Free Radical Biology and Medicine 69 (2014) 108–115
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Original Contribution
Atg5 deficiency-mediated mitophagy aggravates cardiac inflammation and injury in response to angiotensin II Wei Zhao a, Yulin Li a, Lixin Jia a, Lili Pan a, Huihua Li b,n, Jie Du a,nn a Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing 100029, China b Department of Pathology, Capital Medial University, Beijing 100069, China
art ic l e i nf o
a b s t r a c t
Article history: Received 10 October 2013 Received in revised form 1 January 2014 Accepted 3 January 2014 Available online 10 January 2014
Objective: Hypertension induces end-organ damage through inflammation, and autophagy plays a crucial role in the regulation of cellular homeostasis. In the present study, we aimed to define the role of autophagy in the development of inflammation and cardiac injury induced by angiotensin II (Ang II). Methods and Results: Autophagy protein 5 (Atg5) haplodeficiency (Atg5 þ / ) and age-matched wild-type (WT) C57BL/6 J mice were infused with Ang II (1500 ng/kg/min) or saline for 7 days. Heart sections were stained with hematoxylin and eosin (H&E), Masson0 s trichrome, and immunohistochemical stains. Cytokine and LC3 levels were measured using real-time PCR or western blot analysis. After Ang II infusion, the WT mice exhibited marked macrophage accumulation, cytokine expression, and reactive oxygen species (ROS) production compared with saline-infused controls. However, these effects induced by Ang II infusion were aggravated in Atg5 þ / mice. These effects were associated with Atg5-mediated impaired autophagy, accompanied by increased production of ROS and activation of nuclear factor-κB (NF-κB) in macrophages. Finally, increased cardiac inflammation in Atg5 haplodeficient mice was associated with increased cardiac fibrosis. Conclusion: Atg5 deficiency-mediated autophagy increases ROS production and NF-κB activity in macrophages, thereby contributing to cardiac inflammation and injury. Thus, improving autophagy may be a novel therapeutic strategy to ameliorate hypertension-induced inflammation and organ damage. & 2014 Published by Elsevier Inc.
Keywords: Autophagy protein 5 Mitophagy Inflammation Macrophage
Introduction Activation of the rennin-angiotensin system (RAS) plays critical roles in hypertension, cardiovascular homeostasis, and remodeling [1–3]. Angiotensin II (Ang II) is an octapeptide hormone of this system that can also induce inflammation [4,5]. Inflammation plays an important role in organ damage in hypertension [6]. We and others have shown that hypertension results in cardiac infiltration of proinflammatory cells such as activated macrophages. These cells release various proinflammatory cytokines, which stimulate resident cells to produce excessive extracellular matrix components, thus leading to organ injury [4,7–14]. Autophagy is a lysosomal degradation process that is essential for cell differentiation, survival, and homeostasis [15,16]. Autophagy also regulates inflammation by modulating survival or death of immune
n Corresponding author at: Department of Pathology and Pathophysiology, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China. Fax: þ86 10 83950091. nn Corresponding author at: 2 Anzhen Road, Chaoyang District, Beijing Anzhen Hospital, Institute of Heart Lung and Blood Vessel Diseases, Capital Medical University, Beijing 100029, China. Fax: þ 86 10 64456030. E-mail addresses: [email protected] (H. Li), [email protected] (J. Du)
0891-5849/$ - see front matter & 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.freeradbiomed.2014.01.002
cells such as macrophages [17,18]. The immune system utilizes autophagic degradation of cytoplasmic material to restrict intracellular pathogens and regulates adaptive immunity [19]. Autophagy becomes dysfunctional in atherosclerosis and its deficiency promotes atherosclerosis in part through inflammasome hyperactivation [20]. To avoid inflammation, reactive oxygen species (ROS)-generating mitochondria are constantly removed by mitophagy, a specialized autophagic process [21]. Recently, we demonstrated that cathepsin S deficiency results in autophagosomal abnormalities and aggravates cardiac inflammation in response to Ang II infusion [14]. However, it is unknown whether autophagy directly regulates infiltrating leukocyte homeostasis and contributes to cardiac inflammation and organ damage in Ang II-induced hypertension. Autophagy is a highly regulated process by a set of specific proteins. Autophagy protein 5 (Atg5) is an E3 ubiquitin ligase that is necessary for the formation of autophagosomes and autophagy [16]. Furthermore, Atg5-deficient mice show disorganized sarcomere structure and develop cardiac dysfunction and left ventricular dilatation after pressure overload [22]. In this study, we aimed to determine whether autophagy directly regulates macrophage inflammation and participates in cardiac injury after Ang II treatment. We found that Atg5 haplodeficiency increased inflammation and cardiac fibrosis in response to acute hypertension. Impairment in Ang II-induced autophagy leads to increased
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mitochondrial production of ROS and activation of a nuclear factorκB (NF-κB)-driven inflammatory response.
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IL-1β (1:200), TNF-α (1:200), or transforming growth factor (TGF)β (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA). Images were captured and analyzed. Frozen heart sections underwent double immunofluorescence staining were performed as described [14].
Materials and Methods Ethics Statement Male wild-type (WT) and Atg5 þ / mice in a C57BL/6 background were bred and maintained in the Laboratory of Animal Experiments at Beijing Anzhen Hospital, Capital Medical University. The investigations were approved by the Animal Care and Use Committee of Capital Medical University. Mouse Model of Ang II-Induced Hypertension Hypertension was induced in WT and Atg5 þ / mice (n ¼8 per group) by subcutaneous infusion of Ang II at 1500 ng/kg/min for 7 days via an Alzet Mini-osmotic Pump (MODEL 1007D, DURECT, Cupertino, CA) as described [10,12]. Systolic blood pressures were measured by sphygmomanometer (BP-98 A, Softron, Japan) as described [10,12]. Echocardiography in M-mode was performed in triplicate by use of the Vevo 770 High Resolution Imaging System (Visual Sonics Inc. Toronto, Canada) [11,23]. Histology and Immunohistochemistry All animals were euthanized 7 days after Ang II or saline infusion and hearts were processed and fixed as described [10]. To measure fibrotic areas, sectioned hearts were stained with Masson trichrome as described [14]. Immunohistochemical staining was performed by using the antibodies against Atg5 (1:500) (Abcam, Cambridge, MA), collagen I (1:800), α-smooth muscle actin (α-SMA; 1:200), Mac-2 (1:400),
Western Blot Analysis Proteins were extracted and underwent western blot analysis as described [24]. Images were quantified by use of the Odyssey system (LI-COR Biosciences, Lincoln, NE, USA). RNA Extraction and Real-time PCR RNA was extracted from tissues by the Trizol method (Invitrogen, Carlsbad, CA). Real-time quantitative PCR (qPCR) was performed by using Bio-Rad iQ5 system (Bio-Rad) with SYBR Green I (Takara, Shiga, Japan) and primers used for IL-1β, TNF-α, and GAPDH as describe before [25]. Macrophage Culture and transmission electron microscopy Macrophages were isolated from bone marrow of mice and grown in macrophage colony-stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ) as described [26]. For Ang II treatment, cells were washed with PBS and incubated with Ang II (1 μM) for 24 h. Transmission electron microscopy (TEM) in cells were performed as described [14]. Intracellular ROS Production and Mitochondrial ROS Localization MitoTracker (Invitrogen) was used to label mitochondria as described [27]. MitoSOX (Invitrogen) was used to track mitochondria-specific ROS production as described [28].
Fig. 1. Ang II infusion increases Atg5 expression in the mouse heart. (A) The expression of Atg5 mRNA in the heart tissue at 1, 3 and 7 days after Ang II infusion. (B) Immunohistochemical staining of Ang II-induced Atg5 expression in mice and quantification of Atg5 positivity. Bars, 25 μm. (C) Real-time PCR analysis of mRNA expression of Atg5 in WT and Atg5 þ / mouse hearts after Ang II infusion for 7day. Data are mean7SEM (n¼4 per group). (D) Quantification of Atg5 expression in macrophages (Mφ), cardiac fibroblasts (CF) and cardiomyocytes (CM). Data are mean7SEM (n¼8 per group). nPo0.05 vs. saline control; #Po 0.05 vs. Ang II-infused WT mice.
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NF-κB Luciferase Reporter Assay
Results
NF-κB luciferase reporter activity in macrophages was measured as described before [29]. WT or Atg5 þ / macrophages were infected with adenovirus of NF-κB luciferase reporter for 24 h before Ang II stimulation.
Atg5 haplodeficiency significantly reduces Ang II infusion-induced Atg5 expression in the murine heart
Coculture Experiments Cardiac fibroblasts were prepared from the hearts of WT mice as described [13]. Macrophages from WT or Atg5 þ / mice were cocultured with cardiac fibroblasts in 24-well Transwell inserts with or without Ang II treatment for 48 hours as described [12,25]. Statistical Analysis Data are expressed as mean7SEM. Differences between groups were analyzed by Student0 s t test or ANOVA. Po0.05 was considered statistically significant.
To investigate the role of autophagy in hypertensive cardiac injury, we utilized Atg5 þ / mice and examined whether Atg5 expression was reduced in hypertensive hearts. As shown in Fig. 1 A, Atg5 mRNA expression was markedly upregulated in the wild-type (WT) mouse heart tissue at 1, 3, or 7 days after Ang II infusion. Moreover, immunohistochemistry revealed that the number of Atg5-positive cells in WT Ang II-infused hearts was greater than that of saline-infused controls. However, the number of Atg5-positive cells in Ang II-treated Atg5 þ / hearts was significantly less increased (Fig. 1B). After Ang II infusion for 7 days, Atg5 mRNA expression was also markedly decreased in Atg5 þ / hearts than that in WT heart (Fig. 1C). To determine which cells predominantly express Atg5, qPCR was performed in macrophages, cardiac fibroblasts (CF), and cardiomyocytes (CM).
Fig. 2. Atg5 haplodeficiency increases the infiltration of inflammatory cells in the heart. (A) Representative hematoxylin and eosin staining of WT and Atg5 þ / hearts. Bars, 50 μm. (B) Immunohistochemical staining and quantification of Mac-2 positivity. Bars, 25 μm. Real-time PCR analysis of mRNA expression of tumor necrosis factor (TNF)-α (C) and interleukin (IL)-1β (D) in WT and Atg5 þ / mouse hearts. Data are mean7SEM (n¼4 per group). nPo0.05 vs. saline WT control; #Po0.05 vs. Ang II-infused WT mice.
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As shown in Fig. 1D, Ang II infusion markedly increased the Atg5 mRNA level in the macrophages, but not the CFs and CMs compared with the saline controls. Atg5 haplodeficiency increases inflammatory cell infiltration into the heart To examine whether reduced Atg5 expression affects Ang IIinduced hypertension and cardiac function, WT and Atg5 þ / mice were infused with Ang II or saline for 7 days. Blood pressure and heart function were measured using a noninvasive tail-cuff method and echocardiography. Systolic blood pressure and cardiac function reflected by FS% and EF% (data not shown) were also similar between the two groups after saline or Ang II infusion. Since inflammatory cell infiltration is an early response of Ang II-induced cardiac injury [6,8–14], we next examined the effect of
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Atg5 haplodeficiency on the accumulation of proinflammatory cells in the heart using H&E staining. Ang II infusion markedly increased the infiltration of inflammatory cells in WT hearts, and this effect was further increased in Atg5 þ / hearts (Fig. 2A). Moreover, immunohistochemistry demonstrated that the number of Mac-2-positive cells (a marker of macrophages) was significantly higher in Atg5 þ / than that in WT hearts (Fig. 2B). The expression of proinflammatory cytokines such as TNF-α and IL-1β was also markedly higher in Ang II-infused Atg5 þ / than that in WT hearts (Figs. 2C and D). Atg5 haplodeficiency decreases Ang II-induced mitophagy in macrophages Autophagy plays a major role in immunity and the inflammatory response [30,31]. Therefore, to examine whether autophagy is
Fig. 3. Atg5 haplodeficiency decreases the Ang II-induced mitophagy in macrophages. (A) Immunostaining for LC3 and F4/80 in mouse heart. Bars, 10 μm. (B) Real-time PCR analysis of mRNA expression of LC3 in WT and Atg5 þ / mouse hearts. (C) Immunostaining for LC3 and F4/80 in macrophages. Bars, 5 μm. (D) Western blot analysis of LC3 protein expression in macrophages. (E) Representative electron microphotographs of cytoplasmic vacuolization in macrophages treated with saline or Ang II (1 μM) for 24 hr. Bars, 500 nm. N, nucleus; Mi, mitochondrion; G, Golgi apparatus; Lys, lysosome; ER, endoplasmic reticulum; Red arrows point to autophagy vacuoles. (F) Immunostaining for LC3 and mitochondria marker MitoTracker in macrophages. Bars, 2 μm. Data are mean 7 SEM (n¼4 per group). nPo 0.05 vs. saline WT control; #Po 0.05 vs. Ang II-infused WT mice.
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induced in macrophages in response to Ang II, immunofluorescence staining was performed. Ang II infusion induced colocalization of the autophagosomal marker LC3 (Atg8) and macrophage marker F4/80 in WT hearts, and this effect was significantly decreased in Atg5 þ / hearts (Fig. 3A). LC3 mRNA expression was also markedly upregulated in WT hearts relative to that of Atg5 þ / hearts after Ang II infusion (Fig. 3B). Confocal analysis further showed that Ang II treatment markedly stimulated LC3 protein clustering in cultured WT macrophages, which was further reduced in Atg5 þ / macrophages (Fig. 3C). It is known that cleavage of soluble LC3 (LC3-I) to form LC3-II is associated with the extent of autophagosome formation [32,33], thus the ratio of LC3-II/LC3-I was measured. Ang II treatment increased the LC3-II/LC3-I ratio in WT macrophages, which was further decreased with Atg5 haplodeficiency (Fig. 3D). Furthermore, transmission electron microscopy (TEM) revealed cytosolic autophagic vacuole formation in a number of WT macrophages, which was decreased in Atg5 þ / macrophages (Fig. 3E). To examine if Atg5 deficiency is associated with abnormal mitophagy, we used MitoTracker to track accumulation of LC3 in mitochondria. As shown in Figure 3 F, decreased Atg5 expression was associated with decreased mitophagy in macrophages. Reduced mitophagy induces ROS production and NF-κB activation Ang II stimulates mitochondria ROS production and inflammation in vascular cells [34,35]. MitoSOX Red was used to measure mitochondria-specific ROS levels. As shown in Fig. 4A, the level of red fluorescence was significantly increased in the mitochondria of Atg5 þ / macrophages after Ang II (1 μM) treatment. Moreover, flow cytometry was used to quantify the number of MitoSOX Redpositive cells. Knockdown of Atg5 in cells significantly increased the number of MitoSOX Red-positive cells (Fig. 4B), indicating that Atg5 plays an important role in regulating mitochondria-specific ROS production. ROS is an important trigger of inflammatory transcriptional NFκB activation [36]. To examine whether Atg5 knockdown is associated with aggravated cardiac inflammation, we detected phosphorylated NF-κB/p65 using immunofluorescence staining.
Phosphorylation of p65 was increased in Atg5 þ / macrophages than that in WT macrophages following Ang II stimulation (Fig. 4C). To detect NF-κB transcriptional activity, macrophages were infected with a recombinant adenovirus containing a NF-κBLuc reporter. Ang II significantly increased NF-κB luciferase activity in Atg5 þ / macrophages (Fig. 4D). Atg5 haplodeficiency enhances Ang II-induced cardiac damage Since enhanced inflammation contributes to cardiac damage [6,8–14], the extent of cardiac fibrosis in autophagy-deficient hearts was detected using Masson0 s trichrome staining. Ang II infusion markedly increased the fibrotic area size in WT hearts, which was further increased in Ang II-infused Atg5 þ / hearts (Fig. 5A). The expressions of collagen I, TGF-β, and α-SMA (a marker for myofibroblasts) were significantly increased in Ang II-infused WT hearts, which was further enhanced in Ang IIinfused Atg5 þ / hearts (Fig. 5B). Atg5 haplodeficiency in macrophages induces cardiac myofibroblast transformation To investigate the role of macrophage Atg5 activation in cardiac fibrosis, we created an in vitro coculture system of CFs and macrophages. Immunofluorescence analysis revealed that the expression of α-SMA was markedly increased in fibroblasts cocultured with Atg5 þ / macrophages in response to Ang II (Fig. 6A). Real-time PCR further confirmed that coculture of fibroblasts with Atg5 þ / macrophages significantly unregulated the mRNA expression of α-SMA, collagen I, and TGF-β, whereas incubation with WT macrophages attenuated these effects (Fig. 6B–D). Thus, Atg5 þ / macrophages show increased myofibroblast differentiation, thereby inducing cardiac fibrosis.
Discussion In the present study, we established that autophagy regulates Ang II-induced cardiac inflammation and cardiac injury.
Fig. 4. Atg5 haplodeficiency induces ROS production and NF-κB activation. (A) MitoSOX Red fluorescence of mitochondria-specific ROS production were detected by confocal microscopy after Ang II treatment (2 h). Bars, 2 μm. (B) Flow cytometry was used to quantify MitoSOX Red positive macrophages (%). (C) Immunohistochemical staining of NF-κB/p65. Bars, 10 μm. (D) Transcriptional activity of NF-κB. Data are mean7 SEM (n¼ 6 per group). *Po 0.05 vs. WT control macrophages. #Po 0.05 vs. Ang II-treated WT macrophages.
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Fig. 5. Atg5 haplodeficiency increases Ang II-induced cardiac damage. (A) Representative Masson trichrome staining of WT and Atg5 þ / hearts with saline or Ang II infusion and quantitative analysis of fibrotic areas. Bars, 25 μm (B) Immunohistochemical staining and quantification of collagen I, TGF-β and α-SMA in WT and Atg5 þ / hearts with saline or Ang II infusion. Bars, 25 μm. Data are mean 7SEM (n ¼4 per group). nP o0.05 vs. saline WT control; #P o 0.05 vs. Ang II-infused WT mice.
Our results demonstrate that Atg5 haplodeficiency decreases mitophagy, increases mitochondrial ROS production, and activates NF-κB in macrophages, which leads to macrophage infiltration and expression of proinflammatory cytokines, which results in subsequent cardiac injury. Inflammation plays an important role in the initiation and development of cardiac remodeling [8]. Activation of macrophages is a key step in the pathogenesis of cardiac injury [6,7,14]. Ang II can affect the immune response by amplifying the expression of cytokines and chemokines in macrophages, regulating dendritic cell differentiation, and promoting lymphocyte proliferation [5,24,25,37]. Inflammation is regulated by the presence of immune cells such as macrophages and released inflammatory mediators such as cytokines and chemokines [5,13,14]. Cytokines such as IL-1β, IL-6, IL-10, interferon (IFN)-γ, TNF-α, and TGF-β are significantly increased in the Ang II-treated cardiovascular system and exhibit pro- and anti-fibrotic actions [4,24,38]. Atg5 is a key protein that can conjugate to Atg12 and then form a 800-kDa complex with the multimeric protein Atg16. The Atg12-Atg5-Atg16 complex is required for autophagosome formation [39–41]. In the present study, we demonstrated that Atg5 haplodeficiency decreases autophagy but significantly increases Ang II-induced cardiac inflammation (Figs. 2 and 3). These results establish a link among autophagy, activation of macrophages and Ang II-induced cardiac inflammation. It is known that impaired autophagy link to inflammation [42], for example, in response to Salmonella infection, inhibition of mitophagy (a specialized autophagic process) leads to the accumulation of ROS-producing damaged mitochondria and consequentially
to inflammasome activation [21]. We recently reported that protease cathepsin S deficiency led to impaired degradation of lysosomal mitochondria and accumulation of abnormal autophagosomes, leading to increased production of ROS and inflammation [14]. In this study, we showed that Ang II infusion stimulated mitophagy in the heart (Figure3F), and Atg5 haplodeficiency significantly reduced mitophagy (Fig. 3F). Our results also showed that the aforementioned link could be mitochondria ROS production (Figs. 4A and 4B). Damaged mitochondria release ROS, which causes inflammatory responses [43]. ROS are important triggers of NF-κB transcriptional activity and inflammation [44–46]. We found that Ang II-induced ROS production, NF-κB/p65 phosphorylation, and NF-κB activity were increased in the macrophages of Atg5 þ / mice relative to those in WT mouse macrophages (Fig. 4). Atg5 knockdown triggered abnormal mitophagy (mitochondria colocalized with LC3) (Fig. 3F), these results imply a role of Atg5 in regulating ROS production and inflammation. In conclusion, we provide a novel role of autophagy in regulating cardiac injury and the inflammatory response to Ang II via modulation of mitophagy formation, ROS production, and NF-κB activation.
Source of Funding This study was supported by grants from the National Natural Science Foundation of China (81230006, 31090363, 81130001 and 81100094), the Chinese Ministry of Science and Technology (2012CB945104), the Program for Changjiang Scholars and
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Fig. 6. Atg5 þ / macrophages induce cardiac fibroblast transformation. (A) WT or Atg5 þ / macrophages (Mφ) were cocultured with cardiac fibroblasts (CF) with or without angiotensin II (Ang II), then underwent immunofluorescence analysis of myofibroblast differentiation by staining with α-SMA antibody. Bar ¼ 5 μm. Real-time PCR analysis of the mRNA expression of α-SMA (B) and collagen I (C) and TGF-β (D). Data are mean 7 SEM (n ¼4 per group). nPo 0.05 vs. coculture with WT control macrophages. #Po 0.05 vs. coculture with Ang II-treated WT macrophages.
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Original Contribution
Atg5 deficiency-mediated mitophagy aggravates cardiac inflammation and injury in response to angiotensin II Wei Zhao a, Yulin Li a, Lixin Jia a, Lili Pan a, Huihua Li b,n, Jie Du a,nn a Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing 100029, China b Department of Pathology, Capital Medial University, Beijing 100069, China
art ic l e i nf o
a b s t r a c t
Article history: Received 10 October 2013 Received in revised form 1 January 2014 Accepted 3 January 2014 Available online 10 January 2014
Objective: Hypertension induces end-organ damage through inflammation, and autophagy plays a crucial role in the regulation of cellular homeostasis. In the present study, we aimed to define the role of autophagy in the development of inflammation and cardiac injury induced by angiotensin II (Ang II). Methods and Results: Autophagy protein 5 (Atg5) haplodeficiency (Atg5 þ / ) and age-matched wild-type (WT) C57BL/6 J mice were infused with Ang II (1500 ng/kg/min) or saline for 7 days. Heart sections were stained with hematoxylin and eosin (H&E), Masson0 s trichrome, and immunohistochemical stains. Cytokine and LC3 levels were measured using real-time PCR or western blot analysis. After Ang II infusion, the WT mice exhibited marked macrophage accumulation, cytokine expression, and reactive oxygen species (ROS) production compared with saline-infused controls. However, these effects induced by Ang II infusion were aggravated in Atg5 þ / mice. These effects were associated with Atg5-mediated impaired autophagy, accompanied by increased production of ROS and activation of nuclear factor-κB (NF-κB) in macrophages. Finally, increased cardiac inflammation in Atg5 haplodeficient mice was associated with increased cardiac fibrosis. Conclusion: Atg5 deficiency-mediated autophagy increases ROS production and NF-κB activity in macrophages, thereby contributing to cardiac inflammation and injury. Thus, improving autophagy may be a novel therapeutic strategy to ameliorate hypertension-induced inflammation and organ damage. & 2014 Published by Elsevier Inc.
Keywords: Autophagy protein 5 Mitophagy Inflammation Macrophage
Introduction Activation of the rennin-angiotensin system (RAS) plays critical roles in hypertension, cardiovascular homeostasis, and remodeling [1–3]. Angiotensin II (Ang II) is an octapeptide hormone of this system that can also induce inflammation [4,5]. Inflammation plays an important role in organ damage in hypertension [6]. We and others have shown that hypertension results in cardiac infiltration of proinflammatory cells such as activated macrophages. These cells release various proinflammatory cytokines, which stimulate resident cells to produce excessive extracellular matrix components, thus leading to organ injury [4,7–14]. Autophagy is a lysosomal degradation process that is essential for cell differentiation, survival, and homeostasis [15,16]. Autophagy also regulates inflammation by modulating survival or death of immune
n Corresponding author at: Department of Pathology and Pathophysiology, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China. Fax: þ86 10 83950091. nn Corresponding author at: 2 Anzhen Road, Chaoyang District, Beijing Anzhen Hospital, Institute of Heart Lung and Blood Vessel Diseases, Capital Medical University, Beijing 100029, China. Fax: þ 86 10 64456030. E-mail addresses: [email protected] (H. Li), [email protected] (J. Du)
0891-5849/$ - see front matter & 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.freeradbiomed.2014.01.002
cells such as macrophages [17,18]. The immune system utilizes autophagic degradation of cytoplasmic material to restrict intracellular pathogens and regulates adaptive immunity [19]. Autophagy becomes dysfunctional in atherosclerosis and its deficiency promotes atherosclerosis in part through inflammasome hyperactivation [20]. To avoid inflammation, reactive oxygen species (ROS)-generating mitochondria are constantly removed by mitophagy, a specialized autophagic process [21]. Recently, we demonstrated that cathepsin S deficiency results in autophagosomal abnormalities and aggravates cardiac inflammation in response to Ang II infusion [14]. However, it is unknown whether autophagy directly regulates infiltrating leukocyte homeostasis and contributes to cardiac inflammation and organ damage in Ang II-induced hypertension. Autophagy is a highly regulated process by a set of specific proteins. Autophagy protein 5 (Atg5) is an E3 ubiquitin ligase that is necessary for the formation of autophagosomes and autophagy [16]. Furthermore, Atg5-deficient mice show disorganized sarcomere structure and develop cardiac dysfunction and left ventricular dilatation after pressure overload [22]. In this study, we aimed to determine whether autophagy directly regulates macrophage inflammation and participates in cardiac injury after Ang II treatment. We found that Atg5 haplodeficiency increased inflammation and cardiac fibrosis in response to acute hypertension. Impairment in Ang II-induced autophagy leads to increased
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mitochondrial production of ROS and activation of a nuclear factorκB (NF-κB)-driven inflammatory response.
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IL-1β (1:200), TNF-α (1:200), or transforming growth factor (TGF)β (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA). Images were captured and analyzed. Frozen heart sections underwent double immunofluorescence staining were performed as described [14].
Materials and Methods Ethics Statement Male wild-type (WT) and Atg5 þ / mice in a C57BL/6 background were bred and maintained in the Laboratory of Animal Experiments at Beijing Anzhen Hospital, Capital Medical University. The investigations were approved by the Animal Care and Use Committee of Capital Medical University. Mouse Model of Ang II-Induced Hypertension Hypertension was induced in WT and Atg5 þ / mice (n ¼8 per group) by subcutaneous infusion of Ang II at 1500 ng/kg/min for 7 days via an Alzet Mini-osmotic Pump (MODEL 1007D, DURECT, Cupertino, CA) as described [10,12]. Systolic blood pressures were measured by sphygmomanometer (BP-98 A, Softron, Japan) as described [10,12]. Echocardiography in M-mode was performed in triplicate by use of the Vevo 770 High Resolution Imaging System (Visual Sonics Inc. Toronto, Canada) [11,23]. Histology and Immunohistochemistry All animals were euthanized 7 days after Ang II or saline infusion and hearts were processed and fixed as described [10]. To measure fibrotic areas, sectioned hearts were stained with Masson trichrome as described [14]. Immunohistochemical staining was performed by using the antibodies against Atg5 (1:500) (Abcam, Cambridge, MA), collagen I (1:800), α-smooth muscle actin (α-SMA; 1:200), Mac-2 (1:400),
Western Blot Analysis Proteins were extracted and underwent western blot analysis as described [24]. Images were quantified by use of the Odyssey system (LI-COR Biosciences, Lincoln, NE, USA). RNA Extraction and Real-time PCR RNA was extracted from tissues by the Trizol method (Invitrogen, Carlsbad, CA). Real-time quantitative PCR (qPCR) was performed by using Bio-Rad iQ5 system (Bio-Rad) with SYBR Green I (Takara, Shiga, Japan) and primers used for IL-1β, TNF-α, and GAPDH as describe before [25]. Macrophage Culture and transmission electron microscopy Macrophages were isolated from bone marrow of mice and grown in macrophage colony-stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ) as described [26]. For Ang II treatment, cells were washed with PBS and incubated with Ang II (1 μM) for 24 h. Transmission electron microscopy (TEM) in cells were performed as described [14]. Intracellular ROS Production and Mitochondrial ROS Localization MitoTracker (Invitrogen) was used to label mitochondria as described [27]. MitoSOX (Invitrogen) was used to track mitochondria-specific ROS production as described [28].
Fig. 1. Ang II infusion increases Atg5 expression in the mouse heart. (A) The expression of Atg5 mRNA in the heart tissue at 1, 3 and 7 days after Ang II infusion. (B) Immunohistochemical staining of Ang II-induced Atg5 expression in mice and quantification of Atg5 positivity. Bars, 25 μm. (C) Real-time PCR analysis of mRNA expression of Atg5 in WT and Atg5 þ / mouse hearts after Ang II infusion for 7day. Data are mean7SEM (n¼4 per group). (D) Quantification of Atg5 expression in macrophages (Mφ), cardiac fibroblasts (CF) and cardiomyocytes (CM). Data are mean7SEM (n¼8 per group). nPo0.05 vs. saline control; #Po 0.05 vs. Ang II-infused WT mice.
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NF-κB Luciferase Reporter Assay
Results
NF-κB luciferase reporter activity in macrophages was measured as described before [29]. WT or Atg5 þ / macrophages were infected with adenovirus of NF-κB luciferase reporter for 24 h before Ang II stimulation.
Atg5 haplodeficiency significantly reduces Ang II infusion-induced Atg5 expression in the murine heart
Coculture Experiments Cardiac fibroblasts were prepared from the hearts of WT mice as described [13]. Macrophages from WT or Atg5 þ / mice were cocultured with cardiac fibroblasts in 24-well Transwell inserts with or without Ang II treatment for 48 hours as described [12,25]. Statistical Analysis Data are expressed as mean7SEM. Differences between groups were analyzed by Student0 s t test or ANOVA. Po0.05 was considered statistically significant.
To investigate the role of autophagy in hypertensive cardiac injury, we utilized Atg5 þ / mice and examined whether Atg5 expression was reduced in hypertensive hearts. As shown in Fig. 1 A, Atg5 mRNA expression was markedly upregulated in the wild-type (WT) mouse heart tissue at 1, 3, or 7 days after Ang II infusion. Moreover, immunohistochemistry revealed that the number of Atg5-positive cells in WT Ang II-infused hearts was greater than that of saline-infused controls. However, the number of Atg5-positive cells in Ang II-treated Atg5 þ / hearts was significantly less increased (Fig. 1B). After Ang II infusion for 7 days, Atg5 mRNA expression was also markedly decreased in Atg5 þ / hearts than that in WT heart (Fig. 1C). To determine which cells predominantly express Atg5, qPCR was performed in macrophages, cardiac fibroblasts (CF), and cardiomyocytes (CM).
Fig. 2. Atg5 haplodeficiency increases the infiltration of inflammatory cells in the heart. (A) Representative hematoxylin and eosin staining of WT and Atg5 þ / hearts. Bars, 50 μm. (B) Immunohistochemical staining and quantification of Mac-2 positivity. Bars, 25 μm. Real-time PCR analysis of mRNA expression of tumor necrosis factor (TNF)-α (C) and interleukin (IL)-1β (D) in WT and Atg5 þ / mouse hearts. Data are mean7SEM (n¼4 per group). nPo0.05 vs. saline WT control; #Po0.05 vs. Ang II-infused WT mice.
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As shown in Fig. 1D, Ang II infusion markedly increased the Atg5 mRNA level in the macrophages, but not the CFs and CMs compared with the saline controls. Atg5 haplodeficiency increases inflammatory cell infiltration into the heart To examine whether reduced Atg5 expression affects Ang IIinduced hypertension and cardiac function, WT and Atg5 þ / mice were infused with Ang II or saline for 7 days. Blood pressure and heart function were measured using a noninvasive tail-cuff method and echocardiography. Systolic blood pressure and cardiac function reflected by FS% and EF% (data not shown) were also similar between the two groups after saline or Ang II infusion. Since inflammatory cell infiltration is an early response of Ang II-induced cardiac injury [6,8–14], we next examined the effect of
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Atg5 haplodeficiency on the accumulation of proinflammatory cells in the heart using H&E staining. Ang II infusion markedly increased the infiltration of inflammatory cells in WT hearts, and this effect was further increased in Atg5 þ / hearts (Fig. 2A). Moreover, immunohistochemistry demonstrated that the number of Mac-2-positive cells (a marker of macrophages) was significantly higher in Atg5 þ / than that in WT hearts (Fig. 2B). The expression of proinflammatory cytokines such as TNF-α and IL-1β was also markedly higher in Ang II-infused Atg5 þ / than that in WT hearts (Figs. 2C and D). Atg5 haplodeficiency decreases Ang II-induced mitophagy in macrophages Autophagy plays a major role in immunity and the inflammatory response [30,31]. Therefore, to examine whether autophagy is
Fig. 3. Atg5 haplodeficiency decreases the Ang II-induced mitophagy in macrophages. (A) Immunostaining for LC3 and F4/80 in mouse heart. Bars, 10 μm. (B) Real-time PCR analysis of mRNA expression of LC3 in WT and Atg5 þ / mouse hearts. (C) Immunostaining for LC3 and F4/80 in macrophages. Bars, 5 μm. (D) Western blot analysis of LC3 protein expression in macrophages. (E) Representative electron microphotographs of cytoplasmic vacuolization in macrophages treated with saline or Ang II (1 μM) for 24 hr. Bars, 500 nm. N, nucleus; Mi, mitochondrion; G, Golgi apparatus; Lys, lysosome; ER, endoplasmic reticulum; Red arrows point to autophagy vacuoles. (F) Immunostaining for LC3 and mitochondria marker MitoTracker in macrophages. Bars, 2 μm. Data are mean 7 SEM (n¼4 per group). nPo 0.05 vs. saline WT control; #Po 0.05 vs. Ang II-infused WT mice.
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induced in macrophages in response to Ang II, immunofluorescence staining was performed. Ang II infusion induced colocalization of the autophagosomal marker LC3 (Atg8) and macrophage marker F4/80 in WT hearts, and this effect was significantly decreased in Atg5 þ / hearts (Fig. 3A). LC3 mRNA expression was also markedly upregulated in WT hearts relative to that of Atg5 þ / hearts after Ang II infusion (Fig. 3B). Confocal analysis further showed that Ang II treatment markedly stimulated LC3 protein clustering in cultured WT macrophages, which was further reduced in Atg5 þ / macrophages (Fig. 3C). It is known that cleavage of soluble LC3 (LC3-I) to form LC3-II is associated with the extent of autophagosome formation [32,33], thus the ratio of LC3-II/LC3-I was measured. Ang II treatment increased the LC3-II/LC3-I ratio in WT macrophages, which was further decreased with Atg5 haplodeficiency (Fig. 3D). Furthermore, transmission electron microscopy (TEM) revealed cytosolic autophagic vacuole formation in a number of WT macrophages, which was decreased in Atg5 þ / macrophages (Fig. 3E). To examine if Atg5 deficiency is associated with abnormal mitophagy, we used MitoTracker to track accumulation of LC3 in mitochondria. As shown in Figure 3 F, decreased Atg5 expression was associated with decreased mitophagy in macrophages. Reduced mitophagy induces ROS production and NF-κB activation Ang II stimulates mitochondria ROS production and inflammation in vascular cells [34,35]. MitoSOX Red was used to measure mitochondria-specific ROS levels. As shown in Fig. 4A, the level of red fluorescence was significantly increased in the mitochondria of Atg5 þ / macrophages after Ang II (1 μM) treatment. Moreover, flow cytometry was used to quantify the number of MitoSOX Redpositive cells. Knockdown of Atg5 in cells significantly increased the number of MitoSOX Red-positive cells (Fig. 4B), indicating that Atg5 plays an important role in regulating mitochondria-specific ROS production. ROS is an important trigger of inflammatory transcriptional NFκB activation [36]. To examine whether Atg5 knockdown is associated with aggravated cardiac inflammation, we detected phosphorylated NF-κB/p65 using immunofluorescence staining.
Phosphorylation of p65 was increased in Atg5 þ / macrophages than that in WT macrophages following Ang II stimulation (Fig. 4C). To detect NF-κB transcriptional activity, macrophages were infected with a recombinant adenovirus containing a NF-κBLuc reporter. Ang II significantly increased NF-κB luciferase activity in Atg5 þ / macrophages (Fig. 4D). Atg5 haplodeficiency enhances Ang II-induced cardiac damage Since enhanced inflammation contributes to cardiac damage [6,8–14], the extent of cardiac fibrosis in autophagy-deficient hearts was detected using Masson0 s trichrome staining. Ang II infusion markedly increased the fibrotic area size in WT hearts, which was further increased in Ang II-infused Atg5 þ / hearts (Fig. 5A). The expressions of collagen I, TGF-β, and α-SMA (a marker for myofibroblasts) were significantly increased in Ang II-infused WT hearts, which was further enhanced in Ang IIinfused Atg5 þ / hearts (Fig. 5B). Atg5 haplodeficiency in macrophages induces cardiac myofibroblast transformation To investigate the role of macrophage Atg5 activation in cardiac fibrosis, we created an in vitro coculture system of CFs and macrophages. Immunofluorescence analysis revealed that the expression of α-SMA was markedly increased in fibroblasts cocultured with Atg5 þ / macrophages in response to Ang II (Fig. 6A). Real-time PCR further confirmed that coculture of fibroblasts with Atg5 þ / macrophages significantly unregulated the mRNA expression of α-SMA, collagen I, and TGF-β, whereas incubation with WT macrophages attenuated these effects (Fig. 6B–D). Thus, Atg5 þ / macrophages show increased myofibroblast differentiation, thereby inducing cardiac fibrosis.
Discussion In the present study, we established that autophagy regulates Ang II-induced cardiac inflammation and cardiac injury.
Fig. 4. Atg5 haplodeficiency induces ROS production and NF-κB activation. (A) MitoSOX Red fluorescence of mitochondria-specific ROS production were detected by confocal microscopy after Ang II treatment (2 h). Bars, 2 μm. (B) Flow cytometry was used to quantify MitoSOX Red positive macrophages (%). (C) Immunohistochemical staining of NF-κB/p65. Bars, 10 μm. (D) Transcriptional activity of NF-κB. Data are mean7 SEM (n¼ 6 per group). *Po 0.05 vs. WT control macrophages. #Po 0.05 vs. Ang II-treated WT macrophages.
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Fig. 5. Atg5 haplodeficiency increases Ang II-induced cardiac damage. (A) Representative Masson trichrome staining of WT and Atg5 þ / hearts with saline or Ang II infusion and quantitative analysis of fibrotic areas. Bars, 25 μm (B) Immunohistochemical staining and quantification of collagen I, TGF-β and α-SMA in WT and Atg5 þ / hearts with saline or Ang II infusion. Bars, 25 μm. Data are mean 7SEM (n ¼4 per group). nP o0.05 vs. saline WT control; #P o 0.05 vs. Ang II-infused WT mice.
Our results demonstrate that Atg5 haplodeficiency decreases mitophagy, increases mitochondrial ROS production, and activates NF-κB in macrophages, which leads to macrophage infiltration and expression of proinflammatory cytokines, which results in subsequent cardiac injury. Inflammation plays an important role in the initiation and development of cardiac remodeling [8]. Activation of macrophages is a key step in the pathogenesis of cardiac injury [6,7,14]. Ang II can affect the immune response by amplifying the expression of cytokines and chemokines in macrophages, regulating dendritic cell differentiation, and promoting lymphocyte proliferation [5,24,25,37]. Inflammation is regulated by the presence of immune cells such as macrophages and released inflammatory mediators such as cytokines and chemokines [5,13,14]. Cytokines such as IL-1β, IL-6, IL-10, interferon (IFN)-γ, TNF-α, and TGF-β are significantly increased in the Ang II-treated cardiovascular system and exhibit pro- and anti-fibrotic actions [4,24,38]. Atg5 is a key protein that can conjugate to Atg12 and then form a 800-kDa complex with the multimeric protein Atg16. The Atg12-Atg5-Atg16 complex is required for autophagosome formation [39–41]. In the present study, we demonstrated that Atg5 haplodeficiency decreases autophagy but significantly increases Ang II-induced cardiac inflammation (Figs. 2 and 3). These results establish a link among autophagy, activation of macrophages and Ang II-induced cardiac inflammation. It is known that impaired autophagy link to inflammation [42], for example, in response to Salmonella infection, inhibition of mitophagy (a specialized autophagic process) leads to the accumulation of ROS-producing damaged mitochondria and consequentially
to inflammasome activation [21]. We recently reported that protease cathepsin S deficiency led to impaired degradation of lysosomal mitochondria and accumulation of abnormal autophagosomes, leading to increased production of ROS and inflammation [14]. In this study, we showed that Ang II infusion stimulated mitophagy in the heart (Figure3F), and Atg5 haplodeficiency significantly reduced mitophagy (Fig. 3F). Our results also showed that the aforementioned link could be mitochondria ROS production (Figs. 4A and 4B). Damaged mitochondria release ROS, which causes inflammatory responses [43]. ROS are important triggers of NF-κB transcriptional activity and inflammation [44–46]. We found that Ang II-induced ROS production, NF-κB/p65 phosphorylation, and NF-κB activity were increased in the macrophages of Atg5 þ / mice relative to those in WT mouse macrophages (Fig. 4). Atg5 knockdown triggered abnormal mitophagy (mitochondria colocalized with LC3) (Fig. 3F), these results imply a role of Atg5 in regulating ROS production and inflammation. In conclusion, we provide a novel role of autophagy in regulating cardiac injury and the inflammatory response to Ang II via modulation of mitophagy formation, ROS production, and NF-κB activation.
Source of Funding This study was supported by grants from the National Natural Science Foundation of China (81230006, 31090363, 81130001 and 81100094), the Chinese Ministry of Science and Technology (2012CB945104), the Program for Changjiang Scholars and
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Fig. 6. Atg5 þ / macrophages induce cardiac fibroblast transformation. (A) WT or Atg5 þ / macrophages (Mφ) were cocultured with cardiac fibroblasts (CF) with or without angiotensin II (Ang II), then underwent immunofluorescence analysis of myofibroblast differentiation by staining with α-SMA antibody. Bar ¼ 5 μm. Real-time PCR analysis of the mRNA expression of α-SMA (B) and collagen I (C) and TGF-β (D). Data are mean 7 SEM (n ¼4 per group). nPo 0.05 vs. coculture with WT control macrophages. #Po 0.05 vs. coculture with Ang II-treated WT macrophages.
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