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Endocrinology Vol. 147, No. 12 5901-5906
Copyright © 2006 by The Endocrine Society

The Role of the Glucocorticoid Receptor in Mineralocorticoid/Salt-Mediated Cardiac Fibrosis

Amanda J. Rickard, John W. Funder, Peter J. Fuller and Morag J. Young

Dr. Prince Henry’s Institute of Medical Research, Endocrine Genetics, Clayton, Victoria 3168, Australia

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pathophysiological consequences of excess mineralocorticoid for salt status include hypertension, vascular inflammation, and cardiac fibrosis. Mineralocorticoid receptor (MR) blockade can both prevent and reverse established inflammation and fibrosis due to exogenous mineralocorticoids or endogenous glucocorticoid activation of the MR. Glucocorticoids also exert potent antiinflammatory effects via glucocorticoid receptors (GR) in the vascular wall. We propose that GR signaling may ameliorate mineralocorticoid/salt-induced vascular inflammation and fibrosis in the mineralocorticoid/salt model. In the present study, the role of GR in the mineralocorticoid/salt model was explored in uninephrectomized rats that were maintained on 0.9% saline solution to drink and treated as follows: control (CON), no further treatment; deoxycorticosterone (DOC; 20 mg/wk) for 4 wk (DOC4); DOC for 8 wk (DOC8); DOC for 8 wk plus the GR antagonist RU486 (2 mg/d) wk 5–8 (DOC8/RU486); and DOC for 8 wk plus RU486 and the MR antagonist eplerenone (EPL; 50 mg/kg·d) for wk 5–8 (DOC8/RU486+EPL). DOC treatment significantly increased systolic blood pressure, cardiac fibrosis, inflammation (ED-1-positive macrophages and osteopontin), and mRNA for markers of oxidative stress (p22phox, gp91phox, and NAD(P)H-4). GR blockade reduced the DOC-mediated increase in systolic blood pressure and the number of infiltrating ED-1-positive macrophages but had no effect on fibrosis, oxidative stress, or osteopontin mRNA levels. EPL reversed DOC-induced pathology in the absence or presence of GR blockade. Thus, blocking agonist activity at the GR neither enhances nor attenuates the fibrotic response, although it may modulate systolic blood pressure and macrophage recruitment in the mineralocorticoid/salt model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ONE MECHANISM THAT plays a key role in the pathophysiology of heart failure is inappropriate activation of mineralocorticoid receptors (MR). Recent clinical trials, the Randomized Aldactone Evaluation Study (RALES) (1), and the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) (2) have provided convincing clinical data for a role of MR activation in heart failure. RALES showed a 30% reduction in mortality and 35% morbidity with low-dose spironolactone in patients with severe heart failure, and EPHESUS showed substantial improvements in patient outcomes with eplerenone (EPL) after myocardial infarction with systolic dysfunction. In both trials, the MR antagonist was given in addition to current "standard of care" treatment.

The classical effects of MR activation are those in epithelial tissues such as the kidney where aldosterone promotes sodium reabsorption and potassium secretion. MR are also found in nonepithelial tissues such as cardiac myocytes and vascular smooth muscle cells (VSMC), where inappropriate activation of receptors can have pathophysiological consequences. It is now well accepted that administration of aldosterone or deoxycorticosterone (DOC) to uninephrectomized rats on a high-salt diet for 8 wk produces hypertension, cardiac hypertrophy, and a clear increase in perivascular and interstitial cardiac fibrosis (3, 4, 5). More recently, short time-course studies have suggested that an early vascular inflammatory response is a potential mechanism for the initiation of MR-mediated cardiac fibrosis (6, 7, 8, 9). Elevated expression of inflammatory markers such as ED-1-positive macrophages and osteopontin has been shown after 1 wk of mineralocorticoid treatment, a time when no fibrosis is detectable. Similarly, the induction of oxidative stress has been shown to play a role in the establishment and progression of the cardiac pathology in this model (10).

Studies from our laboratory have clearly demonstrated that administration of the selective MR antagonist EPL in a model of established cardiac fibrosis can effectively reverse the MR-mediated cardiac pathology, even in the face of continuing DOC/salt treatment (11). Furthermore, this study also demonstrated that mineralocorticoid withdrawal in an established model of cardiac fibrosis fails to prevent a continuing, submaximal inflammatory and fibrotic response. This effect observed in the withdrawal group may reflect continuing activation of vascular wall MR by endogenous glucocorticoids, in the context of the tissue damage induced by 4 wk of DOC/salt administration.

Glucocorticoids (cortisol in humans and corticosterone in rats) affect a diverse range of physiological processes and possess potent antiinflammatory actions. Glucocorticoid receptors (GR) are ubiquitously distributed, in contrast with the more restricted pattern of MR expression. Glucocorticoids bind MR with high affinity (12, 13) equivalent to that of aldosterone. Given that circulating concentrations of glucocorticoids are commonly three orders of magnitude higher than those of aldosterone, a mechanism that confers aldosterone specificity on epithelial MR is clearly required. In epithelial tissues and VSMC, this specificity is conferred extrinsically by the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (14, 15). This enzyme metabolizes active cortisol and corticosterone to their inactive metabolites cortisone and 11-dehydrocorticosterone and stoichiometrically converts nicotinamide adenine dinucleotide (NAD+) to reduced NAD (NADH). The progression of mineralocorticoid/salt-induced cardiac fibrosis includes a well-described inflammatory response. In the present study, we therefore explored the effect of lowering antiinflammatory activity by administration of the GR antagonist RU486 in an established model of mineralocorticoid-mediated cardiac fibrosis, on the hypothesis that GR blockade might aggravate vascular inflammation and cardiac fibrosis in this mineralocorticoid/salt model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental model
Experiments were performed according to the National Health and Medical Research Council of Australia Code of Practice for the Care and Use of Animals for Scientific Purposes (1997) and were approved by the Monash University Animal Welfare Committee. Male, 8-wk-old (initial weight, 180–200 g) Sprague Dawley rats were anesthetized with Ilium Xylazil (8 mg/kg; Troy Laboratories, Smithfield, New South Wales, Australia) plus ketamine (60 mg/kg; Pfizer Pty. Ltd., Sydney, New South Wales, Australia) and uninephrectomized.

One day after surgery, rats were randomly assigned to one of five treatment groups (n = 7–8 per group). Treatments were administered as sc injections weekly for DOC (Sigma-Aldrich, St. Louis, MO) and daily for RU486 (Mifepristone; Sigma-Aldrich). EPL (Pfizer Pty Ltd.) was incorporated into the rat chow at a concentration such that each animal received approximately 50 mg/kg·d. All groups were maintained on 0.9% NaCl plus 0.4% KCl solution to drink.

Treatment groups were: control (CON), no further treatment (n = 8); DOC (20 mg/wk) for 4 wk (DOC4; n = 7); DOC for 8 wk (DOC8; n = 8); DOC for 8 wk plus the GR antagonist RU486 (2 mg/d) wk 5–8 (DOC8/RU486; n = 7); and DOC for 8 wk plus RU486 and the MR antagonist EPL (50 mg/kg·d) for wk 5–8 (DOC8/RU486+EPL; n = 7).

Systolic blood pressure
Systolic blood pressure of acclimated, warmed rats was measured by tail-cuff plethysmography (ITTC Life Science, Woodland Hills, CA) by a procedure adapted from the ITTC Life Science manual. Briefly, after 15 min of stabilization in the preheated chamber (29 C), pressure was read over three consecutive manual inflation-deflation cycles, and rats were tested twice a week. If the pressure readings differed by more than 5 mm Hg, the readings were discarded, the rats were allowed to rest, and the procedure was repeated until three consistent readings were obtained. All groups were handled daily and to a similar extent throughout the study.

Tissue collection
Animals were killed by CO2 in air at 8 wk, except the DOC4 group, which was killed at 4 wk. An arterial blood sample and the heart were collected and stored for analysis. The apex of the heart was immersion-fixed in 4% paraformaldehyde for histological analysis; the middle section was frozen in OCT compound on dry ice for immunohistochemical analysis, and the third part was snap-frozen in liquid nitrogen for RT-PCR. Cardiac hypertrophy was assessed by wet heart weight to body weight ratio when the rats were killed.

Histological analysis
The extent of fibrosis was determined by dewaxing and staining 5-µm heart sections with 0.1% Sirius red (Sigma Diagnostics, St. Louis, MO) in saturated picric acid (BDH AnalaR, Poole, UK) and quantifying the collagen content with the Analytical Imaging Station (AIS) software package (Version 4.0 ß 1.5, Imaging Research Inc., St. Catharines, Ontario, Canada).

Immunohistochemistry
To characterize the inflammatory response, frozen sections (5 µm) were immunostained with the following primary antibodies: monoclonal antibody against rat monocytes/macrophages, ED-1 (gift from Professor Peter Tipping, Department of Medicine, Monash Medical Centre) [1:200 dilution in 1% Tris-buffered saline (TBS)]; goat polyclonal cyclooxygenase 2 (COX-2) (Santa Cruz Biotechnology, Santa Cruz, CA) (1:200 dilution in 1% TBS); and osteopontin, the MPIIB101 monoclonal antibody (Iowa University Hybridoma Bank, Iowa City, IA) (1:100 dilution in 1% TBS). Briefly, two frozen sections were removed from –80 C storage and fixed in 4 C acetone (ED-1), ethanol (COX-2), or 4% paraformaldehyde (osteopontin) at 4 C. Sections were treated with 0.3% H2O2 in TBS and incubated with horse serum (gift from Professor Ian Clarke, Physiology Department, Monash University) (1:10 dilution in 1% TBS) (ED-1 and osteopontin) or rabbit serum (Vector Laboratories Inc., Burlingame, CA) (1:10 dilution in 1% TBS) (COX-2) and incubated overnight at 4 C with the primary antibody. The following day, sections were washed in 1% TBS and incubated with Vectastain biotinylated universal antibody for ED-1 and osteopontin (1:200 dilution in 1% TBS; Vector Laboratories Inc.) or rabbit-antigoat for COX-2 (1:200 dilution in 1% TBS; Vector Laboratories Inc.) for 45 min, washed in 1% TBS, and treated with the preincubated ABC complex (Vectastain Elite ABC kit, Vector Laboratories Inc.). Filtered diaminobenzidine (Sigma-Aldrich) was applied before counterstaining with 0.1% Mayer’s hemotoxylin solution (Sigma Diagnostics), dehydrating and mounting each section in Depex (BHD Merck, Poole, UK). Infiltrating ED-1-positive macrophages were quantified by an optical dissector method (16), which provides a value for the average number of macrophages per frame (826,890 µm2) rather than per section; more than 80 ED-1-positive macrophages were counted for each rat. COX-2 and osteopontin expression in the vessel wall was quantified by an AIS software package (version 4.0 ß 1.5, Imaging Research Inc.). All vessels in each section were analyzed.

RT-PCR
Total rat heart RNA was prepared with Ultraspec (Fisher Scientific, Pittsburgh, PA). First strand cDNA synthesis from 500 ng total RNA was performed after deoxyribonuclease treatment with avian myeloblastosis virus reverse transcriptase (Roche, Indianapolis, IN) and priming with random hexamers. PCRs were carried out using the primer sets for p22phox (NM_024160), gp91phox (NM_023965), and NOX-4 (NM_053524) as previously described (9, 11) and for osteopontin (5' to 3') sense CCA GCA CAC AAG CAG ACG TT and antisense TCA GTC CAT AAG CCA AGC TAT CAC (NM_012881). 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; V01270 X00133 X00521 X01069). To validate the real-time PCR protocol, gene-specific standard curves for p22phox 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 p22phox and 18S, and gp91phox and NOX-4 were analyzed undiluted. Relative amounts of mRNA were calculated by normalizing reduced NAD phosphate [NAD(P)H] oxidase values to 18S rRNA values.

Statistics
All data sets were analyzed by one-way ANOVA (SPSS statistical software package, version 11.5, Chicago, IL) and Tukey’s comparisons test applied to identify significant effects between groups; differences were considered significant at P < 0.05. All data are reported as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Systolic blood pressure
A one-way ANOVA revealed significant treatment (F(3,27) = 20.256; P ≤ 0.001) main effects. Consistent with previous findings, pairwise comparisons showed that administration of DOC plus salt for 8 wk (DOC8) significantly raises systolic blood pressure over control (178 ± 7 mm Hg vs. 122 ± 3 mm Hg; P ≤ 0.001; Fig. 1AGo). EPL added to DOC in the presence of RU486 for wk 5–8 (DOC8/RU486+EPL) restored systolic blood pressure to control levels (125 ± 7 mm Hg; P ≤ 0.001; Fig. 1AGo). Of interest, blood pressure in the group receiving DOC plus RU486 wk 5–8 (DOC8/RU486) in the absence of EPL was significantly lower than in animals receiving DOC alone for 8 wk (154 ± 8 mm Hg vs. 178 ± 7 mm Hg; P < 0.05; Fig. 1AGo). Systolic blood pressure was determined at 4 wk as well as at 8 wk. However, values obtained at 4 wk are not shown because the rats were not fully acclimated (i.e. control values were elevated and then fell to within the normal range at 8 wk).


Figure 1
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  		FIG. 1. Treatment groups as follows: CON, Control; DOC4, DOC treatment for 4 wk; DOC8, DOC treatment for 8 wk; DOC8/RU486, DOC for 8 wk plus RU486 for wk 5–8; DOC8/RU486+EPL, DOC for 8 wk plus RU486 and EPL for wk 5–8. Values are mean ± SEM. For control and DOC8, n = 8; for DOC4, DOC8/RU486, and DOC8/RU486+EPL, n = 7. A, Systolic blood pressure (SBP) determined by tail-cuff plethysmography. DOC8 increased SBP compared with all other treatments. DOC8/RU486 marginally reduced blood pressure to a level significantly greater than CON and DOC8/RU486+EPL. *, P < 0.001 vs. all other groups; **, P < 0.05 vs. CON and DOC8/RU486+EPL. Data for systolic blood pressure at 4 wk are not shown. B, Perivascular and interstitial cardiac fibrosis. DOC8 significantly increased cardiac collagen content compared with CON and DOC8/RU486+EPL. *, P < 0.05 vs. CON and DOC8/RU486+EPL.

 
Interstitial cardiac fibrosis
Interstitial and perivascular collagen deposition increased after DOC treatment with one-way ANOVA revealing significant treatment (F(4,32) = 4.392; P ≤ 0.001) main effects. In agreement with previous studies, pairwise comparisons showed a significant increase in collagen deposition after 8 wk of DOC plus salt treatment (DOC8 vs. control; P ≤ 0.05; Fig. 1BGo). Consistent with previous studies using this model, administration of EPL wk 5–8 effectively reversed the DOC-mediated increase in fibrosis (P ≤ 0.05; Fig. 1BGo), although in this case in the presence of RU486. In contrast, RU486 treatment over wk 5–8 (DOC8/RU486) had no significant effect on the DOC-induced fibrotic response.

Markers of inflammation
The inflammatory response in the mineralocorticoid/salt model has previously been characterized by an increase in the number of infiltrating ED-1-positive monocytes/macrophages in the cardiac interstitium and increased COX-2 and osteopontin expression in the vessel walls. One-way ANOVA revealed a significant treatment (F(4,32) = 13.624; P ≤ 0.001) main effect for macrophage infiltration. As shown previously, pairwise comparisons showed a significant increased number of infiltrating ED-1-positive monocytes/macrophages after 8 wk of DOC treatment (vs. CON and DOC8/RU486+EPL, P ≤ 0.001; DOC4 and DOC8/RU486, P ≤ 0.01; Fig. 2AGo). One-way ANOVA similarly showed a significant (F(4,30) = 4.916, P ≤ 0.01) osteopontin expression effect, and pairwise comparison a significant increase in osteopontin expression in the vessel wall after 8 wk of DOC treatment (vs. CON P ≤ 0.05; Fig. 2BGo). Furthermore, EPL treatment reversed this DOC-induced increase in the presence of RU486 (DOC8 vs. DOC8/RU486+EPL, P ≤ 0.05; Fig. 2BGo); RU486 treatment alone had no significant effect in osteopontin expression in the vessel wall. One-way ANOVA showed a significant (F(4,29) = 6.304; P ≤ .001) COX-2 expression effect, and pairwise comparison showed a significant increase in COX-2 expression after 4 and 8 wk of DOC treatment (vs. CON P ≤ 0.01; Fig. 2CGo); addition of RU486 wk 5–8 had no effect on the DOC-mediated increase in COX-2 expression (vs. CON P ≤ 0.05; Fig. 2CGo). EPL, which has previously been shown to reverse the DOC-induced increase in COX-2 expression, only partially suppressed the elevated expression when administered with RU486 in the present study.


Figure 2
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  		FIG. 2. Inflammatory markers: treatments as for Fig. 1Go. A, ED-1-positive macrophages. DOC8 increased the number of infiltrating ED-1-positive macrophages compared with all other treatments. *, P < 0.001 vs. CON and DOC8/RU486+EPL; P < 0.01 vs. DOC4 and DOC8/RU486. B, Osteopontin expression in vessel wall. DOC8 significantly increased osteopontin expression in the vessel wall compared with CON and DOC8/RU486+EPL. *, P < 0.05 vs. CON and DOC8/RU486+EPL. C, COX-2 expression in vessel wall. DOC8 and DOC8/RU486 treatment significantly increased COX-2 expression in the vessel wall compared with CON. *, P < 0.05 vs. CON; **, P < 0.01 vs. CON.

 
mRNA levels for the multifunctional protein osteopontin assessed by quantitative RT-PCR were used as an additional marker of inflammation. One-way ANOVA revealed significant (F(4,27) = 3.277; P ≤ 0.05) main effects, and pairwise comparison showed that DOC plus salt for 8 wk increased mRNA expression of osteopontin, although this did not reach significance compared with CON (vs. CON P = 0.06; Fig. 3AGo); addition of EPL wk 5–8 significantly reversed this effect (P ≤ 0.05; Fig. 3AGo). The magnitude of this response was similar to that of previous studies where EPL alone was coadministered with DOC. However, when administered over wk 5–8, RU486 (DOC8/RU486) had no significant effect on osteopontin mRNA levels in response to DOC.


Figure 3
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  		FIG. 3. Treatments as for Fig. 1Go. A, Osteopontin mRNA expression. DOC8 significantly increases osteopontin mRNA expression compared with DOC8/RU486+EPL. *, P < 0.05 vs. DOC8/RU486+EPL. B–D, NAD(P)H oxidase subunit mRNA expression relative to 18S rRNA (p22phox, gp91phox, and NOX-4, respectively). DOC8 significantly increased mRNA expression of the NAD(P)H subunits compared with DOC8/RU486+EPL. DOC4 significantly increased expression of the gp91phox subunit compared with CON and DOC8/RU486+EPL. *, P < 0.05 vs. DOC8/RU486+EPL. **, P < 0.05 vs. CON and DOC8/RU486+EPL.

 
Oxidative stress
To document the role of the oxidative stress in this model, quantitative RT-PCR was used to measure mRNA levels of subunits of the NAD(P)H oxidase system (p22phox, gp91phox, and NOX-4; Fig. 3Go, B–D, respectively). One-way ANOVA revealed significant main effects for p22phox (F(4,30) = 3.121; P ≤ 0.05), gp91phox (F(4,29) = 4.466; P ≤ 0.05), and NOX-4 (F(4,28) = 3.772; P ≤ 0.05). Pairwise comparisons showed in all cases that DOC treatment for 8 wk increased subunit mRNA levels, and administration of EPL over wk 5–8 (P ≤ 0.05; Fig. 3Go, B–D) reversed this DOC-induced increase in NAD(P)H oxidase activity. Administration of RU486 over wk 5–8 had no significant effect on the DOC-induced mRNA levels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows that the pathophysiological consequences of excess mineralocorticoid for salt status are independent of GR activation status and confirms their independence of blood pressure elevation. GR blockade with the synthetic antagonist RU486 clearly did not, as anticipated, increase the established inflammatory response and tissue pathology, but rather modestly and apparently selectively reduced tissue changes in the mineralocorticoid/salt model of inflammation and cardiac fibrosis.

We and others have demonstrated that mineralocorticoid/salt administration induces increased collagen deposition in rats after a prolonged period of time (6–8 wk) (3, 17, 18), although the precise mechanisms that translate inappropriate mineralocorticoids/salt into cardiac fibrosis remain largely unknown. At early time points (1 wk) in the mineralocorticoid/salt model, significant increases in interstitial collagen are not usually detected; progressive coronary vascular and perivascular inflammation with lesions expressing ED-1-positive macrophages, COX-2, and the inflammatory cytokine osteopontin occur over 1–4 wk of treatment (7, 9). These observations clearly support the hypothesis that induction of oxidative stress and vascular inflammatory processes are key intermediate steps in the development of cardiac fibrosis and suggest that the primary action of aldosterone is within the vessel wall.

In addition to being a potent MR agonist, DOC is also a weak GR antagonist (19). Comparing interstitial and perivascular collagen deposition in the mineralocorticoid/salt model in early studies suggested that there may be subtle differences in the pattern of collagen deposition, although overall levels appear similar. Aldosterone-mediated fibrosis appeared to have a more interstitial pattern of deposition, and DOC more perivascular (3). A modest but significant increase in perivascular fibrosis was also seen with RU486 administered alone, providing further support for a possible role of the GR in modifying the fibrotic response (3). In the present study, the collagen fraction reflects the heart as a whole rather than as a ratio of interstitial to perivascular collagen; the present data show that in a model of established tissue fibrosis and vascular damage, addition of the GR antagonist RU486 to continued DOC treatment has no significant effect on collagen deposition compared with DOC alone (DOC8). This would suggest that under these circumstances MR activation can produce a fibrotic response essentially independent of GR signaling pathways.

Hypertension caused by excess glucocorticoids is also well recognized, although the precise mechanisms responsible remain to be elucidated. Previously, in the mineralocorticoid/salt model, the GR agonist corticosterone or the GR antagonist RU486 administered alone for 8 wk produced no change in systolic blood pressure over control (3). In the present study, administration of the GR antagonist RU486 over wk 5–8 of DOC administration (DOC8/RU486) was sufficient to reduce systolic blood pressure, compared with DOC alone (DOC8), to a level that remained, however, above control. Although systolic blood pressure data at 4 wk are not available for the current study, it has been previously shown that 4 wk of DOC administration elevates systolic blood pressure to levels similar to those seen at 8 wk (20). Although it is clear that GR activity can influence systolic blood pressure, it remains unknown how blocking GR in an established model of MR-mediated hypertension reduces systolic blood pressure. Previously, in a model of ACTH-induced hypertension, a high dose of RU486 (70 mg/kg every 3 d) partially reduced increases in systolic blood pressure (21), whereas in a model of cortisol-induced hypertension, RU486 had no such effect (22). Although production of reactive oxygen species through NAD(P)H oxidases has recently been suggested to play an essential role in modulating blood pressure in glucocorticoid-induced hypertension (23, 24), in the present study GR blockade did not significantly reduce DOC-mediated increases in NAD(P)H oxidase mRNA expression.

An early inflammatory response, demonstrated by increased expression of inflammatory markers (COX-2, osteopontin, and ED-1-positive monocytes/macrophages) is now considered a prerequisite in the pathogenesis of mineralocorticoid/salt-induced cardiac fibrosis (6, 7, 8, 9). Consistent with previous studies, DOC treatment alone for 8 wk increased the number of infiltrating macrophages, a response reversed by MR blockade. RU486 administered for wk 5–8 (DOC8/RU486) significantly reduced the number of infiltrating ED-1-positive monocytes/macrophages in the present study compared with DOC alone (DOC8), consistent with a possible role for GR signaling in macrophage recruitment, and perhaps macrophage function.

Previously, a time-course study over 1–4 wk characterized the inflammatory response to mineralocorticoid/salt administration and demonstrated a progressive increase in osteopontin over the study period (7). In an 8-wk study, osteopontin expression increased over the first 4 wk but plateaued from wk 4–8 (11). Inflammatory marker expression in the present study is consistent with these data, showing an increase in mean values for osteopontin mRNA at 4 and 8 wk after DOC treatment and reaching significance, on a population basis, at 8 wk. Although RU486 treatment wk 5–8 (DOC8/RU486) did not significantly affect osteopontin mRNA expression, MR blockade (DOC8/RU486+EPL) completely reversed the DOC-mediated increase, supporting the hypothesis that osteopontin expression is a consequence of inappropriate MR activation via a pathway apparently independent of GR signaling. This finding is consistent with a recent study suggesting that osteopontin plays a key role in the mineralocorticoid-salt response by promoting fibrosis to protect against left ventricular dilation (25).

A number of recent studies have provided increasing evidence supporting a key role for vascular NAD(P)H oxidases in the production of O2- (super oxide) and reactive oxygen species in mineralocorticoid/salt disease models (10, 11). NAD(P)H oxidases generate O2- through the assembly of a multisubunit complex composed of common cytosolic and tissue-specific membrane-associated subunits. We analyzed mRNA expression for three subunits of NAD(P)H oxidase, NOX-4, p22phox, and gp91phox (NOX-2). NOX-4 is associated with VSMC-specific NAD(P)H oxidases (26), p22phox with the vessel wall, and gp91phox (NOX-2) with macrophages and endothelial cells (27, 28). Consistent with our previous findings, mRNA expression for each of the subunits was significantly increased at both 4 and 8 wk of DOC treatment, and these levels returned to control with the addition of EPL for wk 5–8. Coadministration of RU486 did not produce a significant reduction in the expression of any of the transcripts, again suggesting that GR activation has little if any role to play in the regulation of vascular oxidative stress. These findings also reinforce the possibility that MR- and GR-mediated effects occur via largely distinct pathways in this model.

Although the fibrotic response to RU486 observed in previous studies suggested that physiological levels of glucocorticoids act as tonic inhibitors of fibrosis, the marked fibrotic response to 9{alpha}-fludrocortisone (4) would argue against this. The inflammatory responses characterized in the present study, however, indicate that the mechanisms and pathways regulating tissue remodeling may be more complex. We have shown that RU486 given once fibrosis has been established can reduce the number of infiltrating macrophages in response to aldosterone and showed similar trends in terms of mRNA expression of oxidative stress markers. These data are consistent with two possibilities: 1) that GR act via pathways distinct from MR to regulate tissue remodeling, or 2) that there may be effects of RU486 independent of GR in the cardiovascular system. In terms of the second possibility, there is now evidence to suggest that the progesterone receptor may have a role to play in regulating inflammation in the vasculature (29) as well as in breast cancer (30).

Exogenous mineralocorticoids in the context of a high salt intake produce a well-characterized inflammatory response. In the present study, we determined the effect of reduced antiinflammatory activity in this model by administration of RU486 in a model of established mineralocorticoid-mediated cardiac fibrosis. Mean values for all indices were lowered after the addition of RU486 from wk 5 to DOC administration, but this only reached significance for macrophage infiltration and systolic blood pressure. Although the values for the others were not significantly different, this argues against the main hypothesis that the study set out to test— that addition of a potent GR antagonist on top of DOC (a much weaker GR antagonist) would exacerbate the signs of inflammation and tissue remodeling.


   Footnotes
 
Disclosure statement: A.J.R. has nothing to declare. J.W.F. has received consulting fees from Merck, Pfizer, Sankyo, Lilly, Schering-Plough, Wyeth, Exelisis and CBio. P.J.F. has received consulting fees from Merck and lecture fees from Novartis. M.J.Y., P.J.F., and J.W.F. have been the recipients of a previous research grant from Pfizer Inc., and M.J.Y. and J.W.F. from Merck. The present study does not relate to these activities.

First Published Online September 21, 2006

Abbreviations: COX-2, Cyclooxygenase 2; DOC, deoxycorticosterone; EPL, eplerenone; GR, glucocorticoid receptor(s); MR, mineralocorticoid receptor(s); NAD+, nicotinamide adenine dinucleotide; NADH, reduced NAD; NAD(P)H, reduced NAD phosphate; TBS, Tris-buffered saline; VSMC, vascular smooth muscle cell(s).

Received May 18, 2006.

Accepted for publication September 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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