Adiponectin suppresses angiotensin II-induced inflammation and cardiac fibrosis through activation of macrophage autophagy.

RENAL-CARDIAC-VASCULAR

Adiponectin Suppresses Angiotensin II-Induced Inflammation and Cardiac Fibrosis through Activation of Macrophage Autophagy Guan-Ming Qi1†, Li-Xin Jia1†, Yu-Lin Li1, Hui-Hua Li2, and Jie Du1# 1 Beijing Anzhen Hospital, The Key Laboratory of Remodeling-related Cardiovascular Diseases, Capital Medical University, Ministry of Education, Beijing Institute of Heart Lung & Blood Vessel Disease, Beijing 100029, China; 2Department of Pathology, Capital Medical University, Beijing 100069, China

Previous studies have indicated that adiponectin (APN) protects against cardiac remodeling, but the underlying mechanism remains unclear. The present study aimed to elucidate how APN regulates inflammatory responses and cardiac fibrosis in response to angiotensin II (Ang II). Male APN-knockout (APN KO) mice and wild type (WT) C57BL/6 littermates were subcutaneously infused with Ang II at 750 ng/kg per minute. Seven days after Ang II infusion, both APN KO and WT mice developed equally high blood pressure levels. However, APN KO mice developed more severe cardiac fibrosis and inflammation compared to WT mice. This finding was demonstrated by the upregulation of collagen I, ␣-smooth muscle actin, IL-1␤, and TNF-␣ and increased macrophage infiltration in APN KO mice. Moreover, there were substantially fewer LC3-positive autophagosomes in macrophages in the hearts of Ang II-infused APN KO mice. Additional in vitro studies also revealed that globular APN treatment induced autophagy, inhibited Ang II-induced nuclear factor-␬B activity and enhanced the expression of anti-inflammatory cytokines, including IL-10, Mgl-2, Fizzl, and Arg-1, in macrophages. In contrast, APN-induced autophagy and anti-inflammatory cytokine expression was diminished in Atg5-knockdown macrophages or by Compound C, an inhibitor of AMP-activated protein kinase (AMPK). Our study indicates that APN activates macrophage autophagy through the AMPK pathway and suppresses Ang II-induced inflammatory responses, thereby to reducing the extent of cardiac fibrosis.

ypertensive cardiac remodeling is characterized by ventricular hypertrophy and interstitial fibrosis, which are the main causes of heart failure. Emerging evidence suggests that activation of renin-angiotensin II system is a primary cause of cardiac fibrosis in hypertensive heart disease (1, 2), but the underlying mechanism remains unclear. Recent studies demonstrate that inflammation plays a fundamental role in cardiovascular diseases, such as atherosclerosis, hypertension, and infarction (2). Infiltration of inflammatory cells in the heart is an early event in Ang II-induced hypertensive cardiac remodeling. The inflammatory components of this process include macrophages, T cells, and mast cells, which are associated with cytokine secretion, extracellular matrix deposition, and cardiac fibrosis development (3– 8). However, the precise

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inflammatory mechanism and macrophage functions in Ang II-induced cardiac fibrosis remain to be revealed. Recent studies have reported that adipose tissues can secrete numerous cytokines, known as adipokines. APN (also referred to as ACRP30, Adipo Q, and gelatin-binding protein-28) is a 30-kDa adipokine secreted primarily by adipocytes. Total plasma APN levels typically range from 3–30 ␮g/mL in normal human subjects (9). However, the levels of APN is significantly reduced in various diseases, including obesity, type 2 diabetes, hypertension, acute myocardial infarction (MI), and heart failure (9). Although most adipokines induce inflammation, APN exerts anti-inflammatory functions and protects against a variety of cardiovascular diseases such as atherosclerosis, diabetes, vascular remodeling, hypertrophic cardiomyop-

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received November 5, 2013. Accepted March 18, 2014.

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doi: 10.1210/en.2013-2011

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Adiponectin activates macrophage autophagy

athy, and myocardial ischemia-reperfusion injury (9). For example, treatment with recombinant APN inhibits reactive oxygen species (ROS)-induced cardiomyocyte remodeling by activating AMP-activated protein kinase (AMPK) and inhibiting ERK signaling and nuclear factor-␬B (NF␬B) activity (10). APN also protects against Ang II-induced cardiac fibrosis through the AMPK-dependent PPAR-␣ pathway (11). However, the mechanism by which APN regulates inflammatory responses and macrophages function, leading to Ang II-induced cardiac fibrosis, remains unclear. Autophagy is a major intracellular degradation process that delivers cytoplasmic constituents to the vacuole/lysosome and plays essential roles in cell survival or death depending on the cellular context (12). Normally, autophagy occurs at a basal rate in most cells and is critical for maintenance of cellular homeostasis. However, under physiological stress such as starvation or growth factor withdrawal, autophagic activity is upregulated (13). Increasing evidence has shown that autophagic dysfunction is the main reason for numerous pathological states, including aging, neurodegenerative diseases, immune disorders, and cancer (13, 14). Interestingly, activation of autophagy has also been observed in cardiac hypertrophy, ischemia-reperfusion injury, and advanced atherosclerotic plaques (12, 13, 15). In addition, autophagy play an important role in the innate and adaptive immune defense programs leading to increased expression of inflammatory cytokines, (such as TNF-␣ and IFN-␥) (16, 17). A recent study demonstrated that autophagy is stimulated by an essential mediator of APN action, AMPK (18); however, the effect of APN on autophagy in macrophages during Ang II-induced cardiac inflammation and fibrosis remains unknown. In the present study, we investigated the role of APN in Ang II-induced cardiac inflammation and fibrosis and the molecular mechanism involved in the regulation of autophagy by APN. Our results indicated that APN deficiency increased cardiac fibrosis, macrophage infiltration, and inflammatory cytokine expression (TNF-␣ and IL-1␤) in heart tissue. This was associated with decreased autophagy in heart macrophages. These results suggest that APN-mediated increases in macrophage autophagy contribute to the inhibition of cardiac inflammation and fibrosis.

Materials and Methods Antibodies and reagents Anti-Mac-2, anti-IL-1␤, anti-TNF-␣ and anti-GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA). Anticollagen

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anti-F4/80, anti-␣-smooth and I muscle actin (␣-SMA) were obtained from Abcam (Cambridge, MA). Anti-LC3 was from MBL International Operation (Nagoya, Japan). Anti-phosphorAMPK and anti-AMPK were from Cell Signaling Technology (Beverly, MA). FITC or TRITC-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Recombinant globular APN was purchased from BioVision Inc. (Mountain View, CA). Compound C was from Calbiochem (La Jolla, CA). Angiotensin II (Ang II) and other reagents were from Sigma-Aldrich (St. Louis, MO).

Animal model APN knockout (APN KO) mice, Atg 5⫹/- mice and wild-type (WT) C57/BL6 mice (both male and aged 10 –12 weeks) were purchased from Jackson Laboratory. Mice were randomly divided into 4 groups for treatment (n ⫽ 6 – 8 per group): WT⫹saline; KO⫹saline; WT⫹Ang II; KO⫹Ang II. The osmotic minipump (Alzet MODEL 1007D, DURECT, Cupertino, CA) filled with Ang II or acetic acid saline (vehicle) was placed subcutaneously in the intra-scapular area to deliver Ang II at an infusion rate of 750 ng/kg per minute for 7 days as described previously (7, 19). All procedures were performed according to experimental protocols approved by the Capital Medical University Institutional Committee for Use and Care of Laboratory Animals.

Blood pressure measurement and Cardiac Echocardiography Systolic blood pressure (BP) (SBP) and heart rate were measured at the indicated day using a computerized mouse tail cuff system (BP-98A softron, Tokyo, Japan) as we described elsewhere (20, 21). And cardiac echocardiography were performed using Vevo 2000 high resolution micro imaging system (Visual sonic, Toronto, Canada) after anesthetized with isoflurane as we previously described (22, 23).

Histological and immunohistochemical analysis After 7 days of saline or Ang II infusion, Heart tissues were harvested, fixed in 10% formalin, and sectioned at 5-␮m thickness. Hematoxylin/eosin (H&E) and Masson’s trichrome staining were performed using standard procedures (7, 19). Cardiac fibrosis were calculated based upon percentages of collagen stained areas in the total myocardial area (7). Immunohistochemistry was performed as we described (7, 19). The primary antibodies were used: anti-Mac-2 (1:400), anti-IL-1␤ (1:200), anti-TNF-␣ (1:200), anticollagen I (1:800) and anti-␣-SMA (1: 200) and anti-LC3 (1:200). Frozen heart sections (8 ␮m) or cells were stained with primary antibodies, including rabbit anti-LC3 (1:400) and rat antiF4/80 (1:100) at 4°C overnight, and then incubated with FITC or TRITC-conjugated secondary antibodies at room temperature for 1h. Dying macrophages were detected by costaining of F4/80 and TUNEL with the In Situ Cell Death Detection Kit (Promega, Madison, WI) according to the manufacturer’s instructions. Images were viewed and captured using a deconvolution microscope with a Zeiss Plan Apochromat 630 ⫻, 1.4 N.A. objective lens. We randomly counted 100 macrophages of every sample to calculate the total number of autophagosomes and then made statistical analysis. MitoTracker and DCF (Mo-


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lecular Probes, Eugene, OR, USA) were used to examine generation of mitochondrial reactive oxygen species (ROS) (24, 25).


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citrate for 10 minutes. Cells were examined using a JEOL 1230 transmission electron microscope (EM) (JEOL USA. Inc., Peabody, MA).

RNA extraction and real-time PCR analysis Total RNA was isolated by Trireagent (Invitrogen) and realtime PCR was performed as described (20, 21). Primers used in the research were for Collagen I, 5⬘-GAGCGGAGAGTACTGGATCG-3⬘, 5⬘-TACTCGAACGGGAATCCATC-3⬘; ␣-SMA, 5⬘-GCAAACAGGAATACGACGAAGC-3⬘, 5⬘GCTTTGGGCAGGAATGATTTG-3⬘; IL-1␤, 5⬘-CTTCAGGCAGGCAGTATCACTCAT-3⬘, 5⬘-TCTAATGGGAACGTCACACACCAG-3⬘; TNF-␣, 5⬘CATGAGCACAGAAAGCATGATCCG-3⬘, 5⬘AAGCAGGAATGAGAAGAGGCTGAG-3⬘; IL-10, 5⬘-GCTATGCTGCCTGCTCTTACTGA-3⬘, 5⬘-TCATGGCCTTGTAGACACCTTGG-3⬘; Arg-1, 5⬘AGTCCTTAGAGATTATCGGAGCG-3⬘, 5⬘-GGACACAGGTTGCCCATGCAGAT-3⬘; Fizzl, 5⬘-AATGAGGGCTGGTGAGATGA-3⬘, 5⬘-GGACTCTCTCCTTCCACCAT-3⬘; Mgl-2, 5⬘-CCTGTGGCGCATCCTCT-3⬘, 5⬘-GGGAATTTTGGGATCCAAT-3⬘; GAPDH, 5⬘-CCTGGAGAAACCTGCCAAGTATGA-3⬘, 5⬘-TTGAAGTCACAGGAGACAACCTGG-3⬘. Real-time PCR was performed with use of Bio-Rad iQ5.

ELISA assay The protein level of IL-1␤ and TNF ␣ in heart were measured by using a commercial ELISA kit (Xinbosheng, Guangzhou, China) according to the manufacturer’s instructions.

FACS analysis The infiltrated inflammatory cells were quantified by flow cytometry as we described before (20, 21, 26). In Brief, hearts were perfused with cold PBS, then minced and digested with 0.1% collagenase II and 2.4 U/mL dispase II in PBS at 37°C for 15 minutes. Cell suspensions were then centrifuged and stained with a combination of fluorochrome-coupled antibodies to CD45, F4/80 and CD206 (BD Biosciences, San Jose, CA) for 30 minutes in the dark. Data were collected and analyzed by using an EPICS XL Flow Cytometer (Beckman Coulter) and CellQuest (Beckman) respectively.

Cell culture and treatment Bone marrow-derived macrophages (BMDMs) were isolated from tibias and femurs of 3-month-old WT, APN KO and Atg 5⫹/- mice as previously described (27). The adherent BMDMs were pretreated with globular domain of APN (10 ␮g/ml) for 30 minutes and then incubated with Ang II (100 nM) or vehicle for 48 hours. In some experiments, the BMDMs were incubated with Compound C (10 ␮M) to block AMPK pathway before the APN treatment.

Electron microscopy Electron microscopy was performed as reported previously (28). Briefly, cells were fixed with 2.5% (w/v) glutaraldehyde phosphate buffered 2.5% glutaraldehyde (pH 7.4), postfixed for 2 hours with 1% osmium tetroxide, dehydrated through a graded ethanol series, and embedded in Epon medium (Polysciences, Warrington, PA). Ultrathin 60 nm sections were stained with 2% uranyl acetate for 15 minutes, followed by 1% lead

Western Blot Analysis Western blot analysis was performed as we described previously (7, 19). Protein samples from macrophages were separated by SDS-PAGE, transferred onto the membrane (Millipore), and then incubated with primary antibodies, including anti-LC3, anti-phospho-AMPK, anti-AMPK, anti-GAPDH and the secondary antibody for 1 hour at room temperature in dark. The bands were detected by Odyssey system (Li-COR, Lincoln, NE). The ratio of the protein interested was subjected to GAPDH and was analyzed by Odyssey software.

NF-␬B luciferase assay

NF-␬B transcriptional activity was evaluated using a NF-␬B luciferase reporter as described (7, 29). Briefly, the adherents BMDMs were infected with Ad.NF-␬B-Luc at a multiplicity of infection of 5 for 24 hours before treatment with globular-APN (10 ␮g/ml) or Ang II (100 nM). The cells were detected for the NF-␬B activity with luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Relative light units of Firefly luciferase activity were normalized with the luciferase activity of the cells with saline treatment.

Statistical analysis Data were expressed as the mean ⫾ SEM. Differences between groups were performed for statistical significance using Student’s t test or ANOVA followed by Newman-Keuls Multiple Comparison Test from GraphPad Prism 5.0. A P value less than 0.05 denoted the statistically significant difference.

Results APN deficiency enhances the severity of Ang IIinduced cardiac fibrosis in mice To investigate the role of APN in Ang II-induced cardiac injury, we first measured SBP in WT and APN KO mice 7 days after Ang II infusion. The SBP in both APN WT and KO mice was significantly increased after 7 days of Ang II infusion, whereas WT and APN KO mice treated with saline had normal SBP (Supplemental Figure 1), which is consistent with the previous study that APN KO did not affect BP (11). Furthermore, there was no change after Ang II infusion, neither APN KO had any effect (Supplemental Figure 1). Echocardiography was also performed to measure the cardiac function in WT and APN KO mice, and found that there was no difference between APN KO and WT mice 7 days after Ang II infusion (new supplemental Figure 2). To determine whether APN deficiency affects Ang IIinduced cardiac fibrosis, WT and APN KO mice were infused with saline or Ang II for 7 days. The cardiac fibrotic areas were detected by Masson’s trichrome staining. As


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shown in Figure 1A, Ang II infusion significantly increased the heart tissue fibrotic areas in WT mice compared to those infused with saline, and this effect was further enhanced in APN KO mice. Moreover, immunohistochemistry and RT-PCR analysis showed that the levels of collagen I (a major extracellular matrix component) and ␣-SMA (a myofibroblast marker) expression were much higher in APN KO mice than in WT mice after 7 days of Ang II infusion (Figure 1B-D). In contrast, the expression levels of collagen I and ␣-SMA were similar in both WT and APN KO hearts after saline infusion (Figure 1B-D). These results suggest that APN deficiency worsens Ang II-induced cardiac fibrosis.

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APN deficiency promotes Ang II-induced cardiac inflammation To assess the effect of APN on Ang II-induced cardiac inflammation, we performed H&E and immunohistochemical staining and found that there were fewer infiltrating and Mac-2 positive cells in saline-treated WT and APN KO mice (Figure 2A and B). Conversely, Ang II infusion significantly increased the infiltration of inflammatory cells and Mac-2-positive macrophages in the hearts of WT littermates compared to saline-treated WT mice, and number of cells that infiltrated the cardiac tissue was markedly enhanced in APN KO mice after Ang II infusion (Figure 2A and B). The protein levels of IL-1␤ and TNF-␣ were further determined by using an ELISA kit. As is shown in Figure 2F, IL-1␤ and TNF-␣ were increased in the Ang II-treated APN KO mice. To further examine the role of APN in the regulation of cardiac inflammation, we conducted immunohistochemical staining and RT-PCR analysis. As shown in Figure 2C-E, IL-1␤ and TNF-␣ expression were markedly increased in the hearts of APN KO mice relative to WT mice, whereas obvious expression of these cytokines was not detected in WT or APN KO mice after saline infusion. These results suggest that knockout of APN can enhance cardiac inflammation in response to Ang II. Moreover, flow cytometry of WT or APN KO mice hearts was performed after saline or Ang II infusion to further demonstrate the infiltrated macrophages. As is shown in Figure 3, FACS analysis showed that Ang II infusion significantly increased the infiltration of CD45⫹, F4/80⫹, CD206⫹ cells into heart, while APN deficiency led to up-regulation of M2 macrophage markers (F4/ 80⫹CD206⫹) in hearts after Ang II infusion, compared with WT mice.

Figure 1. APN deficiency promotes Ang II–induced cardiac fibrosis. (A) Representative photomicrographs of heart sections from WT and APN KO mice stained with Masson’s trichrome at day 7 of Ang II infusion (left). Magnification: x40 (top), x200 (bottom). Histogram shows quantified fibrotic areas on histological sections (right). (B and C) Representative photomicrographs of heart sections from WT and APN KO mice immunostained for collagen I and ␣-SMA (left). Magnification: x400. Histogram shows quantified collagen I and ␣-SMApositive cells on histological sections (right). (D) Real-time PCR analysis of collagen I (left) and ␣-SMA (right) in the heart of WT and APN KO mice. Data represent the mean⫾SEM (n ⫽ 6 – 8 per group). *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001 vs. saline-treated WT mice; †P ⬍ .05, ††P ⬍ .01 vs. Ang II-treated WT mice.

APN administration inhibits NF␬B-mediated inflammation in macrophages Since NF-␬B is thought to play an important role in the onset of inflammation and development of tissue fibrosis (30), we examined the effect of


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APN on NF-␬B activity in macrophages using a luciferase reporter assay. As shown in Figure 4A, Ang II treatment significantly increased NF-␬B luciferase activity in macrophages, whereas treatment with globular APN markedly attenuated this effect. Global APN alone had no effect. Furthermore, Ang II treatment markedly downregulated the expression of anti-inflammatory cytokines, including IL-10, Mgl-2, Fizzl, and Arg-1, whereas pretreatment with globular APN had an opposite effect. Interestingly, these Ang II-induced alterations were attenuated by globular APN (Figure 4B). These results indicate that increased APN activity can inhibit NF-␬B-mediated inflammation in macrophages. To confirm that whether APN modulates the inflammatory response through the autophagic pathway, we treated Atg5⫹/- macrophages with globular APN and performed RT-PCR analysis to detect anti-inflammatory cy-


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tokine mRNA expression. As shown in Figure 3C, treatment with globular APN markedly downregulated the mRNA levels of IL-10, Mgl-2, Fizzl, and Arg-1 in Atg5deficient macrophages, indicating that APN stimulates autophagy induction, leading to increased expression of anti-inflammatory cytokines.

Effect of APN on Ang II-induced macrophage autophagy Since Ang II induces autophagy in several cell types (31, 32), we next investigated whether APN affects Ang IIinduced cardiac autophagy. Immunohistochemistry with anti-LC3 showed that the expression of LC3 protein, an autophagosome marker (12), in the cardiac tissue of APN KO mice was lower than that in WT mice after Ang II infusion (Figure 5A). To determine whether Ang II-induced autophagy occurs in macrophages, we performed immunostaining in heart sections from WT and APN KO using antibodies against LC3 and F4/80 (a macrophage maker). As shown in Figure 5B, there were fewer cells showing LC3 and F4/80 coexpression in APN KO mice than in WT mice after Ang II infusion. No obvious autophagosomes were observed in the saline-infused WT and APN KO mice. Macrophage apoptosis was also detected by costaining of TUNEL and F4/80. As is shown in Figure 5C, compared with WT mice, APN KO mice exhibited increase in macrophage cell death after Ang II-infusion. As mitochondrial-generated ROS trigger cell autophagy, we further explored the effect of APN in ROS generation in macrophage mitochondria. We pretreated primary mouse macrophages with globular APN in the presence or absence of Ang II, then performed immunofluorescence analysis and observed that MitoTracker colocalized with DCF Figure 2. APN deficiency enhanced Ang II-induced infiltration of inflammatory cells and in Ang II-treated macrophages, expression of inflammatory cytokines. WT and APN KO mice were infused with Ang II (750 ng/kg per minite) for seven days. Heart sections were stained by H&E (A) and immunohistochemistry whereas globular APN treatment dewith macrophage-specific markers Mac-2 (B), TNF-␣ (C) and IL-1␤ (D) (top). Magnification: creased this effect (Figure 5D). To x400. Histogram shows quantified inflammatory cells, Mac-2-, TNF-␣- and IL-1␤-positive cells on further confirm that APN could enhistological sections (bottom). (E) The mRNA levels of IL-1␤ and TNF-␣ in WT and APN KO hearts hance macrophage autophagy, we were detected by RT-PCR (top). Histogram shows densitometric analysis of intensities of IL-1␤ and TNF-␣ relative to that of GAPDH (bottom). (F) Histogram shows the protein expression of detected the autophagy marker LC3 IL-1␤ and TNF-␣ in WT and APN KO heart tissues that were detected by ELISA. Data represent using immunocytochemistry. Ang II the mean⫾SEM (n ⫽ 6 – 8 per group). *P ⬍ .05, ***P ⬍ .001 vs. saline-treated WT mice; †P ⬍ ††† or globular APN stimulated expres.05, P ⬍ .001 vs. Ang II-treated WT mice.


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sion of LC3 in macrophages, and the combination of globular APN and Ang II further enhanced LC3 expression (Figure 5E). Transmission electron microscopy (EM) also demonstrated that macrophages treated with globular APN or Ang II had substantially more autophagic vacuoles compared to control cells (Figure 5F).

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6 vs 5). These results suggest that APN induces autophagy in macrophages via the AMPK pathway.

Discussion

APN has been recognized as an antidiabetic, antiatherosclerotic, and anti-inflammatory protein derived from adiAPN regulates macrophage autophagy through pocytes (33). However, the precise mechanism by which the AMPK pathway AMPK is downstream of APN and plays a critical role APN regulates the extent of cardiac fibrosis has not been in autophagy induction. To determine how APN modu- fully clarified. Our results demonstrate that APN defilates autophagy, we conducted in vitro experiments using ciency caused severe cardiac fibrosis and inflammation but primary mouse macrophages and examined the effect of attenuated autophagy induction in response to Ang II inAPN and Compound C, an inhibitor of AMPK, on AMPK fusion. Treatment of macrophages with APN inhibited the phosphorylation (p-AMPK) and LC3 expression (Figure activity of NF-␬B and upregulated anti-inflammatory cy6A and B). As shown in Figure 6A and B, Ang II treatment tokine expression. Importantly, APN enhanced the exsuppressed activation of AMPK. However, APN signifi- pression of the autophagosome marker LC3 through accantly increased AMPK phosphorylation and LC3 expres- tivation of AMPK in macrophages. Thus, endogenous sion (lanes 2 and 4 vs 1) and the combination of APN and APN exerts a protective effect on cardiac inflammation Ang II further enhanced these effects, whereas such acti- and fibrosis in Ang II-induced hypertension. APN is abundantly expressed in adipocytes and plays vation was abrogated by Compound C (lane 3 vs 2; lane important roles in the development of obesity, diabetes, and cardiovascular diseases (9). And study has shown that APN is not expressed in macrophages (34). Several epidemiological studies demonstrate that circulating APN levels are inversely correlated with cardiovascular disease; for example, plasma APN concentrations are lower in patients with coronary artery disease (CAD) (35, 36). In contrast, high plasma APN levels are associated with a lower risk of MI, type 2 diabetes, and atherosclerosis (35, 37, 38). In addition, APN levels are also reported to rapidly decline following acute MI, type 2 diabetes, hyperlipidemia, and hypertension development (39). Moreover, APN replenishment ameliorates obesity-related hypertension and reduces atherosclerosis in Apo E knockout mice (38, 40). Importantly, APN deficiency results in severe cardiac fibrosis and left ventricular (LV) dysfunction, whereas APN Figure 3. APN deficiency increased Ang II-induced monocytes/macrophages infiltration. WT and overexpression improves these pheAPN KO mice were infused with Ang II (750 ng/kg per minite) for seven days, and then hearts ⫹ notypes (11). Consistent with these were removed and nonmyocytes isolated. Dispersed cells were analyzed for CD45 (A), CD45⫹F4/80⫹ (B), F4/80⫹CD206⫹ (C), and F4/80⫹CD206- (D) expression. (E) Representative findings, our present study showed cytometric diagrams showed distribution of the F4/80⫹CD206⫹ and F4/80⫹CD206- cells. Data that APN protected Ang II-induced represent the mean⫾SEM (n ⫽ 5 per group). *P ⬍ .05 vs. saline-treated WT mice; †P ⬍ .05, †† cardiac fibrosis. It’s known that P ⬍ .01 vs. Ang II-treated WT mice.


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IFN-␥, and transforming growth factor (TGF)-␤, which can act on and promote the metabolism and proliferation of cardiac fibroblasts and myocytes (43). However, the molecular mechanisms that regulate the activation of these cells are not fully understood. In the present study, APN KO increased Ang II induced inflammation in heart compared with the WT group, but there was no difference observed when not infused by Ang II in neither WT nor APN KO mice (Figure 2, 3). We have previously shown that Ang II-induced inflammation in vivo is pressure dependent, as the pressure is required for activation of platelet and inflammation (22, 23). Under normal condition, there is no activation of platelet and inflammation, therefore, lack of APN itself does not initiate inflammatory response. Moreover, Fujita et al also showed that there were no differences observed between WT and KO mice when not challenged with Ang II (11). We and others have shown that Ang II stimulates NF-␬B activity and induces proinflammatory cytokine expression (44 – 46). In this study, we showed that Ang II stimulates ROS production, NF-␬B activation and proinflammatory cytokines expression (Figure 4, 5). APN by stimulating mitophagy prevents mitochondria production of ROS, therefore suppress NF-␬B activation. On the other hand, regulation of expression of IL-4, IL-10 or TGF-␤ is complexed, and is controlled by several transcription factors and regulators, including NFAT, NF-IL6, and EGR-1 (47, 48). Even though NF-␬B is also involved in regulation of anti-inflammatory cytokines, but unlike its role in regulating proinflammatory cytokines, which is primarily regulated by p65/ Figure 4. APN inhibits inflammation in macrophages through autophagic pathway. (A) Primary p50 heterodimer of NF-␬B, in vitro mouse macrophages isolated from WT mice were transfected with NF-␬B luciferase reporter and study indicated that the proximal then treated with globular APN (10 ␮g/ml) in the presence or absence of Ang II (100 nM) for 24 hours. NF-␬B activity was determined by luciferase assay. (B) Primary mouse macrophages NF-␬B binding site in IL-10 proisolated from WT mice are treated with or without APN (10 ␮g/ml) in the presence or absence of moter is regulated predominantly by Ang II (100 nM) for 24 hours. The mRNA levels of anti-inflammatory cytokines including IL-10, p50/50 homodimers that activate Mgl-2, Fizzl, Arg-1 were detected by RT-PCR analysis. (C) Primary mouse macrophages isolated from Atg 5-deficient mice were treated with globular APN (10 ␮g/ml) in the presence or absence IL-10 transcription in macrophages. of Ang II for 24 hours. The mRNA levels of anti-inflammatory cytokines were detected as in B. Moreover, p50/50 homodimers-acData represent the mean⫾SEM (n ⫽ 4 – 6 per group). ##P ⬍ .01, ###P ⬍ .001 vs. untreated tivated IL-10 transcription requires control cells; *P ⬍ .05, **P ⬍ .01 vs. untreated control cells; †P ⬍ .05, ††P ⬍ .01 vs. Ang IItreated cells. the transcriptional coactivator APN is primarily produced by subcutaneous fat, and there are also reports of that APN is expressed in human epicardial adipose tissue, for example, Lacobellis et al have reported that the level of APN expression in epicardial fat is negatively associated with CAD (41). Thus, APN may be a protective factor in the development of cardiac fibrosis induced by Ang II. However, the underlying mechanisms remain to be elucidated. Inflammation plays an important role in the initiation and development of cardiac fibrosis (42). Both macrophages and T cells release cytokines such as IL-1␤, TNF-␣,


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CREB-binding protein (49). Indeed, the p50/p50 homodimers of NF-␬B even inhibits transcription of NF-␬Bdependent proinflammatory genes (49). Moreover, expression of anti-inflammatory cytokine IL-10 in Th1 cells is regulated by STAT4 and STAT1 pathways (50). In Th2 cells, IL-10 expression is induced by

IL-4-STAT6 through GATA3 and NFIL3 (51, 52). In Th17 cells, IL-10 expression is induced by IL-6/TGF-␤STAT3 through c-Maf (53). In Th1 cells, IL-10 is induced by IL-27 through induction of C-Maf (53, 54). Therefore, activation of anti-inflammatory genes is not directly regulated by classic NF-␬B, which could be a reason for lack of inhibitory effect of APN. Accumulating evidence indicates that APN exhibits anti-inflammatory effects in various cells and tissues; for example, APN can inhibit NF-␬B activation, thereby effectively reducing TNF-␣-stimulated expression of adhesion molecules and the anti-inflammatory cytokine IL-8 in endothelial cells (55, 56), decreasing TNF-␣ production in human macrophages (57), and increasing the expression of the anti-inflammatory cytokine IL-10 and the tissue inhibitor of metalloproteinase-1 in macrophages (58). Moreover, APN-deficient mice develop larger infarcts that are associated with increased myocardial cell apoptosis and TNF-␣ expression. In contrast, adenovirus-mediated delivery of APN restores these effects in APN-KO mice (39). The protective action of APN against myocardial ischemiareperfusion injury appears to be mediated by activation of cyclooxygenase-2 and inhibition of TNF-␣ production in cardiac cells (39). Moreover, treatment with globular APN inhibits Ang II-induced NF-␬B activation in neonatal rat ventricular myocytes through AMPK (59). In Figure 5. APN protects against Ang II-induced ROS generation and macrophage autophagy. the present study, our findings dem(A) Representative photomicrographs of heart sections from WT and APN KO mice onstrated that APN deficiency markimmunostained for LC3 expression in Ang II infused hearts (left). Magnification: x400. Histogram edly increased Ang II-induced macshows quantified LC3 expression on histological sections (right). Data represent the mean⫾SEM (n ⫽ 6 – 8 per group). **P ⬍ .01 vs. saline-treated WT mice; †P ⬍ .05 vs. Ang II-treated WT mice. rophage infiltration and expression (B) The expression of macrophage marker F4/80 and autophagosome marker LC3 was detected of TNF-␣ and IL-1␤ in cardiac tissue by immunostaining with anti-F4/80 (red) and anti-LC3 (green) antibodies in Ang II infused hearts. (Figure 2), whereas treatment of Magnification: x630. (C) To detect the colocalization of macrophage marker F4/80 and apoptosis macrophages with globular APN sigmarker by immunostaining with anti-F4/80 antibody (red) and TUNEL (green) in Ang II infused hearts. Magnification: x630. Primary mouse macrophages isolated from WT were treated with nificantly inhibited NF-␬B activity globular APN (10 ␮g/ml) in the presence or absence of Ang II (100 nM) for 24 hours. and upregulated the expression of Representative photomicrographs from macrophages immunostained for (D) mitotracker (red) anti-inflammatory cytokines (Figure and DCF (green); (E) LC3 (red) and DAPI (blue). Magnification: x630. Histogram shows quantified LC3-positive cells from randomly 100 cells. (F) Representative electron 4). Thus, APN may act as a key regmicrophotographs of macrophages treated with globular APN (10 ␮g/ml) in the presence or ulator in macrophage activation and absence of Ang II (100 nM) for 24 hours. Arrows indicate cytoplasmic vacuolization the inflammatory response. (Magnification: x20000). #P ⬍ .05 vs. untreated control cells **P ⬍ .01 vs. untreated control

cells; †††P ⬍ .01 vs. Ang II-treated cells.


doi: 10.1210/en.2013-2011

Autophagy is a lysosome degradation pathway that is critical for maintaining cellular homeostasis under stressful conditions (12). Abnormal autophagy causes accumulation of dead cell components, which activate macrophages and T cells to produce proinflammatory cytokines (60). Although autophagy activation is detected in various cardiovascular diseases including hypertrophy, myocardial injury, and atherosclerosis (12), the responsible mechanisms are not fully understood. We showed in this study that APN increased anti-inflammatory cytokines expression (Figure 4B&C) in an autophagy-dependent fashion. This is consistent with the previous report of that APN has anti-inflammatory actions. Takemura et al have shown that APN could facilitate the uptake of early apoptotic cells by macrophages. APN is capable of opsonizing apoptotic cells, and phagocytosis of cell corpses is mediated by the binding of adiponectin to calreticulin on the macrophage cell surface (61). Voll et al reported that uptake of apoptotic cells by macrophages increases their secretion of the anti-inflammatory IL-10 (62). The mTOR signaling pathway negatively regulates autophagy, while AMPK activation leads to mTOR inhibition, thereby promoting autophagy. AMPK is an important kinase downstream of the APN pathway and activated by APN via APN receptor. However, little is known about the role of APN in regu-

Figure 6. APN induces autophagy through AMPK pathway. (A) Primary mouse macrophages isolated from WT mice were pretreated with globular APN (10 ␮g/ml) or Compound C (10 ␮M) for 30 minutes and then stimulated with or without Ang II (100 nM) for additional 10 minutes. The protein levels of phosphor-AMPK and AMPK are determined by Western blot analysis. Bar graph shows densitometric analysis of intensities of p-AMPK/AMPK relative to that of GAPDH. Ang II treatment down-regulates phosphor-AMPK expression in macrophage. (B) Primary mouse macrophages were cultured as in A and then stimulated with or without Ang II (100 nM) for additional 48 hours. The protein level of LC3 is determined by Western blot analysis. Bar graph shows densitometry analysis of intensities of LC3 relative to that of GAPDH. Data represent the mean⫾SEM (n ⫽ 3– 4 per group). **P ⬍ .01 vs. untreated control cells; ##P ⬍ .01 vs. untreated control cells; ††P ⬍ .01 vs. Ang II-treated cells; **P ⬍ .01 vs. APN⫹Ang IItreated cells.


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lating macrophage autophagy. A striking finding of the present study was that APN deficiency ameliorated Ang II-induced macrophage autophagy characterized by increased expression of LC3, an autophagosome marker, whereas treatment of macrophages with APN enhanced the phosphorylation of AMPK and expression of LC3 protein, which was abolished by Compound C (Figure 6). Our recent study has showed that Atg 5⫹/- deficiency aggravates cardiac inflammation and injury via decreasing macrophage mitophagy in response to Ang II (63). Moreover, Atg5 deficiency attenuated APN-mediated anti-inflammatory cytokine expression (Figure 4). Taken together, these results indicate that APN stimulates macrophage autophagy through activation of the AMPK signaling pathway. The effect of APN on macrophage autophagy we observed could be mediated by both type APN receptors, preferentially type I receptor. Tian et al showed that both AdipoR1 and AdipoR2 were found to have differential effects in diminishing proinflammatory responses. While AdipoR1 was required by APN to suppress tumor necrosis factor (TNF) alpha (TNF-␣) and monocyte chemotactic protein 1 (MCP-1) gene expression, AdipoR2 served as the dominant receptor for APN suppression of scavenger receptor A type 1 (SR-AI) and upregulation of interleukin-1 receptor antagonist (IL-1Ra) (64). In another study, Mandal et al reported that full-length APN, acting via the AdipoR2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype, which was also partially dependent on AMP-activated kinase (65). However, our present study does not allow us to determine which types of adiponectin receptor are involved in APN-induced autophagy in macrophage, therefore, future study is required to identify the type of receptor that mediates the effect. In addition to our observed APN effect on macrophage, there are several reports demonstrate that APN also has direct effect on fibroblasts. Nakasone et al showed that APN stimulated synthesis of extracellular matrix in dermal fibroblasts (66). Defects in APN action have been implicated in the development of cardiac dysfunction in obesity and diabetes. Specifically, in cardiac fibroblast, APN stimulated MT1-MMP translocation, MMP2 activity and cell migration of cardiac fibroblasts, linking to heart failure associated with obesity and diabetes (67). Consistent with our finding, Fujita et al showed that increased cardiac fibrosis were observed in Ang II-infused APN KO mice, they further showed that in cultured cardiac fibroblasts, APN stimulated AMPK activity and suppressed Ang II-induced ERK1/2 activity through AMPKdependent PPAR-␣ activation (11). Along with our present study, APN prevents cardiac fibrosis possibly de-


10


pending on its effects on both macrophages and fibroblasts. In summary, we demonstrated that APN deficiency enhanced Ang II-induced cardiac inflammation and fibrosis and decreased autophagy activation in cardiac tissue. In contrast, treatment of macrophages with APN markedly increased AMPK activation-mediated autophagy, and upregulated anti-inflammatory cytokine expression. Our results suggest that APN protects against Ang II-induced cardiac fibrosis at least in part through AMPK-dependent autophagy activation. But one of limitations of our present study is lack of examination of if exogenous APN supplementation would prevent the effect of pathological phenotypes in APN KO mice. The results of the present study may provide a novel therapeutic target against heart fibrosis.

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Acknowledgments

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None. Address all correspondence and requests for reprints to: # Dr. Jie Du, Beijing Anzhen Hospital, Capital Medical University; Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing 100029, China. E-mail: [email protected], Phone: 86 – 010 – 64456030; Fax: 86 – 010 – 64456094. † The two authors contributed equally to this study. Conflict of interest statement: None. This work was supported by grants from Chinese Ministry of Science and Technology (2012CB517802, 2012CB945104), the National Natural Science Foundation of China (81230006, 31090363, 81100094, and 81300121).

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P‑selectin increases angiotensin II‑induced cardiac inflammation and fibrosis via platelet activation.

DcR3 suppresses influenza virus-induced macrophage activation and attenuates pulmonary inflammation and lethality.

GADD34 suppresses lipopolysaccharide-induced sepsis and tissue injury through the regulation of macrophage activation.

Brazilein Suppresses Inflammation through Inactivation of IRAK4-NF-κB Pathway in LPS-Induced Raw264.7 Macrophage Cells.

Tumor necrosis factor: a mechanistic link between angiotensin-II-induced cardiac inflammation and fibrosis.

Inhibition of Toll-like receptor 2 reduces cardiac fibrosis by attenuating macrophage-mediated inflammation.

2 Phosphorylation.

Hyperglycemia Suppresses Calcium Phosphate-Induced Aneurysm Formation Through Inhibition of Macrophage Activation.

Macrophage migration inhibitory factor deletion exacerbates pressure overload-induced cardiac hypertrophy through mitigating autophagy.

2 activation in mouse macrophage Raw264.7 cells.

Macrophage-derived MCPIP1 mediates silica-induced pulmonary fibrosis via autophagy.

mTOR downregulation and autophagy activation.

Adipocyte-derived PAMM suppresses macrophage inflammation by inhibiting MAPK signalling.

Apamin suppresses biliary fibrosis and activation of hepatic stellate cells.

Mitofusin 2-deficiency suppresses cell proliferation through disturbance of autophagy.

Plasminogen activator inhibitor-1 regulates LPS induced inflammation in rat macrophages through autophagy activation.

SH2B3 Is a Genetic Determinant of Cardiac Inflammation and Fibrosis.

Chronic Activation of the Renin-Angiotensin System Induces Lung Fibrosis.

TRPV1 Activation Attenuates High-Salt Diet-Induced Cardiac Hypertrophy and Fibrosis through PPAR-δ Upregulation.

Macrophage activation in acute exacerbation of idiopathic pulmonary fibrosis.

Isosorbide dinitrate inhibits mechanical stress-induced cardiac hypertrophy and autophagy through downregulation of angiotensin II type 1 receptor.

Perivascular adipose tissue-derived adiponectin inhibits collar-induced carotid atherosclerosis by promoting macrophage autophagy.

Adiponectin expression protects against angiotensin II-mediated inflammation and accelerated atherosclerosis.

Molecular imaging of macrophage enzyme activity in cardiac inflammation.