Basic Res Cardiol (2014) 109:415 DOI 10.1007/s00395-014-0415-z

ORIGINAL CONTRIBUTION

TXNIP mediates NLRP3 inflammasome activation in cardiac microvascular endothelial cells as a novel mechanism in myocardial ischemia/reperfusion injury Yi Liu • Kun Lian • Lijian Zhang • Rutao Wang • Fu Yi • Chao Gao • Chao Xin • Di Zhu • Yan Li • Wenjun Yan • Lize Xiong • Erhe Gao • Haichang Wang • Ling Tao

Received: 17 July 2013 / Revised: 24 April 2014 / Accepted: 7 May 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract NLRP3 inflammasome is necessary for initiating acute sterile inflammation. Recent studies have demonstrated that NLRP3 inflammasome is up-regulated and mediates myocardial ischemia/reperfusion (MI/R) injury. However, the signaling pathways that lead to the activation of NLRP3 inflammasome by MI/R injury have not been fully elucidated. C57BL/6J mice were subjected to 30 min ischemia and 3 or 24 h reperfusion. The ischemic heart exhibited enhanced inflammasome activation as evidenced by increased NLRP3 expression and caspase-1 activity and increased IL-1b and IL-18 production. Intramyocardial NLRP3 siRNA injection or an intraperitoneal injection of BAY 11-7028, an inflammasome inhibitor, attenuated macrophage and neutrophil infiltration and decreased MI/R

Y. Liu, K. Lian and L. Zhang contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00395-014-0415-z) contains supplementary material, which is available to authorized users. Y. Liu  K. Lian  L. Zhang  R. Wang  F. Yi  C. Gao  C. Xin  D. Zhu  Y. Li  W. Yan  H. Wang (&)  L. Tao (&) Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, 15 Changle West Road, Xi’an 710032, China e-mail: [email protected] L. Tao e-mail: [email protected]; [email protected] L. Xiong Department of Anesthesiology, Xijing Hospital, The Fourth Military Medical University, 15 Changle West Road, Xi’an 710032, China E. Gao The Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA

injury, as measured by cardiomyocyte apoptosis and infarct size. The ischemic heart also exhibited enhanced interaction between Txnip and NLRP3, which has been shown to be a mechanism for activating NLRP3. Intramyocardial Txnip siRNA injection also decreased infarct size and NLRP3 activation. In vitro experiments revealed that NLRP3 was expressed in cardiac microvascular endothelial cells (CMECs), but was hardly expressed in cardiomyocytes. Simulated ischemia/reperfusion (SI/R) stimulated NLRP3 inflammasome activation in CMECs, but not in cardiomyocytes. Moreover, CMECs subjected to SI/R injury increased interactions between Txnip and NLRP3. Txnip siRNA diminished NLRP3 inflammasome activation and SI/R-induced injury, as measured by LDH release and caspase-3 activity in CMECs. ROS scavenger dissociated TXNIP from NLRP3 and inhibited the activation of NLRP3 inflammasome in the CMECs. For the first time, we demonstrated that TXNIP-mediated NLRP3 inflammasome activation in CMECs was a novel mechanism of MI/R injury. Interventions that block Txnip/NLRP3 signaling to inhibit the activation of NLRP3 inflammasomes may be novel therapies for mitigating MI/R injury. Keywords Ischemia/reperfusion  TXNIP  NLRP3 inflammasome  Cardiac microvascular endothelial cells

Introduction Inflammasomes are large multiprotein complexes that consist of caspase-1, apoptosis-associated speck-like protein (ASC), and NLRP (nucleotide-binding oligomerization domain-like receptor with a pyrin domain). Apoptosisassociated speck-like protein bridges the interaction between NLRP and caspase-1, therefore making it essential

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for inflammasome activation and the subsequent interleukin-1b (IL-1b) and IL-18 secretion [19]. Increasing evidence indicates that the inflammasome is a key player in sterile inflammation [13]. In particular, IL-1b is a prominent and early mediator for inflammation in myocardial ischemia/reperfusion (MI/R) injury. I/R induces IL-1b expression in the heart, and inhibiting IL-1b prevents myocardial injury after I/R [33]. Apoptosis-associated speck-like protein deletion also protects the myocardium from I/R injury in mice [16]. These results suggest the importance of inflammasomes for the pathophysiology of MI/R injury. To date, four inflammasomes have been identified: NLRP1, NLRP3, NLRC4, and AIM2 [30]. The NLRP3 inflammasome is the most fully characterized and has been associated with a wide range of diseases, including infectious, auto-inflammatory, and autoimmune diseases [20]. Recent study has demonstrated that the NLRP3 inflammasome is up-regulated and mediates myocardial ischemia–reperfusion injury [26]. However, the signaling pathways that lead to the activation of NLRP3 inflammasome by MI/R injury has not been fully elucidated. Many activators of the NLRP3 inflammasome have already been identified, including endotoxins, K? channel openers, and uric acid [14]. Additionally, reactive oxygen species (ROS) have been identified as inflammasome activators for the pathogenesis of MI/R injury [16]. All known NLRP3 activators generate ROS and, conversely, inhibitors of ROS block inflammasome activation [7, 39]. However, the mechanism by which ROS activate the inflammasome is unclear. Thioredoxin-interacting protein (Txnip), a member of the a-arrestin protein superfamily, is a ubiquitously expressed protein in normal tissue cells [23]. Initially, Txnip was identified as an endogenous inhibitor of thioredoxin which, when directly bound, inhibits the ability of thioredoxin to scavenge for ROS [23]. It has been demonstrated that deletion of Txnip in mice protects the myocardium from I/R injury [38]. However, the mechanism by which Txnip mediated myocardial injury has not been fully elucidated. Recently, Txnip has also been demonstrated as essential for the activation of the NLRP3 inflammasome through its direct interaction with NLRP3. Whether Txnip is an activator of NLRP3 inflammasomes in the pathophysiology of MI/R injury is unknown. Inflammasomes are differentially expressed in cardiovascular and other tissues. NLRP3 components are highly expressed in infiltrated inflammatory cells derived from bone marrow. The myocardial ischemic injury induced NLRP3 expression primarily located in non-cardiomyocytes, such as leukocytes, fibroblasts, and endothelial cells within the myocardium [21, 26], and the cardiomyocyte hardly expresses NLRP3 [26]. Existing evidence has indicated the presence of inflammasome activation in cardiac

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fibroblasts during MI/R injury, but not in cardiomyocyte [16, 26]. Microvascular endothelial cells play a crucial role in the pathogenesis of systemic vascular inflammation by actively participating in and regulating of inflammatory processes [5, 24]. Cardiac microvascular endothelial cells (CMECs) are a major component of non-muscle cells in the heart [6]. It has been demonstrated that CMECs play a key role in triggering cardiomyocyte injury during MI/R injury [27]. Cardiac microvasculature injury and its subsequent obstruction by erythrocytes, neutrophils, and debris are associated with adverse ventricular remodeling and patient prognosis [10], and attenuating endothelial injury can improve long-term outcome in patients and animals suffering from myocardial infarction [2, 11, 17]. However, whether cardiac endothelial cells are responsible for initial inflammasome activation in MI/R is not known. Aims of this study were: (1) to further investigate the signaling pathways of MI/R injury that lead to the activation of NLRP3 inflammasomes; (2) to determine the role of NLRP3 inflammasomes in cardiac resident cells of the myocardium in I/R injury; and (3) to investigate potential therapeutic modalities capable of attenuating MI/R injury by utilizing the mechanistic information gleaned from the previous aims.

Research design and methods Experimental protocols All experiments were performed in adherence with the National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Fourth Military Medical University Committee on Animal Care. C57BL/6 mice (aged 8–10 weeks) were used for the present study. Small-interfering RNA (siRNA) was used to knockdown cardiac NLRP3 and Txnip expression. Predesigned NLRP3-specific siRNA (Santa Cruz) or Txnipspecific siRNA (Thermo Scientific) or control scrambled siRNA (Santa Cruz, Thermo Scientific, respectively) were diluted in 5 % glucose and mixed in vivo with jet PEI (polyethyleneimine; Genesee Scientific, San Diego, CA), each scrambled siRNA contains a scrambled sequence of NLRP3 or Txnip that will not lead to the specific degradation of any known cellular mRNA. C57BL/6 mice were anesthetized with 2 % isoflurane, and the heart was exposed via thoracotomy of the left fifth intercostal space. SiRNA were delivered by three separate intramyocardial injections, temporarily blanching the left ventricular free wall. Hearts were subjected to I/R 48 h after injection. In vivo MI/R was performed as previously described [8]. Briefly, male mice were anesthetized with 2 % isoflurane, and MI was induced by temporarily exteriorizing the heart

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via a left thoracic incision while placing a silk suture (6–0) slipknot around the left anterior descending coronary artery. After 30 min of MI, the slipknot was released, and the myocardium was reperfused for 3 or 24 h (for infarct size and cardiac function assays). Additionally, 10 min before reperfusion the non-NLRP3 siRNA-treated mice received BAY 11-7028, an inflammasome inhibitor, through an intraperitoneal (IP) injection. Sham-operated control mice (sham MI/R) underwent identical surgical procedures, but with the suture under the left coronary artery remaining untied. At the conclusion of the reperfusion period, the ligature around the coronary artery was retied, and 2 % Evans Blue dye was injected into the left ventricular cavity. The heart was quickly excised, and the I/R cardiac tissue was isolated and processed as described below. Determination of cardiac function and myocardial infarct size Twenty-four hours after the reperfusion, mice were anesthetized, and cardiac function was determined by echocardiography (VisualSonics VeVo 2100 imaging system). After assessment, cardiectomies were performed, and myocardial infarct size was determined using Evans Blue/ 2, 3, 5-triphenyltetrazolium chloride (TTC) staining as previously described [3]. Determination of myocardial apoptosis Myocardial apoptosis was determined using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining (TUNEL, Roche Ltd., Switzerland) and caspase-3 activity assays (Beyotime Company, Shanghai, China). These methods were applied to the entire I/R region termed ‘‘area-at-risk’’ as previously described [31]. Histology and immunohistochemistry For immunohistochemical analysis, the paraffin-embedded heart sections were incubated with primary antibodies against mouse Ly6C and Mac-3, followed by incubation with biotin-conjugated secondary antibodies, and then treated with avidin-peroxidase. The reaction was developed using the DAB substrate kit, and the sections were then counterstained with hematoxylin. No signals were detected when species-matched IgG was used instead of the primary antibody as negative controls. Quantitative assessment of neutrophils and macrophages was performed by counting the number of positive cells in five different fields of the area (mm2) per heart. All measurements were performed in a double-blind manner by two independent researchers.

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Quantification of superoxide production Superoxide production, an index of oxidative stress, in viable cardiac microvascular endothelial cells (CMECs) was measured by lucigenin-enhanced chemiluminescence as previously described [18]. This was expressed as relative light units per second per milligram protein. Mouse cardiomyocytes and SI/R injury Murine neonatal cardiomyocytes were prepared from the ventricles of 1-day-old mice. Cells were dissociated using 0.25 % trypsin, followed by 0.8 mg/mL collagenase. The cells were then washed and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum. The isolated cardiomyocytes were grown in 10 % FBS-containing DMEM, and primary cardiomyocytes were used for the experiments. For simulated ischemia/reperfusion (SI/R) injury, glucose-free culture medium was first exposed to a hypoxic gas mixture (95 % N2/5 % CO2) for 5 min. Culture media were quickly replaced by the hypoxia–hypoglycemic medium, and cardiomyocytes were placed in a Napco 8000WJhypoxia (1 % O2/5 % CO2/94 % N2) incubator. After 9 h of hypoxia/hypoglycemic exposure, the medium was replaced with medium with a normal glucose concentration. Cells were incubated for an additional 3 h under normoxic conditions in a CO2 incubator before the assays below were performed. All cells were subjected to an identical SI/R procedure. CMECs and SI/R injury Cardiac microvascular endothelial cells were isolated as previously described [22] with minor modifications. Briefly, male C57BL/6J mice (aged 8–10 weeks) were anesthetized with ether, and the heart was rapidly excised and rinsed with PBS supplemented with heparin. After rinsing, the right ventricle, atria, and valvular tissues were removed, and the remaining left ventricle was immersed in 75 % ethanol for 20–30 s to devitalize the epicardial mesothelial cells and endocardial endothelial cells. About one-third of the outer free ventricular wall was dissected to remove the epicardial arteries. The remaining tissue was then minced in PBS and incubated in 0.2 % collagenase for 10 min, followed by 0.2 % trypsin for another 6 min in a 37 °C water bath. Dissociated cells were filtered through a 100-mm mesh filter and centrifuged 1,000 rpm for 10 min. The cells were resuspended in DMEM supplemented with 20 % (v/v) fetal calf serum (FCS) and heparin (20 units/ml) and then plated on laminin-coated (10 lg/ml) dishes. The purity of the CMEM is determined by differential adhesion method. Primary cultures of CMEC were positively

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identified by two endothelial cell markers: factor VIIIrelated antigen and uptake of acetylated low-density lipoprotein. Passage two CMECs were used for the study. After 24 h synchronization, cells were washed with PBS, and non-adherent cells were removed from the culture and were treated with EUK134 (7 lM; Cayman Chemical, Michigan, USA), a peroxynitrite decomposition catalyst. Cells were incubated for 12 h and then subjected to either 10 h of normoxia/normal-glucose incubation or 6 h hypoxia–hypoglycemic plus 4 h normoxia/normal-glucose SI/R injury as previously described [35]. Briefly, oxygen–glucose deprivation injury occurred by placing cells in a hypoxic environment (1 % O2/5 % CO2/94 % N2) in the presence of glucose-free DMEM medium for 6 h. After 6 h, the medium was exchanged for oxygenated and normal-glucose DMEM, and the culture was incubated for 4 h at 37 °C to simulate reperfusion. For IL-1b determination, the CMECs were pretreated with additional LPS (100 ng/ml) for 24 h before the EUK134 treatment.

Nanjing, China), respectively, according to the manufacturer’s instructions.

Plasmid and siRNA transfection

Data are presented as mean ± SEM. The Mann–Whitney test was used to compare the two groups. Comparisons between the CMECs and cardiomyocytes groups were performed by two-way ANOVA followed by Bonferroni correction for post hoc t test. Immunoblotting densities were analyzed with the Kruskal–Wallis test followed by Dunn post hoc test. For the other comparisons, data were analyzed by one-way ANOVA followed by Bonferroni correction for post hoc t test. A P \ 0.05 was considered statistically significant.

To specifically suppress TXNIP and NLRP3 expression in CMECs, cells were transfected with siRNA against TXNIP (Thermo Scientific) and NLRP3 (Santa Cruz Biotechnology, Inc.). Scrambled siRNA [Thermo Scientific and (Santa Cruz Biotechnology, Inc.)] was used as a nonspecific control. Cardiac microvascular endothelial cells were pretreated with either scramble control or TXNIP-specific or NLRP3-specific siRNA for 48 h. Cardiac microvascular endothelial cells were transiently transfected with GFPtagged TXNIP plasmid. Experiments were performed 24 h after transfection. RNA isolation and PCR analysis Total RNA was isolated from CMECs via TRIzol reagent (Invitrogen, Carlsbad, CA, USA). IL-1b and NLRP3 mRNA levels was quantified by real-time PCR via SYBR Green (DRR081A, TaKaRa), and corrected for b-actin mRNA level. Measurement of inflammatory cytokines, LDH release, caspase-3 and caspase-1 activity IL-1b and IL-18 levels in heart and cell extracts were assessed using a mouse ELISA kit (R&D systems) according to the manufacturer’s instructions. Lactate dehydrogenase (LDH) release in supernatant of cultured cells and caspase-3 activity, caspase-1 activity in heart and cell extracts were determined using an enzyme activity assay kit (Nanjing Institute of Jiancheng Bioengineering,

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Immunoblotting Cardiac tissue homogenate proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Western blots were performed using monoclonal antibodies against Txnip (Abcam, Cambridge, MA, USA), NLRP3 (Santa Cruz, Delaware, USA), or caspase-1(Cell Signaling, Boston, MA, USA), E-selectin (R&D Systems) and ICAM-1(R&D Systems). Nitrocellulose membranes were then incubated with HRP-conjugated IgG antibody (Santa Cruz Biotechnology, Inc.) for 1 h. The blot was developed with an ECL-Plus chemiluminescence reagent kit and visualized via UVP Bio-Imaging Systems. Blot densities were analyzed with vision works LS acquisition and analysis software. Statistical analysis

Results NLRP3 inflammasomes were activated in the heart by MI/R injury Inflammasomes act as activators for caspase-1 which subsequently leads to the maturation of the pro-inflammatory cytokines IL-1b and IL-18 [34]. To investigate whether inflammasomes are activated during MI/R injury, C57BL/ 6J mice were subjected to 30 min ischemia and 3 or 24 h reperfusion, and then caspase-1, IL-1b, and IL-18 levels were evaluated at 3- and 24-h reperfusion duration, respectively. NLRP3 (Fig. 1a) expression was increased after 3-h reperfusion duration, which gradually increased after 24-h reperfusion duration. In a consistent fashion, augmented caspase-1 activity (Fig. 1b) and IL-1b (Fig. 1c) and IL-18 content (Fig. 1d) were observed after 3-h reperfusion duration, also gradually increasing until 24-h reperfusion duration. In consistence with previous study [26], our present study indicates that NLRP3

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Fig. 1 The NLRP3 inflammasome was activated in hearts subjected to MI/R injury. C57BL/6J mice were subjected to 30 min ischemia and 3 or 24 h reperfusion. NLRP3 (a) were determined using Western blots (n = 4 per group). Caspase-1 activity (b) was determined by an enzyme activity assay kit (n = 6 per group); IL-1b (c) and IL-18 (d) levels were determined using ELISA (n = 6 per group). The relative caspase-1 activities were calculated as ratios of the cleavage of their substrates in MI/R group relative to sham group. Data were expressed as mean ± SEM. *P \ 0.05, **P \ 0.01 versus Sham group

inflammasomes may be involved in the pathophysiology of MI/R injury. Intramyocardial NLRP3 siRNA and an inflammasome inhibitor attenuated inflammatory cell infiltration and cardiomyocyte apoptosis in hearts subjected to I/R injury A very recent study has investigated the role of NLRP3 in the MI/R injury in ex vivo I/R [26] model. To further clarify the role of the NLRP3 inflammasome in I/R pathogenesis in in vivo I/R model, additional experiments were performed. First, NLRP3 siRNA, administered via intramyocardial injections, were used to knock down cardiacspecific NLRP3 expression. We also used intraperitoneal injection of BAY 11-7028, an inflammasome inhibitor, to inhibit NLRP3 activation. Our method of intramyocardial siRNA delivery was highly successful and markedly decreased basal NLRP3 expression by 55 % (Fig. 2a). Both NLRP3 siRNA and BAY 11-7028 significantly inhibited the I/R-induced cardiac NLRP3 inflammasome activation, as evidenced by a decrease in caspase-1 expression (Fig. 2b), IL-1b production (Fig. 2d), and by decreased macrophage (Mac-3) (Fig. 4g), and neutrophil (Ly6G) (Fig. 4h) infiltration. More importantly, NLRP3 siRNA and BAY 11-7028 decreased caspase-3 activity (Fig. 2f) and cardiomyocyte apoptosis (Fig. 2e).

Intramyocardial NLRP3 siRNA and an inflammasome inhibitor decreased infarct size and restored heart function We further investigated whether NLRP3 siRNA or the inflammasome inhibitor BAY 11-7028 could attenuate infarct size and restore heart function in an ischemic heart. Our data indicated that NLRP3 siRNA and BAY 11-7028 significantly attenuated myocardial infarct size (Fig. 3a) and restored heart function (Fig. 3b). Intramyocardial Txnip siRNA decreased the NLRP3 activation and infarct size It has been demonstrated that deletion of Txnip in mice protects the myocardium from I/R injury. However, the mechanism by which Txnip mediated myocardial injury has not been fully elucidated. Recently, it has been shown that TXNIP is essential for activating the NLRP3 inflammasome by directly interacting with NLRP3 under oxidative stress [39]. Therefore, we hypothesized that the NLRP3 activation may be the mechanism of Txnip-mediated myocardial injury. Firstly, we investigated the interaction between TXNIP and NLRP3 during the MI/R injury. We found that the interaction between TXNIP and NLRP3 was significantly increased during the MI/R injury (Fig. 4a). Secondly, Txnip siRNA was administered via

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Fig. 2 Intramyocardial NLRP3 siRNA and an inflammasome inhibitor attenuated inflammatory cell infiltration and cardiomyocyte apoptosis in hearts subjected to I/R injury. Forty-eight hours after intramyocardial NLRP3 siRNA injection, the heart was subjected to MI/R injury. NonNLRP3 siRNA-treated mice received BAY 11-7028, an inflammasome inhibitor, via IP injection 10 min before reperfusion. Hearts were excised after 3 h reperfusion. NLRP3 (a) and caspase-1 (b, c) expression were determined using Western blots (n = 5 per group). IL-1b (d) was determined using ELISA (n = 6 per group). Caspase-3 activity (f) was determined using an enzyme activity assay kit (n = 6 per group). Cardiomyocytes apoptosis (e) was determined with TUNEL staining

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(n = 4 per group). Representative photographs of heart sections are shown. TUNEL staining (green) indicates apoptotic nuclei, and DAPI counterstaining (blue) indicates total nuclei. TUNEL-positive nuclei (e) were expressed as a percentage of the total number of nuclei and were automatically counted and calculated by Image-Pro Plus software. Inflammatory cell infiltration was determined by immunohistochemistry. The sections were immunohistologically analyzed using staining of antibodies against Ly6G for neutrophils (h) and Mac-3 for macrophages (g) (n = 6 per group). Quantitative analysis of neutrophils and monocytes/macrophages was performed. Data were expressed as mean ± SEM. *P \ 0.05, **P \ 0.01 versus Sham group

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Fig. 3 Intramyocardial NLRP3 siRNA and an inflammasome inhibitor decreased infarct size and restored heart function. Forty-eight hours after intramyocardial NLRP3 siRNA injection, the heart was subjected to the MI/R injury. Non-NLRP3 siRNA-treated mice received BAY 11-7028 via IP injection 10 min before reperfusion. The mice received echo examinations, and the hearts were excised after 24 h reperfusion. Myocardial infarct size (a) was assessed by Evans Blue/TTC double staining (n = 6 per group). Representative

photographs of heart sections are shown (top). Blue-stained areas indicate non-ischemic/normal regions; red-stained areas indicate I/R but viable regions; and negatively stained areas indicate I/R and infarcted regions. Quantification of infarct size was expressed as the ratio of infarct area (Inf) to total I/R area (area-at-risk, AAR) (bottom). Cardiac function (b) was assessed by echocardiography (n = 6 per group). Data were expressed as mean ± SEM. *P \ 0.05 versus Sham group; #P \ 0.05 versus vehicle

intramyocardial injections to knock down myocardial Txnip expression. Our method of intramyocardial siRNA delivery was highly successful and markedly decreased basal Txnip expression by 53 % (Fig. 4b). Our data indicated that Txnip siRNA significantly attenuated myocardial infarct size (Fig. 4c) and restored heart function (Fig. 4d). Txnip siRNA also significantly decreased the activation of NLRP3 inflammasome, evidenced by decreased IL-1b production (Fig. 4e). Txnip siRNA did not significantly decrease the expression of NLRP3 in myocardium (Fig. 4f). Our data indicate that the Txnip-mediated NLRP3 activation possibly mainly involved the increase of interaction between Txnip and NLRP3.

NLRP3 in cardiomyocytes from neonatal mice and CMECs from adult mice. We found that NLRP3 was definitely expressed in CMECs but hardly expressed in cardiomyocytes (Fig. 5a). Next, CMECs and cardiomyocytes were subjected to SI/R injury. SI/R injury slightly increased NLRP3 expression but failed to reach statistical significance (Fig. 5a). SI/R injury did significantly increase IL-1b production in the CMECs (Fig. 5b). However, consistent with a previous study [16], SI/R injury had little effect on NLRP3 expression (Fig. 5a) and Il-1b production (Fig. 5b) in cardiomyocytes. Finally, to elucidate whether the NLRP3 inflammasome was responsible for the IL-1b production in CMECs subjected to SI/R injury, the CMECs were treated with NLRP3 siRNA and then subjected to the SI/R injury. We found that the SI/R-induced IL-1b production was significantly attenuated (Fig. 5c). Our present study demonstrated for the first time that the SI/R-induced activation of NLRP3 inflammasomes occurs in CMECs but not in cardiomyocytes. Additionally, the activation of NLRP3 inflammasomes in CMECs may be a novel cause of myocardial injury during I/R.

SI/R stimulated NLRP3 inflammasome activation in CMECs but not in cardiomyocytes Previous studies have shown that inflammasomes are differently expressed in cardiovascular and other tissues and that the activation of inflammasomes is triggered mainly in non-myocardial cells during MI/R injury, such as fibroblasts [26]. In the myocardium, ASC and NLRP3, two key structural components of the inflammasome, are expressed highest in the endothelial cells and are barely expressed in the cardiomyocytes. As endothelial cells actively participate in inflammation [4], we hypothesized that activation of NLRP3 inflammasomes may also occur in CMECs. To test this hypothesis, we first evaluated the expression of

Txnip siRNA inhibited NLRP3 inflammasome activation and SI/R injury in CMECs To further investigate the role of Txnip in the activation of NLRP3 in the CMECs. The CMECs were treated with Txnip siRNA and subjected to SI/R injury. We found that

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Fig. 4 Intramyocardial Txnip siRNA decreased the NLRP3 activation and infarct size. Forty-eight hours after intramyocardial Txnip siRNA injection, the heart was subjected to the MI/R injury. The mice received echo examinations, and the hearts were excised after 24 h reperfusion. The interaction between Txnip and NLRP3 (a) was tested using immunoprecipitation (n = 5 per group); Txnip (b) and NLRP3 expression (f) were assessed by Western blot (n = 4 animals per group); myocardial infarct size (c) was assessed by Evans Blue/ TTC double staining (n = 6 per group). Cardiac function (d) was assessed by echocardiography (n = 6 per group); IL-1b (e) levels were determined using ELISA (n = 6 per group). Data were expressed as mean ± SEM. *P \ 0.05, **P \ 0.05 versus Sham group; # P \ 0.05 versus scrambled siRNA

interactions between Txnip and NLRP3 increased in the CMECs subjected to SI/R (Fig. 6b). Additionally, Txnip siRNA inhibited SI/R-induced NLRP3 inflammasome

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activation, as evidenced by a decrease in caspase-1 expression (Fig. 6c) and IL-1b mRNA and protein production (Fig. 6d, e). Txnip did not significantly decrease

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Fig. 5 SI/R stimulated NLRP3 inflammasome activation in CMECs, but not in cardiomyocytes. a Mice neonatal cardiomyocytes were subjected to 9 h of hypoxia, followed by 3 h of reoxygenation. CMECs were subjected to 6 h of hypoxia, followed by 4 h of reoxygenation. NLRP3 expression was determined using Western blots (n = 4 per group). b Cardiomyocytes and CMECs were pretreated with LPS (100 ng/ml) for 24 h and then subjected to SI/R injury. IL-1b content was determined by ELISA(n = 6 per group).c CMECs were treated with NLRP3 siRNA for 48 h, followed by 24 h of LPS treatment, and then were subjected to SI/R injury. IL-1b content was determined by ELISA (n = 6 per group). Data were expressed as mean ± SEM. *P \ 0.05 versus Sham group; #P \ 0.05 versus vehicle

the expression of NLRP3 mRNA in CMECs (Fig. 4f). Txnip siRNA also inhibited SI/R-induced LDH release (Fig. 6g) and caspase-3 activity (Fig. 6h). ROS scavenger dissociated TXNIP from NLRP3 and inhibited the activation of NLRP3 inflammasomes in CMECs subjected to SI/R injury Reactive oxygen species has been shown to play a key role in the inflammasome activation triggered by I/R injury in cardiac fibroblasts. To test whether the Txnip-mediated NLRP3 inflammasome activation was dependent on the generation of ROS, the CMECs were treated with EUK134, an ROS scavenger, and then subjected to SI/R injury. Reactive oxygen species production was significantly increased in the CMECs subjected to the SI/R injury (Fig. 7a). Additionally, EUK134 decreased the MI/Rinduced interaction between Txnip and NLRP3 (Fig. 7b). EUK134 also lowered MI/R-induced IL-1b mRNA and protein expression (Fig. 7d, e) and Caspase-1 expression (Fig. 7c). Our results indicate that the Txnip-mediated activation of NLRP3 inflammasomes is also ROS dependent in SI/R.

Discussion Several novel observations were found in this study. Firstly, NLRP3 inflammasomes activation occurred in CMECs but not in cardiomyocytes, indicating that CMECs may mediate inflammation in response to MI/R. Secondly, Txnip activated NLRP3 inflammasomes in MI/R injury in an ROS-dependent fashion. Finally, blocking Txnip/ NLRP3 signaling inhibited the activation of NLRP3 inflammasomes and may provide insight into novel therapies for mitigating MI/R injury. MI/R injury is characterized by a rapid increase in cytokines and chemokines and an influx of leukocytes into the endangered myocardial region [1, 36]. Inflammatory responses after MI/R injury are detrimental for cell survival and extracellular matrix integrity due to their enhanced activation of pro-apoptotic signaling pathways. Interventions that target leukocytes or inflammatory mediators substantially reduce myocardial I/R injury [15, 29]. Therefore, inflammation plays a key role in the pathophysiology of MI/R injury. Increasing evidence indicates that IL-1b is a prominent and early mediator of inflammation in I/R injury [33]. I/R induces IL-1b expression in

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Page 10 of 14 Fig. 6 Txnip siRNA inhibited NLRP3 inflammasome activation and SI/R injury in CMECs. a CMECs were transfected with Txnip siRNA for 48 h, and Txnip expression was determined using Western blots (n = 4 per group); b CMECs were subjected to SI/ R injury. The interaction between Txnip and NLRP3 was determined using immunoprecipitation (n = 6 per group); c–f CMECs were treated with Txnip siRNA for 48 h, followed by 24 h of LPS treatment, and then were subjected to SI/R injury. Caspase-1 expression was determined using Western blots, and IL-1b content was determined using ELISA, and IL-1b mRNA level was determined by real-time PCR (n = 6 per group). g, h CMECs were treated with Txnip siRNA for 48 h and then subjected to SI/R injury (n = 6 per group). LDH release and caspase-3 activity were determined using enzyme activity assay kit. Data were expressed as mean ± SEM. *P \ 0.05, **P \ 0.01 versus Sham group; # P \ 0.05 versus vehicle

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Fig. 7 ROS scavenger dissociated TXNIP from NLRP3 and inhibited the activation of NLRP3 inflammasomes in CMECs subjected to SI/R injury: CMECs were treated with EUK134, an ROS scavenger. After 12 h of incubation, cells were subjected to SI/R injury. a Superoxide production was measured by lucigeninenhanced chemiluminescence (n = 6 per group). b The interaction between Txnip and NLRP3 was tested using immunoprecipitation (n = 5 per group). c, d and e CMECs were treated with EUK134 for 12 h, followed by 24 h of LPS treatment, and then were subjected to SI/R injury. Caspase-1 expression was determined using Western blots (n = 5 per group), and IL-1b content was determined using ELISA (n = 6 per group). IL1b mRNA was determined by real-time PCR (n = 6 per group). Data were expressed as mean ± SEM. *P \ 0.05 versus Sham group; #P \ 0.05 versus vehicle

the heart, and neutralizing IL-1b reduces myocardial injury after I/R [25]. Active, mature IL-1b is produced when caspase-1 cleaves the inactive pro-IL-1b precursor. This cleavage is activated by a large multiprotein complex called the inflammasome [28], which has been shown to mediate the inflammation triggered by tissue damage and endogenous danger signals [30]. So far, four inflammasomes have been described: the NLRP1 inflammasome, the NLRP3 inflammasome, the IPAF inflammasome, and the AIM2 inflammasome. The NLRP-inflammasomes are characterized by cytoplasmic receptors with NACHTdomain and leucine-rich repeats and activate caspase-1 via

the adaptor molecule ASC (apoptosis-associated speck-like protein) which has a caspase recruitment domain [34]. Recently, studies have demonstrated that ASC-/- mice had diminished MI/R injury [16], indicating the close involvement of inflammasomes with the pathophysiology of MI/R injury. The NLRP3 inflammasome is the most fully characterized inflammasomes and has been associated with a wide range of diseases including infectious, autoinflammatory, and autoimmune disorders [20]. A very recent study demonstrates that cardiac contractile function is preserved and infarct size is reduced in hearts from NLRP3-/- mice after ex vivo I/R [26]. In the present

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study, we also found that I/R significantly increased NLRP3 expression and inflammasome activation in a timedependent fashion, as evidenced by increased caspase-1 expression and IL-1b and IL-18 content. Cardiac-specific NLRP3 knockdown and an inflammasome inhibitor also attenuated macrophage (Mac-3) and neutrophil (Ly6G) infiltration, decreased myocardial infarct size, and restored the heart function after in vivo I/R. We further investigated the adhesion molecules expression, which are required for leukocyte recruitment. In an in vitro study, we found that NLRP3 siRNA decreased the ICAM-1 and E-selectin expression in the cardiac microvascular endothelial cells (CMECs) (supplement Fig. 2). These results provide further evidence that the NLRP3 inflammasome plays a key role in the pathogenesis of MI/R injury. Many activators of the NLRP3 inflammasome have already been identified, including endotoxins, K? channel openers, and uric acid [14]. Additionally, reactive oxygen species (ROS) have been identified as inflammasome activators for the pathogenesis of MI/R injury [16]. Reactive oxygen species plays a key role in the activation of inflammasome. All known NLRP3 activators generate ROS and, conversely, inhibitors of ROS block inflammasome activation [7, 39]. However, the mechanism by which ROS activates inflammasomes is poorly understood for MI/ R injury. Thioredoxin-interacting protein (Txnip), a member of the a-arrestin protein superfamily, was identified as an endogenous inhibitor of thioredoxin, a key antioxidant in the human body [23]. Txnip acts by binding to thioredoxin and inhibiting its ability to scavenge ROS [23], which indicates that Txnip is closely related to the production of ROS. Recently, it has been shown that Txnip is essential for activation of the NLRP3 inflammasome via a direct interaction with NLRP3 [39]. In the present study, we found that the interaction between TXNIP and NLRP3 was significantly increased during the MI/R injury. More importantly, Txnip siRNA also significantly decreased the activation of NLRP3 inflammasome. Txnip overexpression exacerbated the IL-1b production and SI/R injury in the CMECs (supplement Fig. 3). Our data indicate that the Txnip-mediated NLRP3 activation possibly mainly involved the increase of interaction between Txnip and NLRP3. To determine whether Txnip-mediated NLRP3 activation was ROS dependent or not, an ROS scavenger was administrated before reperfusion. We found that the ROS scavenger dissociated TXNIP from NLRP3 and inhibited the activation of NLRP3 inflammasomes in CMECs subjected to I/R injury. Our results indicated that Txnip is a novel activator of NLRP3 inflammasomes in MI/ R injury. Moreover, the Txnip-mediated NLRP3 inflammasome activation is in an ROS-dependent fashion. Inflammasomes are differentially expressed in cardiovascular and other tissues [37]. The inflammasome

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components ASC and NALP3 were shown to be highly expressed in infiltrating inflammatory cells derived from bone marrow. In the myocardium, endothelial cells and fibroblasts have the highest NALP3 and ASC expression levels by an immunofluorescence technique [21, 32]. A recent study also demonstrates that cardiomyocyte hardly expresses NLRP3, but can be induced to expression NLRP3 in the mRNA level. In the present study, we found that NLRP3 was definitely expressed in CMECs, but barely expressed in cardiomyocytes in vitro experiment. More importantly, SI/R injury still did not induce the expression of NLRP3 in the protein level. Existing evidence has indicated that inflammasome activation occurs in cardiac fibroblasts, but not cardiomyocytes during the MI/R injury [16]. A very recent study reveals a contribution of cardiac fibroblast NPLR3 to ischemia–reperfusion injury [26]. Microvascular endothelial cells are among the most dynamic and biologically active cellular components of blood vessels, which play a crucial role in the pathogenesis of systemic vascular inflammation [5]. Microvascular endothelial cells at a site of inflammation are both active participants in and regulators of inflammatory processes [24]. Cardiac microvascular endothelial cells are a major component of non-muscle cells in the heart. Each cardiomyocyte is surrounded by at least 3–4 myocardial capillaries [6]. It has been demonstrated previously that reperfusion induces the release of soluble pro-apoptotic mediators from cardiac microvascular endothelial cells that promote myocyte apoptosis [27], which implicates that cardiac microvascular endothelial play a key role in triggering cardiomyocyte injury during ischemia/reperfusion injury. CMECs act as ‘‘sentinel’’ cells that sense danger signals because they express toll-like receptors and possess nucleotide-binding oligomerization domains (NODs), both of which are mediators of innate immunity [9, 12]. Therefore, we supposed that CMECs may play a key role in the activation of NLRP3 inflammasome due to the role of endothelial cells in inflammation. In the present study, we also showed that SI/R injury stimulates interaction between TXNIP and NLRP3 and that Il-1b was detected in CMECs, but not cardiomyocytes subjected to SI/R. Txnip siRNA inhibited SI/R-induced NLRP3 inflammasome activation, as evidenced by a decrease in caspase-1 expression. These observations suggested that NLRP3 inflammasome activation occur in CMECs but not in cardiomyocytes. Attenuating endothelial injury by blocking the activation of NLRP3 inflammasome may reduce cardiomyocyte cell death after ischemia and reperfusion ultimately. Taken together, our results demonstrated, for the first time, that the NLRP3 inflammasome is activated in CMECs but not in cardiomyocytes, and that cardiac-specific NLRP3 knockdown attenuates MI/R injury. Our results indicated that the NLRP3 inflammasome plays an

Basic Res Cardiol (2014) 109:415

essential role in the pathophysiology of MI/R injury and that CMECs may mediate inflammation in this response. Additionally, Txnip was shown to mediate the activation of NLRP3 inflammasomes in an ROS-dependent fashion in CMECs. Interventions that block Txnip/NLRP3 signaling inhibit the activation of NLRP3 inflammasome and may provide novel therapies for mitigating MI/R injury. Acknowledgments This work was supported by program for Chinese National Science Fund for Distinguished Young Scholars (Grant No. 81225001), National Key Basic Research Program of China (973 Program, 2013CB531204), New Century Excellent Talents in University (Grant No. NCET-11-0870), Chinese National Science Funds (Grants Nos. 81070676 and 81170186), Innovation Team Development Grant by China Department of Education (2010CXTD01) and Major Science and Technology Projects of China ‘‘Significant New Drug Development’’ (Grant No. 2012ZX09J12108-06B). Conflict of interest exists.

The authors declare that no conflict of interest

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reperfusion injury.

NLRP3 inflammasome is necessary for initiating acute sterile inflammation. Recent studies have demonstrated that NLRP3 inflammasome is up-regulated an...
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