Amanda J. Rickard

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

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.

Corticosteroid receptors, macrophages and cardiovascular disease

The mineralocorticoid receptor (MR) and glucocorticoid receptor are ligand-activated transcription factors that have important physiological and pathophysiological actions in a broad range of cell types including monocytes and macrophages. While the glucocorticoids cortisol and corticosterone have well-described anti-inflammatory actions on both recruited and tissue resident macrophages, a role for the mineralocorticoid aldosterone in these cells is largely undefined. Emerging evidence, however, suggests that MR signalling may promote pro-inflammatory effects. This review will discuss the current understanding of the role of corticosteroid receptors in macrophages and their effect on diseases involving inflammation, with a particular focus on cardiovascular disease.

Macrophage Mineralocorticoid Receptor Signaling Plays a Key Role in Aldosterone-Independent Cardiac Fibrosis

Mineralocorticoid receptor (MR) activation promotes the development of cardiac fibrosis and heart failure. Clinical evidence demonstrates that MR antagonism is protective even when plasma aldosterone levels are not increased. We hypothesize that MR activation in macrophages drives the profibrotic phenotype in the heart even when aldosterone levels are not elevated. The aim of the present study was to establish the role of macrophage MR signaling in mediating cardiac tissue remodeling caused by nitric oxide (NO) deficiency, a mineralocorticoid-independent insult. Male wild-type (MRflox/flox) and macrophage MR-knockout (MRflox/flox/LysMCre/+; mac-MRKO) mice were uninephrectomized, maintained on 0.9% NaCl drinking solution, with either vehicle (control) or the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (L-NAME; 150 mg/kg/d) for 8 wk. NO deficiency increased systolic blood pressure at 4 wk in wild-type L-NAME/salt-treated mice compared with all other groups. At 8 wk, systolic blood pressure was increased above control in both L-NAME/salt treated wild-type and mac-MRKO mice by approximately 28 mm Hg by L-NAME/salt. Recruitment of macrophages was increased 2- to 3-fold in both L-NAME/salt treated wild-type and mac-MRKO. Inducible NOS positive macrophage infiltration and TNFα mRNA expression was greater in wild-type L-NAME/salt-treated mice compared with mac-MRKO, demonstrating that loss of MR reduces M1 phenotype. mRNA levels for markers of vascular inflammation and oxidative stress (NADPH oxidase 2, p22phox, intercellular adhesion molecule-1, G protein-coupled chemokine receptor 5) were similar in treated wild-type and mac-MRKO mice compared with control groups. In contrast, L-NAME/salt treatment increased interstitial collagen deposition in wild-type by about 33% but not in mac-MRKO mice. mRNA levels for connective tissue growth factor and collagen III were also increased above control treatment in wild-type (1.931 ± 0.215 vs. 1 ± 0.073) but not mac-MRKO mice (1.403 ± 0.150 vs. 1.286 ± 0.255). These data demonstrate that macrophage MR are necessary for the translation of inflammation and oxidative stress into interstitial and perivascular fibrosis after NO deficiency, even when plasma aldosterone is not elevated.

Endothelial Cell Mineralocorticoid Receptors Regulate Deoxycorticosterone/Salt-Mediated Cardiac Remodeling and Vascular Reactivity But Not Blood Pressure

Recent studies have identified novel pathological roles for mineralocorticoid receptors (MR) in specific cell types in cardiovascular disease. The mechanisms by which MR promotes inflammation and fibrosis involve multiple cell-specific events. To identify the role of MR in endothelial cells (EC-MR), the current study explored the vascular responses to aldosterone in wild-type (WT) and EC-null mice (EC-MRKO). Nitric oxide function was impaired in the thoracic aorta and mesenteric arteries of aldosterone-treated WT mice. Although endothelial nitric oxide function was equivalently impaired in the mesenteric arteries of aldosterone-treated EC-MRKO mice, endothelial function was unaffected in the aorta, suggesting a differential role for EC-MR depending on the vascular bed. Second, the contribution of EC-MR to cardiovascular inflammation, fibrosis, and hypertension was determined in WT and EC-MRKO treated with deoxycorticosterone/salt for 8 days or 8 weeks. At 8 days, loss of EC-MR prevented macrophage infiltration and the expression of proinflammatory genes in the myocardium. Increased cardiac fibrosis was not detected in either genotype at this time, mRNA levels of profibrotic genes were significantly lower in EC-MRKO mice versus WT. At 8 weeks, deoxycorticosterone/salt treatment increased macrophage recruitment and proinflammatory gene expression in WT but not in EC-MRKO. Collagen deposition and connective tissue growth factor expression were significantly reduced in EC-MRKO versus WT. Interestingly, systolic blood pressure was equivalently elevated in deoxycorticosterone/salt treated WT and EC-MRKO. Our data demonstrate that (1) EC-MR signaling contributes to vascular nitric oxide function in large conduit arteries but not in resistance vessels and (2) an independent role for EC-MR in the inflammatory and profibrotic response to deoxycorticosterone/salt.

Cardiomyocyte mineralocorticoid receptors are essential for deoxycorticosterone/salt-mediated inflammation and cardiac fibrosis.

Because the role of mineralocorticoid receptors in specific cell types in cardiac remodeling remains unknown, we have compared cardiac responses with deoxycorticosterone/salt in cardiomyocyte mineralocorticoid receptor-null (MyoMRKO) and wild-type (WT) mice at 8 days and 8 weeks. No differences in cardiac function between untreated WT and MyoMRKO mice were found, whereas profibrotic markers were reduced in MyoMRKO hearts at baseline. At 8 days, MyoMRKO showed monocyte/macrophage recruitment equivalent to WT mice in response to deoxycorticosterone/salt but a suppression of markers of fibrosis compared with WT. At 8 weeks, MyoMRKO mice showed no deoxycorticosterone/salt-induced increase in inflammatory cell infiltration and collagen deposition or in proinflammatory gene expression. Although some profibrotic markers were equivalently increased in both genotypes, MyoMRKO mice also showed increased baseline levels of mRNA and protein for the transforming growth factor-&bgr;/connective tissue growth factor inhibitor decorin compared with WT that was accompanied by higher levels of matrix metalloproteinase 2/matrix metalloproteinase 9 activity. These data point to a direct role for cardiomyocyte mineralocorticoid receptor in both deoxycorticosterone/salt-induced tissue inflammation and remodeling and suggest potential mechanisms for the cardioprotective effects of selective mineralocorticoid receptor blockade in cardiomyocytes that may involve regulation of matrix metalloproteinase 2/matrix metalloproteinase 9 activity and the transforming growth factor-&bgr;-connective tissue growth factor profibrotic pathway.

Mineralocorticoid receptor activation and cardiac fibrosis.

MR (mineralocorticoid receptor) activation by either administration of exogenous mineralocorticoids or by allowing endogenous glucocorticoids to activate the MR has been shown to produce oxidative stress and vascular inflammation at the earliest stages of the development of cardiac fibrosis in experimental animals. These studies suggest potential mechanisms for the benefits observed in recent large scale clinical trials investigating the cardioprotective effects of MR antagonists given in conjunction with current best practice therapy for moderate-to-severe heart failure and heart failure post-myocardial infarction. Given that few patients had elevated plasma aldosterone, novel mechanisms involved in activating the MR in the failing heart are now being investigated.

Mineralocorticoid receptors in the heart: lessons from cell-selective transgenic animals

The clinical impact of cardiovascular disease cannot be underestimated. Equally, the importance of cost-effective management of cardiac failure is a pressing issue in the face of an ageing population and the increasing incidence of metabolic disorders worldwide. Targeting the mineralocorticoid receptor (MR) offers one approach for the treatment of heart failure with current strategies for novel MR therapeutics focusing on harnessing their cardio-protective benefits, but limiting the side effects of existing agents. It is now well accepted that activation of the MR in the cardiovascular system promotes tissue inflammation and fibrosis and has negative consequences for cardiac function and patient outcomes following cardiac events. Indeed, blockade of the MR using one of the two available antagonists (spironolactone and eplerenone) provides significant cardio-protective effects in the clinical and experimental setting. Although the pathways downstream of MR that translate receptor activation into tissue inflammation, fibrosis and dysfunction are still being elucidated, a series of recent studies using cell-selective MR (NR3C2)-null or MR-overexpressing mice have offered many new insights into the role of MR in cardiovascular disease and the control of blood pressure. Dissecting the cell-specific roles of MR signalling in the heart and vasculature to identify those pathways that are critical for MR-dependent responses is an important step towards achieving cardiac-selective therapeutics. The goal of this review is to discuss recent advances in this area that have emerged from the study of tissue-selective MR-null mice, and other targeted transgenic models and their relevance to clinical disease.

Cardiac Tissue Injury and Remodeling Is Dependent Upon MR Regulation of Activation Pathways in Cardiac Tissue Macrophages

Macrophage mineralocorticoid receptor (MR) signaling is an important mediator of cardiac tissue inflammation and fibrosis. The goal of the present study was to determine the cellular mechanisms of MR signaling in macrophages that promote cardiac tissue injury and remodeling. We sought to identify specific markers of MR signaling in isolated tissue macrophages (cardiac, aortic) vs splenic mononuclear cells from wild-type and myeloid MR-null mice given vehicle/salt or deoxycorticosterone (DOC)/salt for 8 weeks. Cardiac tissue fibrosis in response to 8 weeks of DOC/salt treatment was found in the hearts from wild-type but not myeloid MR-null mice. This was associated with an increased expression of the profibrotic markers TGF-β1 and matrix metalloproteinase-12 and type 1 inflammatory markers TNFα and chemokine (C-X-C motif) ligand-9 in cardiac macrophages. Differential expression of immunomodulatory M2-like markers (eg, arginase-1, macrophage scavenger receptor 1) was dependent on the tissue location of wild-type and MR-null macrophages. Finally, intact MR signaling is required for the phosphorylation of c-Jun NH2-terminal kinase in response to a proinflammatory stimulus in bone marrow monocytes/macrophages in culture. These data suggest that the activation of the c-Jun NH2-terminal kinase pathway in macrophages after a tissue injury and inflammatory stimuli in the DOC/salt model is MR dependent and regulates the transcription of downstream profibrotic factors, which may represent potential therapeutic targets in heart failure patients.

Cardiomyocyte Mineralocorticoid Receptor Activation Impairs Acute Cardiac Functional Recovery After Ischemic Insult

Loss of mineralocorticoid receptor signaling selectively in cardiomyocytes can ameliorate cardiac fibrotic and inflammatory responses caused by excess mineralocorticoids. The aim of this study was to characterize the role of cardiomyocyte mineralocorticoid receptor signaling in ischemia–reperfusion injury and recovery and to identify a role of mineralocorticoid receptor modulation of cardiac function. Wild-type and cardiomyocyte mineralocorticoid receptor knockout mice (8 weeks) were uninephrectomized and maintained on (1) high salt (0.9% NaCl, 0.4% KCl) or (2) high salt plus deoxycorticosterone pellet (0.3 mg/d, 0.9% NaCl, 0.4% KCl). After 8 weeks of treatment, hearts were isolated and subjected to 20 minutes of global ischemia plus 45 minutes of reperfusion. Mineralocorticoid excess increased peak contracture during ischemia regardless of genotype. Recovery of left ventricular developed pressure and rates of contraction and relaxation post ischemia–reperfusion were greater in knockout versus wild-type hearts. The incidence of arrhythmic activity during early reperfusion was significantly higher in wild-type than in knockout hearts. Levels of autophosphorylated Ca2+/calmodulin protein kinase II (Thr287) were elevated in hearts from wild-type versus knockout mice and associated with increased sodium hydrogen exchanger-1 expression. These findings demonstrate that cardiomyocyte-specific mineralocorticoid receptor–dependent signaling contributes to electromechanical vulnerability in acute ischemia–reperfusion via a mechanism involving Ca2+/calmodulin protein kinase II activation in association with upstream alteration in expression regulation of the sodium hydrogen exchanger-1.

Mineralocorticoid and Epidermal Growth Factor Receptors: Partners In Vivo

See related article, pp 238–244nnIn the decade since the publication of the Randomized Aldactone Evaluation Study (RALES),1 interest in the role of aldosterone and the mineralocorticoid receptor (MR) has increased exponentially. The comfortable notion that mineralocorticoid hypertension was just a matter of salt and water has been supplanted by a recognition that multiple tissues and systems are impacted.2,3nnIt is clear that the adverse cardiovascular consequences of mineralocorticoid-induced hypertension are far in excess of what might be expected for the magnitude and duration of the blood pressure rise.4 These observations have focused research on the consequences of MR activation in nonepithelial tissues.5 This represents somewhat of a renaissance as the consequences for the cardiovascular system of exposure to mineralocorticoids was first noted by Hans Seyle in 1946 and rediscovered by Brilla and Weber with further characterization by Young et al.6 These investigators established a robust rodent model of mineralocorticoid-induced hypertension, cardiac hypertrophy, and cardiac fibrosis resulting from mineralocorticoid/salt administration over a period of weeks. The cardiac fibrosis is preceded by vascular inflammation and is independent of the hypertension, cardiac hypertrophy, angiotensin II, and potassium status. It is blocked by coadministration of MR antagonists (spironolactone or eplerenone) and, more importantly, once established, as in RALES, …