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Eplerenone, But Not Steroid Withdrawal, Reverses Cardiac Fibrosis in Deoxycorticosterone/ Salt-Treated Rats

Prince Henry’s Institute for Medical Research, Clayton, 3168 Victoria, Australia

Address all correspondence and requests for reprints to: Dr. Morag Young, Prince Henry’s Institute for Medical Research, P.O. Box 5152, Clayton, 3168 Victoria, Australia. E-mail: morag.young{at}phimr.monash.edu.au.

	Abstract

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Aldosterone has been thought to act primarily on epithelia to regulate fluid and electrolyte homeostasis. Mineralocorticoid receptors (MR), however, are also expressed in nonepithelial tissues, such as the heart and vascular smooth muscle. Recently, pathophysiological effects of nonepithelial MR activation by aldosterone have been demonstrated in the context of inappropriate mineralocorticoid levels for salt status, including coronary vascular inflammation and cardiac fibrosis. These effects are mostly prevented by the concomitant administration of MR antagonists, but to date, no equivalent studies have determined whether MR blockade can reverse established inflammation and fibrosis. Uninephrectomized rats maintained on 0.9% NaCl solution to drink were treated as follows: group 1 served as controls; group 2 received deoxycorticosterone (DOC; 20 mg/wk) for 4 wk until death, and group 3 received DOC for 8 wk. Group 4 received DOC for 4 wk and no steroid from wk 5–8; group 5 received DOC for 8 wk and eplerenone in their chow during wk 5–8. DOC progressively raised cardiac collagen accumulation at 4 and 8 wk. Rats given DOC for 4 wk and killed at 8 wk showed levels of fibrosis identical to those in animals killed at 4 wk, i.e. persistently elevated above control values. Rats given DOC for 8 wk and eplerenone for the second half of the period showed cardiac collagen levels indistinguishable from control values. Values for inflammatory marker and NAD(P)H oxidase subunit expression in coronary vessels showed a similar pattern of response, with minor variation. Thus, MR antagonists do not only prevent cardiac fibrosis, but also reverse cardiac fibrosis once it is established. In addition, the continuing vascular inflammatory response and fibrosis after DOC withdrawal (group 4) support a role for activation of vascular MR by endogenous glucocorticoids in the context of tissue damage and generation of reactive oxygen species.

	Introduction

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INAPPROPRIATE ACTIVATION of mineralocorticoid receptors (MR) by elevated plasma aldosterone in the context of a high salt intake produces vascular and perivascular inflammatory responses, followed by perivascular and interstitial fibrosis (1, 2, 3). It is now clear that this inflammatory response is a direct humoral effect, rather than secondary to hemodynamic changes. First, the fibrosis is equally marked in right and left ventricles (2, 3). Secondly, coadministered spironolactone (4) or potassium canrenoate (5) blocks cardiac hypertrophy and fibrosis at doses that lower blood pressure minimally if at all. Third, rats infused peripherally with aldosterone and intracerebroventricularly with an MR antagonist (RU28318) remain normotensive, but show cardiac effects indistinguishable from those in hypertensive, aldosterone-infused littermates (6).

Studies addressing the time course of these cardiac responses have shown no increase in collagen deposition until the first 3–4 wk of aldosterone administration (7, 8), although a more rapid time course has been reported after bolus doses of deoxycorticosterone (DOC) (9, 10). From shorter time-course studies using DOC or aldosterone the intramural vasculature was identified as a site for initiation of early responses; primary markers of inflammation, measurable at 1 wk (9, 11), either plateau or continue to increase from 2–4 wk (11). In all instances, the elevations in inflammatory markers and the morphological changes were reduced to near control levels by concomitant MR blockade.

To date investigators have addressed the role of MR antagonists in development of the inflammatory and fibrotic responses (4, 5, 12) rather than investigating a possible role in the reversal of established inflammation and cardiac fibrosis. Studies addressing potential reversal have been reported in the context of progressive heart failure in both humans (13) and dogs (14); those studies in the rat were limited and over a very short time course (15). The current studies were thus designed to explore whether MR blockade can reverse coronary vascular inflammation and established cardiac fibrosis in the mineralocorticoid/salt model of cardiovascular damage, and if so, whether MR blockade is more effective than steroid withdrawal alone.

	Materials and Methods

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Protocols for animal use were approved by the Monash University animal ethics committee. Male Sprague Dawley rats (190–210 g starting weight) were uninephrectomized under anesthesia with ilium xylazil (8 mg/kg; Troy Laboratories, Smithfield, Australia) and ketamine (60 mg/kg; Parke-Davis, Auckland, New Zealand), with carprofen (5 mg/kg, given once sc; Pfizer, New York, NY) for postoperative analgesia. Rats were then maintained on chow ad libitum and 0.9% NaCl solution to drink and were allocated into five treatment groups as follows: 1) control, with no further treatment for 8 wk (n = 8); 2) DOC (20 mg in corn oil, sc) once a week from d 2 for 4 wk (DOC4; n = 7; Sigma-Aldrich Corp., St. Louis, MO); 3) DOC as for group 2, but for 8 wk (DOC8; n = 7); 4) DOC for 4 wk, then no treatment for 4 wk (DOC404; n = 8); and 5) DOC for 8 wk plus eplerenone (100 mg/kg·d) for 5–8 wk (DOC8EPL4; n = 8; Amersham Pharmacia Biotech, Skokie, IL; eplerenone was incorporated into the chow by Glenforest Stock Feeders, Perth, Australia).

Animals were killed by CO₂ in air at 8 wk, except for the DOC4 group, which was killed at 4 wk. Hearts were excised, weighed, and bisected, and one half was fixed in buffered paraformaldehyde for 6 h, then rinsed and stored overnight in PBS before paraffin embedding. Tissue samples stored for longer times before embedding were placed in 70% ethanol. The remainder of the fresh tissue was used for RT-PCR, as detailed below.

Histological analysis
Tissue blocks were sectioned in the midcoronal plane at 5-µm thickness onto glass slides, and myocardial (perivascular plus interstitial) collagen levels were determined by picrosirius red staining analyzed by AIS computer image analysis software (AIS, version 4.0 Beta 1.5, Imaging Research, Inc., St. Catharines, Ontario, Canada). Immunochemistry for inflammatory cytokines used antibodies as previously described (9). The collagen content and level of inflammatory marker expression are expressed as a percentage of area per field analyzed; for each marker, 10–12 fields were analyzed for each rat. Small and medium-sized coronary arteries were scored on a scale of 0–3 for marker expression in vessel wall. Values for individual animals were averaged so that one value per animal was used for statistical comparison by one-way ANOVA.

RT-PCR
Total RNA was prepared from freshly isolated rat heart tissues with Ultraspec (Fisher Scientific, Pittsburgh, PA). First strand cDNA synthesis from 500 ng total RNA was performed after deoxyribonuclease treatment using avian myeloblastosis virus reverse transcriptase (Roche, Indianapolis, IN) primed by random hexamers. PCR reactions were carried out using the following primer sets (all 5'3'): p22^phox: sense, CCC CCG GGG AAA GAG GAA AA; antisense, GCA GGC GAC AGC ACT AAG; gp 91^phox: sense, CCA TTC GGA GGT CTT ACT TTG; antisense, CTG GGC ACT CCT TTA TTT TTC; and NOX-4: sense, GAA CCT CAA CTG CAG CCT GAT C; and antisense, CCT TTG TCC AAC AAT CTT CTT GTT CTC. Expression levels were normalized to those of the 18S ribosomal subunit: (18S sense, CGG CTA CCA CAT CCA AGG AA; antisense, GCT GGA ATT ACC GCC GCT). To validate the real-time PCR protocol, gene-specific standard curves for p22^phox and ribosomal 18S were generated from one in 10 serial dilutions of previously prepared standards. Standards were diluted as follows: from 10 to 0.1 pg/µl for 18S and from 500 to 0.5 fg/µl for all other transcripts. Real-time PCR amplification was performed on the LightCycler (Roche) using SYBR Green reaction mix (Roche) and the primers described above. cDNA samples were diluted 1:20 in water immediately before use for p22^phox and 18S; gp91^phox and NOX-4 were analyzed undiluted.

	Results

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Figure 1 shows levels of the inflammatory markers ED-1 (for estimation of macrophage infiltration, top panel), cyclooxygenase-2 (COX-2; center), and osteopontin in the five groups of animals. The three markers overall show a commonality of response, with minor variations between them in terms of time course, extent of change, and significant differences between groups. For ED-1, for example, the values at 8 wk are higher than those at 4 wk of DOC treatment, whereas no significant difference was seen between 4 and 8 wk for the other two markers studied. For all three markers, the steroid withdrawal group (DOC404) showed levels of staining persistently higher than control values, and no different from those in animals killed after 4 wk of DOC (DOC4). In all three groups, the mean values for the eplerenone-treated group were lower than those in the other treated groups; for COX-2 and osteopontin these values are not significantly different from controls, signifying complete reversal, whereas for ED-1 DOC8E4 levels, though clearly diminished vs. DOC8, remained significantly above the controls (P < 0.05).

View larger version (12K): [in this window] [in a new window]   FIG. 1. Vascular and perivascular inflammatory marker expression in DOC/salt-treated rats as determined by semiquantitative scoring (from 0–3). CON, Control; DOC4, DOC treatment for 4 wk; DOC8, DOC for 8 wk; DOC404 DOC for 4 wk and no steroid during wk 5–8; DOC8EPL4, DOC for 8 wk plus eplerenone during wk 5–8. Staining after steroid withdrawal (DOC404) was significantly lower than that at 8 wk, but not that at 4 wk. *, P < 0.05 vs. CON; **, P < 0.05 vs. all other groups; , P < 0.05 vs. DOC404.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Myocardial NAD(P)H oxidase subunit mRNA expression relative to 18S rRNA. See Fig. 1 for details of groups. Values after DOC withdrawal (DOC404) were significantly lower than those at 8 wk, but not those at 4 wk. *, P < 0.05 vs. CON.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Perivascular and interstitial cardiac fibrosis percent area of left and right ventricles. See Fig. 1 for details of groups. Staining after DOC withdrawal (DOC404) was significantly lower than that at 8 wk, but not that at 4 wk. *, P < 0.05 vs. CON; **, P < 0.05 (vs. all other groups). , P < 0.05 (vs. DOC404).

	Discussion

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In previous studies of the vascular inflammatory response to aldosterone/salt and the effects of coadministered eplerenone, Rocha and co-workers (11) described variations in time course over the first 4 wk. For macrophage inflammatory protein-1, a modest progressive rise was seen from wk 1–2 and from wk 2–4, not inconsistent with our finding of a continued increase between wk 4–8 of DOC/salt treatment. For COX-2, they found a progressive increase over the first 2 wk, but plateau levels between wk 2 and 4, again consistent with our findings of plateau levels from wk 4–8. For osteopontin, levels rose steeply over the course of the previous study, with much higher levels at wk 4 than at wk 2; our finding of not dissimilar levels at wk 4 and 8 suggest that wk 4 may represent maximal levels for this marker, as opposed to wk 2 for COX-2, and (at the earliest) wk 8 for ED-1. What is also noteworthy in comparing the two studies is that concomitant administration of eplerenone (at comparable doses to the present study) reduced the inflammatory response very markedly, but often to levels still above those in control rats. The demonstration in the present study that in the DOC8E4 group levels for some indexes remain above control values is thus not inconsistent with previous studies and suggests that the time course of some markers of inflammation may be longer than that for the other indexes in terms of both increment (e.g. 8 wk for ED-1) and reversal (e.g. >4 wk for osteopontin).

Expression patterns for NAD(P)H oxidase subunits were similar, with elevated mRNA expression at 4 wk and further increases after 8 wk of DOC treatment. For each subunit, mean values were higher in the steroid withdrawal group than in the reversal group, but this was not significant on a group basis. Based on previous studies, we consider this increase in expression to be indicative of a commensurate increase in ROS production (24, 25, 26), which may be responsible for the initiation of vascular damage and inflammatory processes. Further studies are required to determine the signaling pathways responsible for translating MR activation into changes in cellular redox state, but including those on the rapid MR signaling effects on sodium hydrogen exchanger1 activity (27, 28).

The findings for the steroid withdrawal group (DOC404) are of potential importance in two respects. First, they are higher in terms of mean values across the board than those for the eplerenone-treated group (despite continuation of DOC administration in the latter) and are significantly higher on a group basis for some indexes (e.g. fibrosis). Secondly, they are in no case significantly different from values in animals killed after 4 wk of DOC administration. Taken together, these findings suggest that the inflammatory process may continue, more or less at plateau levels, over the 4 wk after steroid withdrawal. The process is clearly not equivalent to continuing administration of DOC, as mean values for the DOC404 group are consistently lower than those for the DOC8 group, in some instances (e.g. fibrosis) significantly so, on a group basis.

A possible explanation for the continuing submaximal inflammatory response in the DOC404 group may be activation of vascular wall MR by endogenous glucocorticoids, in the context of the tissue damage consequent upon 4 wk of DOC/salt administration. In many tissues, under normal circumstances the physiological glucocorticoids occupy MR in a tonic inhibitory mode (29, 30); under other circumstances [11ß-hydroxysteroid dehydrogenase (11ßHSD2) type 2 blockade and tissue damage], glucocorticoids appear capable of not only occupying, but also activating, MR (9, 31). In recent studies we have shown that the vascular inflammatory effects of DOC/salt administration can be mimicked by giving carbenoxolone to block 11ßHSD2; importantly, the effects of carbenoxolone are completely reversed by eplerenone, indicating that the endogenous glucocorticoid effects are via MR activation (9). In previous studies in experimental angioplasty in pigs, we have also shown that eplerenone preserves the coronary artery luminal diameter (31). In these studies the animals were receiving no aldosterone or salt supplement to their regular diet; it is formally possible that the eplerenone is blocking profibrotic effects of normal levels of aldosterone in the absence of added salt, although in previous rat studies even high doses of aldosterone (0.75 µg/h) were shown to be ineffective on a low salt diet (2). A more plausible explanation is that activation of vascular wall MR by endogenous glucocorticoids in the context of tissue damage and generation of reactive oxygen species may alter the intracellular redox state in a similar manner as 11ßHSD2 blockade by carbenoxolone. In the current report we hypothesis that a similar mechanism may be responsible for the sustained inflammatory and fibrotic responses seen in the DOC404 group, i.e. vascular damage and the production of reactive oxygen species may sufficiently change the intracellular environment in terms of NAD⁺:NADH to allow endogenous glucocorticoids to act as agonists at the MR. A fuller exposition of this hypothesis can be found in several recent reviews (32, 33).

If this is the case, the question may be reasonably asked why continued DOC administration is clearly more proinflammatory/profibrotic than steroid withdrawal even if in the latter group the process is at least to some extent being sustained over wk 5–8 by endogenous corticosterone activating vascular wall MR. One possible answer to this question is a countervailing antiinflammatory effect in the DOC404 group, over wk 5–8, of endogenous glucocorticoids via vascular wall glucocorticoid receptors (GR). Such receptors have affinity for corticosterone more than an order of magnitude lower than that of MR and thus will be occupied and activated more or less over the diurnal range of circulating steroid levels. In the DOC8 animals this is much less likely to be the case, as DOC is a GR antagonist as well as an MR agonist. This property of DOC may also contribute to what appears to be a more marked effect on vascular inflammation (3) than that seen with aldosterone, which is a low affinity GR agonist rather than a GR antagonist.

Finally, the salient finding of the study is the ability of MR blockade to reverse the indexes of inflammation (with the exception of osteopontin) and fibrosis to control levels over 4 wk despite continued administration of DOC. Brown and colleagues (15), in a study comparing captopril, candesartan, and spironolactone for 3–4 wk, showed all three to attenuate or reverse some, but not all, of the changes seen in rats given DOC (25 mg/4 d) for 4 wk at both mRNA and functional levels. In a substudy of RALES, Zannad et al. (13) found that reduction of circulating levels of PIIINP (the N-terminal fragment of procollagen III) correlated with a positive effect of spironolactone in terms of survival in severe heart failure; these data were reasonably interpreted as evidence for the possibility that MR blockade decreased collagen turnover in a proportion of patients, which was to their advantage in terms of the progression of disease. In subsequent dog studies, Suzuki et al. (14) demonstrated that eplerenone similarly halted the progression of experimental heart failure on a variety of indexes compared with a vehicle-treated control group over 3 months; on none of these indexes, however, was their any indication of reversal, perhaps not unexpectedly given the context of experimentally induced heart failure.

The present studies represent a time-telescoped model of inappropriate MR activation, and extrapolation to the clinical conditions of aldosterone excess, as in Conn’s syndrome, or other situations predisposing to vasculitis, should be made with considerable caution. Given this caveat, the essentially complete reversal of vasculitis and fibrosis by eplerenone, despite the continuing inappropriate mineralocorticoid/salt status in these animals, indicates the potential importance of extending such studies to the clinical area. The laggardly response to steroid withdrawal compared with MR blockade would appear to underscore the potential importance of the latter in terms of the potential therapeutic impact to reverse established inflammatory effects and remodeling in the cardiovascular system.

	Footnotes

 
Abbreviations: COX, Cyclooxygenase; DOC, deoxycorticosterone; GR, glucocorticoid receptor; 11ßHSD2, 11ß-hydroxysteroid dehydrogenase; MR, mineralocorticoid receptor; ROS, reactive oxygen species.

Received January 5, 2004.

Accepted for publication March 26, 2004.

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