Copyright ©The Histochemical Society, Inc.

Connective Tissue Growth Factor and Cardiac Fibrosis after Myocardial Infarction

Rachael G. Dean, Leanne C. Balding, Riccardo Candido, Wendy C. Burns, Zemin Cao, Stephen M. Twigg and Louise M. Burrell

Department of Medicine, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia (RGD,LCB,RC,ZC,LMB); Diabetic Complications Group, Baker Heart Research Institute, Prahan, Victoria, Australia (WCB); and Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales, Australia (SMT)

Correspondence to: Dr. Rachael Dean, Department of Medicine, University of Melbourne, Austin Health (Repat Campus), Heidelberg, 3081, Victoria, Australia. E-mail: rdean{at}unimelb.edu.au


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The temporal and spatial expression of transforming growth factor (TGF)-ß1 and connective tissue growth factor (CTGF) was assessed in the left ventricle of a myocardial infarction (MI) model of injury with and without angiotensin-converting enzyme (ACE) inhibition. Coronary artery ligated rats were killed 1, 3, 7, 28, and 180 days after MI. TGF-ß1, CTGF, and procollagen {alpha}1(I) mRNA were localized by in situ hybridization, and TGF-ß1 and CTGF protein levels by immunohistochemistry. Collagen protein was measured using picrosirius red staining. In a separate group, rats were treated for 6 months with an ACE inhibitor. There were temporal and regional differences in the expression of TGF-ß1, CTGF, and collagen after MI. Procollagen {alpha}1(I) mRNA expression increased in the border zone and scar peaking 1 week after MI, whereas collagen protein increased in all areas of the heart over the 180 days. Expression of TGF-ß1 mRNA and protein showed major increases in the border zone and scar peaking 1 week after MI. The major increases in CTGF mRNA and protein occurred in the viable myocardium at 180 days after MI. Long-term ACE inhibition reduced left ventricular mass and decreased fibrosis in the viable myocardium, but had no effect on cardiac TGF-ß1 or CTGF. TGF-ß1 is involved in the initial, acute phase of inflammation and repair after MI, whereas CTGF is involved in the ongoing fibrosis of the heart. The antifibrotic benefits of captopril are not mediated through a reduction in CTGF.

(J Histochem Cytochem 53:1245–1256, 2005)

Key Words: connective tissue growth factor • transforming growth factor ß1 • myocardial infarction • fibrosis • angiotensin-converting enzyme


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
AFTER MYOCARDIAL INFARCTION (MI), activation of neurohormonal systems preserves circulatory homeostasis in the short term, but plays a role in the development and progression of cardiac remodelling and heart failure through cytokine activation and cardiac fibrosis in the long term (Eichorn and Bristow 1996Go). Although fibrosis of the scar is an important and necessary response to injury, fibrosis remote to the scar, in the viable non-infarcted myocardium, contributes adversely to tissue structure and function, causing myocardial stiffness, cardiac dysfunction, and, ultimately, heart failure (Weber 1997Go).

Several studies have assessed the role of cytokines on myocyte injury and myocardial function in the acute phase after MI (Ohnishi et al. 1998Go; Ono et al. 1998Go; Sun et al. 1998Go; Lijnen et al. 2000Go; Yu et al. 2001Go; Ahmed et al. 2004Go). Low levels of expression of transforming growth factor (TGF)-ß1, its receptor, and collagen type 1 are seen in normal rat heart and increase after MI. The increase in TGF-ß1 mRNA precedes increases in collagen expression, suggesting a role for TGF-ß1 in cardiac fibrosis and remodelling (Youn et al. 1999Go; Yu et al. 2001Go; Tzanidis et al. 2001Go). Connective tissue growth factor (CTGF) has also been shown to promote fibroblast proliferation and extracellular matrix production in connective tissues, and its overexpression has been observed in wound repair and in fibrotic disorders (Igarashi et al. 1993Go). Acutely after MI, the expression of CTGF increases in parallel with collagen (Ohnishi et al. 1998Go).

The concomitant regional and spatial changes in expression of TGF-ß1 and CTGF after MI have not been assessed. Many studies have assessed either TGF-ß1 or CTGF and most have been short term (<6 weeks) in nature (Ohnishi et al. 1998Go; Youn et al. 1999Go; Tzanidis et al. 2001Go; Yu et al. 2001Go; Ahmed et al. 2004Go). To date, no studies have assessed the expression of TGF-ß1 or CTGF in the chronic stage of remodelling when heart failure has occurred. We have previously shown that significant changes in the spatial distribution of other cytokine systems such as the insulin-like growth factor system can occur for up to 6 months after infarction when heart failure is present (Dean et al. 1999Go). It is possible that sustained production of profibrotic cytokines may underlie the development of fibrosis in the remodelling and failing heart.

We have previously described the benefits of angiotensin-converting enzyme (ACE) inhibition to reduce both cardiac remodelling and fibrosis after MI in the rat (Burrell et al. 2000Go), and now hypothesize that the long-term beneficial effect of ACE inhibitors may be mediated through changes in the profibrotic cytokines TGF-ß1 or CTGF. The aims of this study were 2-fold: (1) to examine the time course and cellular expression of TGF-ß1, CTGF, and collagen in the heart for up to 180 days after MI when heart failure is present and (2) to assess whether one of the mechanisms by which an ACE inhibitor attenuates cardiac remodelling and fibrosis is modulation of either or both of these profibrotic cytokines.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Experimental procedures were performed according to the National Health and Medical Research Council of Australia Guidelines for Animal Experimentation. Rats were housed at 23 to 25C in a 12:12 light:dark cycle, with ad libitum food containing 0.4–0.6% NaCl and water.

Experimental Design
The rat model of MI has been extensively used to examine the efficacy of therapeutic interventions on cardiac remodelling and survival (Pfeffer et al. 1985Go; Burrell et al. 1996Go, 2000Go; Dean et al. 1999Go). Left ventricular free-wall myocardial infarction was induced in female Sprague-Dawley rats (200–250 g) by ligation of the proximal left anterior descending artery as described previously. Sham-operated (control) rats underwent an identical operation, but the suture was not tied.

Study 1: Time Course
At 1, 3, 7, 28, and 180 days after MI, rats (n=4 per time point) were decapitated, the hearts collected, and the left ventricle/interventricular septum (LV) dissected, and fixed in formalin for in situ hybridization histochemistry and histological staining.

Study 2: Captopril Treatment
In a separate study, rats surviving for 24 hr postoperatively were randomized to vehicle (once daily gavage of 5% arabic gum) or captopril (25 mg/kg, twice daily gavage) (n=12/group). Dosage was based on previous studies showing attenuation ofÄ remodelling after MI (Burrell et al. 2000Go). At 180 days, systolic blood pressure was measured by the indirect tail-cuff technique (38L flatbed recorder, model 229 Amplifier; ITCH Life Science, Woodland Hills, CA) in conscious, lightly restrained rats. Rats were then weighed, killed, and their trunk blood collected into EDTA tubes for the measurement of atrial natriuretic peptide (ANP) (Burrell et al. 1990Go) and plasma renin activity. The LV was dissected, weighed, and fixed in 10% buffered formalin for in situ hybridization histochemistry and histological staining.

Infarct Size
The LV was sectioned at four levels from the base to the apex, paraffin embedded, and sections cut and stained with Masson's trichome, and hematoxylin and eosin. The mean epicardial and endocardial scar circumference was compared with total LV circumference to calculate total infarct size (Pfeffer et al. 1985Go).

In Situ Hybridization
In situ hybridization was performed on 4-µm LV sections from rats involved in study 1 and 2 (n=4–5 per time or treatment group) to localize mRNA for TGF-ß1, CTGF, and procollagen {alpha}1(I) as previously described (Wu et al. 1997Go; Yu et al. 1998Go). The 945-bp cDNA probe for TGF-ß1 was cloned into pBluescript KS+ (Stratagene; La Jolla, CA) and linearized with Xba1 to produce an antisense riboprobe with T3 RNA polymerase or EcoRI and T7 to produce a sense riboprobe. The 600-bp cDNA probe for rat procollagen {alpha}1(I) was cloned into pGEM3z (Stratagene) and linearized with Hind III to produce an antisense riboprobe with T7 RNA polymerase or EcoRI and SP6 to produce a sense riboprobe. The 1047-bp human CTGF cDNA sequence (coding for the open reading frame) was inserted in the sense direction into the BamHI and Xho I sites of the pcDNA3 vector (Promega; Sydney, Australia). The vector was digested with KpnI and transcribed with SP6 polymerase to provide the antisense CTGF riboprobe.

Antisense and sense riboprobes labeled with 35S cytidine 5'-T trisphosphate (CTP) were prepared using the Promega transcription system. Between 500 ng and 1 µg of linearized template cDNA was transcribed in a reaction mix containing 1x transcription buffer; 10 mM DTT; 0.6 mM each of ATP, GTP, and UTP; 100 µCi [35S]; 0.5 µl RNasin; and 1 µl of the appropriate RNA polymerase. If the probe length was greater than 400 bp, 4.25 pM of cold CTP was added to the transcription reaction. The reaction was incubated at 37C for 90 min; after this time, 1000 U of DNase (RNase-free) (Boehringer Mannheim; Roche Diagnostics, Sydney, Australia) was added and the reactions incubated for a further 15 min at 37C. The riboprobe was precipitated with ammonium acetate and ethanol using yeast tRNA as carrier, then reconstituted in 10 mM DTT. The length of the purified riboprobe was adjusted to ~150 bp using alkaline hydrolysis, followed by further purification with sodium acetate and ethanol, and resuspension in 10 mM DTT.

Heart sections were dewaxed, rehydrated, digested with Pronase E at 37C, and hybridized overnight at 60C with a buffer containing 2x104 cpm/µl of 35S-labeled riboprobe, 0.72 mg/ml yeast RNA, 50% deionized formamide, 100 mM DTT, 10% dextran sulfate, 0.3 M NaCl, 10 mM Na2HPO4, 10 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0), 0.02% BSA, 0.02% Ficoll 400, and 0.02% polyvinyl pyrrolidone.

After hybridization, sections were stringently washed with 50% formamide, 2xSSC at 55C, incubated with RNase A (150 µg/ml), dehydrated and exposed to Kodak X-Omat autoradiographic film (Eastman Kodak, Rochester, New York) for 1–3 days at room temperature. Slides were dipped in photographic emulsion (Amersham; Buckinghamshire, UK), stored with desiccant at 4C for 14–21 days, developed in Kodak D19, fixed in Ilford Hypam, and stained with hematoxylin and eosin for cellular localization.

Quantitation of Macroscopic In Situ Hybridization Autoradiographs
Quantitation was carried out using a microcomputer imaging device (Imaging Research, Ontario, Canada) run by an IBM PC. Sections from four levels of the LV from each animal were used for quantitation. In MI hearts, the viable myocardium, scar, and border zones were quantitated separately. The border zone is the area of high cellular infiltrate at the edge of the fibrotic scar tissue of the infarct. The optical densities of the autoradiographs were calibrated in terms of radioactivity density as dpm/mm2 by reference to radioactive standards (Amersham, UK) carried through the procedures (Le Moine et al. 1994Go). Quantitation was carried out in the linear region of the curve generated by the radioactive standard. Results are expressed as specific labeling (using antisense probes) minus nonspecific labeling (using sense probes). Nonspecific labeling with the antisense probe was also checked by the use of "negative tissues" for each probe.

Total Collagen Staining
Paraffin sections 4 µm thick were deparaffinized, rehydrated, and then stained with 0.1% Sirius Red (Polysciences Inc.; Warrington, PA) in saturated picric acid (picrosirius red) for 1 hr, differentiated in 0.01% HCl for 30 sec, and rapidly dehydrated. Collagen volume fraction was determined by measuring the area of stained tissue with in a given field. Within the LV, fields containing vessels, artifacts, minor scars, or incomplete tissue were excluded. A total of 15–20 fields were analyzed per animal. The area stained was calculated as a percentage of the total area within a field (Yu et al. 1998Go). Total collagen volume fraction determined by this morphometric approach is closely related to the hydroxyproline concentration in the LV (Weber et al. 1988Go).

Immunohistochemistry for ED1, TGF-ß1, and CTGF
Immunohistochemistry for macrophages and TGF-ß1 was carried out on 4-µm paraffin sections of LV as previously described (Gilbert et al. 1998Go; Dean et al. 1999Go). For CTGF, the primary antibody used was a polyclonal rabbit anti-mouse CTGF antibody (Abcam Ltd, Cambridge, UK) at a concentration of 1:800. Standard techniques were employed and the Elite Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used. The staining was visualized by reaction with 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St Louis, MO). Negative control sections were incubated in the absence of primary antibody.

Immunohistochemical staining for TGF-ß1 and CTGF protein was quantitated (n=4–5 per group) using computerized image analysis (AIS Imaging, Ontario, Canada). All sections used for quantitation were fixed, processed, sectioned, and immunolabeled at the same time and under the same conditions to limit variability. In MI hearts, the non-infarcted viable myocardium, scar, and border regions were quantitated separately. Twelve fields (x20 objective) from each region of the heart were selected for assessment according to a predefined grid pattern. Images were imported into the AIS imaging program using a color video camera and a standard light microscope. The detection level threshold for positively stained areas (brown for DAB staining) was set so that the processed image accurately reflected the positively stained areas as visualized by light microscopy and on the unprocessed digital image. An average intensity for the selected area was then calculated. The percentage area of chromogen staining was determined by calculating the number of selected pixels (positively stained areas) in a given area and expressed as a percentage of the entire image. The average intensity and area of staining were then multiplied to give the final figure (James and Hauer-Jensen 1999Go; Lehr et al. 1999Go; Rimsza et al. 1999Go).

Statistics
Results are expressed as mean ± SEM. Comparisons of tissue weights and vasoactive hormone levels were made by unpaired t-tests. Quantitative in situ hybridization, immunohistochemistry, and picrosirius red staining variables were compared using one- or two-way ANOVA followed by post hoc analysis using the Fisher LSD test. Results were considered significant when p<0.05.


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Study 1: Time Course
TGF-ß1 and Procollagen {alpha}1(I) mRNA Localized to Areas of Inflammation after MI
The macroscopic localization of TGF-ß1, CTGF, and procollagen {alpha}1(I) mRNA is shown in Figure 1. TGF-ß1, CTGF, and procollagen {alpha}1(I) mRNA were localized to the site of myocardial ischemia (scar) and the tissue immediately surrounding this area, the border zone (Figure 1). Light microscopic autoradiography demonstrated the cellular localization of the mRNA.



View larger version (41K):
[in this window]
[in a new window]
 
Figure 1

Representative macroscopic autoradiographs demonstrating levels of signal produced by in situ hybridization histochemistry for transforming growth factor-ß1 (top row), connective tissue growth factor (middle row), and procollagen {alpha}1(I) (bottom row). Increasing levels of signal are demonstrated by a color scale, with red being the highest levels of radiolabeling and blue the lowest. Comparisons can only be made between time points and not individual mRNA species. The scar of the myocardial infarction (MI) left ventricle is present where thinning of the left ventricular wall has occurred. The scar is surrounded by the border zone seen as a ring of high density labeling at 3 days after MI and by 28 days as the junction between the thinned wall and the remaining viable myocardium (white arrows).

 
Silver grains representing procollagen {alpha}1(I) mRNA were seen overlying inflammatory cells throughout the myocardium and surrounding vessels of the scar and border zones of the infarcted LV and to fibroblasts surrounding vessels in all areas of the infarcted LV (not shown). In addition, cords of connective tissue throughout the non-infarcted myocardium demonstrated procollagen {alpha}1(I) labeling (not shown).

TGF-ß1 mRNA localized to the same areas as procollagen {alpha}1(I) mRNA. Immunohistochemical analyses showed the major cell population to be macrophages, the level of labeling tended to decrease over the time course and was at very low levels by 180 days, as shown previously (Dean et al. 1999Go). TGF-ß1 mRNA labeling was low in control LV.

CTGF mRNA and Protein Predominantly Localized to Cardiomyocytes
Early after MI, silver grains representing CTGF mRNA and protein staining were predominantly localized to cardiomyocytes and, to a lesser extent, fibroblasts at the border of the infarct (Figures 2A and 2B). As the scar matured and inflammation became less prominent, the cardiomyocytes in the viable myocardium showed the greatest level of labeling (Figures 2E and 2F). Fibroblasts surrounding vessels were also labeled (not shown).



View larger version (149K):
[in this window]
[in a new window]
 
Figure 2

Micrographs of connective tissue growth factor (CTGF) in situ hybridization (A,C,E) and immunohistochemistry (B,D,F). A and B show the border region 3 days after myocardial infarction; silver grains representing CTGF mRNA are seen overlying cardiomyocytes (A, white arrows) and endothelial cells (A, black arrow) and over the inflammatory infiltrate. In B, brown staining representing CTGF immunoreactivity is seen in the area of the inflammatory infiltrate. C and D show the scar 7 days after myocardial infarction with labeling of the inflammatory infiltrate for both CTGF mRNA (C) and protein (D). E and F show the viable myocardium 180 days after myocardial infarction. White arrows indicate cardiomyocyte labeling of CTGF mRNA (E) and protein (F). All micrographs at magnification x500.

 
Procollagen {alpha}1(I) mRNA Levels Peak in First Week after MI
The macroscopic autoradiographic representation of the induction of procollagen {alpha}1(I) mRNA following MI is shown in Figure 1. Procollagen {alpha}1(I) mRNA was significantly increased, indicating increased type I procollagen synthesis in the border zone at days 1, 3, 7, and 28 (p<0.01) (Figure 3A) and in the scar on days 3, 7, 28, and 180 after MI (p<0.01) (Figure 3B). There was a trend to increased procollagen {alpha}1(I) mRNA in the viable myocardium at 7 days, but over the 180 days, the changes were not significant (Figure 3C).



View larger version (32K):
[in this window]
[in a new window]
 
Figure 3

Expression of procollagen {alpha}1(I) mRNA (A–C) and total collagen protein (D–F) in the border, scar, and viable myocardium at varying time points after myocardial infarction (MI). **p<0.01, 1 day MI vs. control. #p<0.05 and ##p<0.01 vs. 1 day MI.

 
Continuous Increase in Collagen Protein after MI
Total collagen protein gradually increased in all areas of the MI hearts compared with normal hearts over time, with levels in the border zone and scar becoming significant at day 3 and remaining elevated at the 180-day time point (p<0.01) (Figures 3D and 3E). Collagen protein was significantly increased in the non-infarcted viable myocardium at 180 days after MI (Figure 3F).

TGF-ß1 mRNA and TGF-ß1 Protein at Peak Levels in the First Week after MI
Levels of TGF-ß1 mRNA were significantly increased in the border zone at days 1 and 3 (p<0.01) (Figure 4A). In the scar, TGF-ß1 mRNA was significantly increased at days 7, 28, and 180 (all p<0.01) (Figure 4B). TGF-ß1 mRNA was present in non-infarcted viable myocardium, and did not significantly change after MI (Figure 4C). TGF-ß1 protein levels were increased in the border and scar regions at day 1 (p<0.05) and then fell to control levels, paralleling to a large degree the changes at the message level (Figures 4D–4F). The discordant results for TGF-ß1 mRNA and protein apparent in the viable myocardium at days 1 to day 7 and also in the scar at day 1 most likely reflects posttranslational regulation of TGF-ß1.



View larger version (27K):
[in this window]
[in a new window]
 
Figure 4

Expression of transforming growth factor-ß1 mRNA (A–C) and protein (D–F) in the border, scar, and viable myocardium at varying time points after myocardial infarction (MI). *p<0.05 and **p<0.01, 1 day MI vs. control. #p<0.05 and ##p<0.01 vs. 1 day MI.

 
CTGF mRNA and Protein Levels Were Significantly Increased in the Viable Myocardium at 180 Days after MI
CTGF mRNA (p<0.05) and protein (p<0.01) levels were significantly increased in the border zone on day 1 (Figures 5A and 5D) after infarction and remained increased for the entire study period. There were no significant changes in CTGF mRNA and protein levels in the scar (Figures 5B and 5E). CTGF mRNA was significantly increased in the viable myocardium 180 days after infarction (p<0.05) (Figure 5C), which was associated with significant increases at the protein level (p<0.01) (Figure 5F).



View larger version (52K):
[in this window]
[in a new window]
 
Figure 5

Expression of connective tissue growth factor mRNA (A–C) and protein (D–F) in the border, scar, and viable myocardium at varying time points after myocardial infarction (MI). *p<0.05 and **p<0.01, 1 day MI vs. control. #p<0.05 and ##p<0.01 vs. 1 day MI.

 
Study 2: Captopril Treatment
All sham-operated rats (n=10) survived to 24 hr. Of the rats operated on to produce myocardial infarction, ~80% (n=24) were alive at 24 hr and were randomized to vehicle (n=12) or captopril (n=12) treatment groups. At the end of the treatment period (180 days), there were eight vehicle-treated MI and nine captopril-treated MI rats alive. No control animal had evidence of cardiac damage. In MI rats, the average infarct size was 38% and was similar in vehicle and captopril-treated rats (Table 1). There was no significant pretreatment difference in body weight or systolic blood pressure of control and MI rats (not shown). Both control and MI rats gained weight throughout the duration of the study with no effect of treatment on body weight (Table 1). By 180 days after MI, rats had increased relative LV and lung mass (p<0.05) and increased plasma ANP concentrations (p<0.05) compared with control rats (Table 1). In MI rats, captopril reduced systolic blood pressure (p<0.01); LV, lung mass (p<0.05), and plasma ANP (p<0.05); and increased plasma renin activity (p<0.01).


View this table:
[in this window]
[in a new window]
 
Table 1

Infarct size, blood pressure, tissue weights, and blood hormones

 
Long-term Captopril Decreases Total Collagen Protein in the Viable Myocardium
We confirmed our previous findings that 180 days after MI, procollagen {alpha}1(I) mRNA levels were increased in the border and scar zones (p<0.01) (Figures 6A–6C). The levels of procollagen {alpha}1(I) mRNA were not changed by captopril treatment (Figures 6A–6C). Total collagen protein was increased in all areas of the heart 180 days after MI (p<0.01) (Figures 6D–6F). Captopril treatment decreased total collagen protein by 50% in the viable myocardium 180 days after MI (p<0.05) (Figure 6F).



View larger version (28K):
[in this window]
[in a new window]
 
Figure 6

Effect of captopril treatment on the expression of procollagen {alpha}1(I) mRNA (A–C) and total collagen protein (D–F) in the border, scar, and viable myocardium at 180 days after myocardial infarction (MI). **p<0.01 MI vs. control. #p<0.05 captopril vs. vehicle.

 
Captopril Does Not Affect TGF-ß1 mRNA or Protein 180 Days after MI
At 180 days after MI, the level of TGF-ß1 mRNA was returning to control levels in the border zone and scar regions and was not increased in the viable non-infarcted myocardium (Figures 7A–7C). Captopril did not significantly change the levels of TGF-ß1 mRNA and protein (Figures 7D–7F).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 7

Effect of captopril treatment on the expression of transforming growth factor-ß1 mRNA (A–C) and protein (D–F) in the border, scar, and viable myocardium at 180 days after myocardial infarction.

 
Captopril Does Not Affect CTGF mRNA or Protein 180 Days after MI
CTGF mRNA and protein were significantly increased in the border zone and viable myocardium 180 days after MI (Figures 8A–8C). The changes in CTGF, although small, show coordinate CTGF mRNA and protein changes to a level that would be expected to occur in pathophysiological states. These effects were not overtly mediated by captopril (Figure 8).



View larger version (28K):
[in this window]
[in a new window]
 
Figure 8

Effect of captopril treatment on the expression of CTGF mRNA (A–C) and CTGF protein (D–F) in the border, scar and viable myocardium at 180 days following myocardial infarction (MI). *p<0.05, **p<0.01 MI vs. control.

 

    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The main findings of this study in rat after MI are that (1) temporal and regional differences in the expression of TGF-ß1, CTGF, and collagen occur; (2) by 180 days after MI, significant fibrosis is present in the viable myocardium and is also associated with the profibrotic cytokine CTGF but not TGF-ß1; and (3) treatment with an ACE inhibitor attenuates cardiac remodelling, reduces LV mass, and decreases fibrosis in the viable myocardium, but these effects are not mediated by changes in cardiac CTGF expression.

Fibrosis and Cytokine Expression after MI
It has been suggested that TGF-ß1 or CTGF overexpression is involved in cardiac fibrosis and that strategies to reduce these cytokines may be useful in heart failure. In this study, low-level expression of TGF-ß1, CTGF, and collagen mRNA were seen in normal rat hearts. TGF-ß1 increased acutely after MI, preceding increases in collagen expression. The finding that TGF-ß1 and procollagen {alpha}1(I) mRNA expression were localized to infiltrating inflammatory cells and fibroblasts of the scar and border zones early after MI and were not increased in the distant non-infarcted myocardium suggests a regulated response to injury and mechanical stress. It is, however, feasible that localization of procollagen {alpha}1(I) mRNA in inflammatory cells is a nonspecific result and does not result in translation to protein as in fibroblasts. These results extend observations of previous studies that have shown at 2 days after MI there is increased expression of TGF-ß1 mRNA and protein in the border zone (Thompson et al. 1988Go) and at 1 week after MI, TGF-ß1 mRNA is induced in non-myocytes (Yue et al. 1998Go). By 8 weeks after MI, Western blot analysis of cardiac tissue showed the active form of TGF-ß1 was significantly elevated in border and scar tissue, but myocytes remote to the infarct zone expressed comparatively moderate levels of TGF-ß1 only (Hao et al. 2000Go). We found parallel increases in TGF-ß1 mRNA and protein in the early stages after MI. With time, however, the levels of protein fell to control values in all areas of the heart. Interestingly, in the scar and border zone, TGF-ß1 mRNA remained high, but was not translated to protein.

In contrast, CTGF mRNA and protein were increased in myocytes and non-myocytes of the non-infarcted myocardium 180 days after MI, an increase associated with fibrosis. This extends previous reports that demonstrate that CTGF is increased in non-myocytes 42 days after MI (Ahmed et al. 2004Go).

Our data suggest that TGF-ß1 may be involved in the initial induction of procollagen {alpha}1(I) mRNA and total collagen in the early stages after MI, but is not important in the laying down of collagen protein in the continuing remodelling of the heart. The localization of TGF-ß1 mRNA to inflammatory cells suggests that TGF-ß1 has a role beyond collagen production, possibly pleiotropic actions such as wound healing or simply maintaining physiological functioning of the heart (Azhar et al. 2003Go). On the other hand CTGF is involved in remodelling of the viable myocardium in the chronic stage after MI, when the inflammatory response has subsided, but fibrosis is ongoing.

ACE Inhibition after MI
ACE inhibitors are standard therapy after myocardial infarction to attenuate remodelling, delay the progression to heart failure, and decrease mortality (Pfeffer et al. 1992Go). Despite the benefits of ACE inhibitors to slow remodelling, progressive increases in LV dilation and hypertrophy continue to occur, in part because of continued cytokine activation and ongoing fibrosis (Eichorn and Bristow 1996Go). Although it is clear that fibrosis of the scar is an important and necessary response to injury, fibrosis remote to the scar, in the viable non-infarcted myocardium, contributes adversely to tissue structure and function and to the development of heart failure (Weber 1997Go).

Renin-angiotensin System Blockade and TGF-ß1
Both in vitro and in vivo studies have shown Ang II stimulates TGF-ß1 synthesis (Dostal et al. 1996Go; Border and Noble 1998Go). It is well known that fibrosis and activation of the cardiac renin-angiotensin system (RAS) occurs after MI (Passier et al. 1996Go; Duncan et al. 1997Go), and we have shown that the ACE inhibitor captopril reduces LV mass after MI (Burrell et al. 2000Go) and has antifibrotic effects in the heart (Farina et al. 2000Go). Although these data suggest a link between the RAS, fibrosis and the TGF-ß1 system, studies with ACE inhibitors or Ang II type 1 receptor antagonists have produced conflicting results in terms of whether RAS blockade does (Sun et al. 1998Go; Youn et al. 1999Go; Yu et al. 2001Go) or does not (Tzanidis et al. 2001Go; Yu et al. 2001Go) "switch off" cardiac TGF-ß1 expression after MI. In this study, 180 days of treatment with captopril had no effect on TGF-ß1 levels, which may be explained by the fact that the TGF-ß1 levels had already returned to base levels at this time. However, as expected, ACE inhibition did decrease collagen protein in the viable myocardium of MI rats.

RAS Blockade and CTGF
Data on RAS blockade and its effect on CTGF expression are limited. In this study, chronic activation of left ventricular CTGF mRNA and protein was not prevented by ACE inhibition. However, in vascular smooth muscle cells, an Ang II type 1 receptor antagonist blocked Ang II–induced CTGF gene and protein expression (Ruperez et al. 2003Go). In a similar model to that used in our study, the AT1 receptor antagonist losartan prevented the induction of myocardial CTGF mRNA after 25 days of treatment; however, its effect on CTGF protein was not examined (Ahmed et al. 2004Go). This suggests that there may be a difference in treatment outcome between ACE inhibition and angiotensin receptor blockade or that the treatment effect has "worn off" after 6 months of treatment.

TGF-ß1 Induction of CTGF
It was originally believed that the main mechanism for upregulation of CTGF expression was induction by TGF-ß1 in both cardiac (Chen et al. 2000Go) and non-cardiac tissues (Yokoi et al. 2001Go). Data from this study do not support this mechanism because the levels of TGF-ß1 mRNA have returned to baseline levels well before the activation of CTGF. It is now becoming clear that there are alternative mechanisms to induce CTGF expression including activation of a G protein–coupled receptor (Hahn et al. 2000Go), blood pressure–dependent mechanisms (Finkenberg et al. 2003Go), and protein kinase C activation (Way et al. 2002Go). Way et al. demonstrated that, in the fibrotic myocardium of transgenic mice overexpressing protein kinase C, dilating, and undergoing hypertrophy, CTGF, but not TGF-ß expression was increased at all time points studied. Furthermore, consistent with our earlier studies of advanced glycation effects in mesenchymal cells (Twigg et al. 2001Go) in the diabetic rat heart, CTGF, but not TGF-ß expression is increased (Way et al. 2002Go; Candido et al. 2003Go). Alternatively, CTGF has been shown to potentiate TGF-ß1, by facilitating TGF-ß1 binding to the TGF-ß receptor and that, although in this study TGF- ß1 itself does not rise at 180 days, it may well be that TGF-ß1 signaling pathways are used and potentiated by the increase in CTGF protein (Abreu et al. 2002Go).

Limitations
It is possible that small non-significant changes in cytokine levels may lead to significant effects on tissue remodelling and structure. Future in vitro studies may give some insight as to the magnitude of change in CTGF required to modulate collagen production and thus fibrosis, as well as to assess the effect of RAS blockade on this process.

Summary
This study provides evidence that TGF-ß1 plays a role in the acute response to myocardial injury and that CTGF is involved in the late phase of post-MI remodelling when injury and inflammation have abated, but mechanical load remains high and the non-infarcted myocardium is fibrosing, dilating, and hypertrophying. Furthermore, the cardiac benefits of captopril to reduce fibrosis in the viable myocardium are not mediated through changes in CTGF. Identification of a therapeutic intervention that reduces CTGF in the late phase of post-MI remodelling may prove beneficial in the search for improved treatment regimes in cardiovascular disease.


    Acknowledgments
 
This study was supported by the Sir Edward Dunlop Medical Research Foundation.

The authors wish to acknowledge the assistance of Dr. Richard E. Gilbert, Mr. Anthony Gleeson, Mrs. Donna Paxton, and Mrs. Alison Cox.


    Footnotes
 
Received for publication October 25, 2004; accepted May 11, 2005


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Abreu JG, Ketpura NI, Reversade B, De Robertis EM (2002) Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat Cell Biol 4:599–604[Medline]

Ahmed MS, Oie E, Vinge LE, Yndestad A, Oystein Andersen G, Andersson Y, Attramadal T, et al. (2004) Connective tissue growth factor—a novel mediator of angiotensin II-stimulated cardiac fibroblast activation in heart failure in rats. J Mol Cell Cardiol 36:393–404[CrossRef][Medline]

Azhar M, Schultz Jel J, Grupp I, Dorn GW 2nd, Meneton P, Molin DG, Gittenberger-de Groot AC, et al. (2003) Transforming growth factor beta in cardiovascular development and function. Cytokine Growth Factor Rev 14:391–407[CrossRef][Medline]

Border WA, Noble NA (1998) Interactions of transforming growth factor-beta and angiotensin II in renal fibrosis. Hypertension 31:181–188[Abstract/Free Full Text]

Burrell LM, Chan R, Phillips PA, Calafiore P, Tonkin A, Johnston CI (1996) Echocardiographic assessment of cardiac function after myocardial infarction in the rat. Clin Exp Pharmacol Physiol 23:570–572[Medline]

Burrell LM, Farina NK, Balding LC, Johnston CI (2000) Beneficial renal and cardiac effects of vasopeptidase inhibition with S21402 in heart failure. Hypertension 36:1105–1111[Abstract/Free Full Text]

Burrell LM, Palmer J, Charlton JA, Thomas T, Baylis PH (1990) A new radioimmunoassay for human atrial natriuretic peptide and its physiological validation. J Immunoassay 11:159–175[Medline]

Candido R, Forbes JM, Thomas MC, Thallas V, Dean RG, Burns WC, Tikellis C, et al. (2003) A breaker of advanced glycation end products attenuates diabetes-induced myocardial structural changes. Circ Res 92:785–792[Abstract/Free Full Text]

Chen MM, Lam A, Abraham JA, Schreiner GF, Joly AH (2000) CTGF expression is induced by TGF-beta in cardiac fibroblasts and cardiac myocytes: a potential role in heart fibrosis. J Mol Cell Cardiol 32:1805–1819[CrossRef][Medline]

Dean R, Edmondson SR, Burrell LM, Bach LA (1999) Localization of the insulin-like growth factor system in a rat model of heart failure induced by myocardial infarction. J Histochem Cytochem 47:649–660[Abstract/Free Full Text]

Dostal DE, Booz GW, Baker KM (1996) Angiotensin II signalling pathways in cardiac fibroblasts: conventional versus novel mechanisms in mediating cardiac growth and function. Mol Cell Biochem 157:15–21[Medline]

Duncan A-M, Burrell LM, Kladis A, Campbell DJ (1997) Angiotensin and bradykinin peptides in rats with myocardial infarction. J Cardiac Failure 3:41–52[CrossRef][Medline]

Eichorn E, Bristow M (1996) Medical therapy can improve the biological properties of the chronically failing heart. A new era in the treatment of heart failure. Circulation 94:2285–2296[Abstract/Free Full Text]

Farina NK, Johnston CI, Burrell LM (2000) Reversal of cardiac hypertrophy and fibrosis by S21402, a dual inhibitor of neutral endopeptidase and angiotensin converting enzyme in SHRs. J Hypertens 18:749–755[CrossRef][Medline]

Finkenberg P, Inkinen K, Ahonen J, Merasto S, Louhelainen M, Vapaatalo H, Muller D, et al. (2003) Angiotensin II induces connective tissue growth factor gain expression via calcineurin-dependent pathways. Am J Pathol 163:355–366[Abstract/Free Full Text]

Gilbert RE, Cox A, Wu LL, Allen TJ, Hulthen UL, Jerums G, Cooper ME (1998) Expression of transforming growth factor-beta1 and type IV collagen in the renal tubulointerstitium in experimental diabetes: effects of ACE inhibition. Diabetes 47:414–422[Abstract]

Hahn A, Heusinger-Ribeiro J, Lanz T, Zenkel S, Goppelt-Struebe M (2000) Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton. J Biol Chem 275:37429–37435[Abstract/Free Full Text]

Hao JM, Wang BQ, Jones SC, Jassal DS, Dixon IMC (2000) Interaction between angiotensin II and Smad proteins in failing heart and in vitro. Am J Physiol 279:H3020–3030

Igarashi A, Okochi H, Bradham DM, Grotendorst GR (1993) Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell 4:637–645[Abstract]

James J, Hauer-Jensen M (1999) Effects of fixative and fixation time for quantitative computerised image analysis of immunohistochemical staining. J Histotechnol 22:109–111

Le Moine C, Bernard V, Bloch B (1994) Quantitative in situ hybridization using radioactive probes in the study of gene expression in heterocellular systems. In Choo KHA, ed. Methods in molecular biology: in situ hybridization protocols. Totowa, NJ, Humana Press, 301–311

Lehr H-A, van der Loos CM, Teeling P, Gown AM (1999) Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis. J Histochem Cytochem 47:119–125[Abstract/Free Full Text]

Lijnen PJ, Petrov VV, Fagard RH (2000) Induction of cardiac fibrosis by transforming growth factor beta-1. Molecular Genetics and Metabolism 71:418–435[CrossRef][Medline]

Ohnishi H, Oka T, Kusachi S, Nakanishi T, Takeda K, Nakahama M, Doi M, et al. (1998) Increased expression of connective tissue growth factor in the infarct zone of experimentally induced myocardial infarction in rats. J Mol Cell Cardiol 30:2411–2422[CrossRef][Medline]

Ono K, Matsumori A, Shioi T, Furukawa Y, Sasayama S (1998) Cytokine gene expression after myocardial infarction in rat hearts: possible implication in left ventricular remodelling. Circulation 98:149–156[Abstract/Free Full Text]

Passier RC, Smits JF, Verluyten MJ, Daemen MJ (1996) Expression and localization of renin and angiotensinogen in rat heart after myocardial infarction. Am J Physiol 271:H1040–1048[Medline]

Pfeffer M, Braunwald E, Moye L, Basta L, Brown EJ, Cuddy T, Davis B, et al. (1992) Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. Results of the survival and ventricular enlargement trial. The SAVE Investigators. N Engl J Med 327:669–677

Pfeffer MA, Pfeffer JM, Steinberg C, Finn P (1985) Survival after an experimental myocardial infarction: beneficial effects of long-term therapy with captopril. Circulation 72:406–412[Abstract/Free Full Text]

Rimsza LM, Rangel CS, Grogran TM (1999) Image analysis for quantitative evaluation of antigen retrieval efficacy demonstrates increased detection of P-glycoprotein in overfixed cells but decreased detection in optimally fixed cells. J Histotechnology 22:9–14

Ruperez M, Lorenzo O, Blanco-Colio LM, Esteban V, Egido J, Ruiz-Ortega M (2003) Connective tissue growth factor is a mediator of angiotensin II-induced fibrosis. Circulation 108:1499–1505[Abstract/Free Full Text]

Sun Y, Zhang JQ, Zhang J, Ramires FJ (1998) Angiotensin II, transforming growth factor-beta1 and repair in the infarcted heart. J Mol Cell Cardiol 30:1559–1569[CrossRef][Medline]

Thompson NL, Bazoberry F, Speir EH, Casscells W, Ferrans VJ, Flanders KC, Kondaiah P, et al. (1988) Transforming growth factor beta-1 in acute myocardial infarction in rats. Growth Factors 1:91–99[Medline]

Twigg SM, Chen MM, Joly AH, Chakrapani SD, Tsubaki J, Kim HS, Oh Y, et al. (2001) Advanced glycosylation end products up-regulate connective tissue growth factor (insulin-like growth factor-binding protein-related protein 2) in human fibroblasts: a potential mechanism for expansion of extracellular matrix in diabetes mellitus. Endocrinology 142:1760–1769[Abstract/Free Full Text]

Tzanidis A, Lim S, Hannan RD, See F, Ugoni AM, Krum H (2001) Combined angiotensin and endothelin receptor blockade attenuates adverse cardiac remodelling post myocardial infarction in the rat: possible role of transforming growth factor beta-1. J Mol Cell Cardiol 33:969–981[CrossRef][Medline]

Way KJ, Isshiki K, Suzuma K, Yokota T, Zvagelsky D, Schoen FJ, Sandusky GE, et al. (2002) Expression of connective tissue growth factor is increased in injured myocardium associated with protein kinase C beta2 activation and diabetes. Diabetes 51:2709–2718[Abstract/Free Full Text]

Weber KT (1997) Extracellular matrix remodelling in heart failure: a role for de novo angiotensin II generation. Circulation 96:4065–4082[Free Full Text]

Weber KT, Janicki JS, Shroff SG, Pick R, Chen RM, Bashey RI (1988) Collagen remodelling of the pressure-overloaded, hypertrophied nonhuman primate myocardium. Circ Res 62:757–765[Abstract/Free Full Text]

Wu LL, Cox A, Roe CJ, Dziadek M, Cooper ME, Gilbert RE (1997) Transforming growth factor-beta one and renal injury following subtotal nephrectomy in the rat: role of the renin-angiotensin system. Kidney Int 51:1553–1567[Medline]

Yokoi H, Sugawara A, Mukoyama M, Mori K, Makino H, Suganami T, Nagae T, et al. (2001) Role of connective tissue growth factor in profibrotic action of transforming growth factor-beta: a potential target for preventing renal fibrosis. Am J Kidney Dis 38:S134–S138[Medline]

Youn TJ, Kim HS, Oh BH (1999) Ventricular remodelling and transforming growth factor-beta 1 mRNA expression after nontransmural myocardial infarction in rats: effects of angiotensin converting enzyme inhibition and angiotensin II type 1 receptor blockade. Basic Res Cardiol 94:246–253[CrossRef][Medline]

Yu C, Tipoe GL, Wing-Hon Lai K, Lau C (2001) Effects of combination of angiotensin converting enzyme inhibitor and angiotensin receptor antagonist on inflammatory cellular infiltration and myocardial interstitial fibrosis after acute myocardial infarction. Am Coll Cardiol 38:1207–1215[Abstract/Free Full Text]

Yu HC, Burrell LM, Black MJ, Wu LL, Dilley RJ, Cooper ME, Johnston CI (1998) Salt induces myocardial and renal fibrosis in normotensive and hypertensive rats. Circulation 98:2621–2628[Abstract/Free Full Text]

Yue P, Massie BM, Simpson PC, Long CS (1998) Cytokine expression increases in nonmyocytes from rats with postinfarction heart failure. Am J Physiol 275:H250–258[Medline]