern (right). Mean
E/A ratios of consecutive five beats are shown below the panels. (B) Histological analysis
of LVs; upper panel, Masson’s trichrome staining; lower panel, immunohistologic staining
with an anti-CTGF antibody. All scale bars are 50 μm. Arrows indicate CTGF-positive
cardiac myocytes (CM). Asterisks indicate vascular structure.
Supplemental Figure II
(A) Correlation between CTGF/BNP expression ratio and EDPVR in Day 28 sham (n=5)
and AC (n=14) rats.
(B) Correlation between CTGF/BNP expression ratio and COL1A1 mRNA expression level
estimated by quantitative Northern blot in Day 28 sham (n=5) and AC (n=14) rats.
14
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
Supplemental Figure III
Difference in plasma concentration of TGFβ, ET1 and Aldo between higher (filled columns,
n=7) and lower (open columns, n=7) groups of CTGF/BNP expression ratio in Day 28 AC
rats.
Supplemental Figure IV
Immunofluorescent imaging of CTGF (detected by Cy3) and vimentin (detected by FITC)
protein in cultured cardiac cells. Vimentin is an intermediated filament, which has been
shown to be abundant in fibroblasts. A right cell in these panels shows a cardiac fibroblast.
Supplemental Figure V
(A) Northern blot showing the effect of norepinephrine (NE) and angiotensin II (AngII) on
CTGF and BNP mRNA levels in cardiac myocytes.
(B) Northern blot showing the temporal changes of CTGF mRNA levels in CM in
response to synthetic BNP (sBNP: 0.1 μmol/L).
(C) Effect of the protein kinase G inhibitor, KT5823 (1 μmol/L), on the
BNP-mediated suppression of CTGF mRNA levels in CM. Four hours after
15
Koitabashi et al. CTGF vs BNP, and myocardial fibrosis
HYPERTENSION/2006/077537/R4
incubation with synthetic BNP in the presence or absence of KT5823, CTGF
mRNA expression was examined by Northern blot analysis. Experiments were
performed in triplicate.
Supplemental Figure VI
(A) Northern blot showing basal expression of CTGF in cultured neonatal rat cardiac
myocytes (CM) and cardiac fibroblasts (CFB). Cultured rat cardiac fibroblasts were used
after the second passage. Bar graphs show mean values of four independent experiments
relative to the mean level of CTGF mRNA in cardiac myocytes. * P<0.05 vs. cardiac
myocytes.
(B) Northern blot showing the effect of various humoral factors (4 hours) on CTGF mRNA
level in cultured cardiac fibroblasts. Bar graphs show mean values of four independent
experiments. Values in the vehicle-stimulated group are defined as 1. *P<0.05 vs.
vehicle-treated group.
16
Supplemental Table I. Clinical characteristics in patients
Variable
NF
(N=15)
DHF
(N=31)
P
Age
58±15
57±16
NS
9/6
20/11
NS*
HCM/HHD/other
7/3/5
6/12/13
NS*
Prior Medication
2
19
<0.05†
Hypertension(N)
3
12
NS†
Atrial Fibrillation(N)
0
14
<0.01†
Renal Failure(N)
0
2
NS†
Diabetes(N)
3
3
NS†
Prior PCI/CABG (N)
1
0
NS†
PAWP (mmHg)
11.8±3.0
10.5±6.8
NS
LVEDP (mmHg)
15.0±8.8
18.0±8.3
NS
LVEF (%)
62.5±9.6
54.1±12.8
NS
LVEDD (mm)
48.4±8.8
52.3±11.3
NS
LAD
37.5±4.6
43.6±6.3
<0.05
153.6±134.5
189.4±131.6
NS
E/A
0.89 ±0.55
0.79±0.72
NS
DcT (msec)
226.7±24.6
195.0±31.9
NS
BNP (pg/mL)
68.2±73.5
263.5±214.2
<0.05
Sex (Male/Female)
LVM (g)
Prior Medication: angiotensin converting enzyme inhibitor/angiotensin II receptor blocker/aldosterone
blocker/beta
adrenoceptor
blocker;
HHD,
hypertensive
heart
disease;
HCM,
hypertrophic
cardiomyopathy; PCI, percutaneous coronary intervention; CABG, coronary artery bypass graft; PAWP,
mean pulmonary artery wedge pressure; LVEDP, left ventricular end-diastolic pressure; MFA, myocardial
fibrosis area; LVEF, left ventricular ejection fraction; LVEDD, left ventricular end-diastolic diameter; LAD,
left atrial diameter, LVM, left ventricular mass; DcT, deceleration time; P, ANOVA with the exceptions
indicated as follows:* χ2 test; † Fisher’s exact probability test
17
Supplemental Table II. Time-dependent changes in cardiac morphologic and functional parameters after aortic
constriction
Day １
Sham
n=3
Day 4
AC
n=4
Sham
n=4
Day 14
Day 7
AC
n=6
Sham
n=5
AC
n=12
Sham
n=4
Day 28
AC
n=5
Sham
n=5
AC
n=14
BW, g
258±8
280±20
257±1
250±14
281±7
261±7
318±12
318±6
385±12
363±11
HR
342±19
340 ±20
312±11
320 ±18
300 ±19
341 ±18
333 ±26
316 ±19
350 ±11
341 ±18
SBP, mmHg
98 ±10
160 ±25*
103 ±10
155 ±25*
98 ±9
149 ±27*
100 ±5.9
162 ±26*
113±9
141 ±20*
LVEDD, mm
7.25 ±0.25
7.70 ±0.03
7.15 ±0.23
7.72 ±0.08
7.42 ±0.78
7.70 ±0.17
7.90 ±0.21
7.70 ±0.24
8.26 ±0.18
8.03 ±0.17
FS, %
35.0 ±3.0
32.2 ±1.3
32.4 ±2.6
33.0 ±3.3
33.0 ±0.8
31.2 ±1.4
33.5 ±2.5
33.9 ±1.6
34.6 ±1.1
32.3 ±1.6
LVM, mg
466 ±18
457 ±60
526 ±29
637 ±18
540 ±29
788 ±26*
696 ±32
958 ±64*
706 ±27
991 ±84*
1.75±0.10
2.01 ±0.17
2.17 ±0.3
2.13 ±0.04
1.92 ±0.11
2.55 ±0.25
2.09 ±0.26
1.89 ±0.23
2.00 ±0.11
1.88 ±0.23
LVW/BW, mg/g 2.06 ±0.19
2.19 ±0.17
2.03 ±0.11
2.45 ±0.14*
2.00 ±0.06 2.80 ±0.10*
2.06 ±0.07
2.78 ±0.07*
1.86 ±0.04
2.61 ±0.08*
0.89 ±0.11
1.01 ±0.37
1.10 ±0.07
1.38 ±0.15
1.06 ±0.10
0.76 ±0.05
2.66 ±0.19*
1.00 ±0.24
2.85 ±0.64*
E/A
MFA, %
2.93 ±0.71
* P<0.05 vs. Sham
BW, body weight, HR, heart rate, LVEDD, left ventricular end-diastolic diameter, FS, fractional shortening, LVM, left ventricular mass, LVW/BW, left
ventricular weight to body weight ratio, MFA, myocardial fibrosis area
18
Supplemental Table III. Hemodynamic parameters in sham- and AC-operated rats
measured by the Millar pressure-volume conductance catheter system
Variable
Sham
Day 28
N=5
AC
Day 28
N=14
LVESP (mmHg)
108.7±5.7
141.0±10.8*
LVEDP (mmHg)
7.0±1.6
12.0±3.3
EF (%)
50.3±4.9
41.5±2.2
dP/dtmax (mmHg/s)
9229±723
9178±958
dP/dtmin (mmHg/s)
-7971±536
-7810±725
PRSW (mmHg)
123.0±25.3
112.4±17.0
Emax (mmHg/μL)
2.02±0.77
1.27±0.27
0.010±0.004
0.052±0.013*
12.1±0.9
17.3±0.9*
0.002±0.001
0.004±0.003
EDPVR (mmHg/μL)
τ (msec)
κ
* P<0.05 vs. Sham
LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-diastolic
pressure; LVESV, left ventricular end-systolic volume; LVEDV, left ventricular enddiastolic volume; EF, ejection fraction; dP/dtmax, maximal rate of pressure
development; dP/dtmin, maximal rate of pressure decline; PRSW, preload-recruitable
stroke work; Emax, maximal elastance; EDPVR, end-diastolic pressure-volume
relationship; τ, monoexponential time constant of relaxation; κ, constant of chamber
stiffness.
19
Supplemental Table IV. Correlation between CTGF/BNP ratio and
hemodynamic or genetic parameters
Correlation
Coefficient
P value
Hemodynamic parameters
LVESP
LVEDP
dP/dtmax
dP/dtmin
PRSW
Emax
τ
EDPVR
FS*
E/A*
0.246
0.349
0.310
-0.135
0.330
0.204
0.270
0.720
0.161
0.315
0.274
0.113
0.163
0.543
0.159
0.393
0.337
<0.001
0.159
0.009
Gene expressions
COL1A1
COL3A1
TGFβ
SERCA2a
0.458
0.270
0.050
0.179
<0.001
0.006
0.622
0.183
Histomorphological parameter
MFA
0.525
<0.001
Variable
LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular enddiastolic pressure; dP/dtmax, maximal rate of pressure development; dP/dtmin,
maximal rate of pressure decline; PRSW, preload-recruitable stroke work;
Emax, maximal elastance; τ, monoexponential time constant of relaxation;
EDPVR, end-diastolic pressure-volume relationship; FS, fractional shortening;
COL1A1, procollagen type 1α1 mRNA, COL3A1; procollagen type 3 α1 mRNA;
TGFβ, transforming growth factor β1 mRNA; SERCA2a, sarcoplasmic reticulum
Ca2+ ATPase 2a mRNA; MFA, myocardial fibrosis area. * Parameters
estimated by echocardiography
20
Supplemental Figure I
A
Sham day 28
AC day 28
CTGF>BNP
AC day 28
CTGF=BNP
E
A
E/A=1.3
E/A=1.6
B
Sham Day 28
E/A=5.2
AC Day 28
CTGF>BNP
AC Day 28
CTGF=BNP
*
*
*
*
*
*
*
21
*
Supplemental Figure II
B
0.2
5
r=0.720
P<0.001
COL1A1 mRNA
Fold Increase
EDPVR (mmHg/μL)
A
0.15
0.1
0.05
4
3
2
r=0.458
P<0.001
1
0
0
1
2
CTGF/BNP ratio
00
3
22
1
2
CTGF/BNP ratio
3
Supplemental Figure III
CTGF/BNP<1.2
30
20
10
0
6
5
4
3
2
1
0
pg/mL
40
Plasma Aldosterone
Plasma Endothelin 1
pg/mL
ng/mL
Plasma TGFβ
CTGF/BNP>1.2
23
1200
1000
800
600
400
200
0
*
Supplemental Figure IV
Vimentin
Merged
CTGF
24
Supplemental Figure V
NE (μmol/L)
A
0
1
10
AngII (μmol/L)
0
1
10
CTGF
BNP
28S
B
C
sBNP
0
1
2
sBNP
(μmol/L)
4 hrs
CTGF
CTGF
28S
28S
25
Vehicle
0
0.1
KT5823
0.5
0
0.1 0.5
A
Relative Intensity
Supplemental Figure VI
*
3
2
1
0
CM
CFB
CTGF
B
Relative Intensity
28S
1.5
*
1
0.5
0
Veh
TGFβ
(ng/mL)
AngII
(μmol/L)
1
0.1
10
1
CTGF
28S
26
NE
ET1
(μmol/L) (μmol/L)
10
0.01
0.1
Aldo
(μmol/L)
0.1
1