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Abstract

The temporal and spatial expression of transforming growth factor (TGF)-β₁ 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-β₁, CTGF, and procollagen α1(I) mRNA were localized by in situ hybridization, and TGF-β₁ 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-β₁, CTGF, and collagen after MI. Procollagen α1(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-β₁ 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-β₁ or CTGF. TGF-β₁ 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.

Keywords

connective tissue growth factor myocardial infarction fibrosis angiotensin-converting enzyme

A fter 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 1996). 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 1997).

Several studies have assessed the role of cytokines on myocyte injury and myocardial function in the acute phase after MI (Ohnishi et al. 1998; Ono et al. 1998; Sun et al. 1998; Lijnen et al. 2000; Yu et al. 2001; Ahmed et al. 2004). Low levels of expression of transforming growth factor (TGF)-β₁, its receptor, and collagen type 1 are seen in normal rat heart and increase after MI. The increase in TGF-β₁ mRNA precedes increases in collagen expression, suggesting a role for TGF-β₁ in cardiac fibrosis and remodelling (Youn et al. 1999; Yu et al. 2001; Tzanidis et al. 2001). 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. 1993). Acutely after MI, the expression of CTGF increases in parallel with collagen (Ohnishi et al. 1998).

The concomitant regional and spatial changes in expression of TGF-β₁ and CTGF after MI have not been assessed. Many studies have assessed either TGF-β₁ or CTGF and most have been short term (<6 weeks) in nature (Ohnishi et al. 1998; Youn et al. 1999; Tzanidis et al. 2001; Yu et al. 2001; Ahmed et al. 2004). To date, no studies have assessed the expression of TGF-β₁ 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. 1999). 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. 2000), and now hypothesize that the long-term beneficial effect of ACE inhibitors may be mediated through changes in the profibrotic cytokines TGF-β₁ or CTGF. The aims of this study were 2-fold: (1) to examine the time course and cellular expression of TGF-β₁, 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

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. 1985; Burrell et al. 1996, 2000; Dean et al. 1999). 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 of remodelling after MI (Burrell et al. 2000). 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. 1990) 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. 1985).

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-β₁, CTGF, and procollagen α1(I) as previously described (Wu et al. 1997; Yu et al. 1998). The 945-bp cDNA probe for TGF-β₁ 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 α1(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 1 X 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 2 X 10⁴ 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, 2 X SSC 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. 1994). 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. 1998). Total collagen volume fraction determined by this morphometric approach is closely related to the hydroxyproline concentration in the LV (Weber et al. 1988).

Immunohistochemistry for ED1, TGF-β₁, and CTGF

Immunohistochemistry for macrophages and TGF-β₁ was carried out on 4-μm paraffin sections of LV as previously described (Gilbert et al. 1998; Dean et al. 1999). For CTGF, the primary antibody used was a polyclonal rabbit antimouse 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-β₁ 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 1999; Lehr et al. 1999; Rimsza et al. 1999).

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

Study 1: Time Course

TGF-β₁ and Procollagen α1(I) mRNA Localized to Areas of Inflammation after MI. The macroscopic localization of TGF-β₁, CTGF, and procollagen α1(I) mRNA is shown in Figure 1. TGF-β₁, CTGF, and procollagen α1(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.

Silver grains representing procollagen α1(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 α1(I) labeling (not shown).

Figure 1

Representative macroscopic autoradiographs demonstrating levels of signal produced by in situ hybridization histochemistry for transforming growth factor-β₁ (top row), connective tissue growth factor (middle row), and procollagen α1(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).

TGF-β₁ mRNA localized to the same areas as procollagen α1(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. 1999). TGF-β₁ 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).

Procollagen α1(I) mRNA Levels Peak in First Week after MI. The macroscopic autoradiographic representation of the induction of procollagen α1(I) mRNA following MI is shown in Figure 1. Procollagen α1(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 α1(I) mRNA in the viable myocardium at 7 days, but over the 180 days, the changes were not significant (Figure 3C).

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-β₁ mRNA and TGF-β₁ Protein at Peak Levels in the First Week after MI. Levels of TGF-β₁ mRNA were significantly increased in the border zone at days 1 and 3 (p<0.01) (Figure 4A). In the scar, TGF-β₁ mRNA was significantly increased at days 7, 28, and 180 (all p<0.01) (Figure 4B). TGF-β₁ mRNA was present in non-infarcted viable myocardium, and did not significantly change after MI (Figure 4C). TGF-β₁ 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-β₁ 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-β₁.

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.

Figure 3

Expression of procollagen α1(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.

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).

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).

Long-term Captopril Decreases Total Collagen Protein in the Viable Myocardium. We confirmed our previous findings that 180 days after MI, procollagen α1(I) mRNA levels were increased in the border and scar zones (p<0.01) (Figures 6A-6C). The levels of procollagen α1(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).

Captopril Does Not Affect TGF-β₁ mRNA or Protein 180 Days after MI. At 180 days after MI, the level of TGF-β₁ 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-β₁ mRNA and protein (Figures 7D-7F).

Figure 4

Expression of transforming growth factor-β₁ 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.

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).

Discussion

The main findings of this study in rat after MI are that (1) temporal and regional differences in the expression of TGF-β₁, 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-β₁; 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-β₁ 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-β₁, CTGF, and collagen mRNA were seen in normal rat hearts. TGF-β₁ increased acutely after MI, preceding increases in collagen expression. The finding that TGF-β₁ and procollagen α1(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 α1(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-β₁ mRNA and protein in the border zone (Thompson et al. 1988) and at 1 week after MI, TGF-β₁ mRNA is induced in non-myocytes (Yue et al. 1998). By 8 weeks after MI, Western blot analysis of cardiac tissue showed the active form of TGF-β₁ was significantly elevated in border and scar tissue, but myocytes remote to the infarct zone expressed comparatively moderate levels of TGF-β₁ only (Hao et al. 2000). We found parallel increases in TGF-β₁ 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-β₁ mRNA remained high, but was not translated to protein.

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.

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. 2004).

Our data suggest that TGF-β₁ may be involved in the initial induction of procollagen α1(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-β₁ mRNA to inflammatory cells suggests that TGF-β₁ has a role beyond collagen production, possibly pleiotropic actions such as wound healing or simply maintaining physiological functioning of the heart (Azhar et al. 2003). 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. 1992). 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 1996). 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 1997).

Renin-angiotensin System Blockade and TGF-β₁

Both in vitro and in vivo studies have shown Ang II stimulates TGF-β₁ synthesis (Dostal et al. 1996; Border and Noble 1998). It is well known that fibrosis and activation of the cardiac renin-angiotensin system (RAS) occurs after MI (Passier et al. 1996; Duncan et al. 1997), and we have shown that the ACE inhibitor captopril reduces LV mass after MI (Burrell et al. 2000) and has antifibrotic effects in the heart (Farina et al. 2000). Although these data suggest a link between the RAS, fibrosis and the TGF-β₁ 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. 1998; Youn et al. 1999; Yu et al. 2001) or does not (Tzanidis et al. 2001; Yu et al. 2001) “switch off” cardiac TGF-β₁ expression after MI. In this study, 180 days of treatment with captopril had no effect on TGF-β₁ levels, which may be explained by the fact that the TGF-β₁ 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.

Table 1

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

	Control	MI
Parameter	Vehicle	Vehicle	Captopril
n	10	8	9
Infarct size (%)	0	38 ± 0.5	38 ± 0.2
Body weight (g)	288 ± 12	291 ± 20	271 ± 12
Blood pressure (mm Hg)	120 ± 4	118 ± 2	101 ± 4°°
LV/body weight (g/kg)	2.24 ± 0.01	2.58 ± 0.01∗	2.24 ± 0.01°
Lung/body weight (g/kg)	6.26 ± 0.37	8.51 ± 0.87∗	6.63 ± 1.24°
Plasma ANP (pmol/L)	25.7 ± 6.9	90.7 ± 28.0∗	26.8 ± 4.6°
PRA (nmol Ang I/I/hr)	2.3 ± 0.4	4.3 ± 1.5	64.9 ± 17.7°°

Values are expressed as mean ± SEM. MI, myocardial infarction, PRA, plasma renin activity, ANP, atrial natriuretic peptide, LV, left ventricle. ∗p<0.05 and ∗∗p<0.01 MI vehicle vs. control; °p<0.05 and °°p<0.01 MI captopril vs. MI vehicle.

Figure 6

Effect of captopril treatment on the expression of procollagen α1(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.

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. 2003). 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. 2004). 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-β₁ Induction of CTGF

It was originally believed that the main mechanism for upregulation of CTGF expression was induction by TGF-β₁ in both cardiac (Chen et al. 2000) and non-cardiac tissues (Yokoi et al. 2001). Data from this study do not support this mechanism because the levels of TGF-β₁ 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. 2000), blood pressure-dependent mechanisms (Finkenberg et al. 2003), and protein kinase C activation (Way et al. 2002). Way et al. demonstrated that, in the fibrotic myocardium of transgenic mice overexpressing protein kinase C, dilating, and undergoing hypertrophy, CTGF, but not TGF-β expression was increased at all time points studied. Furthermore, consistent with our earlier studies of advanced glycation effects in mesenchymal cells (Twigg et al. 2001) in the diabetic rat heart, CTGF, but not TGF-β expression is increased (Way et al. 2002; Candido et al. 2003). Alternatively, CTGF has been shown to potentiate TGF-β₁, by facilitating TGF-β₁ binding to the TGF-β receptor and that, although in this study TGF- β₁ itself does not rise at 180 days, it may well be that TGF-β₁ signaling pathways are used and potentiated by the increase in CTGF protein (Abreu et al. 2002).

Figure 7

Effect of captopril treatment on the expression of transforming growth factor-β₁ mRNA (A-C) and protein (D-F) in the border, scar, and viable myocardium at 180 days after myocardial infarction.

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.

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-β₁ 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.

Footnotes

Acknowledgements

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.

References

Abreu

Ketpura

Reversade

De Robertis

(2002) Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat Cell Biol 4: 599–604

Ahmed

Oie

Vinge

Yndestad

Oystein Andersen

Andersson

Attramadal

(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

Azhar

Schultz Jel

Grupp

Dorn

2nd Meneton

Molin

Gittenberger-de Groot

(2003) Transforming growth factor beta in cardiovascular development and function. Cytokine Growth Factor Rev 14: 391–407

Border

Noble

(1998) Interactions of transforming growth factor-beta and angiotensin II in renal fibrosis. Hypertension 31: 181–188

Burrell

Chan

Phillips

Calafiore

Tonkin

Johnston

(1996) Echocardiographic assessment of cardiac function after myocardial infarction in the rat. Clin Exp Pharmacol Physiol 23: 570–572

Burrell

Farina

Balding

Johnston

(2000) Beneficial renal and cardiac effects of vasopeptidase inhibition with S21402 in heart failure. Hypertension 36: 1105–1111

Burrell

Palmer

Charlton

Thomas

Baylis

(1990) A new radioimmunoassay for human atrial natriuretic peptide and its physiological validation. J Immunoassay 11: 159–175

Candido

Forbes

Thomas

Thallas

Dean

Burns

Tikellis

(2003) A breaker of advanced glycation end products attenuates diabetes-induced myocardial structural changes. Circ Res 92: 785–792

Chen

Lam

Abraham

Schreiner

Joly

(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

10.

Dean

Edmondson

Burrell

Bach

(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

11.

Dostal

Booz

Baker

(1996) Angiotensin II signalling pathways in cardiac fibroblasts: conventional versus novel mechanisms in mediating cardiac growth and function. Mol Cell Biochem 157: 15–21

12.

Duncan

A-M

Burrell

Kladis

Campbell

(1997) Angiotensin and bradykinin peptides in rats with myocardial infarction. J Cardiac Failure 3: 41–52

13.

Eichorn

Bristow

(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

14.

Farina

Johnston

Burrell

(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

15.

Finkenberg

Inkinen

Ahonen

Merasto

Louhelainen

Vapaatalo

Muller

(2003) Angiotensin II induces connective tissue growth factor gain expression via calcineurin-dependent pathways. Am J Pathol 163: 355–366

16.

Gilbert

Cox

Allen

Hulthen

Jerums

Cooper

(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

17.

Hahn

Heusinger-Ribeiro

Lanz

Zenkel

Goppelt-Struebe

(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

18.

Hao

Wang

Jones

Jassal

Dixon

IMC

(2000) Interaction between angiotensin II and Smad proteins in failing heart and in vitro. Am J Physiol 279: H3020–3030

19.

Igarashi

Okochi

Bradham

Grotendorst

(1993) Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell 4: 637–645

20.

James

Hauer-Jensen

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

21.

Le Moine

Bernard

Bloch

(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

22.

Lehr

H-A

van der Loos

Teeling

Gown

(1999) Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis. J Histochem Cytochem 47: 119–125

23.

Lijnen

Petrov

Fagard

(2000) Induction of cardiac fibrosis by transforming growth factor beta-1. Molecular Genetics and Metabolism 71: 418–435

24.

Ohnishi

Oka

Kusachi

Nakanishi

Takeda

Nakahama

Doi

(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

25.

Ono

Matsumori

Shioi

Furukawa

Sasayama

(1998) Cytokine gene expression after myocardial infarction in rat hearts: possible implication in left ventricular remodelling. Circulation 98: 149–156

26.

Passier

Smits

Verluyten

Daemen

(1996) Expression and localization of renin and angiotensinogen in rat heart after myocardial infarction. Am J Physiol 271: H1040–1048

27.

Pfeffer

Braunwald

Moye

Basta

Brown

Cuddy

Davis

(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

28.

Pfeffer

Steinberg

Finn

(1985) Survival after an experimental myocardial infarction: beneficial effects of long-term therapy with captopril. Circulation 72: 406–412

29.

Rimsza

Rangel

Grogran

(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

30.

Ruperez

Lorenzo

Blanco-Colio

Esteban

Egido

Ruiz-Ortega

(2003) Connective tissue growth factor is a mediator of angiotensin II-induced fibrosis. Circulation 108: 1499–1505

31.

Sun

Zhang

Ramires

(1998) Angiotensin II, transforming growth factor-beta1 and repair in the infarcted heart. J Mol Cell Cardiol 30: 1559–1569

32.

Thompson

Bazoberry

Speir

Casscells

Ferrans

Flanders

Kondaiah

(1988) Transforming growth factor beta-1 in acute myocardial infarction in rats. Growth Factors 1: 91–99

33.

Twigg

Chen

Joly

Chakrapani

Tsubaki

Kim

(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

34.

Tzanidis

Lim

Hannan

See

Ugoni

Krum

(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

35.

Way

Isshiki

Suzuma

Yokota

Zvagelsky

Schoen

Sandusky

(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

36.

Weber

(1997) Extracellular matrix remodelling in heart failure: a role for de novo angiotensin II generation. Circulation 96: 4065–4082

37.

Weber

Janicki

Shroff

Pick

Chen

Bashey

(1988) Collagen remodelling of the pressure-overloaded, hypertrophied nonhuman primate myocardium. Circ Res 62: 757–765

38.

Cox

Roe

Dziadek

Cooper

Gilbert

(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

39.

Yokoi

Sugawara

Mukoyama

Mori

Makino

Suganami

Nagae

(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

40.

Youn

Kim

(1999) Ventricular remodelling and transforming growth factor-beta 1 mRNA expression after non-transmural myocardial infarction in rats: effects of angiotensin converting enzyme inhibition and angiotensin II type 1 receptor blockade. Basic Res Cardiol 94: 246–253

41.

Tipoe

Wing-Hon Lai

Lau

(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

42.

Burrell

Black

Dilley

Cooper

Johnston

(1998) Salt induces myocardial and renal fibrosis in normotensive and hypertensive rats. Circulation 98: 2621–2628

43.

Yue

Massie

Simpson

Long

(1998) Cytokine expression increases in nonmyocytes from rats with postinfarction heart failure. Am J Physiol 275: H250–258

Connective Tissue Growth Factor and Cardiac Fibrosis after Myocardial Infarction

Abstract

Keywords

Materials and Methods

Experimental Design

Study 1: Time Course

Study 2: Captopril Treatment

Infarct Size

In Situ Hybridization

Quantitation of Macroscopic In Situ Hybridization Autoradiographs

Total Collagen Staining

Immunohistochemistry for ED1, TGF-β1, and CTGF

Statistics

Results

Study 1: Time Course

Study 2: Captopril Treatment

Discussion

Fibrosis and Cytokine Expression after MI

ACE Inhibition after MI

Renin-angiotensin System Blockade and TGF-β1

RAS Blockade and CTGF

TGF-β1 Induction of CTGF

Limitations

Summary

Footnotes

Acknowledgements

References

Immunohistochemistry for ED1, TGF-β₁, and CTGF

Renin-angiotensin System Blockade and TGF-β₁

TGF-β₁ Induction of CTGF