© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Raised blood pressure, not renin–angiotensin systems, causes cardiac fibrosis in TGR m(Ren2)27 rats
aCentre for Cardiopulmonary Biochemistry and Respiratory Medicine, University College London Medical School, Rayne Institute, 5 University Street, London WC1E 6JJ, UK
bHatter Institute, University College London Medical School, Rayne Institute, 5 University Street, London WC1E 6JJ, UK
cInstitute of Pharmacology, Christian-Albrechts University of Kiel, Kiel, Germany
* Corresponding author. Tel.: +44-207-209-6974
Received 14 September 1999; accepted 17 February 2000
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Abstract |
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Objectives: Elevated systemic arterial blood pressure is associated with left ventricular hypertrophy and fibrosis. It has been suggested that both circulating and local myocardial renin–angiotensin systems play a role in mediating these responses. Here we describe the natural history of ventricular hypertrophy and fibrosis in the transgenic (mRen2)27 rat — a monogenetic model — which has a high tissue expression of the murine renin transgene, and suffers severe hypertension. We further explored the relative contribution of both hypertensive burden and circulating and tissue renin–angiotensin systems to the fibrotic process. Methods: The transgenic rats were treated from 28 days old with (1) a hypotensive dose of the ACE inhibitor ramipril which inhibited both tissue and circulating ACE activity, (2) the calcium antagonist amlodipine, or (3) a non-hypotensive dose of ramipril which inhibited about 60% of tissue ACE activity with little effect on circulating ACE. Normotensive Sprague–Dawley rats were used as controls. Results: The transgenics developed left ventricular hypertrophy along with perivascular and interstitial fibrosis which became progressively worse up to 24 weeks of age. Both the high dose of ramipril and amlodipine prevented the hypertrophy and fibrosis, whereas tissue ACE inhibition without lowering blood pressure had no effect, and actually led to a worsening of the fibrosis by 24 weeks. Conclusions: These results suggest that the development of left ventricular hypertrophy and fibrosis in the transgenic (mRen2)27 rat are regulated by blood pressure and not activity of the renin–angiotensin systems and that progression of fibrosis at 24 weeks involves a mechanism unrelated to local renin–angiotensin activity.
KEYWORDS ACE inhibitors; Angiotensin; Blood pressure; Fibrosis; Hypertension
This article is referred to in the Editorial by C.G. Brilla (pages 1–3) in this issue.
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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In both human and animal models of hypertension, pressure overload leads to cardiovascular remodelling comprising myocardial and vascular hypertrophy. An increase in collagen deposition frequently occurs resulting in fibrosis [1]. This remodelling of the collagen network is associated with increased myocardial stiffness [2] and abnormalities of cardiac function in both animal models and clinically [3]. Indeed, it has been suggested that a major factor determining the progression of left ventricular hypertrophy (LVH) to failure is the presence of cardiac fibrosis [4].
Although of great importance, the mechanisms that regulate the hypertrophic and fibrotic responses remain poorly understood. Mechanical burden per se may be a critical factor since mechanical loading in vitro stimulates both myocyte hypertrophy and matrix deposition by fibroblasts [5,6]. However, the development and regression of LVH in animal models appears to correlate poorly with systolic blood pressure [7]. Despite the fact that a number of classes of antihypertensive agents are able to reverse the hypertrophic response, the efficacy of treatment appears to depend on other pharmacological properties of the agents [8]. In this respect the renin–angiotensin systems (RAS) may prove important. The endocrine RAS plays a role in circulatory homeostasis, with angiotensin-converting enzyme (ACE or kininase II) degrading kinins and converting angiotensin I (AngI) to the vasoconstrictor angiotensin II (AngII). In addition, it has been suggested that a local tissue cardiac renin–angiotensin system [9] may influence myocyte growth and matrix deposition [10,11]. With regard to the fibrotic response, there have been few studies separating the relative roles of such local systems and of systemic blood pressure burden itself [7]. In a previous study where markers of fibrosis were determined in TGRs, blocking the local system in vivo had some blood pressure lowering effect [12].
The TGR(mRen-2d)27 rat (TGR) represents a well documented genetic model of hypertension. The transgenic rat was produced by introduction of the murine Ren-2 gene into the rat genome [13] and provides a monogenic model of hypertension to study the role of the local RAS specifically in cardiovascular remodelling since there is local expression of the transgene in cardiovascular tissues, including the heart [14]. The aim of this study was to characterise the development of hypertension, LV hypertrophy and, particularly, fibrosis in this model, and to investigate the relative roles of BP and local cardiac RAS in the cardiac fibrotic response.
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2 Methods |
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All chemicals were of reagent grade and purchased from BDH/Merck (Lutterworth, UK) unless otherwise indicated.
2.1 Experimental groups
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Transgenic rats homozygous for the Ren2 gene derived from pure Hanover stock (line 27) were used. Male heterozygotes were derived from homozygous TGR(mRen2)27 male rats crossed with female Edinburgh Sprague–Dawleys (SD). Heterozygotes were studied as homozygocity is associated with fatal severe hypertension in the absence of treatment (lisinopril dihydrate (Zeneca Pharmaceuticals, Cheshire, UK) 25 mg/l added to drinking water). Offspring were weaned by 28 days when treatment was begun with either the ACE inhibitor, ramipril, kindly donated by Hoechst-Marion-Roussel (Frankfurt, Germany) at a dose yielding a BP similar to that in control animals (1 mg/kg/day) or with ramipril at a non-hypotensive dose (5 µg/kg/day) (Fig. 1). The calcium channel blocker, amlodipine (Pfizer UK, Sandwich, Kent) at 10 mg/kg/day was also used to further define the role of blood pressure. Drugs were administered in drinking water from weaning (changed three times a week). Water consumption was measured and drug concentration was adjusted every 2 days. Animals were housed three to four per cage on wood shavings, with free access to food and tap water. Animals were killed at 10, 16 or 24 weeks of age. Since up to 50% of untreated TGRs die by 10 weeks of age the number of animals allocated to these groups was increased to take this into account. Sprague–Dawley rats of the same strain as the TGRs were used as normotensive controls. Over the whole study, 19 out of 39 untreated TGRs (48%) survived. In all other groups a survival rate of 100% was achieved.
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–), TGR — hypertensive transgenic (mRen2)27 rats (n=14; –
–), RH — TGRs treated with ramipril at 1 mg/kg/day (n=11; –
–) and RL — TGRs treated with ramipril at 5 µg/kg/day (n=12; –
–). (b) The effect of amlodipine. SD n=12, TGRs n=19, A10 — TGRs treated with amlodipine at 10 mg/kg/day. n=7; –
–. All values represent mean±S.E.M. The data in (a) and (b) were obtained from two sets of animals with the two experiments (i.e. a and b) performed on separate occasions.2.2 Measurements
2.2.1 Blood pressure and body weights
Under light general anaesthesia, weight and systolic blood pressure were assessed (tail cuff photoplethysmography: mean of five readings per animal: MacLab Bioamplifier ML-130, AD Instruments Pty Ltd., Australia: Apple Macintosh LC475 Computer: IITC Mod 29 Pulse Amplifier and tail cuff system IITC Life Sciences, Woodland Hills, CA, USA).
2.2.2 Tissue fixation
Under anaesthetic, animals were exsanguinated through an aortic cannula and then perfused with either fixative (paraformaldehyde 4% w/v, glutaraldehyde 0.1% w/v), or saline (0.9% NaCl w/v) at 4°C (160 mmHg for TGRs and RL and 120 mmHg for controls and RH). The heart was excised and trimmed, and the left ventricle wall plus septum (LV) separated from the right ventricle (RV). The tissues were blotted dry and weighed.
2.2.3 Collagen content and concentration
A portion of perfusion-fixed ventricle (
50 mg) was dried to a constant weight and hydrolysed in hydrochloric acid (1 ml, 6 mol/l) at 110°C for 16 h. The collagen content (total mg collagen per ventricle) was determined by a reverse phase HPLC assay for hydroxyproline [15]. Collagen concentration was calculated as mg collagen/g wet weight of ventricle.
2.2.4 Histology
Coronal sections of perfusion fixed LV and RV were processed and paraffin embedded; 5-µm sections were stained with the connective tissue stain, Masson's trichrome, and examined by light microscopy.
2.2.5 Plasma and tissue ACE activity
At exsanguination, blood was drawn into chilled lithium heparin blood tubes (Vacutainer, Beckton Dickinson, NJ, USA) through a sterile 24-G cannula, centrifuged at 2000xg for 12 min at 4°C (Heraeus Sepatech Biofuge 22R) and the plasma stored at –70°C. Animals were perfused with cold saline until the kidneys and heart were pallid (60–90 s). The heart was rapidly removed, the atria and great vessels trimmed away and the LV separated from the RV. ACE activity was measured as previously described [16,17] and expressed as nmol HisLeu/ml/min for plasma samples and nmol HisLeu/mg protein/min for tissue samples. Care was taken to ensure that dilutions of the samples achieved optimal assay conditions and maximized the estimation of the effectiveness of ACE inhibition [18].
2.2.6 Statistical analysis
Data are presented as mean±S.E.M. Statistical analysis was performed using an unpaired Student's t-test for single group comparisons and Newman–Keuls ANOVA for multiple group comparisons [19]. Differences were considered statistically significant at P<0.05.
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3 Results |
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3.1 Development of hypertension in TGRs and the effect of ramipril and amlodipine treatment
From the earliest time point measured (6 weeks) blood pressure in untreated TGRs was increased compared with controls (P<0.01, Fig. 1). BP rose further to reach a maximum in the TGRs by 8 weeks and remained elevated compared with controls, although there was gradual decrease with age. Blood pressure in the TGRs treated with the low dose of ramipril (RL) were comparable to untreated TGRs throughout the study (Fig. 1a). In contrast, BP in the high dose ramipril (RH) treatment group was consistently comparable to normotensive controls. The amlodipine treatment (A10), introduced in an attempt to lower BP to control levels by a method independent of ACE activity, significantly lowered BP compared with untreated TGRs. However, BP remained significantly higher than controls (P<0.01, Fig. 1b). Since control blood pressure could not be achieved, the A10 treatment was not continued for the 24 weeks — 20 mg/kg/day amlodopine had no further effect on BP (data not shown).
3.2 Effect of treatment on plasma ACE activity
Plasma ACE activity in controls was only different from the TGRs at 10 weeks (when it was higher, P<0.05, Table 1). At all time points, treatment with RH led to a significant reduction (46–65%) in plasma ACE activity compared with TGRs (all P<0.05). Treatment with RL led to an increase in plasma ACE activity compared with TGRs at 10 weeks (P<0.05), and a small (17 and 27%) but significant reduction at 16 and 24 weeks (both P<0.05, Table 1). At 10 weeks, treatment with A10 led to a rise in plasma ACE activity compared with TGRs (P<0.05) although this level was not significantly different from controls. At 16 weeks, A10 had a similar effect on plasma ACE activity as RL (P<0.05 compared to TGRs, Table 1).
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3.3 Effect of treatment on tissue ACE activity
There was no significant difference between ACE activity in the ventricles of controls compared with TGRs at either time point (Table 1). Treatment with RH led to a significant reduction (approximately 90%) in ACE activity in both ventricles compared with TGRs (all P<0.05). RL treatment also led to a significant fall in ventricular ACE activity (57–68%) compared with TGRs (all P<0.05).
3.4 Development of LVH in TGRs and the effect of drug treatment
TGRs had established LVH by 10 weeks when expressed as an increased LV wt, LV wt normalized to Bwt (LV:Bwt ratio, both P<0.01, Table 2) or the LV:RV ratio (Fig. 2). There was no further increase in LVH of TGRs relative to controls. The non-hypotensive dose of ramipril had no effect on LVH, apart from a small lowering effect on LV:Bwt at 10 weeks. Treatment with the high dose of ramipril prevented LVH and, despite not reducing BP to control levels, amlodipine treatment also completely prevented the appearance of LVH.
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P<0.01 vs. SD, 
P<0.05, ||P<0.01 vs. TGR, ¶P<0.01 vs. RH.3.5 LV fibrosis in TGRs and effect of drug treatment
At 10 weeks there was no significant difference between the LV collagen content of the TGRs compared with controls (Fig. 3a). At 16 weeks the LV collagen content of the TGRs was increased by 70% compared with controls (P<0.01) and by 24 weeks had increased further still (P<0.01). Collagen concentration at this later time was also increased significantly compared to the controls (7.14±0.28 mg/g wet weight of tissue vs. 5.28±0.26 mg/g, P<0.05). Perivascular fibrosis, in the form of enlarged areas of connective tissue surrounding coronary arterioles in the LV of TGRs compared with controls, was present at all times, becoming more severe with age (Figs. 4 and 5
). At 16 (not shown) and 24 weeks (Fig. 5), the fibrotic tissue spread into the surrounding interstitium, where the interstitial fibrosis was associated with replacement scarring.
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At 10 weeks there was no significant difference between the LV collagen content of the TGRs and all the treatments (Fig. 3a). However, treatment with RH completely prevented the development of perivascular fibrosis (Fig. 4) and at 16 and 24 weeks, also the interstitial and replacement fibrosis (not shown). At 16 weeks the LV collagen content of the RH group was reduced to control (Fig. 3). LV sections taken from RH treated animals were indistinguishable from those of the SD controls (Fig. 4). In the RL group at 16 weeks, the LV collagen content was higher than control and not significantly different from untreated TGRs (Fig. 3). Histologically, the ventricles of RL treated animals appeared the same as those of the TGRs at this time. In the A10 group, however, LV collagen content was lower than in the TGRs and not significantly different from normotensive controls. Similarly the sections appeared indistinguishable from controls. At 24 weeks, the LV collagen content of the RH group was significantly lower than in untreated TGRs (P<0.05), whereas in the RL group it was significantly higher (P<0.01). At 24 weeks, the extent of fibrosis in RL treated animals appeared more severe than in untreated TGRs and foci of replacement scarring appeared more prevalent (Fig. 5). At this time collagen concentration was also significantly greater in the RL group than in the untreated TGRs (9.77±0.57 mg/g wet weight of tissue vs. 7.14±0.28 mg/g, P<0.01).
3.6 RV weight and RV to body weight ratio in the study groups
At every time point studied, there was no significant difference between the RV wt of the study groups, except at 16 weeks, where the RV wt of the amlodipine treated group was greater than that of the controls and untreated TGRs (Table 2). However RV wt normalized to Bwt (RV:Bwt) ratio was not different from the untreated TGRs.
3.7 RV fibrosis in TGRs and effect of drug treatment
There was no significant difference in RV collagen content between the controls and TGRs at any time point (Fig. 3b). However, at all time points there was histological evidence of perivascular fibrosis accompanied by an increase in the thickness of the tunica media of coronary arterioles in the RV of TGRs compared with controls (10 weeks shown in Fig. 4). There was no evidence of interstitial fibrosis or replacement scarring in the RV of TGRs at any time. At 10 weeks RH, RL and A10 had no effect on RV collagen content (Fig. 3b). At 16 weeks the RV collagen content of the RH and A10 groups were lower than that of the TGRs, whereas RL had no effect. RH treatment prevented the increase in tunica media thickness and perivascular fibrosis seen in the untreated TGRs, with RH RV sections being indistinguishable from control sections at all time points (Fig. 4). Treatment with RL did not affect vascular thickening and fibrosis, with RL RV sections being indistinguishable from untreated TGR RV sections at all times. Treatment with amlodipine prevented the changes seen in RVs of untreated TGRs.
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4 Discussion |
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We have used the recently developed TGR(mRen2)27 rat which harbours the mouse Ren2 gene and is characterised by an activation of tissue RAS and a depressed plasma and kidney renin activity [13]. The advantage of this model is the monogenic pathogenesis of hypertension, which allows investigation of phenotypic changes in various organs caused by a genetic perturbation of one system — the RAS. This approach was also useful to overcome problems inherent in the choice of the appropriate controls in genetic models of spontaneous hypertension [20].
Until now, the cardiac hypertrophic response in the TGR(mRen-2d)27 rat has not been well characterised, with several studies reporting the effects at one time point only [12,21,22]. We have documented here the presence of significant LVH in the TGR rat up to 6 months of age. The hypertrophic response in this model is associated with considerable vascular changes, with intramyocardial arterioles demonstrating marked medial hypertrophy. Such medial thickening is a recognised association of sustained hypertension and has been demonstrated in other tissues in the same model [23,24].
Although the hypertrophy in these animals appears established by 10 weeks, both the perivascular and intersitital fibrosis continue to advance with age. Age was also associated with the appearance of microinfarcts. The early development of perivascular fibrosis was not apparent from the biochemical analysis, the hydroxyproline measurement clearly not sensitive enough to detect the subtle, extremely localized enhanced collagen deposition at this time. However, at the other time points collagen content correlated well with the histology. Collagen content was used for analysis in preference to concentration since it reflects better the dynamics of collagen turnover — the latter being influenced by the degree of hypertrophy in the tissue. For example, there may be local areas of fibrosis but extensive hypertrophy in the remaining myocardium. This will be detected as an increase in collagen content (therefore reflecting collagen deposition) whereas collagen concentration may not increase (see review Ref. [25]).
The histological evidence of extensive perivascular fibrosis in the RV was most fascinating and to our knowledge has not been reported previously in the TGR. We have shown that although there is no RV hypertrophy, there is excessive deposition of collagen around the coronary vessels. This suggests a pressure related response, since the coronary vessels supplying the RV will be subjected to elevated systemic pressures. Moreover, the interstitial fibrosis and microinfarcts are seen only in the pressure-overloaded LV and not in the normotensive RV. Given the ‘in-series’ arrangement of the ventricles, circulating factors cannot therefore be responsible for the appearance of interstitial fibrosis and microinfarcts, since we would have expected such changes in the RV also if this were the case.
In order to dissociate the effects of RAS activity and systolic pressure burden on LV growth and fibrosis we manipulated BP by both high dose ACE inhibition (1 mg/kg/day being the lowest dose needed to lower BP to control), reducing net ACE activity, and the calcium channel blocker, amlodipine, which had only a small effect on plasma ACE. In investigating the role of the local RAS in the development of LVH and fibrosis we used a low dose of ramipril (5 µg/kg/day identified as the highest non-hypotensive dose for these animals, data not shown) which, while an effective inhibitor of tissue ACE activity (approximately 60% inhibition), did not lower BP. There was a small inhibition of plasma ACE activity by 27 and 17% at 16 and 24 weeks only, respectively, compared to inhibitions of 65, 58 and 46% by the high dose of ramipril at each consecutive time point. This low dose of ramipril in fact had very similar effects on plasma ACE as amlodipine treatment.
Treatment of TGRs with the high dose ACE inhibitor lowered BP to control values throughout life and prevented the development of LVH and the appearance of perivascular and interstitial fibrosis. LV weight and collagen content did not differ significantly from the SD control at each time point. Although amlodipine treatment failed to lower BP to control values,its effects appeared to be sufficient to prevent the development of LVH and the appearance of fibrosis. This suggests that there may be a threshold BP above which cardiac remodelling occurs. These results also indicate that LVH and deposition of LV collagen is regulated by ventricular loading and not RAS in the TGR, although one cannot exclude a direct effect of calcium channel blockade by amlodipine on procollagen synthesis [26].
The low dose of ramipril did not affect BP, neither did it prevent the appearance of LVH and fibrosis suggesting that the development of LVH and fibrosis in the TGR(mRen2)27 rat is driven by BP rather than the prevalent local RAS activity. Plasma ACE activities were similar in the amlodipine and RL treated animals yet BP was lower with amlodipine than RL. The effect of amlodipine on LV and fibrosis is therefore likely related to the reduction in BP than plasma ACE activity. Tissue ACE activity was not obtained in the amlodipine group, since, as discussed above, we had initially considered this group not suitable for further investigation as it did not reduced BP to control levels.
In this study, therefore, using a very low dose ACE inhibitor we have dissociated the effects of tissue RAS from plasma RAS; and separated the effects of plasma ACE from BP. The very low levels of tissue ACE remaining after treatment with the low dose inhibitor are conceivably still functional in the TGRs, producing sufficient AngII to affect myocyte growth and fibroblast function. However, higher levels of activity were found in the normal SD rats and yet did not drive hypertrophy and fibrosis independent of BP.
ACE inhibition also prevents bradykinin degradation. This pathway appears to play a role in reducing cardiac hypertrophy only in models of hypertension which are dependent on an activated RAS, such as rats with aortic banding [7], but not in SHR or SHRSP [27,28]. It was suggested that these rats are more susceptible to bradykinin, since the kinins system appears to counteract, in part, the overstimulated RAS. Also, bradykinin has been shown in vitro to reduce cardiac fibroblast procollagen synthesis [29]. Thus one cannot rule out the possibility that ACE inhibition is preventing fibrosis through the action of enhanced levels of bradykinin. Due to the required delivery systems for the bradykinin inhibitor HOE140 it was not considered feasible for the young animals and the lengthy time frame involved. AngII receptor blockers would have provided an alternative means of answering this question, however, the animals are so exquisitely sensitive to these agents with regard to their blood pressure it would be difficult to establish a dose that effectively blocked AngII activity without influencing blood pressure. Also, Gohlke et al. [30] have recently demonstrated that AT1 receptor antagonists can indirectly lead to bradykinin dependent effects by an overstimulation of unblocked AT2 receptors.
Several studies of the TGR rat have employed specific inhibitors of the RAS, such as ACE inhibitors or AngII receptor antagonists, and demonstrated regression of LVH and a reduction in the levels of markers of fibrosis, even at low doses that caused no or a slight reduction in BP, whereas direct vasodilators failed to exhibit similar cardioprotective effects despite efficient BP reduction [12,22,31]. However, each of these studies were concerned with regression, rather than prevention of LVH, as treatment in the former studies did not commence until hypertension and LVH were already established. In our study, treatment was aimed at preventing the rise in BP, by applying the anithypertensive drugs prior to the establishment of hypertension and the development of hypertrophy. However, no LV weight measurements were made on animals before the first BP measurements could be taken to prove that there was no hypertrophy at this very early age, but since the earliest BPs were lower than the amlodipine treated animals, in which there was no hypertrophy, we assume that at 4 weeks hypertrophy would be minimal, if present at all. This suggests that distinct mechanisms may regulate the regression and prevention of LVH and fibrosis in this model.
At 24 weeks, the LV collagen content of the RL group was higher than in the TGRs. The difference was due to increased interstitial fibrosis and the number of microinfarcts in the RL group compared with untreated TGRs. This effect may be due to the increased survival rate in this group. We confirmed here previous observations of development of malignant hypertension (MH) and ensuing death in 66–81% of the heterozygote animals by 100 days of age [32]. Treatment with the low dose of ramipril completely prevented the appearance of MH in these animals despite the lack of hypotensive effects. Consequently this appeared to prevent the high mortality rate seen in untreated TGRs [24]. All RL treated animals survived for examination whereas only a select (possibly less severely affected) subgroup survived in the untreated group. The fibrosis in the myocardium of the animals treated with the low dose of ramipril occurs despite inhibition of tissue ACE activity.
The relationship between LVH and collagen deposition is complex. Although LVH appears established at 10 weeks, the collagen deposition continues to increase until 24 weeks. This may reflect the physiopathology of the disease. The initial reaction to pressure overload is a need for the heart to pump more efficiently — achieved in part by an increase in muscle mass. The fibroblasts — the collagen producing cells — clearly affected by enhanced load (see review Ref. [33]), initially deposit more collagen in the perivascular regions, where fibroblasts are more abundant and the major stimulus for activity may be vascular load. As the disease progresses collagen is deposited as a scarring process in response to regions of micro-infarcts possibly reflecting a drop out of pressure injured arterioles.
In summary, we have demonstrated that cardiac hypertrophy in the TGR(mRen2)27 rat is accompanied by the development of extensive cardiac fibrosis. We have also shown that perivascular fibrosis occurs in the normotensive RV as well as the pressure overloaded LV. Furthermore, we have presented evidence that elevated BP and not plasma or local RAS activity regulates the development of both LVH and cardiac fibrosis in this model.
Time for primary review 22 days.
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Acknowledgements |
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This work was supported by the Wellcome Trust (Jill Bishop is a Wellcome Senior Research Fellow, Linda Kiernan held a Wellcome Prize PhD Studentship). Dr Montgomery was supported by a British Heart Foundation Junior Fellowship.
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Notes |
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1 Jill Bishop and Linda Kiernan contributed equally to the preparation of this paper.
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