Skip main navigation

Deletion of Mineralocorticoid Receptors From Macrophages Protects Against Deoxycorticosterone/Salt-Induced Cardiac Fibrosis and Increased Blood Pressure

Originally publishedhttps://doi.org/10.1161/HYPERTENSIONAHA.109.131110Hypertension. 2009;54:537–543

Abstract

Increased mineralocorticoid levels plus high salt promote vascular inflammation and cardiac tissue remodeling. Mineralocorticoid receptors are expressed in many cell types of the cardiovascular system, including monocytes/macrophages and other inflammatory cell types. Although mineralocorticoid receptors are expressed in monocytes/macrophages, their role in regulating macrophage function to date has not been investigated. We, thus, used the Cre/LoxP-recombination system to selectively delete mineralocorticoid receptors from monocytes/macrophages with the lysozyme M promoter used to drive Cre expression (MRflox/flox/LysMCre/− mice). Male mice from each genotype (MRflox/flox or wild-type and MRflox/flox/LysMCre/− mice) were uninephrectomized, given 0.9% NaCl solution to drink, and treated for 8 days or 8 weeks with either vehicle (n=10) or deoxycorticosterone (n=10). Equivalent tissue macrophage numbers were seen for deoxycorticosterone treatment of each genotype at 8 days; in contrast, plasminogen activator inhibitor type 1 and NAD(P)H oxidase subunit 2 levels were increased in wild-type but not in MRflox/flox/LysMCre/− mice given deoxycorticosterone. Baseline expression of other inflammatory genes was reduced in MRflox/flox/LysMCre/− mice compared with wild-type mice. At 8 weeks, deoxycorticosterone-induced macrophage recruitment and connective tissue growth factor and plasminogen activator inhibitor type 1 mRNA levels were similar for each genotype; in contrast, MRflox/flox/LysMCre/− mice showed no increase in cardiac fibrosis or blood pressure, as was seen in wild-type mice at 8 weeks. These data demonstrate the following points: (1) mineralocorticoid receptor signaling regulates basal monocyte/macrophage function; (2) macrophage recruitment is not altered by loss of mineralocorticoid receptor signaling in these cells; and (3) a novel and significant role is seen for macrophage signaling in the regulation of cardiac remodeling and systolic blood pressure in the deoxycorticosterone/salt model.

The clinical use of mineralocorticoid receptor (MR) antagonists added to the current standard of care reduces morbidity and mortality in patients with congestive heart failure1,2 and reduces blood pressure and proteinuria as monotherapy in essential hypertension.3 Although the precise mechanism for this protection remains to be determined, considerable insights have been obtained from experimental models of mineralocorticoid/salt-mediated cardiac fibrosis4–6; hypertension, cardiac hypertrophy, and fibrosis are key responses to the administration of aldosterone or deoxycorticosterone (DOC) concurrently with a high salt intake for 8 weeks. Importantly, the pathogenesis of cardiac fibrosis is independent of hypertension and cardiac hypertrophy in this model, suggesting a direct role for MR activation in driving the onset and progression of cardiovascular disease.4–6

We and others have previously identified vascular inflammation (ie, osteopontin and plasminogen activator inhibitor type 1 [PAI-1] expression) and an increased macrophage infiltration in the myocardium before the onset of fibrosis, suggesting that these are key players in the initiation and progression of MR-mediated cardiac fibrosis.7–10 Oxidative stress has also been shown to play a key role in MR-mediated cardiac pathology.11 The NAD(P)H oxidoreductase system is widely expressed throughout the cardiovascular system and is a major source of reactive oxygen species in the vessel wall.12,13 Expression of the NAD(P)H oxidase subunit 2 (NOX2; also called gp91phox) and p22phox subunits of NAD(P)H oxidase are increased after DOC/salt treatment from 1 to 2 weeks,14 whereas a potential role specifically for macrophage-MR signaling in oxidative stress has been suggested by increased levels of p22phox and PAI-1 in human monocytes and increased macrophage NAD(P)H oxidase activity after aldosterone treatment in vivo.15,16 Changes in NO signaling in response to the onset of inflammation also contributes to the production of superoxide and vascular dysfunction in this model.11,17 It is our hypothesis that MR signaling, specifically, in monocytes/macrophages, represents an important and novel mechanism in the pathology of cardiovascular disease.

Macrophages contain both MRs and glucocorticoid receptors (GR) but not the aldosterone specificity-conferring enzyme 11β-hydroxysteroid dehydrogenase type 2, indicating that MRs in macrophages will be normally occupied by glucocorticoids (cortisol in humans and corticosterone in rodents).18,19 The relative contribution of GRs and MRs and their respective ligands in the control of macrophage phenotype and activation has not been determined in the context of cardiovascular disease.

The aim of the current study is to investigate whether mineralocorticoid activation of monocytes/macrophages plays a unique role in the cardiac pathology of the mineralocorticoid/salt model. Conventional MR knockout (KO) mice are available but show high neonatal lethality because of their inability to concentrate urinary sodium20; hippocampal MR selective KO mice have also been described.21 We, therefore, generated macrophage MR null (MRflox/flox/LysMCre/−) mice using the Crelox approach and investigated cardiovascular response to acute (8 days) and chronic (8 weeks) administration of DOC/salt.

Methods

Additional materials and methods are provided in the online Data Supplement (please see http://hyper.ahajournals.org).

Generation of Monocyte/Macrophage MR Null Mice

Mice containing the MRflox allele (kindly provided by Pfizer Inc) with mice expressing Cre recombinase under the control of the myeloid lineage-specific promoter, lysozyme M (LysM), were crossed to generate mice in which the MR was deleted in monocytes/macrophages.22 The presence of the MRflox/flox and LysM Cre transgene was determined by PCR analysis of genomic DNA from tail tips. MR deletion from the myeloid lineage was validated by Western blot analysis of bone marrow macrophages from KO and MRflox/flox control mice using the MR1–18 monoclonal antibody hybridoma supernatant (1:250; a gift from Prof Celso Gomez-Sanchez23).

DOC/Salt Model of Cardiac Fibrosis

Mice ≈8 weeks of age (n=10 per group) were uninephrectomized and given standard chow and 0.9% NaCl/0.4% KCl solution to drink. Mice of each genotype were randomly assigned to one of the following treatments, resulting in a total of 4 groups of mice for each time point: (1) control treatment for 8 days, (2) continuous DOC treatment for 8 days and control treatment for 8 weeks, and (3) continuous DOC treatment for 8 weeks.24,25 Mice receiving DOC treatment received an SC 7-mg, 21-day release pellet that was replaced every 3 weeks.

Systolic Blood Pressure

Systolic blood pressure (SBP) was measured by tail-cuff plethysmography (ITTC Life Science) biweekly for 3 weeks before SBP recording.26–28

Tissue Collection and Analysis

Animals were killed by CO2 in air at 8 days or 8 weeks with an arterial blood sample, and the heart was collected and stored for analysis, along with plasma radioimmunoassay for aldosterone (MP Biomedical), histological analysis for collagen content (0.1% Sirius red, Sigma-Aldrich),5,28 immunohistochemistry for CD68+ monocytes/macrophages (1:200, Sigma-Aldrich),9,28,29 and quantitative RT-PCR for mRNA expression, as detailed in Table S1 (available in the online Data Supplement).7,11,28,30

Statistics

All of the data were analyzed by 1-way ANOVA (Prism statistical software package, Graph Pad version 5.0A), and Bonferroni’s comparisons test applied to identify significant effects between groups. Mean differences were considered significant at P≤0.05. All of the data are reported as mean±SEM. One cohort of animals (10 per group) was the subject of the current study.

Results

Macrophage-MR Null Mice

MRflox/−/LysMCre/− and MRflox/flox (WT) mice used to breed macrophage MR null mice showed normal fertility and litter size. Mice genotyped as MRflox/flox/LysMCre/− showed normal phenotype, body, and heart weight (Figure S1A and Table S2), as well as the expected plasma aldosterone levels at 8 days for mice drinking 1% saline (aldosterone picograms per 100 μL: WT control, 12.4±1.6; WT DOC, 8.1±1.3; KO control, 12.4±2.2; KO DOC, 6.9±0.5). Resident macrophage numbers in the hearts of MRflox/−/LysMCre/− mice were equivalent to those in MRflox/flox mice, indicating no obvious systemic deletion of monocytes or macrophages (Figure 1A). Deletion of MRs from monocytes/macrophages was confirmed by Western blot analysis of MR expression in expanded bone marrow macrophages (Figure S1B). An antibody directed to the N terminus of the MR protein shows a band in WT lysates (lane 4) equivalent to the positive control purified human MR protein (lane 2) and whole mouse kidney (lane 3), whereas the sample from MRflox/flox/LysMCre/− mice showed no band (lane 5).

Figure 1. Macrophage recruitment and collagen deposition at 8 days. Treatment groups as follows: WT CON, untreated WT mice; WT DOC, WT mice treated with DOC for 8 days; KO CON, untreated macrophage-specific MR-null mice; KO DOC, macrophage-specific null mice treated with DOC for 8 days. Values are mean±SEM; n=8. A, Average number of FA/11-positive macrophages in heart tissue at 8 days. DOC treatment for 8 days significantly increased the number of infiltrating FA/11-positive macrophages in WT (WT DOC) and macrophage MR-null mice (KO DOC) vs untreated mice (*P<0.05 vs WT CON and KO CON for each). B, Cardiac collagen. DOC treatment for 8 days did not significantly alter cardiac collagen content in WT (WT DOC) or macrophage MR-null mice (KO DOC).

Role of Macrophage MRs in a Model of Acute DOC-Mediated Cardiac Pathology: 8 day Study

Macrophage Infiltration at 8 Days

To assess the role of the macrophage MRs in acute DOC-induced macrophage recruitment, the number of infiltrating CD68-positive monocytes/macrophages was assessed by immunohistochemistry. As shown previously in WT mice, DOC treatment for 8 days significantly increased the number of infiltrating macrophages in the heart; this effect was not altered by specific deletion of the MRs from macrophages (Figure 1A).

Cardiac Fibrosis at 8 Days

The role of macrophage MRs in DOC-induced collagen deposition was determined by staining with Sirius red (Figure S2). As expected, DOC treatment for 8 days did not significantly alter cardiac collagen content in either genotype at 8 days (Figure 1B).

Expression of Proinflammatory Genes at 8 Days

(To further explore to role of macrophage MRs in macrophage recruitment, mRNA levels of the chemoattractant monocyte chemotactic protein 1 MCP-1) were assessed by quantitative RT-PCR. DOC treatment for 8 days significantly increased mRNA levels of MCP-1 in both genotypes (P<0.05; Figure 2A).

Figure 2. mRNA levels for MCP-1 and PAI-1 at 8 days, relative to 18S rRNA. Treatments are as for Figure 1. A, MCP-1, DOC treatment for 8 days significantly increased MCP-1 mRNA levels in WT (WT DOC) and macrophage MR-null mice (KO DOC) vs untreated mice (*P<0.05 vs WT CON and KO CON); B, PAI-1; DOC treatment for 8 days significantly increased PAI-1 mRNA in WT (WT DOC) but not in KO DOC. Data represent the average of 2 separate reverse-transcription and PCR experiments. *P<0.05.

To investigate the role of macrophage MRs in the early pathological events of the mineralocorticoid/salt model, expression of known proinflammatory/fibrogenic genes was determined (Figure S3A through S3G). For PAI-1 (Figure 2B) and NOX2 (Figure S3G), gene expression DOC/salt treatment in WT, but not MR null, mice significantly increased mRNA levels. Values for mRNA expression of osteopontin, transforming growth factor (TGF)-β1, eNOS, connective tissue growth factor, collagen 1, and NOX2 in untreated MRflox/flox/LysMCre/− mice were significantly reduced compared with values for untreated WT mice at baseline (P<0.05; Figure S3A through S3D and S3H). In contrast, DOC treatment for 8 days did not significantly alter the expression of these genes over baseline for either genotype. No significant change in mRNA levels at 8 days of DOC/salt treatment was seen for glucose-6-phosphate dehydrogenase, p22phox (Figure S3E and S3F), fibronectin, and procollagen III (data not shown).

Role of Macrophage MRs in a Model of Chronic DOC-Mediated Cardiac Pathology: 8-Week Study

Macrophage Infiltration

As expected, DOC treatment for 8 weeks significantly increased the number of infiltrating CD68-positive monocytes/macrophages in WT mice, and this was not altered by deletion of MRs from macrophages (Figure 3A and Figure S2).

Figure 3. Macrophage recruitment and collagen deposition at 8 weeks. Treatments are as for Figure 1. Values are mean±SEM; n=8. A, Average number of FA/11-positive macrophages in heart tissue at 8 weeks. DOC treatment for 8 weeks increased the number of infiltrating FA/11-positive macrophages in WT (WT DOC) and macrophage MR-null mice (KO DOC) vs untreated mice. B, Cardiac collagen. DOC treatment for 8 weeks significantly increased cardiac collagen content in WT mice (WT DOC) but not macrophage MR-null mice (KO DOC). *P<0.05 vs WT CON and KO CON.

Cardiac Fibrosis

In agreement with previous studies, 8 weeks of DOC treatment in WT mice significantly increased interstitial and perivascular collagen. In contrast, MR deletion from monocytes/macrophages protected against the DOC-induced increase in cardiac fibrosis (Figures 3B and S2).

Expression of Proinflammatory Genes

DOC treatment for 8 weeks increased the mRNA levels of PAI-1 (P<0.05; Figure 4A), inducible NO synthase, and connective tissue growth factor (Figure S4A and S4B) in both genotypes, whereas deletion of MRs from macrophages reduced baseline TGF-β1 mRNA levels (P<0.05; Figure 4B). Values for osteopontin for the KO DOC mice were increased over KO control mice, whereas the equivalent values in WT mice did not reach significance (Figure S4C). In contrast, mRNA levels of collagen 1, endothelial NO synthase, glucose-6-phosphate dehydrogenase, MCP-1, NOX2 (Figure S4D through S4H), fibronectin, and procollagen III (data not shown) were not altered at 8 weeks by DOC treatment or deletion of MRs from macrophages.

Figure 4. Expression of genes associated with inflammation, oxidative stress, and tissue remodeling, relative to 18S rRNA, at 8 weeks. Treatments are as for Figure 1. A, PAI-1. B, TGF-β1. DOC treatment for 8 weeks increased PAI-1 mRNA in WT (WT DOC) and macrophage MR-null mice (KO DOC) vs untreated controls. Deletion of the MR from macrophages reduced baseline mRNA levels of TGF-β1 at 8 weeks (WT CON vs KO CON). *P<0.05 vs WT CON and KO CON.

Systolic Blood Pressure

The effect of deletion of MRs from macrophages on DOC-mediated increases in SBP was assessed at 4 and 8 weeks (Figure 5). As shown previously, DOC treatment for 4 weeks significantly increased SBP in WT mice (103±2 mm Hg for WT control versus 112±3 mm Hg for WT DOC mice; P≤0.05; Figure 5A), whereas the specific deletion of MRs from macrophages attenuated this effect (103±1 mm Hg for KO DOC, not different from 100±2 mm Hg for KO control mice; Figure 5A). DOC induced a further increase in SBP at 8 weeks in WT mice (103±3 mm Hg for WT control versus 121±3 mm Hg for WT DOC mice; P≤0.05; Figure 5B), whereas, at 8 weeks, SBP in macrophage MR-null mice was 109±3 mm Hg for KO DOC mice, not significantly different from 101±2 mm Hg for KO CON (P=0.06; Figure 5B).

Figure 5. SBP determined by tail-cuff plethysmography at 4 and 8 weeks. Treatments are as for Figure 1. DOC treatment for 4 and 8 weeks significantly increased SBP in WT mice (WT DOC) vs untreated (WT CON; no significant differences were seen for KO mice given DOC/salt at either time point vs KO CON and WT CON. *P<0.05 vs WT CON, KO CON, and KO DOC.

Discussion

The present study shows that selective deletion of the MRs from the monocyte/macrophage cell lineage protects against chronic mineralocorticoid/salt-induced cardiac fibrosis and increased SBP after DOC administration for 8 weeks. Short time-course studies (8 days) investigating potential mechanisms underlying these responses revealed altered basal expression of known proinflammatory/profibrotic genes restored to baseline at 8 weeks except for TGF-β1. Taken together these studies demonstrate an important role for macrophage MR signaling in the maintenance of normal macrophage phenotype and function and for the development of the cardiovascular sequelae of inappropriate mineralocorticoid-salt status. In support of these data, we have shown previously that the vascular and interstitial inflammatory and fibrotic responses to DOC/salt are MR specific in that the effects of DOC are not altered by administration of GR antagonists.28

Cardiac collagen deposition is increased in experimental animals after DOC/salt treatment for periods of 6 to 8 weeks.4,31,32 Tissue remodeling in this and similar models (angiotensin II/salt administration) has been clearly correlated with reduced cardiac function, although the present study is focused on the mechanisms of tissue remodeling rather than changes in cardiac parameters. The number of macrophages recruited at 8 days and 8 weeks and the onset of fibrosis at 8 weeks are consistent with previous rat studies of DOC/salt-induced cardiac fibrosis.4,5 We note, however, that, whereas some markers (eg, endothelial NO synthase) were regulated in our previous rat studies by DOC-salt treatment, this was not always the case for the mice in the present study. Although our data are generally consistent with other reports of DOC/salt treatment in mice, they also highlight some species differences.

Role of MRs in Macrophage Physiology and Pathophysiology

Given that monocytes/macrophages express MRs but not 11β-hydroxysteroid dehydrogenase type 2,18,19 these receptors will be predominantly occupied by endogenous glucocorticoids. A number of previous studies have suggested differential effects of corticosteroids on macrophages and similar cells types, in particular, microglia, where very low doses of corticosteroids (1 nM) promote expression of inflammatory indices, whereas higher doses (100 nM) lower expression of the same factors,33 reflecting MR-only occupancy at the lower concentrations and GR occupancy at higher levels. This pattern of response contrasts with classic epithelial responses for MRs but is consistent with other nonepithelial, MR-expressing tissues, including cardiac myocytes and specific nuclei in the brain34–36; it is well accepted that, in nonepithelial tissues, glucocorticoids normally do not mimic the effects of aldosterone mediated by the MRs but antagonize responses to coadministered aldosterone.5 Our data for markers of inflammation at 8 days strongly suggest that, as described for an increasing number of tissues, MRs may not only play an important role in regulating basal functions previously ascribed solely to the GR, but that the role of MRs in inflammation is in direct contrast to that of GR.

Macrophages and Fibrosis

The recruitment of macrophages is clearly important for the onset of the cardiac remodeling processes and the development of fibrosis, as indicated by studies in osteopontin null mice and MCP-1 null mice. In these animals, a markedly reduced monocyte/macrophage infiltration in cardiac and renal disease models is accompanied by a substantial reduction in the fibrosis37,38; in contrast, our data show no tissue remodeling with normal macrophage recruitment. The protection from tissue fibrosis observed in the MRflox/flox/LysMCre/− mice is evidence for a direct role for MR activation in macrophage function.

Our data show that MR signaling in macrophages is required to elicit a full fibrotic response in the heart. Macrophages play a key role in initiating fibroblast differentiation into myofibroblasts, important collagen-producing cells, via secretion of profibrotic stimuli, including angiotensin II, TGF-β, and cytokines.39–41 Examination of a panel of markers for inflammation and tissue remodeling showed significant induction of MCP-1, macrophage number, NOX2, and PAI-1 at 8 days of DOC treatment in the WT mice; increased NOX2 and PAI-1 expression were blocked by loss of MR signaling in macrophages, consistent with a role for inflammation and activated macrophages in the early stages of the pathology. PAI-1 levels were, however, increased at 8 weeks in the MRflox/flox/LysMCre/− mice, the reason for which remains to be explored. Moreover, a key finding at 8 days was that, for most markers, the baseline expression of mRNA was consistently significantly lower in MRflox/flox/LysMCre/− mice. The combination of lower levels of marker expression may represent a loss of the classic activation phenotype (M1) in both the infiltrating and resident macrophages.41,42 Whether macrophages in these mice are able to fully differentiate into one or both of the classic polarized phenotypes remains to be determined.

A second key finding in the current study is that there is a mismatch between the normal recruitment of macrophages and loss of the fibrotic response. These data indicate that MR signaling in other cell types (ie, endothelial cells, vascular smooth muscle cells, and cardiac myocytes) remains intact, enabling normal recruitment of macrophages to the myocardium; in contrast, the ability of macrophages to mount a normal inflammatory response to DOC/salt-mediated tissue injury is lost. This observation is important in that it indicates that the initial tissue response to DOC/salt, including macrophage recruitment, is mediated by a cell type(s) other than the macrophage. The cardiovascular injury response and macrophage recruitment have been well described by our laboratory and others and appear to involve, at least in part, 11β-hydroxysteroid dehydrogenase type 2–protected MRs, as found in vascular smooth muscle cells and endothelial cells. Analysis of remodeling and inflammatory responses after selective deletion of the MRs from the other cell types in the heart will be informative in this regard.

Macrophages and SBP: Regulation by Inflammatory Cells

The unexpected finding that loss of MR signaling in macrophages blocked the DOC-induced rise in SBP suggests a role for inflammatory cells in blood pressure regulation. In the current study, the magnitude of response to DOC/salt administration at 8 weeks is less than that demonstrated previously for rat studies (≈180 to 200 mm Hg at 8 weeks). Although this may reflect the fact that C57Bl6 mice are less susceptible to hypertension than some other strains, eg, 129/sv mice,43,44 the data are consistent with previous mouse studies using DOC pellets over 6 to 8 weeks.24,25 Vascular inflammation, however, does not necessarily result in elevated blood pressure; eg, overexpression of endothelin 1 in endothelial cells is characterized by increased vascular inflammation but not by increased SBP.45

Although the MR-mediated inflammatory response appears to be involved in the genesis of the hypertension seen in this model, numerous studies have shown that cardiac fibrosis in the DOC/salt model is not secondary to hypertension.5,46 In aldosterone/salt-treated rats, increased blood pressure was blocked by intracerebroventricular infusion of the MR antagonist RU28318, despite volume expansion consequent to the epithelial actions of administered aldosterone.46 Subsequently, in DOC/salt-treated rats infused with RU28318 intracerebroventricular, the cardiac fibrosis response to systemic DOC/salt was equivalent to that in intracerebroventricular vehicle–infused rats, despite no elevation of blood pressure.5 Moreover, it is also well accepted that equivalent tissue remodeling and inflammatory responses occur not only in the left ventricle but also in the right ventricle, which is not subject to changes in systemic blood pressure,4,5,7,11 and that subpressor doses of MR antagonists provide marked cardiovascular protection.1,2,47 Thus, the fact that the macrophage-MR null mice were protected from elevated SBP suggests that the hypertension typically seen in the DOC/salt model may have a macrophage component in its etiology.

Recently, a critical role for T cells has been demonstrated in the angiotensin II/salt and DOCA/salt models.48 Replacement of T- and B-cell populations in RAG1−/− (T- and B-cell null) mice, which are resistant to angiotensin II/salt and DOCA/salt-induced hypertension, identified T cells as the critical cell type for restoration of the hypertensive phenotype. It is also clear from these studies that low-grade inflammatory changes in the adventitia and perivascular spaces of resistance vessels can regulate blood pressure. Given that subpopulations of T cells play an important role in the activation of macrophages, it remains to be determined in the present study whether the MR null macrophages remain responsive to T cells.

Perspectives

Mice in which MRs have been selectively deleted from macrophages show baseline differences in gene expression compared with WT mice, evidence for a putative role of glucocorticoid-bound MRs in maintaining normal macrophage function. These mice also show cardiovascular protection from the administration of DOC/salt, consistent with a distinct role for these receptors in the resultant phenotype in WT mice. Specifically, our data show that mice with selective deletion of macrophage MRs do not, as anticipated, respond to DOC/salt by increasing cardiac inflammation and fibrosis. Interestingly, the effect of macrophage-MR deletion extended to the SBP response, which, in contrast to WT mice, was not different from control mice, suggesting that MR signaling in macrophages may contribute to blood pressure responses. Although elevated blood pressure in response to DOC/salt treatment is commonly held to reflect volume expansion, central and vascular mechanisms have also been shown to play key roles. Thus, the present studies suggest critical roles for macrophage MRs under basal conditions, in terms of macrophage function, and in determining the inflammatory and fibrotic responses to DOC/salt. They also suggest a hitherto-unexpected role for macrophage MRs in the hypertensive effects of DOC/salt by mechanisms that await further exploration.

We are grateful to Pfizer Inc for the generous gift of MRflox/flox mice and to Prof Celso Gomez-Sanchez (University of Mississippi) for the kind gift of the MR 1-18 antibody. We thank Dr David Nikolic-Paterson (Monash University) for his constructive input into the preparation of this article.

Sources of Funding

This work is supported by grant 388914 from the National Health and Medical Research Council of Australia. A.J.R. is supported by a Monash Graduate Scholarship.

Disclosures

J.W.F. has received consulting fees from Merck, Pfizer, Sankyo, Lilly, Schering-Plough, Bayer, and CBio. P.J.F. has received consulting fees from Merck and lecture fees from Bayer, Pfizer, and 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.

Footnotes

Correspondence to Morag J. Young, Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton 3168, Australia. E-mail

References

  • 1 Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, Palensky J, Wittes J. The effect of spironolactone on morbidity and mortality in patients with severe heart failure: Randomized Aldactone Evaluation Study Investigators. N Engl J Med. 1999; 341: 709–717.CrossrefMedlineGoogle Scholar
  • 2 Pitt B, Williams G, Remme W, Martinez F, Lopez-Sendon J, Zannad F, Neaton J, Roniker B, Hurley S, Burns D, Bittman R, Kleiman J. The EPHESUS trial: eplerenone in patients with heart failure due to systolic dysfunction complicating acute myocardial infarction–Eplerenone Post-AMI Heart Failure Efficacy and Survival Study. Cardiovasc Drugs Ther. 2001; 15: 79–87.CrossrefMedlineGoogle Scholar
  • 3 Pitt B, Reichek N, Willenbrock R, Zannad F, Phillips RA, Roniker B, Kleiman J, Krause S, Burns D, Williams GH. Effects of eplerenone, enalapril, and eplerenone/enalapril in patients with essential hypertension and left ventricular hypertrophy: the 4E-left ventricular hypertrophy study. Circulation. 2003; 108: 1831–1838.LinkGoogle Scholar
  • 4 Young M, Fullerton M, Dilley R, Funder J. Mineralocorticoids, hypertension, and cardiac fibrosis. J Clin Invest. 1994; 93: 2578–2583.CrossrefMedlineGoogle Scholar
  • 5 Young M, Head G, Funder J. Determinants of cardiac fibrosis in experimental hypermineralocorticoid states. Am J Physiol. 1995; 269: E657–E662.MedlineGoogle Scholar
  • 6 Brilla CG, Weber KT. Mineralocorticoid excess, dietary sodium, and myocardial fibrosis. J Lab Clin Med. 1992; 120: 893–901.MedlineGoogle Scholar
  • 7 Rocha R, Rudolph AE, Frierdich GE, Nachowiak DA, Kekec BK, Blomme EA, McMahon EG, Delyani JA. Aldosterone induces a vascular inflammatory phenotype in the rat heart. Am J Physiol Heart Circ Physiol. 2002; 283: H1802–H1810.CrossrefMedlineGoogle Scholar
  • 8 Rocha R, Stier CT Jr, Kifor I, Ochoa-Maya MR, Rennke HG, Williams GH, Adler GK. Aldosterone: a mediator of myocardial necrosis and renal arteriopathy. Endocrinology. 2000; 141: 3871–3878.CrossrefMedlineGoogle Scholar
  • 9 Young MJ, Moussa L, Dilley R, Funder JW. Early inflammatory responses in experimental cardiac hypertrophy and fibrosis: effects of 11 beta-hydroxysteroid dehydrogenase inactivation. Endocrinology. 2003; 144: 1121–1125.CrossrefMedlineGoogle Scholar
  • 10 Fujisawa G, Dilley R, Fullerton MJ, Funder JW. Experimental cardiac fibrosis: differential time course of responses to mineralocorticoid-salt administration. Endocrinology. 2001; 142: 3625–3631.CrossrefMedlineGoogle Scholar
  • 11 Sun Y, Zhang J, Lu L, Chen SS, Quinn MT, Weber KT. Aldosterone-induced inflammation in the rat heart: role of oxidative stress. Am J Pathol. 2002; 161: 1773–1781.CrossrefMedlineGoogle Scholar
  • 12 Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.CrossrefMedlineGoogle Scholar
  • 13 Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J Clin Invest. 1996; 97: 1916–1923.CrossrefMedlineGoogle Scholar
  • 14 Wilson P, Morgan J, Funder JW, Fuller PJ, Young MJ. Mediators of mineralocorticoid receptor-induced profibrotic inflammatory responses in the heart. Clin Sci (Lond). 2009; 116: 731–739.CrossrefMedlineGoogle Scholar
  • 15 Calo LA, Zaghetto F, Pagnin E, Davis PA, De Mozzi P, Sartorato P, Martire G, Fiore C, Armanini D. Effect of aldosterone and glycyrrhetinic acid on the protein expression of PAI-1 and p22(phox) in human mononuclear leukocytes. J Clin Endocrinol Metab. 2004; 89: 1973–1976.CrossrefMedlineGoogle Scholar
  • 16 Keidar S, Kaplan M, Pavlotzky E, Coleman R, Hayek T, Hamoud S, Aviram M. Aldosterone administration to mice stimulates macrophage NADPH oxidase and increases atherosclerosis development: a possible role for angiotensin-converting enzyme and the receptors for angiotensin II and aldosterone. Circulation. 2004; 109: 2213–2220.LinkGoogle Scholar
  • 17 Sun Y, Ahokas RA, Bhattacharya SK, Gerling IC, Carbone LD, Weber KT. Oxidative stress in aldosteronism. Cardiovasc Res. 2006; 71: 300–309.CrossrefMedlineGoogle Scholar
  • 18 Lim HY, Muller N, Herold MJ, van den Brandt J, Reichardt HM. Glucocorticoids exert opposing effects on macrophage function dependent on their concentration. Immunology. 2007; 122: 47–53.CrossrefMedlineGoogle Scholar
  • 19 Armanini D, Strasser T, Weber PC. Binding of agonists and antagonists to mineralocorticoid receptors in human peripheral mononuclear leucocytes. J Hypertension. 1985; 3 (suppl): S157–S159.Google Scholar
  • 20 Berger S, Bleich M, Schmid W, Cole TJ, Peters J, Watanabe H, Kriz W, Warth R, Greger R, Schutz G. Mineralocorticoid receptor knockout mice: pathophysiology of Na+ metabolism. Proc Natl Acad Sci U S A. 1998; 95: 9424–9429.CrossrefMedlineGoogle Scholar
  • 21 Berger S, Wolfer DP, Selbach O, Alter H, Erdmann G, Reichardt HM, Chepkova AN, Welzl H, Haas HL, Lipp HP, Schutz G. Loss of the limbic mineralocorticoid receptor impairs behavioral plasticity. Proc Natl Acad Sci U S A. 2006; 103: 195–200.CrossrefMedlineGoogle Scholar
  • 22 Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 1999; 8: 265–277.CrossrefMedlineGoogle Scholar
  • 23 Gomez-Sanchez CE, de Rodriguez AF, Romero DG, Estess J, Warden MP, Gomez-Sanchez MT, Gomez-Sanchez EP. Development of a panel of monoclonal antibodies against the mineralocorticoid receptor. Endocrinology. 2006; 147: 1343–1348.CrossrefMedlineGoogle Scholar
  • 24 Artunc F, Amann K, Nasir O, Friedrich B, Sandulache D, Jahovic N, Risler T, Vallon V, Wulff P, Kuhl D, Lang F. Blunted DOCA/high salt induced albuminuria and renal tubulointerstitial damage in gene-targeted mice lacking SGK1. J Mol Med. 2006; 84: 737–746.CrossrefMedlineGoogle Scholar
  • 25 Handtrack C, Cordasic N, Klanke B, Veelken R, Hilgers KF. Effect of the angiotensinogen genotype on experimental hypertension in mice. J Mol Med. 2007; 85: 343–350.CrossrefMedlineGoogle Scholar
  • 26 Walsh GM. Blood pressure measurements in the conscious intact rat. In: Proceedings of a Workshop on Blood Pressure Measurements in Hypertensive Animal Models. Bethesda, MD: US Department of Health, Education, and Welfare,National Institutes of Health; 1977: 1Google Scholar
  • 27 Rickard AJ, Funder JW, Fuller PJ, Young MJ. The role of the glucocorticoid receptor in mineralocorticoid/salt-mediated cardiac fibrosis. Endocrinology. 2006; 147: 5901–5906.CrossrefMedlineGoogle Scholar
  • 28 Rickard AJ, Funder JW, Morgan J, Fuller PJ, Young MJ. Does glucocorticoid receptor blockade exacerbate tissue damage after mineralocorticoid/salt administration? Endocrinology. 2007; 148: 4829–4835.CrossrefMedlineGoogle Scholar
  • 29 Wreford NG. Theory and practice of stereological techniques applied to the estimation of cell number and nuclear volume in the testis. Microsc Res Tech. 1995; 32: 423–436.CrossrefMedlineGoogle Scholar
  • 30 Brown NJ. Aldosterone and end-organ damage. Curr Opin Nephrol Hypertens. 2005; 14: 235–241.CrossrefMedlineGoogle Scholar
  • 31 Brilla CG, Weber KT. Reactive and reparative myocardial fibrosis in arterial hypertension in the rat. Cardiovasc Res. 1992; 26: 671–677.CrossrefMedlineGoogle Scholar
  • 32 Robert V, Silvestre JS, Charlemagne D, Sabri A, Trouve P, Wassef M, Swynghedauw B, Delcayre C. Biological determinants of aldosterone-induced cardiac fibrosis in rats. Hypertension. 1995; 26: 971–978.CrossrefMedlineGoogle Scholar
  • 33 Tanaka J, Fujita H, Matsuda S, Toku K, Sakanaka M, Maeda N. Glucocorticoid- and mineralocorticoid receptors in microglial cells: the two receptors mediate differential effects of corticosteroids. Glia. 1997; 20: 23–37.CrossrefMedlineGoogle Scholar
  • 34 Qin W, Rudolph AE, Bond BR, Rocha R, Blomme EA, Goellner JJ, Funder JW, McMahon EG. Transgenic model of aldosterone-driven cardiac hypertrophy and heart failure. Circ Res. 2003; 93: 69–76.LinkGoogle Scholar
  • 35 Mihailidou AS, Funder JW. Nongenomic effects of mineralocorticoid receptor activation in the cardiovascular system. Steroids. 2005; 70: 347–351.CrossrefMedlineGoogle Scholar
  • 36 Gomez-Sanchez EP, Venkataraman MT, Thwaites D, Fort C. ICV infusion of corticosterone antagonizes ICV-aldosterone hypertension. Am J Physiol. 1990; 258: E649–E653.CrossrefMedlineGoogle Scholar
  • 37 Persy VP, Verhulst A, Ysebaert DK, De Greef KE, De Broe ME. Reduced postischemic macrophage infiltration and interstitial fibrosis in osteopontin knockout mice. Kidney Int. 2003; 63: 543–553.CrossrefMedlineGoogle Scholar
  • 38 Dewald O, Zymek P, Winkelmann K, Koerting A, Ren G, Abou-Khamis T, Michael LH, Rollins BJ, Entman ML, Frangogiannis NG. CCL2/monocyte chemoattractant protein-1 regulates inflammatory responses critical to healing myocardial infarcts. Circ Res. 2005; 96: 881–889.LinkGoogle Scholar
  • 39 Brilla CG, Maisch B, Zhou G, Weber KT. Hormonal regulation of cardiac fibroblast function. Eur Heart J. 1995; 16: C45–C50.CrossrefMedlineGoogle Scholar
  • 40 Vaughan MB, Howard EW, Tomasek JJ. Transforming growth factor-beta1 promotes the morphological and functional differentiation of the myofibroblast. Exp Cell Res. 2000; 257: 180–189.CrossrefMedlineGoogle Scholar
  • 41 Lupher ML Jr, Gallatin WM. Regulation of fibrosis by the immune system. Adv Immunol. 2006; 89: 245–288.CrossrefMedlineGoogle Scholar
  • 42 Song E, Ouyang N, Horbelt M, Antus B, Wang M, Exton MS. Influence of alternatively and classically activated macrophages on fibrogenic activities of human fibroblasts. Cell Immunol. 2000; 204: 19–28.CrossrefMedlineGoogle Scholar
  • 43 Hartner A, Cordasic N, Klanke B, Veelken R, Hilgers KF. Strain differences in the development of hypertension and glomerular lesions induced by deoxycorticosterone acetate salt in mice. Nephrol Dial Transplant. 2003; 18: 1999–2004.CrossrefMedlineGoogle Scholar
  • 44 Karatas A, Hegner B, de Windt LJ, Luft FC, Schubert C, Gross V, Akashi YJ, Gurgen D, Kintscher U, da Costa Goncalves AC, Regitz-Zagrosek V, Dragun D. Deoxycorticosterone acetate-salt mice exhibit blood pressure-independent sexual dimorphism. Hypertension. 2008; 51: 1177–1183.LinkGoogle Scholar
  • 45 Amiri F, Virdis A, Neves MF, Iglarz M, Seidah NG, Touyz RM, Reudelhuber TL, Schiffrin EL. Endothelium-restricted overexpression of human endothelin-1 causes vascular remodeling and endothelial dysfunction. Circulation. 2004; 110: 2233–2240.LinkGoogle Scholar
  • 46 Gomez-Sanchez EP, Fort CM, Gomez-Sanchez CE. Intracerebroventricular infusion of RU28318 blocks aldosterone-salt hypertension. Am J Physiol. 1990; 258: E482–E484.MedlineGoogle Scholar
  • 47 Rocha R, Chander PN, Khanna K, Zuckerman A, Stier CT Jr. Mineralocorticoid blockade reduces vascular injury in stroke-prone hypertensive rats. Hypertension. 1998; 31: 451–458.CrossrefMedlineGoogle Scholar
  • 48 Guzik TJ, Hoch NE, Brown KA, McCann LA, Rahman A, Dikalov S, Goronzy J, Weyand C, Harrison DG. Role of the T cell in the genesis of angiotensin II induced hypertension and vascular dysfunction. J Exp Med. 2007; 204: 2449–2460.CrossrefMedlineGoogle Scholar

eLetters(0)

eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.

Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.