Ryan A. Frieler

Myeloid mineralocorticoid receptor controls macrophage polarization and cardiovascular hypertrophy and remodeling in mice.

Inappropriate excess of the steroid hormone aldosterone, which is a mineralocorticoid receptor (MR) agonist, is associated with increased inflammation and risk of cardiovascular disease. MR antagonists are cardioprotective and antiinflammatory in vivo, and evidence suggests that they mediate these effects in part by aldosterone-independent mechanisms. Here we have shown that MR on myeloid cells is necessary for efficient classical macrophage activation by proinflammatory cytokines. Macrophages from mice lacking MR in myeloid cells (referred to herein as MyMRKO mice) exhibited a transcription profile of alternative activation. In vitro, MR deficiency synergized with inducers of alternatively activated macrophages (for example, IL-4 and agonists of PPARgamma and the glucocorticoid receptor) to enhance alternative activation. In vivo, MR deficiency in macrophages mimicked the effects of MR antagonists and protected against cardiac hypertrophy, fibrosis, and vascular damage caused by L-NAME/Ang II. Increased blood pressure and heart rates and decreased circadian variation were observed during treatment of MyMRKO mice with L-NAME/Ang II. We conclude that myeloid MR is an important control point in macrophage polarization and that the function of MR on myeloid cells likely represents a conserved ancestral MR function that is integrated in a transcriptional network with PPARgamma and glucocorticoid receptor. Furthermore, myeloid MR is critical for blood pressure control and for hypertrophic and fibrotic responses in the mouse heart and aorta.

Coxibs interfere with the action of aspirin by binding tightly to one monomer of cyclooxygenase-1

Pain associated with inflammation involves prostaglandins synthesized from arachidonic acid (AA) through cyclooxygenase-2 (COX-2) pathways while thromboxane A2 formed by platelets from AA via cyclooxygenase-1 (COX-1) mediates thrombosis. COX-1 and COX-2 are both targets of nonselective nonsteroidal antiinflammatory drugs (nsNSAIDs) including aspirin whereas COX-2 activity is preferentially blocked by COX-2 inhibitors called coxibs. COXs are homodimers composed of identical subunits, but we have shown that only one subunit is active at a time during catalysis; moreover, many nsNSAIDS bind to a single subunit of a COX dimer to inhibit the COX activity of the entire dimer. Here, we report the surprising observation that celecoxib and other coxibs bind tightly to a subunit of COX-1. Although celecoxib binding to one monomer of COX-1 does not affect the normal catalytic processing of AA by the second, partner subunit, celecoxib does interfere with the inhibition of COX-1 by aspirin in vitro. X-ray crystallographic results obtained with a celecoxib/COX-1 complex show how celecoxib can bind to one of the two available COX sites of the COX-1 dimer. Finally, we find that administration of celecoxib to dogs interferes with the ability of a low dose of aspirin to inhibit AA-induced ex vivo platelet aggregation. COX-2 inhibitors such as celecoxib are widely used for pain relief. Because coxibs exhibit cardiovascular side effects, they are often prescribed in combination with low-dose aspirin to prevent thrombosis. Our studies predict that the cardioprotective effect of low-dose aspirin on COX-1 may be blunted when taken with coxibs.

Immune Cell and Other Noncardiomyocyte Regulation of Cardiac Hypertrophy and Remodeling

Cardiac hypertrophy and remodeling are pathological features of many cardiac diseases, with underlying causes including hypertension, cardiomyopathy, valvular dysfunction, and myocardial infarction. In these diseases, ventricular hypertrophy occurs in response to pathological stimuli such as pressure and volume overload, sarcomere gene mutations, and neurohumoral activation, and a major consequence of prolonged and uncontrolled hypertrophic remodeling is cardiac dysfunction, which can lead to heart failure or cardiac arrest resulting from arrhythmia. Despite the various pathological stimuli, there are many common features in the hypertrophic response in different cardiac diseases. In addition to increased cardiomyocyte mass, sarcomere rearrangement, and extracellular matrix deposition, other common features have recently been appreciated, including inflammatory signaling and immune cell activation. Numerous cell types are involved in orchestrating this complex pathological response. The heart consists of a heterogeneous population of cells, including cardiomyocytes and noncardiomyocytes, and it is now clear that intercellular signaling and communication between these cell types are critical in the pathophysiology of ventricular hypertrophy and remodeling (Figure 1). Figure 1. Overview of cardiomyocyte and noncardiomyocyte interactions during cardiac hypertrophy and remodeling. Cardiomyocytes respond to pathogenic stimuli by secreting inflammatory cytokines, chemokines, and damage-associated molecular pattern molecules (DAMPs), which are recognized by local noncardiomyocyte cells. This induces activation and expansion of resident macrophages and fibroblasts and recruits bone marrow–derived immune cells from the circulation. Activated immune cells and fibroblasts secrete both prohypertrophic and profibrotic cytokines, which induce cardiomyocyte hypertrophy and promote fibroblast differentiation, matrix deposition, and interstitial fibrosis. ECM indicates extracellular matrix. Noncardiomyocytes display phenotypic changes during the development of cardiac hypertrophy. There is still much to be revealed about the specific roles of these cell types and their overall contribution to the hypertrophic response. Inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and transforming growth factor-β (TGF-β) and neurohumoral factors such as …

Myeloid-Specific Deletion of the Mineralocorticoid Receptor Reduces Infarct Volume and Alters Inflammation During Cerebral Ischemia

Background and Purpose— Mineralocorticoid receptor (MR) antagonists have protective effects in rodent models of ischemic stroke, but the cell type-specific actions of these drugs are unknown. In the present study, we examined the contribution of myeloid cell MR during focal cerebral ischemia using myeloid-specific MR knockout mice. Methods— Myeloid-specific MR knockout mice were subjected to transient (90 minutes) middle cerebral artery occlusion followed by 24 hours reperfusion (n=5 to 7 per group). Ischemic cerebral infarcts were identified by hematoxylin and eosin staining and quantified with image analysis software. Immunohistochemical localization of microglia and macrophages was performed using Iba1 staining, and the expression of inflammatory markers was measured after 24 hours of reperfusion by quantitative reverse transcription-polymerase chain reaction. Results— Myeloid-specific MR knockout resulted in a 65% reduction in infarct volume (P=0.005) after middle cerebral artery occlusion. This was accompanied by a significant reduction in activated microglia and macrophages in the ischemic core. Furthermore, myeloid-specific MR knockout suppressed classically activated M1 macrophage markers tumor necrosis factor-&agr;, interleukin-1&bgr;, monocyte chemoattractant protein-1, macrophage inflammatory protein-1&agr;, and interleukin-6 at the same time as partially preserving the induction of alternatively activated, M2, markers Arg1, and Ym1. Conclusions— These data demonstrate that myeloid MR activation exacerbates stroke and identify myeloid MR as a critical target for MR antagonists. Furthermore, these data indicate that MR activation has an important role in controlling immune cell function during the inflammatory response to stroke.

Myeloid mineralocorticoid receptor deficiency inhibits aortic constriction-induced cardiac hypertrophy in mice

Mineralocorticoid receptor (MR) blockade has been shown to suppress cardiac hypertrophy and remodeling in animal models of pressure overload (POL). This study aims to determine whether MR deficiency in myeloid cells modulates aortic constriction-induced cardiovascular injuries. Myeloid MR knockout (MMRKO) mice and littermate control mice were subjected to abdominal aortic constriction (AAC) or sham operation. We found that AAC-induced cardiac hypertrophy and fibrosis were significantly attenuated in MMRKO mice. Expression of genes important in generating reactive oxygen species was decreased in MMRKO mice, while that of manganese superoxide dismutase increased. Furthermore, expression of genes important in cardiac metabolism was increased in MMRKO hearts. Macrophage infiltration in the heart was inhibited and expression of inflammatory genes was decreased in MMRKO mice. In addition, aortic fibrosis and inflammation were attenuated in MMRKO mice. Taken together, our data indicated that MR deficiency in myeloid cells effectively attenuated aortic constriction-induced cardiac hypertrophy and fibrosis, as well as aortic fibrosis and inflammation.

Myeloid mineralocorticoid receptor during experimental ischemic stroke: effects of model and sex.

Background Mineralocorticoid receptor (MR) antagonists have protective effects in the brain during experimental ischemic stroke, and we have previously demonstrated a key role for myeloid MR during stroke pathogenesis. In this study, we explore both model- and sex-specific actions of myeloid MR during ischemic stroke. Methods and Results The MR antagonist eplerenone significantly reduced the infarct size in male (control, 99.5 mm3; eplerenone, 74.2 mm3; n=8 to 12 per group) but not female (control, 84.0 mm3; eplerenone, 83.7 mm3; n=6 to 7 per group) mice after transient (90-minute) middle cerebral artery occlusion. In contrast to MR antagonism, genetic ablation of myeloid MR in female mice significantly reduced infarct size (myeloid MR knockout, 9.4 mm3 [5.4 to 36.6]; control, 66.0 mm3 [50.0 to 81.4]; n=6 per group) after transient middle cerebral artery occlusion. This was accompanied by reductions in inflammatory gene expression and improvement in neurological function. In contrast to ischemia-reperfusion, myeloid MR–knockout mice were not protected from permanent middle cerebral artery occlusion. The infarct size and inflammatory response after permanent occlusion showed no evidence of protection by myeloid MR knockout in photothrombotic and intraluminal filament models of permanent occlusion. Conclusions These studies demonstrate that MR antagonism is protective in male but not female mice during transient middle cerebral artery occlusion, whereas genetic ablation of myeloid MR is protective in both male and female mice. They also highlight important mechanistic differences in the role of myeloid cells in different models of stroke and confirm that specific myeloid phenotypes play key roles in stroke protection.

Smooth muscle protein 22 alpha-Cre is expressed in myeloid cells in mice.

BACKGROUND Experiments using Cre recombinase to study smooth muscle specific functions rely on strict specificity of Cre transgene expression. Therefore, accurate determination of Cre activity is critical to the interpretation of experiments using smooth muscle specific Cre. METHODS AND RESULTS Two lines of smooth muscle protein 22 α-Cre (SM22α-Cre) mice were bred to floxed mice in order to define Cre transgene expression. Southern blotting demonstrated that SM22α-Cre was expressed not only in tissues abundant of smooth muscle, but also in spleen, which consists largely of immune cells including myeloid and lymphoid cells. PCR detected SM22α-Cre expression in peripheral blood and peritoneal macrophages. Analysis of SM22α-Cre mice crossed with a recombination detector GFP mouse revealed GFP expression, and hence recombination, in circulating neutrophils and monocytes by flow cytometry. CONCLUSIONS SM22α-Cre mediates recombination not only in smooth muscle cells, but also in myeloid cells including neutrophils, monocytes, and macrophages. Given the known contributions of myeloid cells to cardiovascular phenotypes, caution should be taken when interpreting data using SM22α-Cre mice to investigate smooth muscle specific functions. Strategies such as bone marrow transplantation may be necessary when SM22α-Cre is used to differentiate the contribution of smooth muscle cells versus myeloid cells to observed phenotypes.

Depletion of macrophages in CD11b diphtheria toxin receptor mice induces brain inflammation and enhances inflammatory signaling during traumatic brain injury.

Immune cells have important roles during disease and are known to contribute to secondary, inflammation-induced injury after traumatic brain injury. To delineate the functional role of macrophages during traumatic brain injury, we depleted macrophages using transgenic CD11b-DTR mice and subjected them to controlled cortical impact. We found that macrophage depletion had no effect on lesion size assessed by T2-weighted MRI scans 28 days after injury. Macrophage depletion resulted in a robust increase in proinflammatory gene expression in both the ipsilateral and contralateral hemispheres after controlled cortical impact. Interestingly, this sizeable increase in inflammation did not affect lesion development. We also showed that macrophage depletion resulted in increased proinflammatory gene expression in the brain and kidney in the absence of injury. These data demonstrate that depletion of macrophages in CD11b-DTR mice can significantly modulate the inflammatory response during brain injury without affecting lesion formation. These data also reveal a potentially confounding inflammatory effect in CD11b-DTR mice that must be considered when interpreting the effects of macrophage depletion in disease models.

Nuclear receptor control of opposing macrophage phenotypes in cardiovascular disease.

Macrophages have important physiological roles and display a high degree of heterogeneous phenotypes in response to a variety of stimuli. In particular, the spectrum of alternatively activated macrophages has been a focus because many lines of evidence indicate a cardioprotective role for this macrophage phenotype. This phenotype is controlled in part by opposing nuclear transcription factors including the PPARs that stimulate alternative activation and the recently recognized role of the mineralocorticoid receptor in stimulating classically activated macrophages. This review highlights some of the recent findings involving alternatively activated macrophages and these nuclear receptors in cardiovascular disease.

Genetic neutrophil deficiency ameliorates cerebral ischemia-reperfusion injury

&NA; Neutrophils respond rapidly to cerebral ischemia and are thought to contribute to inflammation‐mediated injury during stroke. Using myeloid Mcl1 knockout mice as a model of genetic neutrophil deficiency, we investigated the contribution of neutrophils to stroke pathophysiology. Myeloid Mcl1 knockout mice were subjected to transient middle cerebral artery occlusion and infarct size was assessed by MRI after 24 h reperfusion. Immune cell mobilization and infiltration was assessed by flow cytometry. We found that myeloid Mcl1 knockout mice had significantly reduced infarct size when compared to heterozygous and wild type control mice (MyMcl1+/+: 78.0 mm3; MyMcl1+/−: 83.4 mm3; MyMcl1−/−: 55.1 mm3). This was accompanied by a nearly complete absence of neutrophils in the ischemic hemisphere of myeloid Mcl1 knockout mice. Although myeloid Mcl1 knockout mice were protected from cerebral infarction, no significant differences in neurological deficit or the mRNA expression of inflammatory genes (TNF&agr;, IL‐1&bgr;, and MCP1) were detected. Inhibition of neutrophil chemotaxis using CXCR2 pepducin treatment partially reduced neutrophil mobilization and recruitment to the brain after stroke, but did not reduce infarct size 24 h after transient MCA occlusion. These data confirm that neutrophils have an important role in infarct development during stroke pathophysiology, and suggest that complete deficiency, but not partial inhibition, is necessary to prevent neutrophil‐mediated injury during stroke. HighlightsMyeloid Mcl1 knockout mice are neutrophil deficient and lack neutrophil mobilization and recruitment during strokeGenetic neutrophil deficiency in myeloid Mcl1 knockout mice decreased infarct size during strokeGenetic neutrophil deficiency did not suppress inflammatory gene mRNA expression during strokeInhibition of neutrophil chemotaxis with CXCR2 pepducin was not sufficient to reduce infarct size during stroke