Increased Connective Tissue Growth Factor Relative to ...hyper.ahajournals.org/content/hypertensionaha/49/5/1120.full.pdf · in AC rats, RNA isolation and Northern blot analysis,
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Increased Connective Tissue Growth Factor Relative toBrain Natriuretic Peptide as a Determinant of
Abstract—Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was toinvestigate 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 thedevelopment of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsysamples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiacmyocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. Ina rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportionto one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP waspositively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression.Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cellsdemonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, Gprotein–coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereasaldosterone and transforming growth factor-� preferentially induced expression of the CTGF gene. Finally, exogenousBNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase inCTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis anddiastolic heart failure. (Hypertension. 2007;49:1120-1127.)
Epidemiological studies have established that 40% to 50%of patients with heart failure have normal or minimally
impaired left ventricular (LV) ejection fraction, a clinicalsyndrome that is commonly referred to as diastolic heartfailure (DHF). These patients typically have cardiac hyper-trophy that is induced by long-standing hypertension or byprimary hypertrophic cardiomyopathy, as well as increasedpassive LV stiffness.1 Among various molecular mechanismsthat regulate LV stiffness,2 abnormalities in the transcrip-tional or posttranscriptional regulation of the collagen genecan result in the disproportionate accumulation of fibroustissue and elevation of stiffness in the hypertrophied heart.2,3
Recent studies have shown that, in addition to mechanicalload, autocrine, paracrine, and endocrine factors, such asangiotensin II, aldosterone (Aldo), endothelin-1 (ET1), natri-uretic peptides, osteopontin, and transforming growthfactor-�1 (TGF-�), play important roles in the development
of myocardial hypertrophy and fibrosis.4,5 However, the precisemolecular mechanisms that initiate and promote myocardialfibrosis and increases in ventricular stiffness remain largelyunknown.
Connective tissue growth factor (CCN2/CTGF) belongs tothe CCN (Cyr61, CTGF, and Nov) family of immediate earlygenes, which are highly conserved among species.6 Thiscysteine-rich secreted protein may contribute to progressivefibrosis and excessive scarring in various systemic and localfibrotic diseases.6 Further, CTGF expression is increased inthe hypertrophied and failing myocardium of experimentalanimal models.7,8 CTGF is also an essential mediator for thebiological actions of TGF-�6 and its downstream signal trans-duction elements.9 However, a recent in vitro study dem-onstrated that CTGF is 1 of the earliest growth factorstranscriptionally induced by hypertrophic stimuli in cardiacmyocytes (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 ofMedicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
Correspondence to Masashi Arai, Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-22Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail [email protected]
In this study, to confirm the involvement of CTGF in themyocardial fibrosis, we first investigated CTGF protein produc-tion in myocardial biopsy samples of patients with DHF.Secondly, by using the pressure overload rat model with asuprarenal abdominal aortic constriction (AC), which mimics amodel of DHF,5 we determined and compared the temporalchanges of CTGF, TGF-�, and an antifibrotic peptide, brainnatriuretic peptide (BNP).11 Because the collagen accumulationlevel is reflected by the balance of profibrotic factors andantifibrotic factors,12 we investigated their functional interac-tions, especially between CTGF and BNP, in the development ofmyocardial fibrosis and DHF.
MethodsAn expanded Methods section is available online at http://hyper.ahajournals.org.
Forty-six consecutive patients with normal or minimally impairedLV ejection fraction (�40%), estimated by echocardiography, whounderwent endomyocardial biopsy of the LV-free wall in GunmaUniversity Hospital were enrolled in this study (Table S1). All of thepatients were clinically stable when the biopsy was performed. Ofthese patients, 31 patients who had a previous history of overt heartfailure within the preceding year in the absence of impaired systolicfunction as estimated by echocardiography were designated as theDHF group. Another 15 patients without a previous history of heartfailure were designated as the nonfailing (NF) group. The clinicaldiagnosis and the exclusion criteria are described in the expandedMethods section.
AC was established with a 21G silver clip13 in male Wistar rats(Charles River, Japan) weighing 250 to 300 g. Cell culture, histo-chemical analysis and immunostaining, hemodynamic measurementsin AC rats, RNA isolation and Northern blot analysis, Westernblotting, and statistical analysis are described in the expanded Methodssection online.
ResultsElevated Levels of CTGF Protein in CMCorrelates With Myocardial Interstitial Fibrosis inPatients With Preserved Ejection FactorClinical characteristics of the NF and DHF groups aresummarized in Table S1. There were no significant differ-ences in age, sex, clinical diagnosis, and frequency ofcomplicated disease, except for atrial fibrillation, when com-paring the 2 groups. Sixty-one percent of DHF patients hadbeen given previous medication, including angiotensin-converting enzyme inhibitors and/or �-adrenoceptor block-ers, because of their previous history of congestive heartfailure. Pulmonary artery wedge pressure and LV end-dia-stolic pressure were not different when comparing the 2groups. Furthermore, there were no significant differences inechocardiographic parameters when comparing the 2 groupsexcept for left atrial dimension. Plasma BNP concentrationwas significantly elevated in the DHF group. RepresentativeLV biopsy samples taken from a patient from the NF groupwith hypertension and a patient from the DHF group are
Figure 1. Myocardial fibrosis and CTGFprotein in endomyocardial biopsy sam-ples. A through D, Main panels show high-power fields (�400). Left small panelsshow low-power fields (�100). All scalebars are 50 �m. A and C, Masson’strichrome staining; B and D, immunostain-ing for CTGF; A and B, NF group: A77-year–old man with mild LV hypertrophyand chronic hypertension, without previ-ous history of pulmonary congestion. HisLV ejection fraction was 45%. PlasmaBNP concentration was 76.2 pg/mL. Cand D, DHF group: A 74-year–old manwith mild LV hypertrophy, chronic atrialfibrillation, and hypertension. His LV ejec-tion fraction was 75%. Plasma BNP con-centration was 135 pg/mL. E, Comparisonbetween NF (n�15) and DHF (n�31) inMFA estimated by Masson’s trichromestaining in NF (n�15) and DHF (n�31). F,Comparison between NF (n�15) and DHF(n�31) in positive-stained area with CTGFantibody. G, Correlation between MFA andCTGF-stained area among all of thepatients enrolled (n�46). �, NF; ●, DHF.
Koitabashi et al CTGF vs BNP and Myocardial Fibrosis 1121
by guest on June 25, 2018http://hyper.ahajournals.org/
illustrated in Figure 1. Biopsies from the NF patient showedmild hypertrophic myocytes but no interstitial fibrosis byMasson’s trichrome staining (Figure 1A). CTGF immuno-staining of serial sections showed a small amount of CTGFprotein in the myocytes (Figure 1B). By contrast, biopsiesfrom 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 inDHF patients (Figure 1E and 1F). Interestingly, the CTGF-stained area correlated with MFA (r�0.638; P�0.001;Figure 1G).
CTGF and BNP Gene Expression AreCoordinately Induced Early in the Development ofCardiac Hypertrophy and FibrosisTo investigate the role of CTGF for the development ofcardiac fibrosis, we created a rat pressure overload cardiachypertrophy model by constricting the abdominal aorta. Inaccordance with the increase of systolic blood pressure, on
day 4 after AC operation and until day 28, LV weight/bodyweight ratio, a parameter of LV hypertrophy, significantlyincreased (Table S2). Furthermore, MFA was significantlyincreased on day 14 after AC.
Quantitative Northern blot analysis revealed that CTGFmRNA levels peaked on day 1, whereas TGF-� mRNA levelsincreased gradually and peaked on day 7 (Figure 2A).Furthermore, procollagen type 1�1 (COL1A1) mRNA levelswere significantly increased on day 7 and continued toincrease until day 28. Interestingly, the temporal course ofchanges 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 AssociatedWith Myocardial Fibrosis and VentricularStiffness at a Later Stage of Cardiac HypertrophyAlthough a correlation between CTGF and BNP mRNAexpression was observed during the entire experimentalperiod (N�69; r�0.804; P�0.001; Figure not shown), the
Figure 2. Gene expressions in hypertro-phied rat hearts. A, Temporal expressionpatterns of mRNAs normalized to 18SrRNA. S-0, rat euthanized immediatelyafter sham operation; AC-1, 4, 7, 14, and28, rats euthanized on designated dayafter AC or sham operation; *P�0.05 vssham-operated rats at same postoperativeperiods. B and C, Correlation betweenCTGF and BNP mRNA levels in AC rats ondays 1, 4, and 7 (B) and on days 14 and28 (C). D and E, Representative Northernblot analysis of CTGF, BNP, TGF-�, andCOL1A1 (procollagen type 1�1) mRNAsand 28S and 18S rRNAs on day 4 (D) andday 28 (E).
1122 Hypertension May 2007
by guest on June 25, 2018http://hyper.ahajournals.org/
correlation was weaker between day 14 and day 28 (N�19;r�0.645; P�0.001; Figure 2C and 2E) when compared withthe early stage of cardiac hypertrophy (day 1 to day 7;r�0.836; Figure 2B). As shown in Figure 2C, some ratsexpressed disproportionately abundant CTGF mRNA. Fur-thermore, those rats with high CTGF mRNA levels relative toBNP mRNA levels also showed marked upregulation ofCOL1A1 mRNA (Figure 2E, lanes 4 and 6). By contrast, ratswith proportional increases in both CTGF and BNP mRNAlevels showed only mild upregulation of COL1A1 mRNA(Figure 2E, lanes 3 and 5). Finally, rats with low CTGFmRNA levels relative to BNP mRNA levels showed lowlevels of COL1A1 mRNA (Figure 2E, lane 7).
Hemodynamic analysis was performed in rats on day 28.LV contractility indices, calculated using the pressure-volumerelationship, were comparable when comparing sham-operated and AC rats (Table S3). Diastolic indexes, that is,time constant of relaxation (�) and the slope of end-diastolicpressure-volume relationship (EDPVR slope), were signifi-cantly higher in the AC rats than in the sham-operated rats.
Representative hemodynamic data and histochemicalstaining of CTGF in a sham-operated and 2 AC rats withcomparable or disproportionate mRNA levels for CTGF andBNP are illustrated in Figure 3A and Figure S1. A rat withhigh CTGF levels related to BNP mRNA levels showed asteeper slope of EDPVR (Figure 3A), a high E/A ratio inDoppler echocardiography (Figure S1A), severe interstitialfibrosis, and positive immunostaining against CTGF in CM(Figure S1B) when compared with a sham-operated rat and arat with comparable mRNA levels of CTGF and BNP.
To further characterize hearts with high CTGF mRNAlevels relative to BNP mRNA levels, AC rats were classified
according to the upper or lower 50th percentile groups of theCTGF/BNP expression ratio on day 28. The mean ratio of theCTGF/BNP mRNA level was 1.2 (Figure 3B). AC rats witha higher CTGF/BNP mRNA ratio (n�7) showed elevatedEDPVR slope, higher E/A ratio, and increased MFA (Figure3B) relative to AC rats with the lower CTGF/BNP ratio(n�7). By contrast, LV relaxation (�), contractility (ejectionfactor), or LV hypertrophy (LV weight/body weight ratio)was similar when comparing the 2 groups (Figure 3B). Theprotein content of sarcomeric �-actin was also similar whencomparing 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 significantlycorrelated with the E/A ratio, expression levels of procollagentype 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 notcorrelated with the ratio (Table S4). Finally, plasma concentra-tion of Aldo was significantly elevated in AC rats with a higherCTGF/BNP ratio (Figure S3), whereas plasma TGF-� and ET-1concentration was not significantly different when comparingthe 2 groups.
CTGF Is Secreted From CMThe molecular basis of the production of CTGF in the heartand the functional interaction with other neurohumoral fac-tors was investigated using rat neonatal primary CM andcardiac fibroblasts (CFBs). Immunofluorescent study withanti-CTGF antibody revealed production of CTGF in culturedCMs (Figure 4A) and CFBs (vimentin-positive cells; FigureS4). Administration of recombinant CTGF resulted in a
Figure 3. LV diastolic function and myo-cardial fibrosis in rats with different ratiosof CTGF and BNP mRNAs. Representativepressure–volume loops (A) and echocardio-grams (B). Left, central and right panelsshow sham, an AC rat with comparablemRNA levels of CTGF and BNP, and an ACrat with disproportionate increase of CTGFagainst BNP, respectively. A, End-diastolicrelationships are depicted in broken lines.B, Differences in cardiac parametersbetween upper and lower 50th percentilegroups of CTGF/BNP expression ratio inday 28 AC rats. In each subset, � repre-sents the lower group (n�7), and f repre-sents the higher group (n�7). EDPVR,slope of end-diastolic pressure volumerelationship; �, monoexponential time con-stant of relaxation; LVW/BW, ratio of LVweight to body weight at sacrifice; Sarco-meric �-actin, the amount of sarcomeric�-actin in the heart extracts estimated byWestern blot.
Koitabashi et al CTGF vs BNP and Myocardial Fibrosis 1123
by guest on June 25, 2018http://hyper.ahajournals.org/
dose-dependent increase in COL1A1 mRNA levels in cul-tured CFB (Figure 4B). Profibrotic stimulation with TGF-�,ET1, and Aldo resulted in increased CTGF production andrelease into the culture medium from the myocytes (Figure 4C).Furthermore, conditioned medium from these CMs enhancedCOL1A1 mRNA levels in CFBs (Figure 4C), suggesting thatCMs may regulate collagen production in CFBs. When theTGF-�–treated medium was preincubated with anti-CTGF an-tibody, COL1A1 mRNA induction was abolished in CFBs(Figure 4D). Pretreatment of the CM-cultured medium with bothanti-CTGF and anti–TGF-� antibodies further suppressed theCOL1A1 mRNA, suggesting that the induction of the COL1A1gene by TGF-� and even the basal expression level of theCOL1A1 gene in CFBs are mediated through TGF-�–dependentand CTGF-dependent pathways.
Common and Uncommon Stimuli TriggeringCTGF and BNP Gene Transcription in CMsTo further characterize the correlation between CTGF andBNP mRNA induction in AC rats, the role of mechanohmoral
and neurohumoral stimuli on CTGF and BNP induction wasinvestigated. Cyclic stretch induced a rapid increase in CTGFand BNP mRNA levels (Figure 5A). Furthermore, G protein–coupled receptor ligands, such as ET1 (Figure 5B), norepi-nephrine, and angiotensin II (Figure S5A), increased CTGFand BNP levels in a dose-dependent manner. By contrast,Aldo and TGF-� stimulation resulted in increases in CTGFmRNA levels and decreases or no effect on BNP mRNAlevels (Figure 5B). The differential effect of TGF-� and Aldoon CTGF and BNP mRNA levels was also confirmed bycomparing the temporal induction pattern of these genes byTGF-� and Aldo with that induced by ET1 (Figure 5C).
BNP Suppresses CTGF Expression in CMsBecause BNP and TGF-� have opposing biological effects,14
the effect of BNP on CTGF expression was investigated.CTGF mRNA levels decreased 2 hours after administrationof synthetic BNP (Figure S5B). Synthetic BNP-mediatedinhibition of CTGF expression was completely blocked bythe protein kinase G inhibitor KT5823, suggesting that the
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 CTFGin designated concentrations for 24 h. The result was confirmed by triplicate experiments. C, COL1A1 mRNA levels in CFB 24 h afterthe 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 mediaof CM, respectively. Lower panel: Northern blot showing COL1A1 mRNA levels in CFB simulated by conditioned medium. Experimentswere performed in triplicate. D, The effect of an anti-CTGF neutralizing antibody on COL1A1 mRNA levels in CFB. Experimental condi-tions 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 bargraphs show mean mRNA levels based on 4 independent experiments. *P�0.05 versus Veh-medium with normal IgG; †P�0.05 versusTGF�-treated-medium with normal IgG.
1124 Hypertension May 2007
by guest on June 25, 2018http://hyper.ahajournals.org/
BNP–cGMP–protein kinase G pathway plays a critical role inregulating CTGF expression in CM (Figure S5C). Further-more, the effect of BNP was also evident in the context ofenhanced production of the CTGF protein in response toprofibrotic stimuli, such as TGF-�, ET1, and Aldo (Figure 5D).
DiscussionDHF, Fibrosis, and CTGFIn the present study, patients with DHF had greater amountsof interstitial fibrosis when compared with patients without aprevious history of congestive heart failure. Excessive colla-gen deposition contributes to abnormal passive diastolicventricular stiffness3 and leads to pulmonary edema.1 Impor-tantly, MFA, the degree of the interstitial fibrosis, signifi-cantly correlated with the abundance of CTGF-positive CMs(Figure 1G). By contrast, neither MFA nor the percentage ofCTGF-positive CMs was correlated with LV ejection frac-tion, an index of systolic function, in our study subjects (datanot shown). Endomyocardial biopsy can merely disclosehistological changes of a limited portion of whole heart, andthis immunohistochemical analysis is not a quantitative mea-surement of CTGF protein. The amount of biopsy sampleswas not enough to isolate protein for Western blotting.However, multiple samplings from different portions of thesame heart and the staining of serial section with normal IgGas a reference of nonspecific staining minimized samplingand technical variations. Our study suggests that excess
fibrosis, through an increase of CTGF, significantly contrib-utes to the development of DHF. Based on the stainingpattern with CTGF antibody, strong staining was mainlyobserved in CMs rather than in the interstitium (Figure 1D),suggesting that CMs are largely responsible for the produc-tion of CTGF in the DHF heart.
CMs Produce CTGFCTGF is overexpressed in numerous fibrotic diseases, and thedegree of overexpression correlates with the severity ofdisease.6 Collagen is mainly produced by fibroblasts in manyorgans. However, various other cells, including fibroblasts,secrete humoral factors to initiate collagen production infibroblasts.4,5 Although previous reports mainly focused onCFBs as CTGF-producing cells in the heart,15 the presentstudy demonstrated that a significant amount of CTGF isproduced by CMs in the hypertrophied rat heart and in thehearts of patients with DHF.
Interestingly, cultured CFB had higher basal levels ofCTGF mRNA than CM (Figure S4A). However, unlike thesignificant induction in CM, CTGF mRNA levels in CFBswere only minimally affected by extrinsic stimuli (CFBs,Figure S4B; CMs, Figure 5B), which is consistent withobservations by Kemp et al.10 The inducibility of CTGF inCMs, along with the fact that BNP, an antifibrotic factor, isalso produced by CMs, raises the possibility that CTGFproduced in CMs regulates collagen production in CFBs.
Figure 5. Effects of various hypertrophy-associated stimuli on the CTGF and BNPexpression in CMs. A and B, Northern blotshowing the effect of cyclic cell stretching(A) and various humoral factors (4 hours;B) on CTGF and BNP mRNA levels inCMs. Experiments were performed at leastin triplicate. C, Temporal patterns of CTGFand BNP mRNA levels in response toTGF-� (10 ng/mL), ET1 (0.1 �mol/L), orAldo (1 �mol/L) stimulation. Each dot indi-cates the mean intensity of 4 independentexperiments relative to the value in thevehicle-treated sample in each time point.D, Western blot showing the effect ofTGF-�, ET1, and Aldo and the inhibitoryeffect of BNP on CTGF protein levels. Syn-thetic BNP (sBNP) was added with TGF-�,ET1, or Aldo, and cells were incubated for24 hours. Bar graphs show mean CTGFprotein levels based on 4 independentexperiments. *P�0.01 vs vehicle, †P�0.05vs maximal level of CTGF protein inducedby these stimuli.
Koitabashi et al CTGF vs BNP and Myocardial Fibrosis 1125
by guest on June 25, 2018http://hyper.ahajournals.org/
Indeed, the present study demonstrated that CTGF wassecreted into the cultured medium of CMs and that thisconditioned medium induced increases in COL1A1 mRNAlevels in CFBs (Figure 4C). In addition, neutralization ofconditioned medium with CTGF antibody sufficientlyblunted the fibrotic signal from CMs to CFBs (Figure 4D).These data suggest that there is molecular communication ina paracrine manner between CMs and CFBs and that thisprocess regulates production of collagen.
CTGF/BNP Balance Regulates Cardiac FibrosisBecause we could not obtain a well-working antibody forBNP immunostaining, we were unable to estimate the BNPprotein level in myocardial biopsy samples. However, thepercentage of CTGF-positive staining cells in biopsy samplescorrelated with the plasma BNP level (r�0.41; P�0.05;Figure not shown). A close correlation between CTGF andBNP mRNA induction was also seen in the pressure overloadrat heart and in cultured CMs under cell stretch or stimulationwith G protein–coupled receptor ligands. Although the pre-cise mechanisms for this induction were not investigated inthis study, preliminary studies demonstrated that the ET1-induced increase in CTGF and BNP mRNA levels wasblocked 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 CTGFand BNP genes may be mediated by these signalingpathways.
Most importantly, the CTGF/BNP ratio in CM significantlycorrelated with indices of fibrosis and diastolic function, suchas the slope of EDPVR, E/A ratio, COL1A1 mRNA levels,and MFA (Figure 3 and Table S4). Furthermore, AC rats withcomparable levels of CTGF mRNA and BNP mRNA expres-sion showed mild production of CTGF protein and sparsefibrosis in the myocardium. Sarcomeric �-actin content wasnot different between the high CTGF/BNP ratio group andthe comparable ratio group, which suggests that myocyte lossis not responsible for the change of the ratio (Figure 3B).SERCA2a is a principal protein responsible for the initiationof the diastolic phase through its ability to remove cytoplas-mic Ca2�.2 However, SERCA2a mRNA level was not corre-lated with the CTGF/BNP ratio (Table S4). These datasuggest that the CTGF/BNP ratio does not associate with LVdiastolic function, which is related to Ca2� removal fromcytoplasm.
The identity of upstream factors responsible for the dispro-portionate expression of CTGF and BNP in CMs remainsunclear. AC causes severe hypertension and increases in thelevels of various neurohumoral factors, such as renin andangiotensin II.16 Interestingly, rats with a higher CTGF/BNPratio had a higher plasma Aldo concentration and a tendencytoward higher plasma TGF-� and ET1 concentrations thanthe rats with a lower CTGF/BNP ratio (Figure S3). Inaddition, in vitro study demonstrated that Aldo and TGF-�induced increases in CTGF mRNA but not in BNP mRNA incontrast to the response to cell stretch or G protein–coupledreceptor ligands (Figure 5A through 5C). Therefore, at leastin the present model, Aldo may be an upstream factorresponsible for disproportionate CTGF expression.
In addition to the effect on body fluid homeostasis andblood pressure control, BNP can exert antihypertrophic andantifibrotic effects in the stressed myocardium.11 The presentstudy demonstrated that BNP suppressed basal CTGF expres-sion level in CMs via its effects on protein kinase G (FigureS4C). The effect of BNP on CTGF expression was alsoobserved under various profibrotic stimuli, such as ET1,Aldo, and TGF-� (Figure 5D). Thus, the increase of CTGFand/or decrease of BNP in CMs may play a central role in theinduction of excessive myocardial fibrosis and abnormaldiastolic function (Figure 6).
To dissect the role of CTGF in the development of DHF,we used a rat pressure-overloaded model as a preservedsystolic but impaired diastolic function model. Given thatcollagen accumulation is regulated by a balance of its synthesisand degradation, the pressure-overloaded model may becharacterized as a “synthesis”-dominant model.12 On theother hand, myocardial infarction is a “accelerated synthesisand accelerated degradation” model with respect to collagenturnover.17 Myocardial infarction is another leading cause toprovoke cardiac fibrosis. Therefore, our hypothesis should bealso tested in the ischemic heart model, as well as thepressure-overloaded cardiac hypertrophy model.
PerspectivesCTGF is a secreted protein, and plasma CTGF concentrationcorrelates with the severity of several systemic fibroticdisorders.18 Measurement of plasma CTGF concentrations iseasier and less invasive than assessment of CTGF levels inbiopsy samples. Furthermore, the present data suggest thatplasma concentration of CTGF or the ratio of plasma con-centration of CTGF:BNP may be a diagnostic marker formyocardial fibrosis. In addition, our data showing the induc-ibility of CTGF in CMs and the myocardial responsiveness to
Figure 6. A scheme illustrating that an abundance of CTGF rela-tive to BNP in CMs promotes pathogenic collagen production inCFBs.
1126 Hypertension May 2007
by guest on June 25, 2018http://hyper.ahajournals.org/
exogenously administered CTGF suggest that CTGF plays anactive role in cardiac progressive fibrosis and, thus, becomesa good candidate molecule as a target of antifibrotic therapy.
ConclusionsThe present study demonstrated the following: (1) productionof CTGF from CMs is associated with the myocardialinterstitial fibrosis and DHF; (2) increased CTGF expressionrelative to BNP expression triggers excessive cardiac fibrosisvia BNP-mediated suppression of CTGF expression; and (3)Aldo and TGF-� induce a disproportionate induction ofCTGF and BNP expression, whereas a mechanical stretch ofCM and G protein–coupled receptor ligands induces propor-tionate CTGF and BNP expression. These data suggest thatCTGF is a key molecule in the process of cardiac fibrosis andthat it may serve as a diagnostic marker and therapeutic targetfor cardiac fibrosis and DHF.
AcknowledgmentsWe are grateful to Miki Yamazaki for her technical assistance withthe cardiac myocytes culture and Yoshiko Nonaka for her excellentpreparation of histological samples.
Sources of FundingThis work was supported in part by a Grant-in-Aid for ScientificResearch (KAKENHI B-17390224 and S-15109010) from the JapanSociety for the Promotion of Science.
DisclosuresNone.
References1. Zile MR, Baicu CF, Gaasch WH. Diastolic heart failure–abnormalities in
active relaxation and passive stiffness of the left ventricle. N EnglJ Med. 2004;350:1953–1959.
2. Kass DA, Bronzwaer JG, Paulus WJ. What mechanisms underlie diastolicdysfunction in heart failure? Circ Res. 2004;94:1533–1542.
3. Mundhenke M, Schwartzkopff B, Strauer BE. Structural analysis ofarteriolar and myocardial remodelling in the subendocardial region ofpatients with hypertensive heart disease and hypertrophic cardiomyopa-thy. 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.
5. Kai H, Kuwahara F, Tokuda K, Imaizumi T. Diastolic dysfunction inhypertensive hearts: roles of perivascular inflammation and reactive myo-cardial fibrosis. Hypertens Res. 2005;28:483–490.
6. Blom IE, Goldschmeding R, Leask A. Gene regulation of connectivetissue 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, DoiM, Murakami T, Ninomiya Y, Takigawa M, Tsuji T. Increased expressionof connective tissue growth factor in the infarct zone of experimentallyinduced myocardial infarction in rats. J Mol Cell Cardiol. 1998;30:2411–2422.
8. Matsui Y, Sadoshima J. Rapid upregulation of CTGF in cardiac myocytesby 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-tissuegrowth factor (CTGF) modulates cell signalling by BMP and TGF-beta.Nat Cell Biol. 2002;4:599–604.
11. Cameron VA, Ellmers LJ. Minireview: natriuretic peptides during devel-opment 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 inhypertensive heart disease. Nat Clin Pract Cardiovasc Med. 2005;2:209–216.
13. Takizawa T, Arai M, Yoguchi A, Tomaru K, Kurabayashi M, Nagai R.Transcription of the SERCA2 gene is decreased in pressure-overloadedhearts: 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 peptideexerts broad functional opposition to transforming growth factor-beta inprimary 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 growthfactor–a novel mediator of angiotensin II-stimulated cardiac fibroblastactivation in heart failure in rats. J Mol Cell Cardiol. 2004;36:393–404.
16. Parker FB Jr, Streeten DH, Farrell B, Blackman MS, Sondheimer HM,Anderson GH Jr. Preoperative and postoperative renin levels in coarc-tation of the aorta. Circulation. 1982;66:513–514.
17. Jugdutt BI. Ventricular remodeling after infarction and the extracellularcollagen 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 thefibrotic phenotype in scleroderma. Qjm. 2005;98:485–492.
Koitabashi et al CTGF vs BNP and Myocardial Fibrosis 1127
by guest on June 25, 2018http://hyper.ahajournals.org/
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/HYPERTENSIONAHA.106.0775372007;49:1120-1127; originally published online March 19, 2007;Hypertension.
http://hyper.ahajournals.org/content/49/5/1120World Wide Web at:
The online version of this article, along with updated information and services, is located on the
is online at: Hypertension Information about subscribing to Subscriptions:
http://www.lww.com/reprints Information about reprints can be found online at: Reprints:
document. Permissions and Rights Question and Answer this process is available in the
click Request Permissions in the middle column of the Web page under Services. Further information aboutOffice. Once the online version of the published article for which permission is being requested is located,
can be obtained via RightsLink, a service of the Copyright Clearance Center, not the EditorialHypertensionin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:
by guest on June 25, 2018http://hyper.ahajournals.org/