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ACE inhibitor suppresses cardiac remodeling after myocardial infarction by regulating dendritic cells and AT2 receptor-mediated mechanism in mice
Biomedicine & Pharmacotherapy 114 (2019) 108660
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Original article
ACE inhibitor suppresses cardiac remodeling after myocardial infarction by regulating dendritic cells and AT2 receptor-mediated mechanism in mice Yuanji Maa,b,1, Jie Yuanb,1, Jialu Hua,b,1, Wei Gaoa,b,1, Yunzeng Zoua,b, Junbo Gea,b, a b
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Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai, China Shanghai Institute of Cardiovascular Diseases, Shanghai, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Dendritic cell ACE inhibitor Myocardial infarction AT2 receptor Inflammation
Dendritic cells (DCs) play a complex role in the progression of myocardial infarction (MI). The impact of angiotensin-converting enzyme (ACE) inhibitor therapy, partly via affecting DCs maturation and recruitment, was tested on a MI mouse model. Furthermore, the cardioprotective effects of ACEI were enhanced through attenuating migration of DCs from the spleen into peripheral circulation, thereby inhibiting DCs maturation and tissue inflammation. ACEI repress DCs immune inflammatory response through down-regulating DCs maturation surface markers and regulating inflammatory cytokines, which led to a higher survival rate, improved function and remodeling through decreased inflammatory response after MI. However, inhibition of AT2R activation, resulted in a reduction of ACEI effects on DCs. The potent anti-inflammatory effect of ACEI can partially be attributed to its impact on DCs through activation of AT2R, which may provide a new target mechanism for ACEI therapy after MI.
1. Introduction Dendritic cell (DC) is a potent central immunoregulator that orchestrates various types of inflammatory cells in innate and adoptive immunity [1–3]. Emerging evidence suggest that DCs are infiltrated into infarcted hearts in experimental myocardial infarction (MI) models [4]. Recent studies reported that an increased number of DCs in the infarcted heart is associated with deterioration of left ventricular (LV) remodeling [5] and blunting DCs mobilization after MI has favorable effects on survival and LV remodeling [6]. These observations indicate the potential role of DCs in inflammation and consequent tissue remodeling after MI. On the other hand, DCs can express angiotensin (Ang) 2/vasopressin and angiotensin-converting enzyme (ACE) receptors, Ang2 type 1 receptor (AT1R) and Ang2 type 2 receptor (AT2R) [7], while the proinflammatory cytokine inhibitory function of ACE inhibitor (ACEI) in DCs can be induced by gene expression suppression of ACE [8]. Subsequently, Ang2, drastically increased in circulation after MI, is a proinflammatory peptide that closely interacts with the immune system, including monocytes and macrophages [9,10]. However, interaction between DCs and the renin–angiotensin system (RAS) during the healing process after MI has not been established. The RAS plays an important role in inflammatory regulation [11] and is implicated in post-MI cardiac remodeling [12]. Elevated Ang2 in
the plasma or myocardium have a widespread deleterious effect on systemic inflammatory response, tissue fibroblast proliferation and maladaptive tissue repair via activation of AT1R [13,14]. Recent studies have demonstrated stimulation of AT2R by Ang2 might evoke overall cardioprotective effects via bradykinin, nitric oxide, or prostacylin pathways, and thereby counteract the deleterious effects of AT1R [15–17]. Furthermore, the density of AT2Rs in the perivascular, endocardial and infarcted regions of an ischemic heart was markedly increased after MI [18]. In addition, ACEI diminished formation of Ang2, decreased hypertrophy and fibrosis through direct effects on heart tissues [19]. However, the exact mechanism of how ACEI affects AT2R remains unknown. The present study is aimed at exploring the possible involvement of AT2R-mediated mechanism in the cardio-protective effects of ACEI on post-MI remodeling via DCs. 2. Materials and methods 2.1. Animals C57BL/6 mice, with an average age of 12 weeks and fed on a control diet, were obtained from the Animal Administration Center of Fudan University. Mice were treated with lisinopril at a dose of 100 mg/L [20,21] in the drinking water, which was initiated 2 days before MI and
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Corresponding author. E-mail address: [email protected] (J. Ge). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.108660 Received 13 November 2018; Received in revised form 31 January 2019; Accepted 1 February 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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hematoxylin. The same methods were performed without the primary antibodies, serving as negative controls.
continued for 7 days thereafter. Additional groups of mice were treated with AT1R antagonist candesartan cilexetil (25 mg/L [22]; water drinking; AstraZeneca) and the AT2R antagonist PD123319 (1 mg/kg/ day; intraperitoneal injection; Sigma Aldrich, United States), alone or in combination with lisinopril, for 7 days after MI. MI was induced by permanent coronary ligation at 10–12 weeks of age. All procedures and protocols were approved by the Institutional Review Board of Zhongshan Hospital, Fudan University, Shanghai Institutes for Cardiovascular Diseases (A5894-01) and were conducted in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals.
2.7. Preparation of cardiac, splenic, myeloid and peripheral blood cells Mice were sacrificed on day 7 after MI (n = 5–6 mice per group). Spleens and bone marrow (femurs) were removed, triturated in HBSS (Mediatech, Inc.) at 4 °C with the end of a 3 ml syringe and filtered through a 100 μm nylon mesh (BD Biosciences). The cell suspension was centrifuged at 300g for 10 min at 4 °C. Heart tissue was harvested, minced and digested by collagenase II (Sigma, USA) at a concentration of 0.5 mg/ml in 37 °C for 30 min. Single-cell suspension was made by screening with 40 μm cell strainers (BD Biosciences, USA). The cells were washed and resuspended by HBSS containing 2% BSA. Total spleen and cardiac cell numbers were determined with Trypan blue (Mediatech, Inc.). Peripheral blood was drawn via cardiac puncture and subjected to red cells lysis using ACK lysing buffer (150 mM NH4Cl, 10 mM KHCO3, 10 mM EDTA) and three washes with PBS buffer (PBS containing 1% FCS and 5 mM EDTA).
2.2. Cell culture and treatments Bone marrow-derived dendritic cells (BMDCs) obtained from C57BL/6 mice (about 12 weeks) were cultured in RPMI 1640 media supplemented with 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1 ng/ml IL-4 at 37 °C in 5% humidified CO2 for 4 h. Non-adherent cells were replaced with fresh medium every 2 days. On culture day 7, the cells were treated with Ang2 (100 nM; Sigma–Aldrich, USA) for 24 h alone or in the presence of lisinopril (10 μM; Sigma-Aldrich). Phosphate buffer solution (PBS) was used as control. In the inhibitor experiment, the cells were exposed to Ang2 (100 nM) for 24 h after pretreatment with AT2R inhibitor (100 nM PD123319; Sigma–Aldrich, USA) for 1 h.
2.8. Flow cytometry A subset of six mice per group was used for flow cytometric analysis. Cell suspensions were incubated with a mixture of antibodies (antiCD11c-PE, anti-CD45-FITC, CD40, CD83, CD80 and CD86; BD Biosciences) at 4 °C to determine the percentage of DCs in hearts, spleens, bone marrow and peripheral blood, respectively. Then, samples were loaded after two washes with PBS (2% BSA) and the raw data were analyzed using a flow cytometer instrument (Beckman, Germany).
2.3. Myocardial infarction protocol Mice were anesthetized by inhalation of isoflurane and intubated with a 22-G intravenous catheter, followed by full anesthetization with 1.0–2.0% isoflurane gas and mechanical ventilation with a positive pressure ventilator. The heart was exposed through a left thoracotomy and MI was induced by ligation of the left coronary artery with an 8-0 nylon suture. Successful ligation was confirmed when the anterior wall of the left ventricle turned pale. Mice that died within 24 h after the operation were excluded from the analysis. Sham-operated animals underwent the same procedure without ligation of the coronary artery.
2.9. Western blotting Proteins were extracted in a RIPA buffer from the whole mouse heart. Samples were fractionated with 12% SDS-PAGE (Invitrogen, USA), then transferred into polyvinylidene fluoride membranes (Millipore, USA). The membranes with blotted protein were blocked, followed by probing with AT2R antibody (Abcam, Cambridge, MA) at 4 °C overnight. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were identified using Super Signal West Pico Chemiluminescence Substrate (Thermo, USA). Densitometric analysis of western blots was performed with the use of Image J software. GAPDH was used as loading control.
2.4. Echocardiography Mice were placed on controlled heating pads and the core temperature was monitored via a rectal probe. Core temperature was maintained at 37 °C. M-mode was performed with Visual Sonics system (Vevo770, Visual Sonic Inc., CA) equipped with a linear 30-MHz probe (RMV 707B) on mice anesthetized with isoflurane/oxygen (1.0%/ 100%). All measurements were performed by two double-blinded, independent researchers.
2.10. Enzyme-linked immunosorbent assay 2.5. Morphometric analysis The supernatant of the cultured BMDCs was harvested and stored at −70 °C. The cytokine concentrations of tumornecrosisfactor-α (TNF-α), interleukin (IL)-10, and IL-6 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Ang2 concentration of cardiac tissue was determined with an Ang2 ELISA kit (Cayman Chemical) according to the manufacturer's instructions.
Heart tissue was fixed in cold methanol, embedded in paraffin, cut into 5 μm-thick sections, then stained with Masson staining to determine the infarct size. Tissue sections were observed under Leica DMRE microscope and analyzed via LeicaQwin software (Leica Imaging Systems, Cambridge, UK). 2.6. Immunohistochemistry Heart tissue sections (5 μm) were air-dried and fixed in acetone at room temperature for 10 min or in 4% paraformaldehyde at RT for 20 min. After inhibiting endogenous peroxidase activity, the sections were incubated with primary anti-Mac-3, anti-CD45, and anti-CD11c (BD Pharmingen) overnight at 4 °C. After incubation with primary antibodies, the Vectastain ABC elite Kit (Vector Labs) was used according to the manufacturer's instructions. Following visualization with 3,3′diaminobenzidine, the sections were ultimately counterstained with
2.11. Statistical analyses Data were presented as mean ± SD with P < 0.05 considered significant. Statistical comparisons between two groups were evaluated by Student's t-test and corrected by ANOVA for multiple comparisons. Survival rates were compared by Kaplan–Meier method and analyzed by log-rank test. All statistical analyses were performed with SPSS 11.5. 2
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Fig. 1. Cardiac function measured by echocardiography on day 7 post-surgery. (A) Representative M-mode images from individual groups. Measurements shown are LVEDD, LVESD, and LVEF in the different treatment groups. Data are presented as the mean ± (SD) (n = 12). (B) Kaplan–Meier survival analysis. Percentage of surviving mice after sham operation or MI were plotted. Difference between groups was tested by log-rank test. *P < 0.05 vs Sham; $P < 0.05 vs Saline MI; & P < 0.05 vs ACEI MI. PD: PD123319. (C) Myocardial infarct size expressed as a percentage of the left ventricle. The data are shown as mean ± (SD) (n = 5).
3. Results
To evaluate whether the effect of ACEI on MI size is medicated by AT2R, representative photomicrographs of mid-ventricular cross-sections of Masson-stained hearts are shown in Fig. 1C. Consistent with the improvement in cardiac function, the photomicrographs displayed a smaller infarct size (% of LV) 7 days after MI in the lisinopril-treated group, which was reversed by PD123319. However, there was no significant difference in infarct size among lisinopril, lisinopril + candesartan and lisinopril + candesartan + PD123319 groups (not shown).
3.1. Improved cardiac function after MI Acute MI in mice caused a myriad of hemodynamic stresses, which triggered left ventricular remodeling and eventual functional decompensation and heart failure. Echocardiographic analysis revealed decreased cardiac dilatation and improved systolic function in the lisinopril-treated group compared to saline-treated infarcted hearts at day 7 (Fig. 1A). ACEI significantly increased left ventricular ejection fraction (LVEF), decreased left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD) in postMI mice. However, these effects were not observed along with PD123319. Although no significant difference was observed in LVEDD, LVESD and LVEF between lisinopril alone or with candesartan, the reverse phenomenon of PD123319 was not prominent when lisinopril was combined with candesartan (not shown).
3.3. ACEI reduced the infiltration of CD45+ leukocytes, Mac-3+ macrophages, and CD11c+ DCs into the injured myocardium On day 7, CD45+ leukocyte infiltration into the infarcted site was reduced in the lisinopril-treated group compared with saline-treated group. Interestingly, fewer CD11c+ DCs was also observed in the infarcted myocardium of lisinopril-treated mice compared with salinetreated mice. Similarly, infiltrating Mac-3+ macrophages tend to be fewer in lisinopril-treated than in saline-treated mice. However, these effects of lisinopril on leukocytes, macrophages and DCs were repressed when treated with AT2R inhibitor PD123319 (Fig. 2A–D). To elucidate the effects of lisinopril on immune activation and infiltration in the infarcted heart, immune cells in the heart were quantified with DCs surface markers CD11c and CD45. Flow cytometry analysis showed that CD45+CD11c+ DCs were significantly increased in the heart after infarction, compared to the sham group. However, lisinopril treatment significantly reduced the recruitment of CD45+CD11c+ DCs as seen in the saline group, which was suppressed by PD123319. The number of DCs was also significantly increased in
3.2. Survival rate and limited MI size All sham-operated mice survived throughout the study. Survival was significantly higher in the lisinopril-treated (16 of 20, 80%) group compared to saline-treated (17 of 31, 55%) group on day 7 after MI. However, the survival rate was lowered to 71% (15 of 21) in the group treated with PD123319 and lisinopril (Fig. 1B). PD123319 in combination with lisinopril plus candesartan displayed a slightly higher perioperative mortality rate than PD123319 with lisinopril alone, but the differences were not significant (not shown). 3
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Fig. 2. Effect of ACEI medicated by AT2 receptor on the infiltration of CD45+ leukocytes, Mac-3+ macrophages, and CD11c+ DCs into the infarcted hearts of mice. (A) Representative microscopy images of respective stains on day 7. Scale bar = 50 μm. (B–D) Quantitative analysis of the extent and intensity of respective staining on day 7 after MI. The brown color indicates positive staining. The data are shown as mean ± (SD) (n = 5); $P < 0.05 vs saline MI; &P < 0.05 vs ACEI MI. PD: PD123319.
CD83, CD80 and CD86 were upregulated after Ang2 stimulation, but only CD80 and CD86 were attenuated when treated with lisinopril via flow cytometry. However, when combined with PD123319, the inhibitory effects of lisinopril on CD80 and CD86 were moderated. The expressions of CD40 and CD83 were similar between strains in Ang2, lisinopril, and lisinopril plus PD123319 groups. Treatment with PD123319 alone had no obvious effect on the maturation of DCs (Fig. 4A). These results indicate that lisinopril could suppress Ang2induced BMDCs maturation via AT2R. Analysis of cytokine secretions in BMDCs, such as TNF-α, IL-10, and IL-6, indicated inflammatory response in BMDCs. Secretions of TNF-α, IL-6 and IL-10 secretion were significantly increased with Ang2. While, secretions of inflammatory cytokines were suppressed by lisinopril.
the blood after infarction. However, administration of lisinopril, reduced the number of circulating CD45+CD11c+ DCs compared to the saline + MI group. Additionally, lisinopril resulted in more CD45+CD11c+ DCs in the reservoir spleen of lisinopril + MI group, compared with saline + MI group. With the addition of AT2R inhibitor PD123319, the influence of lisinopril on the recruitment of DCs were abrogated (Fig. 3). 3.4. Role of AT2R in Ang2-induced BMDCs maturation and inflammatory cytokine secretion There are many mature markers on the surface of DCs, such as CD40, CD83, CD80 and CD86. The expressions of cell-surface CD40, 4
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Fig. 3. Impact of ACEI on the recruitment of dendritic cells via AT2 receptor in MI mice. Representative dot plots of CD45-FITC/CD11c-PE profile of mice 7 days after MI. Numbers on the plots indicate the percentage of CD45+CD11c+ DCs of total living cells in the heart, spleen and blood. Quantitation of CD45+CD11c+ DCs by flow cytometry in the heart, spleen and blood after MI. The data are shown as mean ± (SD) (n = 5–6); *P < 0.05. PD: PD123319.
Fig. 4. AT2R is involved in the ACEI-mediated inhibition of BMDC maturation and inflammatory responses. (A) CD40, CD80, CD86, and CD83 expression as maturation marker was assessed by flow cytometry. (B) Expression of cytokines in BMDCs analyzed by ELISA. *P < 0.05 versus medium alone; $P < 0.05 versus Ang2; &P < 0.05 versus Ang2 + Lisinopril. (C) Representative immunoblots and the results of quantitative analysis of AT2R expression in the infarcted heart. (D) Cardiac Ang2 levels in mice treated with saline, lisinopril, or candesartan, alone or in combination, for 7 days. Data represent mean ± SD (n = 3). *P < 0.05. 5
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fully matured DCs. CD83 is preferentially expressed on mature DCs [31], while CD40 is a member of the TNF receptor family, which is transiently expressed on T cells under inflammatory conditions [32]. We found that lisinopril down-regulates the expression of CD80 and CD86. The inflammatory activation of DCs is reflected by the differentially expressed cytokine. An increase of proinflammatory cytokines IL-10, IL-6 and TNF-α expression after Ang2 stimulation was observed. In contrast, lisinopril inhibited Ang2-induced increase in IL-6 and TNFα. Surprisingly, all these effects on both DCs maturation and inflammation were ameliorated with application of AT2R inhibitor PD123319. Thus, it is inferred that AT2R may be involved in the process of DCs maturation and inflammatory reaction suppressed by lisinopril. The deleterious role of increases in Ang2 in cardiovascular disease is well established [33,34]. However, most of the recognized effects of Ang2 are mediated through AT1R, while physiologic functions of AT2R in myocardium remain under scrutiny. It has been demonstrated that baseline LV function is improved and post-MI remodeling is attenuated with cardiac overexpression of AT2R [38]. Furthermore, the activation of AT2R counteracts AT1R-mediated actions, which in most cases is believed to be cardiac-beneficial [35]. Nevertheless, conflicting results regarding AT2R overexpression in cardiomyocytes have been reported as well. Several studies found that ventricle-specific expression of AT2R caused cardiac hypertrophy and aggravated the development of dilated cardiomyopathy and heart failure [36,37]. In the present study, we found that mice with lisinopril had a higher expression of AT2R and better-preserved cardiac function compared with saline-MI mice. Both AT2R expression and Ang2 concentration in cardiac tissue were measured. Acute administration of ACEI did not alter the expression of AT2R in sham operated mice. However, after MI, low to moderate levels of Ang2 promoted AT2R expression, but high levels of Ang2 decreased AT2R and blunted its cardioprotective effects. Co-treatment with candesartan, which has a negative feedback on the RAS axis, blocked AT1R, increased renin, Ang1, and Ang2 levels [38]. Increased Ang2 levels may affect the expression of AT2R, thus inhibiting the cardioprotective effect of ACEI. Some limitations apply to the data obtained in the current analysis. First, the dose of 100 mg/L ACEI in the drinking water may not be the ideal dose in mice, despite its proven benefits in mice model [20,21]. Second, the mean infarct sizes in the current study were quite large, which may have overridden any potential benefit of ACEI at the studied dosage. Future studies should include a range of infarct sizes, as well as a range of different ACEI dosages. In addition, there is currently no valid explanation as to why the expression of AT2R is related to the levels of Ang2 in post-MI tissue, which warrants further investigation. The present study demonstrated that ACEI substantially reduced the mobilization of DCs from the spleen, lowered their numbers in the peripheral circulation and infarct region in mice models via AT2R. Moreover, ACEI repressed DCs maturation and inflammatory response by regulating AT2R expression. Thus, the role of AT2R in the process of DCs regulation by ACEI could provide new therapeutic strategies.
Nevertheless, AT2R antagonist PD123319 significantly attenuated the inhibitory effect of lisinopril on the secretions of inflammatory cytokine in BMDCs (Fig. 4B). 3.5. AT2R protein expression in post-MI cardiac tissue The level of AT2R protein expression in the heart was very low in the sham group, and was further decreased after MI. However, lisinopril significantly augmented the expression of AT2R in the injured myocardium. Interestingly, there was a slight increase in AT2R expression with candesartan alone or co-treatment with lisinopril (Fig. 4C). 3.6. Ang2 levels in post-MI cardiac tissue The concentration of Ang2 in cardiac tissue was measured by ELISA. Fig. 4D illustrated a significant increase in Ang2 in the injured myocardium, compared with the sham group. Candesartan had no effect on cardiac Ang2 levels. In contrast, lisinopril caused nearly 5-folds decrease in cardiac Ang2 levels. Interestingly, in mice treated with a combination of lisinopril and candesartan, cardiac Ang2 levels were only 2-folds lower than concentrations in saline-treated mice. Thus, candesartan may attenuate lisinopril-induced decrease in cardiac Ang2 levels. 4. Discussion Our study identified ACEI repression of DCs immune inflammatory response through down-regulating DCs maturation surface markers and regulating inflammatory cytokines, leading to a higher survival rate and improved function through decreased inflammation after MI. However, inhibition of AT2R activation reduced the cardioprotective effects of ACEI. The present study identified a new mechanism that may enhance the beneficial effects of early ACEI therapy after MI. Apart from acute inflammation after MI, a persistent autoimmune response against cardiac proteins is responsible for further myocardial damage leading to LV remodeling and progressive heart failure [23]. In turn, the degree of inflammatory response is an important determinant of host outcome [24,25]. DCs, the most professional antigen-presenting cells, play a complex role in the initiation and progression of MI. DCs are characterized by high capability for antigen capture and processing, migration to lymphoid organs, and expression of various co-stimulatory molecules. Additionally, bone marrow and splenic precursors and circulating monocytes can differentiate into DCs [26] and exert various influences on the immune system [9]. Our findings are consistent with the recent report by Naito K and colleagues in which mature DCs infiltrated into the infarcted heart and border areas, peaking on day 7 [5]. Moreover, we found that lisinopril could protect against ischemic myocardial injury by reducing the recruitment of CD45+CD11c+ DCs to the myocardium and inhibiting the migration of these cells from the splenic reservoir. The renin–angiotensin system is related to various biological processes, such as apoptosis, vascular remodeling, and inflammation [27,28]. Ang2 is involved in several steps of the inflammatory process. ACE binds specific receptors, namely AT1R and AT2R, to convert Ang1 into Ang2 on cell membranes. Previously, we reported inflammation induced by Ang2 in DCs [29], and that DCs could express receptor of Ang2/vasopressin [7,30], angiotensinogen, and ACE. Therefore, it is conceivable that under appropriate conditions, Ang2 is formed, and occupancy of its receptor on DCs occurs in vivo. Moreover, other studies showed that ACEI could suppress the production of DCs-derived proinflammatory cytokines [8]. We hereby displayed that stimulation of Ang2 allowed BMDCs, in the context of preexisting inflammation, to imitate the circumstances after MI and modulate functional features of DCs with lisinopril. In this study, the influence of lisinopril on BMDCs maturation and cytokine production after MI was investigated. All CD83, CD40, CD80, and CD86 are characteristic surface markers for
Conflict of interest The authors declare no competing financial interests. Disclosure The abstract of this manuscript has been presented at 2018 AHA Scientific Session, Chicago, Illinois, USA, November 10–12.
Acknowledgment This project was supported by the National Natural Science Foundation of China, Grant 81600280. 6
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Original article
ACE inhibitor suppresses cardiac remodeling after myocardial infarction by regulating dendritic cells and AT2 receptor-mediated mechanism in mice Yuanji Maa,b,1, Jie Yuanb,1, Jialu Hua,b,1, Wei Gaoa,b,1, Yunzeng Zoua,b, Junbo Gea,b, a b
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Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai, China Shanghai Institute of Cardiovascular Diseases, Shanghai, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Dendritic cell ACE inhibitor Myocardial infarction AT2 receptor Inflammation
Dendritic cells (DCs) play a complex role in the progression of myocardial infarction (MI). The impact of angiotensin-converting enzyme (ACE) inhibitor therapy, partly via affecting DCs maturation and recruitment, was tested on a MI mouse model. Furthermore, the cardioprotective effects of ACEI were enhanced through attenuating migration of DCs from the spleen into peripheral circulation, thereby inhibiting DCs maturation and tissue inflammation. ACEI repress DCs immune inflammatory response through down-regulating DCs maturation surface markers and regulating inflammatory cytokines, which led to a higher survival rate, improved function and remodeling through decreased inflammatory response after MI. However, inhibition of AT2R activation, resulted in a reduction of ACEI effects on DCs. The potent anti-inflammatory effect of ACEI can partially be attributed to its impact on DCs through activation of AT2R, which may provide a new target mechanism for ACEI therapy after MI.
1. Introduction Dendritic cell (DC) is a potent central immunoregulator that orchestrates various types of inflammatory cells in innate and adoptive immunity [1–3]. Emerging evidence suggest that DCs are infiltrated into infarcted hearts in experimental myocardial infarction (MI) models [4]. Recent studies reported that an increased number of DCs in the infarcted heart is associated with deterioration of left ventricular (LV) remodeling [5] and blunting DCs mobilization after MI has favorable effects on survival and LV remodeling [6]. These observations indicate the potential role of DCs in inflammation and consequent tissue remodeling after MI. On the other hand, DCs can express angiotensin (Ang) 2/vasopressin and angiotensin-converting enzyme (ACE) receptors, Ang2 type 1 receptor (AT1R) and Ang2 type 2 receptor (AT2R) [7], while the proinflammatory cytokine inhibitory function of ACE inhibitor (ACEI) in DCs can be induced by gene expression suppression of ACE [8]. Subsequently, Ang2, drastically increased in circulation after MI, is a proinflammatory peptide that closely interacts with the immune system, including monocytes and macrophages [9,10]. However, interaction between DCs and the renin–angiotensin system (RAS) during the healing process after MI has not been established. The RAS plays an important role in inflammatory regulation [11] and is implicated in post-MI cardiac remodeling [12]. Elevated Ang2 in
the plasma or myocardium have a widespread deleterious effect on systemic inflammatory response, tissue fibroblast proliferation and maladaptive tissue repair via activation of AT1R [13,14]. Recent studies have demonstrated stimulation of AT2R by Ang2 might evoke overall cardioprotective effects via bradykinin, nitric oxide, or prostacylin pathways, and thereby counteract the deleterious effects of AT1R [15–17]. Furthermore, the density of AT2Rs in the perivascular, endocardial and infarcted regions of an ischemic heart was markedly increased after MI [18]. In addition, ACEI diminished formation of Ang2, decreased hypertrophy and fibrosis through direct effects on heart tissues [19]. However, the exact mechanism of how ACEI affects AT2R remains unknown. The present study is aimed at exploring the possible involvement of AT2R-mediated mechanism in the cardio-protective effects of ACEI on post-MI remodeling via DCs. 2. Materials and methods 2.1. Animals C57BL/6 mice, with an average age of 12 weeks and fed on a control diet, were obtained from the Animal Administration Center of Fudan University. Mice were treated with lisinopril at a dose of 100 mg/L [20,21] in the drinking water, which was initiated 2 days before MI and
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Corresponding author. E-mail address: [email protected] (J. Ge). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.108660 Received 13 November 2018; Received in revised form 31 January 2019; Accepted 1 February 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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hematoxylin. The same methods were performed without the primary antibodies, serving as negative controls.
continued for 7 days thereafter. Additional groups of mice were treated with AT1R antagonist candesartan cilexetil (25 mg/L [22]; water drinking; AstraZeneca) and the AT2R antagonist PD123319 (1 mg/kg/ day; intraperitoneal injection; Sigma Aldrich, United States), alone or in combination with lisinopril, for 7 days after MI. MI was induced by permanent coronary ligation at 10–12 weeks of age. All procedures and protocols were approved by the Institutional Review Board of Zhongshan Hospital, Fudan University, Shanghai Institutes for Cardiovascular Diseases (A5894-01) and were conducted in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals.
2.7. Preparation of cardiac, splenic, myeloid and peripheral blood cells Mice were sacrificed on day 7 after MI (n = 5–6 mice per group). Spleens and bone marrow (femurs) were removed, triturated in HBSS (Mediatech, Inc.) at 4 °C with the end of a 3 ml syringe and filtered through a 100 μm nylon mesh (BD Biosciences). The cell suspension was centrifuged at 300g for 10 min at 4 °C. Heart tissue was harvested, minced and digested by collagenase II (Sigma, USA) at a concentration of 0.5 mg/ml in 37 °C for 30 min. Single-cell suspension was made by screening with 40 μm cell strainers (BD Biosciences, USA). The cells were washed and resuspended by HBSS containing 2% BSA. Total spleen and cardiac cell numbers were determined with Trypan blue (Mediatech, Inc.). Peripheral blood was drawn via cardiac puncture and subjected to red cells lysis using ACK lysing buffer (150 mM NH4Cl, 10 mM KHCO3, 10 mM EDTA) and three washes with PBS buffer (PBS containing 1% FCS and 5 mM EDTA).
2.2. Cell culture and treatments Bone marrow-derived dendritic cells (BMDCs) obtained from C57BL/6 mice (about 12 weeks) were cultured in RPMI 1640 media supplemented with 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1 ng/ml IL-4 at 37 °C in 5% humidified CO2 for 4 h. Non-adherent cells were replaced with fresh medium every 2 days. On culture day 7, the cells were treated with Ang2 (100 nM; Sigma–Aldrich, USA) for 24 h alone or in the presence of lisinopril (10 μM; Sigma-Aldrich). Phosphate buffer solution (PBS) was used as control. In the inhibitor experiment, the cells were exposed to Ang2 (100 nM) for 24 h after pretreatment with AT2R inhibitor (100 nM PD123319; Sigma–Aldrich, USA) for 1 h.
2.8. Flow cytometry A subset of six mice per group was used for flow cytometric analysis. Cell suspensions were incubated with a mixture of antibodies (antiCD11c-PE, anti-CD45-FITC, CD40, CD83, CD80 and CD86; BD Biosciences) at 4 °C to determine the percentage of DCs in hearts, spleens, bone marrow and peripheral blood, respectively. Then, samples were loaded after two washes with PBS (2% BSA) and the raw data were analyzed using a flow cytometer instrument (Beckman, Germany).
2.3. Myocardial infarction protocol Mice were anesthetized by inhalation of isoflurane and intubated with a 22-G intravenous catheter, followed by full anesthetization with 1.0–2.0% isoflurane gas and mechanical ventilation with a positive pressure ventilator. The heart was exposed through a left thoracotomy and MI was induced by ligation of the left coronary artery with an 8-0 nylon suture. Successful ligation was confirmed when the anterior wall of the left ventricle turned pale. Mice that died within 24 h after the operation were excluded from the analysis. Sham-operated animals underwent the same procedure without ligation of the coronary artery.
2.9. Western blotting Proteins were extracted in a RIPA buffer from the whole mouse heart. Samples were fractionated with 12% SDS-PAGE (Invitrogen, USA), then transferred into polyvinylidene fluoride membranes (Millipore, USA). The membranes with blotted protein were blocked, followed by probing with AT2R antibody (Abcam, Cambridge, MA) at 4 °C overnight. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were identified using Super Signal West Pico Chemiluminescence Substrate (Thermo, USA). Densitometric analysis of western blots was performed with the use of Image J software. GAPDH was used as loading control.
2.4. Echocardiography Mice were placed on controlled heating pads and the core temperature was monitored via a rectal probe. Core temperature was maintained at 37 °C. M-mode was performed with Visual Sonics system (Vevo770, Visual Sonic Inc., CA) equipped with a linear 30-MHz probe (RMV 707B) on mice anesthetized with isoflurane/oxygen (1.0%/ 100%). All measurements were performed by two double-blinded, independent researchers.
2.10. Enzyme-linked immunosorbent assay 2.5. Morphometric analysis The supernatant of the cultured BMDCs was harvested and stored at −70 °C. The cytokine concentrations of tumornecrosisfactor-α (TNF-α), interleukin (IL)-10, and IL-6 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Ang2 concentration of cardiac tissue was determined with an Ang2 ELISA kit (Cayman Chemical) according to the manufacturer's instructions.
Heart tissue was fixed in cold methanol, embedded in paraffin, cut into 5 μm-thick sections, then stained with Masson staining to determine the infarct size. Tissue sections were observed under Leica DMRE microscope and analyzed via LeicaQwin software (Leica Imaging Systems, Cambridge, UK). 2.6. Immunohistochemistry Heart tissue sections (5 μm) were air-dried and fixed in acetone at room temperature for 10 min or in 4% paraformaldehyde at RT for 20 min. After inhibiting endogenous peroxidase activity, the sections were incubated with primary anti-Mac-3, anti-CD45, and anti-CD11c (BD Pharmingen) overnight at 4 °C. After incubation with primary antibodies, the Vectastain ABC elite Kit (Vector Labs) was used according to the manufacturer's instructions. Following visualization with 3,3′diaminobenzidine, the sections were ultimately counterstained with
2.11. Statistical analyses Data were presented as mean ± SD with P < 0.05 considered significant. Statistical comparisons between two groups were evaluated by Student's t-test and corrected by ANOVA for multiple comparisons. Survival rates were compared by Kaplan–Meier method and analyzed by log-rank test. All statistical analyses were performed with SPSS 11.5. 2
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Fig. 1. Cardiac function measured by echocardiography on day 7 post-surgery. (A) Representative M-mode images from individual groups. Measurements shown are LVEDD, LVESD, and LVEF in the different treatment groups. Data are presented as the mean ± (SD) (n = 12). (B) Kaplan–Meier survival analysis. Percentage of surviving mice after sham operation or MI were plotted. Difference between groups was tested by log-rank test. *P < 0.05 vs Sham; $P < 0.05 vs Saline MI; & P < 0.05 vs ACEI MI. PD: PD123319. (C) Myocardial infarct size expressed as a percentage of the left ventricle. The data are shown as mean ± (SD) (n = 5).
3. Results
To evaluate whether the effect of ACEI on MI size is medicated by AT2R, representative photomicrographs of mid-ventricular cross-sections of Masson-stained hearts are shown in Fig. 1C. Consistent with the improvement in cardiac function, the photomicrographs displayed a smaller infarct size (% of LV) 7 days after MI in the lisinopril-treated group, which was reversed by PD123319. However, there was no significant difference in infarct size among lisinopril, lisinopril + candesartan and lisinopril + candesartan + PD123319 groups (not shown).
3.1. Improved cardiac function after MI Acute MI in mice caused a myriad of hemodynamic stresses, which triggered left ventricular remodeling and eventual functional decompensation and heart failure. Echocardiographic analysis revealed decreased cardiac dilatation and improved systolic function in the lisinopril-treated group compared to saline-treated infarcted hearts at day 7 (Fig. 1A). ACEI significantly increased left ventricular ejection fraction (LVEF), decreased left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD) in postMI mice. However, these effects were not observed along with PD123319. Although no significant difference was observed in LVEDD, LVESD and LVEF between lisinopril alone or with candesartan, the reverse phenomenon of PD123319 was not prominent when lisinopril was combined with candesartan (not shown).
3.3. ACEI reduced the infiltration of CD45+ leukocytes, Mac-3+ macrophages, and CD11c+ DCs into the injured myocardium On day 7, CD45+ leukocyte infiltration into the infarcted site was reduced in the lisinopril-treated group compared with saline-treated group. Interestingly, fewer CD11c+ DCs was also observed in the infarcted myocardium of lisinopril-treated mice compared with salinetreated mice. Similarly, infiltrating Mac-3+ macrophages tend to be fewer in lisinopril-treated than in saline-treated mice. However, these effects of lisinopril on leukocytes, macrophages and DCs were repressed when treated with AT2R inhibitor PD123319 (Fig. 2A–D). To elucidate the effects of lisinopril on immune activation and infiltration in the infarcted heart, immune cells in the heart were quantified with DCs surface markers CD11c and CD45. Flow cytometry analysis showed that CD45+CD11c+ DCs were significantly increased in the heart after infarction, compared to the sham group. However, lisinopril treatment significantly reduced the recruitment of CD45+CD11c+ DCs as seen in the saline group, which was suppressed by PD123319. The number of DCs was also significantly increased in
3.2. Survival rate and limited MI size All sham-operated mice survived throughout the study. Survival was significantly higher in the lisinopril-treated (16 of 20, 80%) group compared to saline-treated (17 of 31, 55%) group on day 7 after MI. However, the survival rate was lowered to 71% (15 of 21) in the group treated with PD123319 and lisinopril (Fig. 1B). PD123319 in combination with lisinopril plus candesartan displayed a slightly higher perioperative mortality rate than PD123319 with lisinopril alone, but the differences were not significant (not shown). 3
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Fig. 2. Effect of ACEI medicated by AT2 receptor on the infiltration of CD45+ leukocytes, Mac-3+ macrophages, and CD11c+ DCs into the infarcted hearts of mice. (A) Representative microscopy images of respective stains on day 7. Scale bar = 50 μm. (B–D) Quantitative analysis of the extent and intensity of respective staining on day 7 after MI. The brown color indicates positive staining. The data are shown as mean ± (SD) (n = 5); $P < 0.05 vs saline MI; &P < 0.05 vs ACEI MI. PD: PD123319.
CD83, CD80 and CD86 were upregulated after Ang2 stimulation, but only CD80 and CD86 were attenuated when treated with lisinopril via flow cytometry. However, when combined with PD123319, the inhibitory effects of lisinopril on CD80 and CD86 were moderated. The expressions of CD40 and CD83 were similar between strains in Ang2, lisinopril, and lisinopril plus PD123319 groups. Treatment with PD123319 alone had no obvious effect on the maturation of DCs (Fig. 4A). These results indicate that lisinopril could suppress Ang2induced BMDCs maturation via AT2R. Analysis of cytokine secretions in BMDCs, such as TNF-α, IL-10, and IL-6, indicated inflammatory response in BMDCs. Secretions of TNF-α, IL-6 and IL-10 secretion were significantly increased with Ang2. While, secretions of inflammatory cytokines were suppressed by lisinopril.
the blood after infarction. However, administration of lisinopril, reduced the number of circulating CD45+CD11c+ DCs compared to the saline + MI group. Additionally, lisinopril resulted in more CD45+CD11c+ DCs in the reservoir spleen of lisinopril + MI group, compared with saline + MI group. With the addition of AT2R inhibitor PD123319, the influence of lisinopril on the recruitment of DCs were abrogated (Fig. 3). 3.4. Role of AT2R in Ang2-induced BMDCs maturation and inflammatory cytokine secretion There are many mature markers on the surface of DCs, such as CD40, CD83, CD80 and CD86. The expressions of cell-surface CD40, 4
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Fig. 3. Impact of ACEI on the recruitment of dendritic cells via AT2 receptor in MI mice. Representative dot plots of CD45-FITC/CD11c-PE profile of mice 7 days after MI. Numbers on the plots indicate the percentage of CD45+CD11c+ DCs of total living cells in the heart, spleen and blood. Quantitation of CD45+CD11c+ DCs by flow cytometry in the heart, spleen and blood after MI. The data are shown as mean ± (SD) (n = 5–6); *P < 0.05. PD: PD123319.
Fig. 4. AT2R is involved in the ACEI-mediated inhibition of BMDC maturation and inflammatory responses. (A) CD40, CD80, CD86, and CD83 expression as maturation marker was assessed by flow cytometry. (B) Expression of cytokines in BMDCs analyzed by ELISA. *P < 0.05 versus medium alone; $P < 0.05 versus Ang2; &P < 0.05 versus Ang2 + Lisinopril. (C) Representative immunoblots and the results of quantitative analysis of AT2R expression in the infarcted heart. (D) Cardiac Ang2 levels in mice treated with saline, lisinopril, or candesartan, alone or in combination, for 7 days. Data represent mean ± SD (n = 3). *P < 0.05. 5
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fully matured DCs. CD83 is preferentially expressed on mature DCs [31], while CD40 is a member of the TNF receptor family, which is transiently expressed on T cells under inflammatory conditions [32]. We found that lisinopril down-regulates the expression of CD80 and CD86. The inflammatory activation of DCs is reflected by the differentially expressed cytokine. An increase of proinflammatory cytokines IL-10, IL-6 and TNF-α expression after Ang2 stimulation was observed. In contrast, lisinopril inhibited Ang2-induced increase in IL-6 and TNFα. Surprisingly, all these effects on both DCs maturation and inflammation were ameliorated with application of AT2R inhibitor PD123319. Thus, it is inferred that AT2R may be involved in the process of DCs maturation and inflammatory reaction suppressed by lisinopril. The deleterious role of increases in Ang2 in cardiovascular disease is well established [33,34]. However, most of the recognized effects of Ang2 are mediated through AT1R, while physiologic functions of AT2R in myocardium remain under scrutiny. It has been demonstrated that baseline LV function is improved and post-MI remodeling is attenuated with cardiac overexpression of AT2R [38]. Furthermore, the activation of AT2R counteracts AT1R-mediated actions, which in most cases is believed to be cardiac-beneficial [35]. Nevertheless, conflicting results regarding AT2R overexpression in cardiomyocytes have been reported as well. Several studies found that ventricle-specific expression of AT2R caused cardiac hypertrophy and aggravated the development of dilated cardiomyopathy and heart failure [36,37]. In the present study, we found that mice with lisinopril had a higher expression of AT2R and better-preserved cardiac function compared with saline-MI mice. Both AT2R expression and Ang2 concentration in cardiac tissue were measured. Acute administration of ACEI did not alter the expression of AT2R in sham operated mice. However, after MI, low to moderate levels of Ang2 promoted AT2R expression, but high levels of Ang2 decreased AT2R and blunted its cardioprotective effects. Co-treatment with candesartan, which has a negative feedback on the RAS axis, blocked AT1R, increased renin, Ang1, and Ang2 levels [38]. Increased Ang2 levels may affect the expression of AT2R, thus inhibiting the cardioprotective effect of ACEI. Some limitations apply to the data obtained in the current analysis. First, the dose of 100 mg/L ACEI in the drinking water may not be the ideal dose in mice, despite its proven benefits in mice model [20,21]. Second, the mean infarct sizes in the current study were quite large, which may have overridden any potential benefit of ACEI at the studied dosage. Future studies should include a range of infarct sizes, as well as a range of different ACEI dosages. In addition, there is currently no valid explanation as to why the expression of AT2R is related to the levels of Ang2 in post-MI tissue, which warrants further investigation. The present study demonstrated that ACEI substantially reduced the mobilization of DCs from the spleen, lowered their numbers in the peripheral circulation and infarct region in mice models via AT2R. Moreover, ACEI repressed DCs maturation and inflammatory response by regulating AT2R expression. Thus, the role of AT2R in the process of DCs regulation by ACEI could provide new therapeutic strategies.
Nevertheless, AT2R antagonist PD123319 significantly attenuated the inhibitory effect of lisinopril on the secretions of inflammatory cytokine in BMDCs (Fig. 4B). 3.5. AT2R protein expression in post-MI cardiac tissue The level of AT2R protein expression in the heart was very low in the sham group, and was further decreased after MI. However, lisinopril significantly augmented the expression of AT2R in the injured myocardium. Interestingly, there was a slight increase in AT2R expression with candesartan alone or co-treatment with lisinopril (Fig. 4C). 3.6. Ang2 levels in post-MI cardiac tissue The concentration of Ang2 in cardiac tissue was measured by ELISA. Fig. 4D illustrated a significant increase in Ang2 in the injured myocardium, compared with the sham group. Candesartan had no effect on cardiac Ang2 levels. In contrast, lisinopril caused nearly 5-folds decrease in cardiac Ang2 levels. Interestingly, in mice treated with a combination of lisinopril and candesartan, cardiac Ang2 levels were only 2-folds lower than concentrations in saline-treated mice. Thus, candesartan may attenuate lisinopril-induced decrease in cardiac Ang2 levels. 4. Discussion Our study identified ACEI repression of DCs immune inflammatory response through down-regulating DCs maturation surface markers and regulating inflammatory cytokines, leading to a higher survival rate and improved function through decreased inflammation after MI. However, inhibition of AT2R activation reduced the cardioprotective effects of ACEI. The present study identified a new mechanism that may enhance the beneficial effects of early ACEI therapy after MI. Apart from acute inflammation after MI, a persistent autoimmune response against cardiac proteins is responsible for further myocardial damage leading to LV remodeling and progressive heart failure [23]. In turn, the degree of inflammatory response is an important determinant of host outcome [24,25]. DCs, the most professional antigen-presenting cells, play a complex role in the initiation and progression of MI. DCs are characterized by high capability for antigen capture and processing, migration to lymphoid organs, and expression of various co-stimulatory molecules. Additionally, bone marrow and splenic precursors and circulating monocytes can differentiate into DCs [26] and exert various influences on the immune system [9]. Our findings are consistent with the recent report by Naito K and colleagues in which mature DCs infiltrated into the infarcted heart and border areas, peaking on day 7 [5]. Moreover, we found that lisinopril could protect against ischemic myocardial injury by reducing the recruitment of CD45+CD11c+ DCs to the myocardium and inhibiting the migration of these cells from the splenic reservoir. The renin–angiotensin system is related to various biological processes, such as apoptosis, vascular remodeling, and inflammation [27,28]. Ang2 is involved in several steps of the inflammatory process. ACE binds specific receptors, namely AT1R and AT2R, to convert Ang1 into Ang2 on cell membranes. Previously, we reported inflammation induced by Ang2 in DCs [29], and that DCs could express receptor of Ang2/vasopressin [7,30], angiotensinogen, and ACE. Therefore, it is conceivable that under appropriate conditions, Ang2 is formed, and occupancy of its receptor on DCs occurs in vivo. Moreover, other studies showed that ACEI could suppress the production of DCs-derived proinflammatory cytokines [8]. We hereby displayed that stimulation of Ang2 allowed BMDCs, in the context of preexisting inflammation, to imitate the circumstances after MI and modulate functional features of DCs with lisinopril. In this study, the influence of lisinopril on BMDCs maturation and cytokine production after MI was investigated. All CD83, CD40, CD80, and CD86 are characteristic surface markers for
Conflict of interest The authors declare no competing financial interests. Disclosure The abstract of this manuscript has been presented at 2018 AHA Scientific Session, Chicago, Illinois, USA, November 10–12.
Acknowledgment This project was supported by the National Natural Science Foundation of China, Grant 81600280. 6
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