increased connective tissue growth factor relative to...

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 1 and the elevation of plasma BNP

2


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


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


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


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


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


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


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


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


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


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


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


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


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


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

Age

Sex (Male/Female)

HCM/HHD/other

Prior Medication

Hypertension(N)

Atrial Fibrillation(N)

Renal Failure(N)

Diabetes(N)

Prior PCI/CABG (N)

PAWP (mmHg)

LVEDP (mmHg)

LVEF (%)

LVEDD (mm)

LAD

LVM (g)

E/A

DcT (msec)

BNP (pg/mL)

58±15

9/6

7/3/5

2

3

0

0

3

1

11.8±3.0

15.0±8.8

62.5±9.6

48.4±8.8

37.5±4.6

153.6±134.5

0.89 ±0.55

226.7±24.6

68.2±73.5

57±16

20/11

6/12/13

19

12

14

2

3

0

10.5±6.8

18.0±8.3

54.1±12.8

52.3±11.3

43.6±6.3

189.4±131.6

0.79±0.72

195.0±31.9

263.5±214.2

VariableNF

(N=15)DHF

(N=31)

NS

NS*

NS*

<0.05†

NS†

<0.01†

NS†

NS†

NS†

NS

NS

NS

NS

<0.05

NS

NS

NS

<0.05

P

Supplemental Table I. Clinical characteristics in patients

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

Shamn=5

ACn=12

Shamn=5

ACn=14

Day 7 Day 28

BW, g

HR

SBP, mmHg

LVEDD, mm

FS, %

LVM, mg

E/A

LVW/BW, mg/g

MFA, %

257±1

312±11

103 ±10

7.15 ±0.23

32.4 ±2.6

526 ±29

2.17 ±0.3

2.03 ±0.11

1.10 ±0.07

250±14

320 ±18

155 ±25*

7.72 ±0.08

33.0 ±3.3

637 ±18

2.13 ±0.04

2.45 ±0.14*

1.38 ±0.15

Shamn=4

ACn=5

Day 14

Shamn=4

ACn=6

Day 4

281±7

300 ±19

98 ±9

7.42 ±0.78

33.0 ±0.8

540 ±29

1.92 ±0.11

2.00 ±0.06

1.06 ±0.10

261±7

341 ±18

149 ±27*

7.70 ±0.17

31.2 ±1.4

788 ±26*

2.55 ±0.25

2.80 ±0.10*

2.93 ±0.71

318±12

333 ±26

100 ±5.9

7.90 ±0.21

33.5 ±2.5

696 ±32

2.09 ±0.26

2.06 ±0.07

0.76 ±0.05

318±6

316 ±19

162 ±26*

7.70 ±0.24

33.9 ±1.6

958 ±64*

1.89 ±0.23

2.78 ±0.07*

2.66 ±0.19*

385±12

350 ±11

113±9

8.26 ±0.18

34.6 ±1.1

706 ±27

2.00 ±0.11

1.86 ±0.04

1.00 ±0.24

363±11

341 ±18

141 ±20*

8.03 ±0.17

32.3 ±1.6

991 ±84*

1.88 ±0.23

2.61 ±0.08*

2.85 ±0.64*

* 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

258±8

342±19

98 ±10

7.25 ±0.25

35.0 ±3.0

466 ±18

1.75±0.10

2.06 ±0.19

0.89 ±0.11

280±20

340 ±20

160 ±25*

7.70 ±0.03

32.2 ±1.3

457 ±60

2.01 ±0.17

2.19 ±0.17

1.01 ±0.37

Shamn=3

ACn=4

Day １

Supplemental Table II. Time-dependent changes in cardiac morphologic and functional parameters after aortic constriction

18

Supplemental Table III. Hemodynamic parameters in sham- and AC-operated rats measured by the Millar pressure-volume conductance catheter system

ShamDay 28

N=5

ACDay 28N=14

LVESP (mmHg)

LVEDP (mmHg)

EF (%)

dP/dtmax (mmHg/s)

dP/dtmin (mmHg/s)

PRSW (mmHg)

Emax (mmHg/μL)

EDPVR (mmHg/μL)

τ (msec)

κ

108.7±5.7

7.0±1.6

50.3±4.9

9229±723

-7971±536

123.0±25.3

2.02±0.77

0.010±0.004

12.1±0.9

0.002±0.001

141.0±10.8*

12.0±3.3

41.5±2.2

9178±958

-7810±725

112.4±17.0

1.27±0.27

0.052±0.013*

17.3±0.9*

0.004±0.003

* 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 end-

diastolic 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

Variable

VariableCorrelationCoefficient P value

Hemodynamic parametersLVESPLVEDPdP/dtmax

dP/dtmin

PRSWEmaxτEDPVRFS*E/A*

Gene expressionsCOL1A1COL3A1TGFβSERCA2a

Histomorphological parameterMFA

0.2460.3490.310-0.1350.3300.2040.2700.7200.1610.315

0.4580.2700.0500.179

0.525

0.2740.1130.1630.5430.1590.3930.337<0.0010.1590.009

<0.0010.0060.6220.183

<0.001

Supplemental Table IV. Correlation between CTGF/BNP ratio and hemodynamic or genetic parameters

LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-

diastolic 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

Sham day 28 AC day 28CTGF=BNP

AC day 28CTGF>BNPA

E/A=1.6 E/A=1.3 E/A=5.2

Sham Day 28 AC Day 28CTGF>BNP

AC Day 28CTGF=BNP

B

*

*

*

*

*

*

*

*

EEAA

Supplemental Figure I

21

A B

r=0.720P<0.001

0

0.05

0.1

0.15

0.2

0 1 2 3CTGF/BNP ratio

EDPV

R (m

mH

g/μL

)

r=0.458 P<0.001C

OL1

A1

mR

NA

Fold

Incr

ease

0 1 2 3CTGF/BNP ratio

5

4

3

2

1

0

Supplemental Figure II

22

Plasma TGFβ

0

10

20

30

40

ng/m

L

Plasma Endothelin 1

0123456

pg/m

L

Plasma Aldosterone

*

0200400600800

10001200

pg/m

L

CTGF/BNP<1.2 CTGF/BNP>1.2

Supplemental Figure III

23

Supplemental Figure IV

CTGFVimentin Merged

24

NE (μmol/L)0 1010 101

AngII (μmol/L)

Supplemental Figure V

1 2 4

sBNPhrs0

CTGF

28S

0 0.5sBNP(μmol/L) 0.1 0 0.50.1

KT5823Vehicle

CTGF

28S

B C

A

28S

BNP

CTGF

25

CM CFB

CTGF

28S

0

1

2

3

Rel

ativ

e In

tens

ityA *

Veh 1 10 0.1 1 10 0.01 0.1 0.1 1

TGFβ(ng/mL)

AngII(μmol/L)

NE(μmol/L)

ET1(μmol/L)

Aldo(μmol/L)

CTGF

28S

0

0.5

1

1.5 *

Rel

ativ

e In

tens

ityB

Supplemental Figure VI

26

increased connective tissue growth factor relative to...

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