Interleukin-6 plays a critical role in aldosterone-induced macrophage recruitment and infiltration in the myocardium

Journal Pre-proof Interleukin-6 plays a critical role in aldosterone-induced macrophage recruitment and infiltration in the myocardium

Che-Wei Liao, Chia-Hung Chou, Xue-Ming Wu, Zheng-Wei Chen, Ying-Hsien Chen, Yi-Yao Chang, Vin-Cent Wu, Stefan Rose-John, Chi-Sheng Hung, Yen-Hung Lin, the TAIPAI Study Group PII:

S0925-4439(19)30355-2

DOI:

https://doi.org/10.1016/j.bbadis.2019.165627

Reference:

BBADIS 165627

To appear in:

BBA - Molecular Basis of Disease

Received date:

5 March 2019

Revised date:

22 November 2019

Accepted date:

26 November 2019

Please cite this article as: C.-W. Liao, C.-H. Chou, X.-M. Wu, et al., Interleukin-6 plays a critical role in aldosterone-induced macrophage recruitment and infiltration in the myocardium, BBA - Molecular Basis of Disease(2019), https://doi.org/10.1016/ j.bbadis.2019.165627

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© 2019 Published by Elsevier.

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Interleukin-6 plays a critical role in aldosterone-induced macrophage recruitment and infiltration in the myocardium Short title: IL-6 and macrophage infiltration Che-Wei Liaoa*, Chia-Hung Choub*, Xue-Ming Wuc, Zheng-Wei Chend, Ying-Hsien Chene, Yi-Yao Changf, Vin-Cent Wue, Stefan Rose-Johng, Chi-Sheng Hunge,h†,

Department of Internal Medicine, National Taiwan University Hospital Hsin-Chu

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Yen-Hung Line†, the TAIPAI Study Group

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Branch, Hsin-Chu, Taiwan; bDepartment of Obstetrics and Gynecology and Internal

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Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan; eDepartment of Internal Medicine, Taoyuan

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General Hospital, Taoyuan, Taiwan; dDepartment of Internal Medicine, National

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Taiwan University Hospital Yun-Lin Branch, Yun-Lin, Taiwan; eDepartment of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan; fDivision of Cardiovascular Medical Center, Far Eastern Memorial Hospital, New Taipei City, Taiwan; g Institute of Biochemistry, Kiel University, Olshausenstrasse 40, Kiel, Germany; hTelehealth Center, National Taiwan University Hospital, Taipei, Taiwan; *† These two authors contributed equally. Corresponding author

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Chi-Sheng Hung, MD, PhD, Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, 100, Taiwan Tel: + 886-2-23123456 ext. 62152, Fax: + 886-2-23515811

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E-mail: [email protected]

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Yen-Hung Lin, MD, PhD,

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Department of Internal Medicine, National Taiwan University Hospital,

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No. 7, Chung-Shan South Road, Taipei, 100, Taiwan Tel: + 886-2-23123456 ext. 62152 Fax: + 886-2-23515811

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Word count: 3954

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E-mail: [email protected]

Table: 0 Figure: 7

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ABSTRACT Macrophages play an important role in aldosterone-induced myocardial fibrosis, in which the first key steps are macrophage recruitment and infiltration. We hypothesized that IL-6 may be a key mediator of aldosterone-induced macrophage

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recruitment and infiltration. To test this hypothesis, we designed cell studies with a

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human monocytic cell line THP-1 that with monocyte/macrophage functions to

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explore the signaling pathway of aldosterone-induced macrophage infiltration, and

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further investigated the phenomenon and consequent pathway in aldosterone-infused

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mice studies. The results showed that aldosterone induced the expression of IL-6 via

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mineralocorticoid receptors, and enhanced THP-1 cell migration and infiltration.

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Further experiments using a protease array and siRNA revealed that expressions of MMP-1 and MMP-9 were associated with aldosterone-induced macrophage infiltration. In addition, aldosterone-induced MMP-1 and MMP-9 expressions were mediated via cyclooxygenase-II and prostaglandin E2/EP-2 and EP-4 receptors. In aldosterone-infused mice, mRNA expressions of MMP-1, MMP-9 and COX-2 in peripheral blood monocytic cells were significantly increased. Moreover, the number of mouse macrophage-restricted F4/80 protein-positive cells in the myocardium was significantly higher in the aldosterone-infused mice compared with control mice. The increase in F4/80-positive cells in the myocardium was suppressed in the 3

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aldosterone-infused mice with the aldosterone antagonist eplerenone or anti-IL-6 antibody treatment. In conclusion, interleukin-6 played an important role in aldosterone-induced macrophage recruitment and infiltration in the myocardium.

Key words:

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aldosterone, interleukin 6, macrophage, matrix metalloproteinase

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Introduction Primary aldosteronism (PA) is characterized by excess aldosterone production and is one of the most common causes of secondary hypertension [1]. Patients with PA have been shown to have a higher risk of adverse cardiovascular outcomes

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compared to patients with blood pressure-matched essential hypertension (EH) [2],

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and also to have higher rates of left ventricular hypertrophy and fibrosis than patients

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with EH [3, 4]. In patients with adenomatous PA, left ventricular hypertrophy and

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fibrosis can be improved by adrenalectomy [3-5].

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Recent studies also support that aldosterone induces cardiac fibrosis

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independently of its effect on blood pressure. In an animal study, aldosterone infusion

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was shown to induce ventricular hypertrophy and interstitial fibrosis in both right and left ventricles [6]. Further studies have suggested that aldosterone-induced reactive oxygen species generation and low-grade inflammation cause aldosterone-induced cardiac fibrosis [7, 8]. Aldosterone has also been shown to promote monocyte and macrophage infiltration into the myocardium before the development of myocardial or vascular fibrosis [8, 9], during which increased expressions of inflammatory markers such as cyclo-oxygenase-2, osteopontin and monocyte chemoattractant protein-1 have been reported. Similar changes in pathophysiology have also been demonstrated in aldosterone-induced renal fibrosis [10]. The exact mechanism of inflammatory cell 5

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infiltration into the myocardium before the development of fibrosis is important to allow for modification or inhibition of the process. Aldosterone infusion has been shown to lead to increased production of the inflammatory mediator interleukin-6 (IL-6) in the kidneys and heart, and IL-6 has been proposed to be an important mediator in aldosterone-induced renal and cardiac

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fibrosis [10, 11]. We previously showed that IL-6 can induce collagen synthesis in

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cardiac fibroblasts [12], however, whether IL-6 plays a role in the infiltration of

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macrophages into the myocardium has not previously been investigated. We

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hypothesized that aldosterone-induced IL-6 secretion could promote macrophage infiltration into the myocardium. To test this hypothesis, we conducted this

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pre-clinical study and investigated the underlying signaling pathways.

Materials and Methods

Cell cultures of human THP-1 cells THP-1 cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 μM β-mercaptoethanol (Invitrogen, USA) and 10% fetal calf serum for cell growth. 6

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Reagents and chemical inhibitors Aldosterone, eplerenone, SB203580, PD98059, LY294002, Ro318220 and SP600125 were purchased from Sigma (Sigma, St. Louis, MO, USA). Eplerenone was dissolved in ethanol, and aldosterone and the other chemical inhibitors were dissolved in DMSO. Recombinant human PGE2, mouse IL-6 neutralizing antibody,

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and control IgG were purchased from R&D Systems (R&D Systems Inc., Minneapolis,

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USA).

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Western blotting

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Total protein of the THP-1 cells was extracted using a Total Protein Extraction Kit (Merck Millipore), and the protein concentration was measured using a Bio-Rad

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protein assay. Protein samples (50 µg) were separated using 10% sodium dodecyl

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sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVD) membranes. The membranes were blocked with 5% fat-free milk for 30 minutes, and then immunoblotted with anti-COX-2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour at room temperature. The bound antibodies on the membranes were detected using peroxidase-coupled secondary antibodies for 30 minutes. Signals were detected by adding commercial chemiluminescent detection reagents (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were stripped in stripping buffer (37.5 mM Tris, pH 6.8, 2% SDS, 1%

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β-mercaptoethanol) at 56°C for 20 minutes, washed three times with phosphate-buffered saline (PBS) containing Tween (10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20), and then immunoblotted with anti-GAPDH antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). A digital imaging system (Bio Pioneer Tech Co.) was used to detect the signals, which were further analyzed using

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Image J® software.

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Quantitative real-time reverse transcription polymerase chain reaction

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(qRT-PCR)

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Total RNA was isolated from THP-1 cells and macrophages using RNAzol B

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reagent according to the instructions of the manufacturer, and then cDNA was prepared from 2 µg of total RNA with random hexamer primers according to the cDNA synthesis ImProm-II protocol (Promega). Specific gene mRNA expressions under various conditions were measured using a fluorescein quantitative real-time PCR detection system (Light Cycler DNA Master SYBR Green I; Roche Molecular Biochemicals, Indianapolis, IN). The primer pairs were as follows: human -actin, 5′ -AAATCTGGCACCACACCTTC-3′ and 5′ -GGGGTGTTGAAGGTCTCAAA-3′; human IL-6, 5′CTTCGGTCCAGTTGCCTTCT-3′ and 5′-AGGAACTCCTTAAAGCTGCG-3′; 8

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human MMP-1, 5′-AGCTAGCTCAGGATGACATTGATG-3′ and 5′ -GCCGATGGGCTGGACAG-3′; human MMP-9, 5′ -GCACGACGTCTTCCAGTACC-3′ and 5′ -CAGGATGTCATAGGTCACGTAGC-3′; mouse β-actin 5′ -AAATCTGGCACCACACCTTC-3′ and 5′

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-CCTCTGGTCTTCTGGAGTACC-3′ and 5′

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-GGGGTGTTGAAGGTCTCAAA-3′; mouse IL-6, 5′

-CGACCTCAGTGTCTTCCTCA-3′; mouse COX-2, 5′

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-AAGAGCATCGCAGAGGT-3 and 5’-CCCATTAGCAGCCAGTT-3’; mouse MMP-9, 5′-AACCACAGCCGACAGCACCT-3 and 5’-

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ATCCAGTACCAACCGTCCTTGAAG-3’; mouse MMP-1, 5′-

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GCTAACCTTTGATGCTATAACTACGA-3′ and 5′TTTGTGCGCATGTAGAATCTG-3′.

The amplification program consisted of one cycle with initial incubation at 60°C for 20 minutes to fully activate the DNA polymerase, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 55°C for 10 seconds, and extension at 72°C for 10 seconds. The relative mRNA level was calculated using the 2−ΔΔCt method. The amount of mRNA was normalized to that of β-actin mRNA and

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presented in arbitrary units, with 1 U corresponding to the value in cells treated with a vehicle control.

RNA interference Small interfering RNA (siRNA) duplexes were purchased from Santa Cruz

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Biotechnology (Santa Cruz, CA, USA) for the MR gene (sc-38836), GR gene

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(sc-35505), control siRNA (sc-37007), IL-6 gene (sc-130326), EP-1 (sc-40169), EP-2

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(sc-40171), EP-3 (sc-35314), EP-4 (sc-40173), MMP-1 (sc-41552), MMP-3

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(sc-29399) and MMP-9 (sc-29400). THP-1 cells were transfected with siRNAs at a

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concentration of 25 nM in serum-free Opti-MEM using the Oligofectamine method

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(Invitrogen, Carlsbad, CA, USA). The inhibition efficiency of each siRNA on

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knocked down its indicated protein was confirmed by Western blot or ELISA (supplementary Figure 1 and 2).

Synthesis and treatment of decoy oligodeoxynucleotides (ODNs) Synthetic double-stranded ODNs were used as “decoy” elements to block the binding of nuclear factors to promoter regions of the targeted genes. The following phosphorothioate ODN sequences were used: NF-B decoy ODN: 5′ -CCTTGAAGGGATTTCCCTCC-3′ and 3′ -GGAACTTCCCTAAAGGGAGG-5′; AP-1 decoy ODN: 5′ -TGTCTGACTCATGTC-3′ and 3′-CAGACTGAGTACA-5′; scrambled decoy 10

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ODN: 5′-TTGCCGTACCTGACTTAGCC-3′ and 3′ -AACGGCATGGACTGAATCGG-5′. The ODNs were mixed with an equal volume (10:1) of TransFast™ (Promega) for 15 minutes, and then incubated with the cells in serum-free medium. NF-κB binding site-driven luciferase reporter assay

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Transfection of nuclear factor (NF)-κB binding site-driven luciferase plasmids (BD

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Bioscience, Palo Alto, CA) into THP-1 cells was performed in six-well plates using

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the TransFast™ Transfection Reagent (Promega) method. At 24 hours after

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transfection, the THP-1 cells were processed for various experimental conditions, and also transfected with pSV-β-galactosidase to confirm the transfection efficiency and

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for data normalization after all transient transfections.

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IL-6, PGE2, MMP-1, MMP-3 and MMP-9 detection Levels of IL-6, PGE2, MMP-1, MMP-3 and MMP-9 in the THP-1 culture supernatant were determined by ELISA . Levels of IL-6 in mouse serum was determined using mouse IL-6 Quantikine ELISA kits which were purchased from R&D Systems (R&D Systems Inc., Minneapolis, USA).

Transwell migration and infiltration assays

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For migration assays, THP-1 cells were seeded into the inserts of transwell (8-μm pore) dishes. These cells were allowed to migrate for 16 hours towards the lower chamber. Non-migrated cells were removed from the upper surface of the membrane, while cells that had migrated to the lower surface were fixed and stained with crystal violet (fixing/staining solution: crystal violet (0.05% w/v) and

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formaldehyde (4%) in 1x PBS). The migrated cells were photographed using a digital

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camera and counted per high power field (HPF). For infiltration assays, THP-1 cells

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were seeded into the inserts of transwell (8-μm pore) dishes that were coated with

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Matrigel (Corning Matrigel matrix; 10 mg/ml) for 30 minutes at 37°C. These cells were allowed to infiltrate for 48 hours towards the lower chamber. Non-infiltrated

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cells were removed from the upper surface of the membrane, while cells that had

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infiltrated to the lower surface were fixed and stained with crystal violet. Infiltrated cells were photographed using a digital camera and counted per HPF. Protease array analysis

A Proteome Profiler Human Protease Array Kit (Catalog # ARY021B, R&D Systems) was used to detect proteases according to the manufacturer's instructions. Briefly, THP-1 cell culture supernatants were first mixed with a detection antibody cocktail at room temperature for 1 hour prior to being added to the array membrane. The membrane was then incubated overnight at 2–8°C on a shaker. After washing,

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horseradish peroxidase-conjugated streptavidin was added to the membrane followed by 30 minutes of incubation at room temperature on a shaker. After washing, signal development was achieved by adding commercial chemiluminescent detection reagents. A digital imaging system (Bio Pioneer Tech Co.) was used to detect the signals, which were further analyzed using Image J® software.

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Animal model

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Eight-week-old C57BL/6 male mice were purchased from the Animal Center of

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the Medical College of National Taiwan University and kept in standard animal

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housing conditions. The protocol of this experiment was approved by the Animal Care and Use Committee of the Medical College of National Taiwan University (No.

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20060181). The animal experiments were performed in a laboratory at the Medical

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College of National Taiwan University. Mice with a similar body weight (25–30 g) were used in this experiment. In the aldosterone infusion group, a 21-day continuous aldosterone release pellet (Innovative Research of America, Sarasota, FL, USA, 0.25 mg/pellet, 21-day release, 0.11 mg/day) was implanted subcutaneously. In the placebo group, a placebo pellet (vehicle only) was implanted. For the 8-week continuous aldosterone infusion model, the pellets were changed every 20 days. The mice were then given eplerenone (100 mg/kg/day) by oral gavage with or without intraperitoneal injections of a recombinant neutralizing polyclonal antibody specific for murine IL-6

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(10 mg/kg; R&D Systems; Minneapolis, MN AF-406) or an isotype control antibody (R&D Systems) three times per week for 6 weeks. The mice were sacrificed at the indicated time points by deep anesthesia with 5% isoflurane gas. In the peripheral blood mononuclear cell (PBMC) collection experiments, the mice were then processed for cardiac puncture, and then euthanized by carbon dioxide inhalation. In

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our previous report, this aldosterone-infused model developed significantly higher

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heart weight to body weight ratio, higher degree of interstitial and perivascular

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fibrosis at 12 weeks compared with placebo group [12].

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PBMC isolation

After the mice had been sacrificed, whole blood was collected by cardiac

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puncture in heparin sodium prefilled syringes, and PBMCs were isolated using a

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density gradient technique. Mouse whole blood was mixed with an equal volume of 1x PBS and the mixed blood solution was overlaid on top of Ficoll-Paque PLUS solution (GE Healthcare Life Sciences). Centrifugation was performed at 400 x g for 30 minutes at room temperature. The PBMCs floating over the Ficoll-plasma interface were collected and washed twice with 1x PBS at 300 x g at 4°C, and were then used for RNA isolation. Macrophage isolation

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After the mice had been sacrificed, heart tissue was isolated and washed with 20 ml RPMI 1640 medium (Invitrogen, CA, USA). The tissue was then cut into pieces and treated with 2.5 ml 0.1% type II collagenase in RPMI at 37°C for 2 hours. The cell suspension was washed in 5 ml Hank's balanced salt solution containing 10% FCS and processed to isolate F4/80-expressing macrophages using a MagniSort™

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Mouse F4/80 Positive Selection Kit (Thermo Fisher Scientific). F4/80-positive cells

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were used for RNA isolation. The M1 or M2 phenotype of the isolated macrophage

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was determined by measuring the mRNA level of M1(iNOS and TNF-a) and

Immunohistochemistry

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M2(arginase and IL-10) biomarkers [13]

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Paraffin sections of mouse heart tissue were deparaffinized, rehydrated, and

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microwaved in 0.01 M citrate buffer (pH 6.0) for antigen retrieval. Serial sections of each tissue sample were blocked with goat serum and incubated with anti-mouse F4/80 antibodies (Santa Cruz Biotechnology, Texas, USA). Immunoreactivity was visualized using an ABC staining system (Vector Laboratories, Burlington, CA, USA), according to the manufacturer’s instructions. The sections were counterstained with Mayer’s hematoxylin, and the F4/80-positive cells were counted in 10 HPF (400× magnification). Statistical analysis

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Data were expressed as mean ± SD or number (percentage). In the cell studies, each experiment was performed in triplicate and all experiments were independently repeated at least three times. In the animal studies, each group contained 6-8 animals. One- or two-way ANOVA was used to analyze the data, with repeated measurements as appropriate. Bonferroni correction was used to control for multiple comparisons.

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Simes method was used to calculate q-values from the original unadjusted p-values in

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the protease array. Statistical analyses were performed using Stata software version

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13.0 (StataCorp. College Station, TX: StataCorp LP). A two-tailed p-value < 0.05 was

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Results

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considered to indicate statistical significance.

Aldosterone induced the expression of IL-6 in the human monocytic cell line THP-1

To test whether aldosterone directly induced the expression of IL-6 in macrophages, we treated human monocytic THP-1 cells with 10-9~10-6 M aldosterone for 24 hours. The supernatant was collected and the concentration of IL-6 was measured. The results revealed that aldosterone at a concentration of 10-8~10-7 M induced THP-1 cells to secrete IL-6 (Figure 1A). To determine the time course of aldosterone-induced IL-6 expression in the THP-1 cells, we measured the mRNA and 16

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protein level of IL-6 in cell and cell supernatant, respectively, at 0, 4, 8, 16, 24, 30 and 36 hours after 10-7 M aldosterone treatment. The results showed an increase in IL-6 mRNA up to 16 hours after aldosterone treatment (Figure 1B), and this increase remained significant until 24 hours. There was also an increase in IL-6 protein level at 4 hours after aldosterone treatment, which was correlated with the timing of IL-6

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mRNA expression (Figure 1C).

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We used siRNAs to test whether aldosterone induced IL-6 protein secretion in

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the THP-1 cells through mineralocorticoid (MR) or glucocorticoid (GR) receptors.

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The results showed that aldosterone-induced IL-6 protein expression could be suppressed by MR siRNA, but not by GR siRNA. We confirmed this result by

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treatment with the aldosterone antagonist eplerenone. These results suggested that

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aldosterone induced the expression of IL-6 protein in the THP-1 cells via MR (Figure

We used chemical inhibitors to analyze downstream signal transduction, and the results showed that aldosterone-induced IL-6 protein expression could be suppressed by the PI3K/AKT inhibitor LY294002 and the p38 MAPK inhibitor SB203580. These results suggested that the aldosterone-induced IL-6 protein expression in the THP-1 cells was mediated by PI3K/AKT and p38 MAPK signaling (Figure 1E).

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We used decoy ODNs to test the involvement of the transcription factors NF-kB and AP-1 in the aldosterone-induced IL-6 protein expression, and the results showed that aldosterone induced the expression of IL-6 protein via activation of the transcription factor NF-kB (Figure 1F). We then performed an experiment on the effects of PI3K and MAPK on NF-kB promotor activity. The results showed that both

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LY294002 and SB203580 inhibited the aldosterone-induced enhancement of NF-kB

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promotor activity (Figure 1G). Taken together, these data suggested that

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aldosterone-induced IL-6 expression is mediated through MR-induced PI3K and p38

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mRNA expression.

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MAPK signaling pathways, both of which act on NF-kB and the final increase in IL-6

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Aldosterone enhanced migration and infiltration in the human monocytic cell

To test whether aldosterone induced macrophage migration and infiltration, we used the THP-1 in transwell and Matrigel-coated transwell experiments. The migration of THP-1 cells through the transwell membrane was significantly increased after 10-8~10-7 M aldosterone treatment (Figure 2A, 2B), and the infiltration of THP-1 cells into Matrigel-coated transwell dishes was also increased significantly after 10-8~10-7 M aldosterone treatment (Figure 2C, 2D). These results suggested that

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aldosterone could enhance both macrophage migration and infiltration into the extracellular matrix (ECM). We then tested whether the aldosterone-induced macrophage infiltration into the ECM was influenced by IL-6. After pretreating the culture with IL-6 siRNA, the expression of IL-6 was markedly suppressed (Figure 2E) and the number of cells that

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infiltrated through the Matrigel-coated transwell membrane was also significantly

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suppressed (Figure 2F). These results showed that IL-6 signaling was involved in

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aldosterone-induced macrophage infiltration into the ECM.

Protease arrays to explore changes in aldosterone-induced protein expression

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To explore changes in protease expression after aldosterone treatment of

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macrophages, we performed protease array analysis of human THP-1 cells. The results showed that MMP-1, 3, and 9 were significantly increased (2.5-, 2.7- and 2.0-fold, respectively) in the THP-1 cells treated with 10-7 M aldosterone compared to those treated with 10-10 M aldosterone, after controlling for false discovery rate (Figure 3A, 3B). We confirmed these protein array findings by directly measuring MMP-1, 3 and 9 protein concentrations in the THP-1 cells after treatment with 10-10 and 10-7 M aldosterone (Figure 3C-E). We used siRNA inhibition to test whether MMP-1, 3 or 9

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was involved in the macrophage infiltration into the ECM. The results showed that MMP-1 and MMP-9 siRNA suppressed the number of cells that infiltrated into the Matrigel-coated transwell dishes (Figure 3F), whereas the suppression of MMP-3 by siRNA did not (Figure 3F). These results suggested that MMP-1 and MMP-9 were downstream mediators in the aldosterone-induced macrophage infiltration into the

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ECM.

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Aldosterone induced MMP-1 and MMP-9 via IL-6/COX-2/PGE2 signaling

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We hypothesized that aldosterone-induced MMP-1 and MMP-9 protein expressions may be mediated through COX-2 and PGE2 signaling. We tested this

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hypothesis by using IL-6 siRNA, NS398 (COX-2 inhibitor) and AG490 (JAK2

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inhibitor). The results showed that aldosterone-induced MMP-1, MMP-9 and COX-2 protein expressions could all be suppressed by IL-6 siRNA, NS398 and AG490 (Figure 4A-C).

We further tested whether PGE2, a downstream mediator of COX-2 signaling, could enhance the expressions of MMP-1 and MMP-9 in THP-1 cells. We first tested whether aldosterone could induce PGE2 expression, and the results showed that while aldosterone could induce the expression of PGE2, this effect was suppressed by the inhibitors NS398 and AG490 (Figure 4D). We then added PGE2 directly into THP-1

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cells, and the results showed that by adding PGE2 at 200 to 500 ug/mL the mRNA and protein expressions of MMP-1 and MMP-9 were significantly increased (Figure 4E, 4F). These results suggested that PGE2 induced the expression of MMP-1 mainly through EP-2 and EP-4 receptors, while PGE2 induced the expression of MMP-9

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mainly through the EP-4 receptor (Figure 4G).

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Aldosterone induced IL-6, MMP-1, MMP-9 and COX-2 expressions in murine

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PBMCs

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We used a mouse aldosterone infusion model to test whether the aforementioned signaling existed in vivo. The expression of IL-6 mRNA in PBMCs from the

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aldosterone-infused mice was significantly higher than that in the placebo mice 2 to 6

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weeks after aldosterone infusion (Figure 5A). In addition, the expressions of MMP-1, MMP-9 and COX-2 mRNA in the PBMCs were also significantly higher in the aldosterone-infused mice at 6 weeks after aldosterone infusion (Figure 5B-D). These results suggested that aldosterone did induce IL-6/COX-2 and MMP-1/MMP-9 in the in vivo model.

Aldosterone induced macrophage infiltration in the myocardium, and this could be suppressed by eplerenone or IL-6 antibodies

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We then measured the number of macrophages in the mouse myocardium. The number of F4/80-positive cells per HPF in the myocardium was significantly higher in the aldosterone-infused mice 4 to 8 weeks after aldosterone infusion (Figure 6A and 6 B). We further isolated F4/80-positive cells from ventricular tissue using a

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MagniSort™ Mouse F4/80 Positive Selection Kit. The expressions of IL-6, MMP-1,

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MMP-9 and COX-2 mRNA in the F4/80-positive cells from ventricular tissue were

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also significantly higher in the aldosterone-infused mice at 6 weeks after aldosterone

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infusion (Figure 6C-F).

In the aldosterone-infused mice, the co-treatment with IL-6 antibodies or

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eplerenone suppressed the aldosterone-induced increase in F4/80-positive cells

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(Figure 6G). We further isolated the macrophages that had infiltrated into the myocardium to determine their M1 or M2 phenotype, by measuring the mRNA level of M1(iNOS and TNF-a) and M2(arginase and IL-10) biomarker [13]. The results showed that these macrophages isolated from aldosterone-infused mice expressed significantly higher levels of iNOS and TNF-a, which suggested an M1 phenotype (Figure 7A-D).

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Taken together, these results suggested that the aldosterone-induced macrophage infiltration into the myocardium was mediated by MR and IL-6 signaling pathways in vivo (Figure 8).

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Discussion

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In this study, we investigated the hypothesis and underling mechanism of

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aldosterone-induced macrophage infiltration into the myocardial interstitial area. Our

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results demonstrated that: (1) aldosterone promoted the recruitment, migration and

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infiltration of macrophages into the myocardium; and (2) this aldosterone-induced

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macrophage infiltration was mediated by the MR/IL-6/COX2 and MMP-1 and

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MMP-9 signaling pathways. Our results support the notion that IL-6 has a direct effect on the movement of macrophages, and that it can activate macrophages to secrete MMPs to facilitate their infiltration into the ECM. Macrophages play an important role in myocardial fibrosis through their effects on the overall synthesis and degradation of the ECM. Macrophage infiltration into the myocardium has been demonstrated to be a crucial step in the fibrotic process in angiotensin-II-induced heart failure [14], in a pressure overload model [15], and in a myocardial infarction model. Only a few studies have investigated the mechanism of macrophage infiltration into the myocardium under conditions of excessive 23

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aldosterone concentration. In an early study, aldosterone infusion in rats was shown to induce cardiac hypertrophy and interstitial macrophage infiltration, probably through the upregulation of oxidative stress [16]. In a recent study using macrophage-specific MR knockout mice, the authors demonstrated that MR activation played a central role in the development of interstitial and perivascular fibrosis in the myocardium [17].

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We also previously demonstrated that aldosterone-induced galectin-3 secretion in

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macrophages was crucial for collagen synthesis in cardiac fibroblasts [18]. Based on

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these findings, we further investigated the mechanism of aldosterone-induced

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macrophage infiltration in the current study. Our results are consistent with prior experiments showing that aldosterone-induced macrophage infiltration was MR

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dependent. Furthermore, we delineated the signaling pathway regulating the process

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of infiltration. We demonstrated that aldosterone increased both the migration and infiltration function of macrophages via the IL-6/JAK/COX-2/PGE2 pathway, and finally via the secretion of MMP-1 and MMP-9 to degrade the ECM and thereby facilitating the movement of macrophages into the myocardium. IL-6 infusion has been shown to induce cardiac fibrosis and hypertrophy in rats [11]. In that study, the cardiac remodeling induced by IL-6 infusion was similar to that in a pressure overload model, and could be explained by the effect of IL-6 on cardiac fibroblasts causing increased collagen synthesis. IL-6 also induced cardiac

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fibrosis in mice treated with angiotensin II and high salt. The absence of IL-6 has been shown to prevent angiotensin-induced macrophage infiltration into the myocardium and cardiac fibrosis in IL-6 knockout mice [19]. Our results expand the knowledge further in that IL-6 induced cardiac fibrosis through the upregulation of PGE2 and MMPs in macrophages in the current study. Although our findings with

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regards to the signaling pathways were in THP-1 cells, we also demonstrated that

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aldosterone induced the expressions of IL-6, MMP-1 and MMP-9 in circulating

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PBMCs and macrophages isolated from mice ventricles. It is therefore highly likely

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that this signaling pathway occurs in vivo. Moreover, after we transfected IL-6 siRNA into THP-1 cells, the infiltration of THP-1 cells through transwell membranes was not

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totally suppressed. Therefore, it is possible that other downstream signaling of

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aldosterone may also promote the infiltration of macrophages. In another study by Ma et al [20], the co-culture of macrophages and cardiac fibroblasts resulted in significantly increased IL-6 and collagen I production in cardiac fibroblasts. Considering these and our results, we conclude that IL-6-induced macrophage infiltration is involved in the initiation of the fibrotic process. The infiltrated macrophages then coordinate with cardiac fibroblasts to increase the production of collagen and IL-6, which may serve as an autocrine/paracrine stimulus for cardiac fibroblasts to amplify the fibrotic process.

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Matrix metalloproteinases are zinc-dependent proteases that can degrade components of the ECM including collagen and fibronectin [21]. Macrophages secrete different combinations of MMPs in various pathological conditions, and it has been shown that MMP-mediated ECM degradation facilitates the movement of macrophages in the interstitial space during tuberculosis infection [22],

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smoke-induced emphysema [23], abdominal aortic aneurysm [24], and

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macrophage-associated tumor metastasis [25]. Recent studies have demonstrated the

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role of macrophage-secreted MMP-9 in myocardial fibrosis. In a study on aging mice,

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MMP-9 knockout mice were shown to have less collagen accumulation in the left ventricle and less diastolic dysfunction compared with wild-type mice [26]. In

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addition, a study using macrophage-specific MMP-9 overexpression in transgenic

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mice showed that increased MMP-9 led to a higher collagen content in the left ventricles of these mice [27]. These results are consistent with our finding that MMP-9 was secreted from macrophages and was related to the interstitial fibrosis of the myocardium. However, the precise mechanisms of MMP-9 upregulation are not totally clear. Our data suggest that IL-6/JAK/COX-2/PGE2 is a possible upstream pathway to upregulate the expressions of MMPs. This upstream pathway may serve as a target to manipulate macrophage infiltration into the myocardium and as a candidate target for the treatment of myocardial fibrosis and diastolic dysfunction.

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There are several limitations to this study. First, we examined the effect of IL-6 on macrophage infiltration, however other mediators may also play a role in this process. Second, the infiltration and migration cell studies were performed using THP-1 cells and animal models, and are therefore somewhat limited for the interpretation of human diseases. Third, although our data showed for the first time

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that M1 subpopulation of macrophage is predominant in aldosterone infusion model,

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further study is warranted to determine the role of different subpopulation of

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macrophage in different stage of aldosterone induced myocardial fibrosis.

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Conclusion

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In this study, we demonstrated that aldosterone induced macrophage infiltration

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into the myocardium. First, we showed that IL-6 had a direct effect on the migration and infiltration of macrophages. Second, we found that the aldosterone-induced macrophage infiltration involved an MR/IL-6/COX2/PGE2/EP2,4/MMP-1,9 signaling pathway. Third, we demonstrated a corresponding phenomenon in an animal model. Our data provide a detailed mechanism of aldosterone-induced macrophage infiltration into the myocardium, and provide a potential upstream target for macrophage-coordinated myocardial fibrosis.

Clinical perspectives

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

Macrophage recruitment and infiltration are the initial steps in aldosterone-induced myocardial fibrosis. The underlying mechanism is not totally understood.

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Our study showed that aldosterone-induced macrophage infiltration was mediated by MR/IL-6/COX2 and MMP-1 and MMP-9 signaling pathways. Our study provides a potential upstream target for macrophage-coordinated

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myocardial fibrosis.

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Acknowledgements

The authors would like to thank the staff of the Second Core Lab of Department of

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Medical Research in National Taiwan University Hospital for their great support.

Competing Interests

The authors declare that there is no conflict of interests associated with this manuscript.

Funding: This study was supported by the Department of Health, Executive Yuan, R.O.C. (PTH 107-44), National Taiwan University Hospital (NTUH 107-A141, UN106-072,

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UN107-059, 105-S3044, 106-S3406, 106-S3471, 107-S3812, 107-S3957), National Taiwan University Hospital, Hsin-Chu branch (105-HCH035, 106-HCH013), and the Ministry of Science and Technology (MOST 105-2314-B-002-122-MY3, MOST 106-2314-B-002-048 -MY3, MOST 105-2314-B-002-123, MOST 106-2314-B-002-169-MY3). The funders had no role in study design, data collection

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and analysis, decision to publish, or preparation of the manuscript.

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Author Contributions

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YHL, VCW, CSH and CHC conceived and designed this study. CHC, CWL, XMW performed the experiments. YHC, YYC and ZWC performed data analysis. YHL. SRJ,

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YHC, YHL, CSH, and CHC discussed and interpreted the experiment results. YHL

manuscript.

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and CSH wrote the original manuscript. YHL, CSH, CHC and SRJ edited the

Appendix Membership of the Taiwan Primary Aldosteronism Investigation (TAIPAI) Study Group: Che-Hsiung Wu, MD (Chi-Taz Hospital, PI of Committee); Vin-Cent Wu, MD (NTUH, PI of Committee); Yen-Hung Lin, MD (NTUH, PI of Committee); Yi-Luwn Ho, MD, PhD (NTUH, PI of Committee); Hung-Wei Chang, MD, PhD (Far

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Eastern Hospital, PI of Committee); Lian-Yu Lin MD, PhD (NTUH, PI of Committee); Fu-Chang Hu, MS, ScD, (Harvard Statistics, Site Investigator); Kao-Lang Liu, MD (NTUH, PI of Committee); Shuo-Meng Wang, MD (NTUH, PI of Committee); Kuo-How Huang, MD (NTUH, PI of Committee); Yung-Ming Chen, MD (NTUH, PI of Committee); Chin-Chi Kuo; MD (Yun-Lin, PI of Committee),

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Chin-Chen Chang, MD (NTUH, PI of Committee); Shih-Cheng Liao, MD (NTUH, PI

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of Committee); Ruoh-Fang Yen, MD, PhD (NTUH, PI of Committee); and

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Kwan-Dun Wu, MD, PhD (NTUH, Director of Coordinating Center).

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Conflict of interest: none declared.

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5. Catena, C., Colussi, G., Lapenna, R., Nadalini, E., Chiuch, A., Gianfagna, P. et al. (2007) Long-term cardiac effects of adrenalectomy or mineralocorticoid antagonists in patients with primary aldosteronism. Hypertension. 50, 911-918. 6.

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11. Melendez, G.C., McLarty, J.L., Levick, S.P., Du, Y., Janicki, J.S. and Brower, G.L. (2010) Interleukin 6 mediates myocardial fibrosis, concentric hypertrophy, and diastolic dysfunction in rats. Hypertension. 56, 225-231. 12. Chou, C.H., Hung, C.S., Liao, C.W., Wei, L.H., Chen, C.W., Shun, C.T. et al. (2018) IL-6 trans-signalling contributes to aldosterone-induced cardiac fibrosis. Cardiovasc Res. 114, 690-702.

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13. Ka, M.B., Daumas, A., Textoris, J. and Mege, J.L. (2014) Phenotypic diversity and emerging new tools to study macrophage activation in bacterial infectious diseases. Front Immunol. 5, 500. 14. Sopel, M.J., Rosin, N.L., Lee, T.D. and Legare, J.F. (2011) Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor

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Pressure-Overload Heart Failure. PLoS One. 12, e0170781. 16. Yoshida, K., Kim-Mitsuyama, S., Wake, R., Izumiya, Y., Izumi, Y., Yukimura, T.

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et al. (2005) Excess aldosterone under normal salt diet induces cardiac hypertrophy

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and infiltration via oxidative stress. Hypertens Res. 28, 447-455. 17. Bienvenu, L.A., Morgan, J., Rickard, A.J., Tesch, G.H., Cranston, G.A., Fletcher, E.K. et al. (2012) Macrophage mineralocorticoid receptor signaling plays a key role in aldosterone-independent cardiac fibrosis. Endocrinology. 153, 3416-3425. 18. Lin, Y.H., Chou, C.H., Wu, X.M., Chang, Y.Y., Hung, C.S., Chen, Y.H. et al. (2014) Aldosterone induced galectin-3 secretion in vitro and in vivo: from cells to humans. PLoS One. 9, e95254.

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19. Gonzalez, G.E., Rhaleb, N.E., D'Ambrosio, M.A., Nakagawa, P., Liu, Y., Leung, P. et al. (2015) Deletion of interleukin-6 prevents cardiac inflammation, fibrosis and dysfunction without affecting blood pressure in angiotensin II-high salt-induced hypertension. J Hypertens. 33, 144-152. 20. Ma, F., Li, Y., Jia, L., Han, Y., Cheng, J., Li, H. et al. (2012)

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(2017) Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Are Regulated by Integrin alphaVbeta3 in Mycobacterium tuberculosis Infection. J Immunol. 199, 982-991.

23. Hautamaki, R.D., Kobayashi, D.K., Senior, R.M. and Shapiro, S.D. (1997) Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science. 277, 2002-2004.

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24. Gong, Y., Hart, E., Shchurin, A. and Hoover-Plow, J. (2008) Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice. J Clin Invest. 118, 3012-3024. 25. Kessenbrock, K., Plaks, V. and Werb, Z. (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 141, 52-67.

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fibrosis. Am J Physiol Heart Circ Physiol. 312, H375-H383.

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Figure legends Figure 1. Aldosterone enhanced IL-6 expression in the human monocytic cell line THP-1. (a) THP-1 cells (1x106 /ml) were treated with different amounts of aldosterone (Aldo). After 24 hours, cell culture supernatants were collected for IL-6

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detection using an EIA kit, n=6; from 3 experiments run in duplicate. ***p<0.001,

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compared with the vehicle using one-way ANOVA with Bonferroni correction. (b)

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THP-1 cells were treated with 10-7 M of Aldo or vehicle for different time periods.

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Total RNA was purified for IL-6 mRNA detection using qRT-PCR, n=6; from 3

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experiments run in duplicate. p<0.001, between Aldo and vehicle groups; ***p<0.001

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between Aldo and vehicle groups at 4, 8, 16 and 24 hours, using ANOVA with

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Bonferroni correction. (c) THP-1 cells were treated with 10-7 M of Aldo or vehicle for different time periods. Cell culture supernatants were collected for IL-6 detection by EIA, n=6; from 3 experiments run in duplicate. p<0.001, between Aldo and vehicle groups; ***p<0.001 between Aldo and vehicle groups at 4, 8, 16 and 24 hours, using ANOVA with Bonferroni correction. (d) THP-1 cells were treated with MR or GR siRNA for 24 hours or eplerenone (10-6 M) for 1 hour prior to 10-7 M of Aldo treatment. After 24 hours, cell culture supernatants were collected for IL-6 detection by EIA, n=6; from 3 experiments run in duplicate. ***p<0.001, compared with the vehicle, or compared between the two groups indicated by the line underneath, using 36

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one-way ANOVA with Bonferroni correction. (e) THP-1 cells were treated with the indicated chemical inhibitors (PD98059 50 μg/ml, LY294002 20 μg/ml, SB203580 5 μg/ml, Ro318220 5 nM, SP600125 10 μM) for 1 hour prior to 10-7 M Aldo treatment. After 24 hours, cell culture supernatants were collected for IL-6 detection by EIA, n=6; from 3 experiments run in duplicate. p-value as indicated, using one-way

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ANOVA with Bonferroni correction. (f) THP-1 cells were treated with 5 uM of the

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indicated decoy ODNs for 24 hours prior to 10-7 M Aldo treatment. After 24 hours,

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cell culture supernatants were collected for IL-6 detection by EIA, n=6; from 3

Bonferroni correction. (g)

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experiments run in duplicate. p-value as indicated, using one-way ANOVA with

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***p<0.001, compared with the vehicle, or compared between the two groups

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indicated by the line underneath.

Figure 2. Aldosterone enhanced THP-1 migration and infiltration. (a) THP-1 cells (1x105 /well) were cultured in transwell dishes and treated with different amounts of aldosterone (Aldo). After 16 hours, migrated cells were fixed and photographed. (b) Quantitative results of (a); n=6; from 3 experiments run in duplicate. (c) THP-1 cells (1x105 /well) were cultured on Matrigel-coated transwell dishes and treated with different amounts of Aldo. After 48 hours, infiltrated cells were fixed and

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photographed. (d) Quantitative results of (c); n=6; from 3 experiments run in duplicate. (e) THP-1 cells were treated with 25 nM IL-6 siRNA or control siRNA for 24 hours prior to 10-7 M Aldo treatment. After 24 hours, cell culture supernatants were collected for IL-6 detection by EIA, n=6; from 3 experiments run in duplicate. (f) THP-1 cells were treated with 25 nM IL-6 siRNA or control siRNA for 24 hours, and

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were then cultured on Matrigel-coated transwell dishes and treated with 10-7 M Aldo

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for 48 hours in an infiltration assay. Data are presented as the quantitative results, n=6;

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from 3 experiments run in duplicate. ***p-value <0.001, compared with the vehicle,

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using one-way ANOVA with Bonferroni correction.

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Figure 3. Aldosterone enhanced proteome profiler of human protease expression

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in THP-1 cells. THP-1 cells (1x106 /ml) were treated with 10-10 M or 10-7 M aldosterone (Aldo). After 24 hours, cell culture supernatants were collected for protease array determination. (a) Representative array image. (b) Intensity of each spot was quantified. Data are presented as the relative intensity compared between 10-7 M and 10-10 M Aldo-treated groups. n=3; from 3 separated experiments. *unadjusted p<0.05 (adjusted q-value = 0.193 for MMP-1; 0.059 for MMP-3; and 0.007 for MMP-9). (c) THP-1 cells (1x106 /ml ) were treated with 10-10 M or 10-7 M Aldo. After 24 hours, cell culture supernatants were collected for MMP-1, (d) MMP-3

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and (e) MMP-9 detection by EIA, n=6 from 3 experiments run in duplicate. ***p-value <0.001 (c-e) , using the Student’s T-test. (f) THP-1 cells were treated with 25 nM of the indicated siRNAs for 24 hours and then processed for 48 hours in an infiltration assay. Infiltrated cells were counted. Data are presented as the quantitative results, n=6 from 3 experiments run in duplicate. ***p<0.001, using one-way ANOVA

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with Bonferroni correction, compared with the vehicle in the graph, or compared

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between the two groups indicated by the line underneath

Figure 4. Aldosterone enhanced MMP-1 and MMP-9 protein expressions

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in THP-1 cells through IL-6 and COX-2/PGE2 signaling. (a) THP-1 cells (1x106 /ml) were treated with IL-6 siRNA for 24 hours or the indicated chemical inhibitors

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(NS398 10 uM, AG490 10 uM) for 1 hour prior to aldosterone (Aldo) treatment. After

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24 hours, cell culture supernatants were collected for (a) MMP-1 and (b) MMP-9 detection by EIA, n=6; from 3 experiments run in duplicate. (c) THP-1 cells (1x106 /ml) were treated with IL-6 siRNA for 24 hours or the indicated chemical inhibitors for 1 hour prior to Aldo treatment, cell lysates were collected for COX-2 protein level determination by Western blot and quantification (n=6); from 6 separated experiments. (d) THP-1 cells (1x106 /ml) were treated with the indicated chemical inhibitors for 1 hour prior to Aldo treatment. After 24 hours, cell culture supernatants were collected for PGE2 detection by EIA, n=6 from 3 experiments run in duplicate. (e) THP-1 cells

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were treated with different amounts of PGE2 for 24 hours. Cell culture supernatants were then collected for MMP-1 and MMP-9 detection by EIA, n=6 from 3 experiments run in duplicate. (f) THP-1 cells were treated with different amounts of PGE2 for 8 hours. Total RNA was then purified for MMP-1 and MMP-9 mRNA determination by qRT-PCR, n=6 from 3 experiments run in duplicate. (g) THP-1 cells

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were treated with EP(1-4) or control siRNA for 24 hours prior to Aldo treatment. After

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24 hours, cell culture supernatants were collected for MMP-1 and MMP-9 detection

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by EIA, n=6 from 3 experiments run in duplicate. ***p<0.001, compared with the

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vehicle, or compared between the two groups indicated by the line underneath. Figure 5. Aldosterone infusion significantly enhanced IL-6, COX-2, MMP-1 and

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MMP-9 expressions in PBMCs in a mouse model. (a) Six-week-old C57BL/6

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wild-type male mice with a similar body weight (25–30 g) were implanted subcutaneously with a 21-day continuous aldosterone (Aldo) release pellet, and the vehicle mice received a placebo pellet. Mice were sacrificed every two weeks, and PBMCs were collected for IL-6 mRNA determination by qRT-PCR, n=6; from 2 experiments run in triplicate. *p<0.05, by one-way ANOVA with Bonferroni correction between Aldo and placebo groups at 2 weeks and 4 weeks; **p<0.01, by one-way ANOVA, between Aldo and placebo groups. (b) PBMCs from control or aldosterone release pellet treated mice for 6 weeks were processed for COX-2, (c)

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MMP-9 (d) MMP-1 mRNA determination by qRT-PCR, n=6; from 2 experiments run in triplicate. ***p-value <0.001, **p-value <0.01, *p-value <0.05, by the Student`s T-test, compared with placebo. Figure 6. Blockade of aldosterone by eplerenone and IL-6 Ab significantly reduced macrophage infiltration in ventricle tissue in an aldosterone infusion

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mouse model. (a) Six-week-old C57BL/6 wild-type male mice with a similar body

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weight (25–30 g) were implanted subcutaneously with a 21-day continuous

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aldosterone (Aldo) release pellet, and the vehicle mice received a placebo pellet. Mice

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were sacrificed every two weeks, and ventricle tissues were processed for paraffin sectioning. The macrophage infiltration in ventricle tissue was determined by F4/80

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IHC. Data are presented as the quantitative results of each time period. n=6; from 2 ***p-value <0.001, by two-way ANOVA with

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experiments run in triplicate.

Bonferroni correction, compared between Aldo and placebo groups. (b) Representative image of F4/80 IHC ventricle tissue sections of the vehicle (control) or aldosterone release pellet treated mice for 6 weeks. (c) As described in (a), mice were sacrificed 6 weeks after Aldo release pellet or vehicle treatment, and the macrophage infiltration in ventricle tissue was measured using a MagniSort™ Mouse F4/80 Positive Selection Kit. The expressions of IL-6, (d) COX-2, (e) MMP-1 and (f) MMP-9 mRNA in infiltrated macrophages were determined by qRT-PCR. n=6; from

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2 experiments run in triplicate.

***p-value <0.001 in (c-f), by the Student`s T-test,

compared between Aldo and placebo groups. (g) As described in (a), the mice were implanted subcutaneously with a 21-day continuous aldosterone release pellet; treated with IL-6 monoclonal antibody or control Ab (10 mg/kg) three times per week intraperitoneally, and oral gavage administration of eplerenone (100 mg/kg body

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weight/day) or placebo for a total of 6 weeks. The macrophage infiltration in ventricle

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tissue was determined and quantified by F4/80 IHC. n=6; from 2 experiments run in

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triplicate. ***p<0.001, compared with placebo, or compared between the two groups

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indicated by the line underneath, by one-way ANOVA with Bonferroni correction. Figure 7 Phenotype of the aldosterone-induced macrophage infiltration into the

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myocardium

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The expressions of (a) iNOS, (b) TNF-α, (c) arginase and (d) IL-10 mRNA in infiltrated macrophages were determined by qRT-PCR. n=6; from 2 experiments run in triplicate. ***p<0.001, by the Student`s T test, compared with placebo; NS, non-significant. Figure 8 Summary of the signaling pathway of aldosterone-induced macrophage infiltration into the myocardium. Aldosterone (Aldo) induced the expression of interleukin-6 (IL-6) by binding to mineralocorticoid receptors (MRs) and the subsequent activation of PI3K/Akt and P38, then NF-kB in monocytes/macrophages.

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IL-6 increased the expressions of matrix metalloproteinase (MMP)-1 and MMP-9 via JAK/ cyclooxygenase (COX)-2/ prostaglandin E2 (PGE2) signaling. PGE2 activated monocytes/macrophages to express MMP-1 and MMP-9 by binding to EP-2 or EP-4 receptors. MMP-1 and MMP-9 may facilitate the breakdown of the extracellular

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matrix to help the infiltration of monocytes/macrophages into the myocardium.

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Macrophage recruitment and infiltration are the initial steps in aldosterone-induced myocardial fibrosis. The underlying mechanism is not totally understood.

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Our study showed that aldosterone-induced macrophage infiltration was

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Our study provides a potential upstream target for macrophage-coordinated

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myocardial fibrosis.

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mediated by MR/IL-6/COX2 and MMP-1 and MMP-9 signaling pathways.

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