Reduction in Left Ventricular Messenger RNA for Transforming Growth Factor β1 Attenuates Left Ventricular Fibrosis and Improves Survival Without Lowering Blood Pressure in the Hypertensive TGR(mRen2)27 Rat
Abstract—Angiotensin II recruits transforming growth factor β1 (TGFβ1) and is related to left ventricular fibrosis. However, it is unclear whether chronic in vivo reduction in left ventricular TGFβ1 expression blunts fibrosis and improves outcome in angiotensin II–dependent hypertension. Four-week-old male hypertensive TGR(mRen2)27 (Ren2) rats received either normal food, low-dose losartan (0.5 mg · kg−1 · d−1), or tranilast (a nonspecific TGFβ inhibitor; 400 mg · kg−1 · d−1) (n=10 for each group) for 12 weeks and were compared with Sprague-Dawley control rats. The effect of tranilast on survival was evaluated in 34 additional untreated homozygous Ren2 rats. Tranilast or low-dose losartan did not lower blood pressure. However, the increase in left ventricular weight (Ren2 versus SD 3.1±0.16 versus 2.1± 0.06 mg/g body wt; P<0.05) was significantly (P<0.05) blunted by both tranilast (2.7±0.05) and losartan (2.7±0.07). Both drugs prevented the increase in left ventricular TGFβ1 mRNA and fibronectin mRNA and blunted the increase in hydroxyproline content and the increase in perivascular fibrosis. The perivascular fibrosis score correlated significantly with the level of expression of TGFβ1 (r=0.62; P=0.019). In situ hybridization demonstrated increases in TGFβ1 mRNA, predominantly in perivascular and nonmyocyte areas. Both drugs did not prevent the decrease in systolic or diastolic dP/dt, but tranilast significantly improved the survival of untreated Ren2 rats (P=0.029). In conclusion, TGFβ1 mRNA expression is increased predominantly in nonmyocyte regions in the hypertrophied left ventricle in this angiotensin II–dependent model of hypertension. This increase is probably due to high angiotensin II levels rather than to hypertension. This is the first study to suggest that chronic inhibition of TGFβ1 expression attenuates left ventricular hypertrophy and fibrosis, even without lowering blood pressure.
Hypertension-related left ventricular hypertrophy is associated with an adverse outcome,1 although hypertrophy is a normal adaptation to increased loading and is invariably found in every rat model of hypertension.2 This suggests that only part of the hypertrophic process is maladaptive. It is thought that this maladaptive part of cardiac hypertrophy is related to increased expression of growth factors in the heart, which eventually lead to excess fibrosis. Consequently, it has been proposed that a reduction in growth factors could prevent such adverse changes.3 A central role in this process has been proposed for transforming growth factor β1 (TGFβ1). Although all 3 isoforms of TGFβ (TGFβ1, -β2, and -β3) are present in the heart, the level of the type 1 isoform seems to be particularly related to the development of left ventricular hypertrophy.4 TGFβ1 is secreted by most cell types and has complex actions, depending on the cell type that is involved: it inhibits the proliferation of many cells but also stimulates the growth of mesenchymal cells and stimulates the formation of extracellular matrix.5 The level of TGFβ1 mRNA is increased in left ventricular hypertrophy.6 In spontaneously hypertensive rats (SHR) with heart failure, this increase in left ventricular TGFβ1 expression, accompanied by increased fibronectin expression, has been suggested to mark the transition from stable hypertrophy to failure.6 In that study, the expression of TGFβ1 was not increased in nonfailing SHR. However, an increased expression of TGFβ1 has also been described7 in nonfailing hypertrophy as occurs after abdominal aortic coarctation. This suggests that the particular type of hypertension may determine the relative importance of TGFβ1 expression in the development of hypertrophy and fibrosis. It has been demonstrated in vitro that angiotensin II induces TGFβ1 in smooth muscle cells8 and in cardiac cells and fibroblasts,9 10 which is underlined by the finding that TGFβ1 expression is also increased in the hypertrophied left ventricle of the TGR(mRen2)27 (Ren2) rat,11 12 a hypertensive model characterized by increased cardiac angiotensin II.13 Interestingly, the report by Villarreal et al11 demonstrated only increased perivascular fibrosis and no diffuse fibrosis in this model. Angiotensin II infusion also increases the expression of TGFβ1, although this could not be dissected from a direct pressor effect.14
Although increased TGFβ1 has been associated with its adverse, profibrotic actions, there also is evidence that TGFβ1 can have protective, beneficial effects in human atherosclerotic disease.15 16 As a result, it is yet unclear whether beneficial or detrimental effects should be expected from an in vivo reduction in the expression of TGFβ1 in hypertensive heart disease. Therefore, we evaluated the cardiac effects of a reduction in TGFβ1 mRNA with tranilast in a rat model of hypertension. Tranilast, a drug that was originally used for the treatment of allergic and dermatological diseases, was recently reported to inhibit TGFβ-mediated collagen formation.17 18 This compound is now gaining interest in human cardiovascular disease as a possible therapy for restenosis.19
The hypothesis we sought to test is that in the hypertensive Ren2 rat, the increased angiotensin II level, independent of its hypertensive effect, augments left ventricular TGFβ1 expression and thereby increases collagen content, which leads to impaired left ventricular performance and decreased survival. Therefore, we also assessed the effects of tranilast in direct comparison with a nonhypotensive dose of losartan, a specific angiotensin II type 1 (AT1) receptor antagonist. To assess the capacity of either drug to prevent left ventricular changes, treatment was initiated before hypertension was established.
All rats were purchased from the Moellegaard Breeding Company. Directly after being weaned at 4 weeks of age, 30 male Ren2 rats were randomly assigned to receive no treatment, losartan (0.5 mg · kg−1 · d−1), or tranilast (400 mg · kg−1 · d−1) (n=10 for each group). Tranilast was a generous gift from Dr T. Hama (Kissei Laboratories, Nagano, Japan). The dosage of tranilast was based on previous studies that had shown that the same dosage in Sprague-Dawley (SD) rats significantly decreased the hydroxyproline content in granulation tissue.20 This dosage is expected to result in serum levels of ≈100 μmol/L, which is comparable to the serum concentration in human clinical trials.21 Because we did not anticipate an effect of tranilast on blood pressure, the dosage of losartan was chosen to not affect blood pressure as well. The drugs were mixed with the rat chow with an assumption of a daily intake of 30 g chow · rat−1 · d−1. We verified that the rats maintained a normal food intake during the trial. Nontransgenic SD littermates served as controls and were randomized to receive either no treatment (n=8) or tranilast (400 mg · kg−1 · d−1, n=8).
Treatment was started at the age of 4 weeks and continued until death at 16 weeks. Blood pressure was measured weekly with the tail-cuff method, with the rats under light ether anesthesia.
At the end of the treatment period, each rat was anesthetized with pentobarbital (50 mg/kg IP). The right carotid artery was then cannulated with a 3F Millar microtip catheter, which was connected to a personal computer that automatically displayed and stored the data (TSE Biosystems). The catheter was advanced into the left ventricle for measurement of left ventricular end-diastolic pressure (LVEDP) and the first derivative of pressure over time, dP/dt. LVEDP was measured on a separate channel with an amplified scale.
Thereafter, the heart was rapidly excised and blotted dry, and the right and left ventricles were separated, weighed, and immediately frozen in liquid nitrogen. A transverse slice was immersed in a formalin solution for future histological studies.
The aorta was excised and cleansed of surrounding tissue, and rings were cut of the thoracic aorta. These were suspended individually in organ baths to determine the dose-response curve to increasing concentrations of angiotensin II (0.1 nmol/L to 1 μmol/L) in the presence of NG-monomethyl-l-arginine, as we described earlier.22
The left carotid artery was excised, immersed in formaldehyde, and embedded in paraffin, and sections were cut and stained with hematoxylin-eosin. Because these sections were not perfusion fixed at the respective pressure of the rats, these measurements serve only to provide an indication of whether treatment affected medial thickness. Sections were photographed, and these photographs were coded so that the investigator who took the measurements (Y.M.P.) was unaware of the origin of the photograph. Thickness was measured at 4 locations in the carotid artery, all separated by 45°, in 3 photographs of each artery.
Total RNA was extracted from the left ventricle with TRIzol (GIBCO) reagent, according to the instructions of the manufacturer. Total RNA (15 μg) was denatured with formamide/formaldehyde, size separated with gel electrophoresis, transferred to a nylon membrane (Hybond N; Amersham), and fixed to the membrane through UV cross-linking. The membranes were hybridized with a 32P-labeled 825-bp BamHI fragment of the atrial natriuretic factor (ANF) cDNA cloned in pGEM-4Z (kindly provided by Dr Sigrid Hoffmann, Ruprecht-Karls University Heidelberg, Heidelberg, Germany), a 445-bp polymerase chain reaction–generated cDNA probe for TGFβ1 (primers available on request), and an 800-bp fragment of the fibronectin cDNA (kindly provided by Dr Kenneth Boheler, Gerontology Research Center, Baltimore, Md).
The membranes were hybridized in Quickhyb (Stratagene) solution for 1 hour at 68°C. Thereafter, the membranes were washed in a mixture of 0.1% SDS and decreasing concentrations of standard saline citrate (SSC) at a final temperature of 55°C. Then, the bands were visualized through exposure to x-ray film (Kodak, Germany) for 24 to 48 hours and quantified through digital scanning with the aid of the publicly available NIH Image program. The density of the bands was compared with that of the bands obtained through subsequent hybridization to a GAPDH probe. Results were displayed as the ratio between the density of the target band, relative to the GAPDH band (in arbitrary units).
Hydroxyproline content of the left ventricle was determined according to a previously described method.23 In brief, a sample of the left ventricle (≥100 mg) was hydrolyzed in HCl, after which Ehrlich’s reagent was added. The resultant extinction was compared with a standard curve for the determination of hydroxyproline content.
The transverse midsection of the left ventricle was immersed in formaldehyde and embedded in paraffin, sections were cut and stained with hematoxylin-eosin, and separate sections were stained with Sirius Red to stain for collagen. The left carotid artery was similarly treated but only stained with hematoxylin-eosin.
Sections were photographed, and these photographs were coded so that the investigator who made the measurements (Y.M.P.) was unaware of the origin of the photograph.
To analyze whether treatment affected myocyte thickness, the thickness of individual myocytes was measured on 3 different spots. To analyze the changes in fibrotic areas, we digitized the Sirius Red–stained sections and measured the proportion of stained collagen within a predefined, fixed square. This was done directly adjacent to an artery for the measurement of perivascular fibrosis. It was also done in an area covered by myocytes to measure interstitial fibrosis.
Thickness of the carotid artery was measured at 4 locations in the carotid artery, all separated by 45°, in 3 photographs of each artery.
In Situ Hybridization
Cryosections of left ventricular samples were incubated for 15 minutes in 0.2N HCl and treated with 20 μg/mL Proteinase K for 3 minutes. Then, they were refixed in 4% paraformaldehyde/PBS for 20 minutes and acetylated with 25 mmol/L acetic anhydride in 100 mmol/L triethanolamine for 10 minutes. After being washed twice in PBS, sections were dehydrated with ethanol. Air-dried sections were hybridized under coverslips with 4*108 cpm 33P-labeled RNA probe/mL hybridization mixture (50% formamide, 3 mol/L NaCl, 20 mmol/L Tris, 5 mmol/L EDTA, 10 mmol/L NaH2PO4, 10% dextran sulfate, 1× Denhardt’s solution, 0.5 mg/mL yeast total RNA) for 20 hours at 55°C in a humidified chamber.
After hybridization, coverslips were removed in 5× SSC. The sections were then washed at 55°C in 2× SSC; incubated at 37°C in a buffer composed of 0.5 mol/L NaCl, 10 mmol/L Tris, and 5 mmol/L EDTA containing RNase A (10 μg/mL) for 30 minutes; and washed for 30 minutes at 55°C, followed by dehydration through ethanol containing 0.3 mol/L ammonium acetate. After drying, the sections were dipped in radiographic emulsion (Amersham), exposed for 2 to 3 weeks at 4°C, and developed according to the manufacturer’s instructions. Counterstaining was performed with hematoxylin-eosin.
RNA Probe for TGFβ1
A 338-bp portion of the 3′ portion of the murine TGFβ1 gene was amplified from total mouse embryonic cDNA and cloned into a modified pBluescript SK vector. The construct was linearized with appropriate restriction enzymes, and RNA probes were transcribed in the presence of 33P-UTP with T7 polymerase to prepare the antisense probe and T3 polymerase to generate the sense probes. All sections were hybridized with both the antisense and the sense probe.
Because tranilast treatment appeared to decrease mortality rates in the heterozygous Ren2 rats, in a separate experiment we assessed the effects of tranilast on survival of untreated homozygous Ren2 rats. If untreated with an ACE inhibitor, the homozygous rats are known to have a very high mortality rate after 8 to 12 weeks.24 Thirty-four homozygous, male untreated Ren2 rats were randomized to receive either tranilast (400 mg · kg−1 · d−1) or no treatment. Treatment was started at the age of 4 weeks (immediately after weaning) and continued for 10 weeks.
All results are shown as mean±SEM. Differences between groups were tested by a 1-way ANOVA, corrected where appropriate by Duncan’s multiple range test for multiple comparisons (all with SPSS for Windows 7.5). The effect of tranilast on survival was tested in the Kaplan-Meier survival analysis with a log-rank test. Correlations were tested by a Pearson correlation test.
Neither losartan nor tranilast affected the expected increase in blood pressure, so blood pressure reached similar hypertensive levels in all 3 Ren2 groups (Figure 1⇓). In the untreated Ren2 rat group, 3 rats died, whereas in each of the losartan- and tranilast-treated groups, 1 rat died.
Both systolic and diastolic dP/dt values were significantly decreased in untreated Ren2 rats compared with SD rats (Table⇓); LVEDP was not increased. Neither losartan nor tranilast attenuated the decrease in systolic or diastolic dP/dt. Heart rate and body weights were similar among all groups.
Left ventricular weight was significantly increased in the untreated hypertensive Ren2 rats, and both losartan and tranilast significantly attenuated this increase in left ventricular weight (Figure 2⇓). Tranilast did not significantly affect left ventricular function parameters or left ventricular weight in normal SD rats.
Aortic Dose-Response to Angiotensin II
The dose-response curve to angiotensin II was not significantly altered in aortic rings from untreated hypertensive Ren2 rats compared with untreated SD rats (data not shown). Furthermore, tranilast treatment did not affect the response to angiotensin II. However, low-dose losartan significantly decreased the maximal response to angiotensin II compared with untreated Ren2 rats (Figure 3⇓).
Myocyte thickness was significantly increased in the untreated Ren2 rats, but this was not affected by either treatment (Figure 4⇓, top). The measurement of fibrosis revealed that perivascular fibrosis was significantly increased in the untreated Ren2 rats (Figure 5⇓, bottom), whereas interstitial fibrosis was unaltered (data not shown). Both losartan and tranilast (Figure 5⇓, bottom) blunted the increase in perivascular fibrosis. The degree of perivascular fibrosis correlated significantly with the level of expression of TGFβ (r=0.62; P=0.019) and correlated weakly and nonsignificantly with left ventricular weight–to–body weight ratio (r=0.46; P=0.08). Furthermore, in situ hybridization showed that the increased expression in the left ventricle of the untreated Ren2 rat was located mainly in areas that were not occupied by myocytes, such as the perivascular fibrotic areas, and, to a lesser extent, between myocytes in interstitial areas (Figure 6⇓).
The thickness of the media of the carotid artery was significantly increased in the untreated hypertensive Ren2 rats. Both losartan and tranilast failed to affect media thickness (Figure 4⇑, bottom).
Expression of TGFβ1 mRNA was significantly increased in left ventricular tissue from untreated hypertensive Ren2 rats. This increase was prevented by both losartan and tranilast and to a similar extent (Figure 7A⇓, left). This was also reflected by similar changes in the expression of fibronectin (Figure 7A⇓, right). The expression of ANF was significantly increased in untreated hypertensive Ren2 rats, which was significantly blunted (but not normalized) by both tranilast and losartan (Figure 7B⇓).
Total left ventricular hydroxyproline content was increased in untreated Ren2 rats versus SD rats. Both tranilast and losartan blunted this increase (Figure 5⇑, top), so hydroxyproline was not significantly increased in the groups treated with tranilast or losartan.
Effects of Tranilast on Survival
The Kaplan-Meier curves in Figure 8⇓ demonstrate the effect of tranilast compared with untreated Ren2 rats. Overall, survival was higher than anticipated for the homozygous rats, because these have been reported to not survive at all without ACE inhibitor treatment. We found a mortality rate of only 41% after 3 months in the untreated homozygous rats. Nevertheless, overall survival was significantly improved in the tranilast treated rats compared with the untreated Ren2 rats (P=0.029).
In the present study, we tested the hypothesis that increased angiotensin II levels in the hypertensive Ren2 model directly augment left ventricular TGFβ1 expression and thereby increase collagen content, which leads to impaired left ventricular performance and decreased survival. We postulated that this adverse cascade might be initiated by angiotensin II independent of the increased blood pressure. To address this hypothesis, we compared tranilast, a nonspecific inhibitor of TGFβ expression,17 18 with the effects of a nonhypotensive dose of the angiotensin II type 1 (AT1) receptor antagonist losartan.
Our study is the first to report that tranilast reduces left ventricular TGFβ1 expression, collagen accumulation and perivascular fibrosis, and left ventricular hypertrophy and improves survival in a blood pressure–independent manner. A nonhypotensive dose of losartan similarly blunted the expression of left ventricular TGFβ1 and had similar effects on collagen and perivascular fibrosis. Interestingly, neither tranilast nor losartan altered myocyte thickness, nor did they improve left ventricular function, suggesting a specific role for TGFβ1 in nonmyocyte processes. This notion is underlined by our finding that the increase in mRNA for left ventricular TGFβ1 seems in large part confined to areas outside the myocytes, where we also noted fibrosis. There are only a few studies that have reported the localization of cardiac mRNA for TGFβ1. Li et al25 showed increased TGFβ1 mRNA and protein in samples obtained from patients with hypertrophic cardiomyopathy or aortic stenosis. However, the authors of this study also noted an increase in TGFβ1 in these human cardiac myocytes, which we did not observe in our rat model. In 2 very different rat models of hypertension, the mRNA for TGFβ1 was initially found only in cultured nonmyocyte cardiac cells, but in this study, the induction of TGFβ1 was noted in the cardiomyocytes cultured from hypertrophied left ventricles.26 The paucity of reports on the localization of cardiac TGFβ1 mRNA expression and the different models in which it was investigated do not allow us to draw definite conclusions on the comparability of our findings with those of others. However, the current data do suggest that the increase in TGFβ1 mRNA colocalizes with cardiac fibrosis.
Taken together, our findings suggest that in the hypertensive Ren2 rat, increased angiotensin II levels augment the expression of left ventricular TGFβ1 in noncardiomyocytes and that this augments perivascular fibrosis. This is in line with recent data from a separate study that also suggested that the effects of angiotensin II on cardiac fibroblasts might be mediated in large part by TGFβ.27 It does not confirm the notion that impaired left ventricular performance is related to the increased expression of measured molecular markers of hypertrophy or to perivascular fibrosis.
Mechanism of Action of Tranilast
Tranilast was not originally developed to inhibit TGFβ1 or its effects. Our findings are in agreement with what was described for its mode of action in cultured cells. The present data suggest that tranilast mainly inhibits proliferation of noncardiomyocytes but may not affect the hypertrophic process of myocytes. This is in agreement with the suggestion that tranilast suspends the cell cycle of fibroblasts at the G0/G1 phase.28 29
Other studies suggest that tranilast mainly inhibits activated cells, in that tranilast suppressed collagen synthesis in cultured fibroblasts derived from scar tissue but not from healthy skin.17 This is in accordance with our findings, in which we also noted that tranilast reduced the expression of TGFβ1 only in the hypertensive rats and not in normal rats. Concurrent with this idea, a recent study suggested that tranilast inhibits the increased expression of TGFβ1 in injured rat arteries by inhibiting transcriptional mechanisms.30 Tranilast does not seem to attain its effect through the blockade of angiotensin receptors, because it did not affect the dose-response curve to angiotensin II.
Reduction in TGFβ1-Mediated Collagen Synthesis Improves Survival but Does Not Prevent Left Ventricular Dysfunction
Our data suggest that early reduction in TGFβ-mediated perivascular fibrosis can attenuate hypertensive left ventricular hypertrophy and that this is associated with improved survival. However, chronic reduction in TGFβ expression did not affect myocyte thickening. This suggests that an increased expression of TGFβ mainly affects the noncardiomyocyte compartment. We underlined this idea with the in situ analysis, which demonstrated that the mRNA for TGFβ1 was expressed in areas of fibrosis and was hardly found to be expressed in cardiomyocytes but rather was found in areas adjacent to these cells or in the perivascular area. This clearly suggests that noncardiomyocyte cells are largely responsible for the formation of TGFβ1 and may be the most important cell type responsible for the regulation of TGFβ1.
Reduction in TGFβ1 expression failed to prevent left ventricular dysfunction, indicating that other mechanisms may be responsible for the improved survival. TGFβ affects many other tissues besides the heart, so one might propose that the described effects could be due to vascular or renal protection. However, tranilast did not prevent thickening of the carotid media (Figure 4⇑), nor did it prevent albuminuria or endothelial dysfunction in this experiment (data not shown). Also in favor of a protective effect on the left ventricle are other recent findings from our group. In a preliminary study, we pretreated rats before experimental myocardial infarction with either tranilast or control food. Tranilast tended to decrease infarction-related mortality rats (from 48% to 16%, P=NS) but did not improve left ventricular function (unpublished data).
The improved survival seen in the present study may be due to protection against complications other than loss of contractility, such as arrhythmias. Abundant evidence suggests a relation between cardiac fibrosis and (lethal) cardiac arrhythmias.31 In hypertensive patients, serum procollagen type III amino-terminal peptide was related to the incidence and severity of arrhythmias.32
Losartan and Tranilast Have Strikingly Similar Effects
Losartan and tranilast had strikingly similar effects on most parameters studied. This corroborates the suggestion that a large part of the effects of angiotensin II are mediated via increased expression of TGFβ. This confirms cell culture data that suggest angiotensin II exerts structural effects through the recruitment of growth factors like TGFβ.8 33
As can be concluded from the preceding paragraphs, the present study relates the prevention of increased left ventricular TGFβ1 expression to cardiac effects. However, we cannot definitely answer whether the association is causal. Even a nonhypotensive dose of losartan may interfere with many other mechanisms besides angiotensin blockade to attenuate left ventricular hypertrophy. Similarly, tranilast may decrease hypertrophy and mortality rates via undiscovered mechanisms. It is also conceivable that changes in TGFβ1 mRNA translate into complex changes in the amounts of active and latent TGFβ1. Therefore, it remains to be determined how changes in the expression of TGFβ1 translate into changes in TGFβ1 and related peptides. Furthermore, other components of the TGFβ1 system might also be affected.
In the present study, we did not assess the effects of a higher dose of losartan. Recently, we evaluated treatment with losartan started in the Ren2 rat at the same age but in a dose that prevented the development of high blood pressure.34 There, we demonstrated that this type of treatment prevented all studied changes in the heart. Others have also described this,12 so it was known at the start of this experiment that normalization of blood pressure with high-dose angiotensin II receptor blockade sufficiently prevents end-organ changes. We are not the first to show that in this model a nonhypotensive dose of an angiotensin receptor blocker attenuates the development of left ventricular hypertrophy,35 although left ventricular function was not assessed in the cited study.
In conclusion, it was until now unclear whether chronic in vivo reduction in TGFβ1 would have beneficial effects in hypertensive heart disease. The present study is the first to suggest that regardless of the mechanism involved, a reduction in TGFβ1 may be beneficial even in the absence of blood pressure–lowering effects: it attenuated the increase in left ventricular weight, it attenuated the accumulation of collagen, and it improved survival. Surprisingly, a reduction in the expression of TGFβ1 did not prevent impairment of left ventricular function, so we speculate that a reduction in left ventricular fibrosis may improve survival via an alternative mechanism, such as a reduction in arrhythmias.
Dr Pinto received an Interuniversity Cardiology Institute Netherlands (ICIN) 1996–1997 molecular cardiology fellowship. This study was funded in part by the BIOMED-II program TRANSGENEUR from the European Community and a grant from the German Ministry for Science and Technology (BMBF) to Dr Paul (Clinical Pharmacology Association Berlin-Brandenburg) and a DFG grant La 1028/2–1 to Dr Lauster. We thank Dr Andrea Vortkamp for technical advice regarding the in situ hybridization.
- Received March 8, 2000.
- Revision received April 24, 2000.
- Accepted May 8, 2000.
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