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TXNIP mediates NLRP3 inflammasome activation in cardiac microvascular endothelial cells as a novel mechanism in myocardial ischemia/reperfusion injury

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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-1β 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 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.

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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).

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Authors and Affiliations

  1. Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, 15 Changle West Road, Xi’an, 710032, China

    Yi Liu, Kun Lian, Lijian Zhang, Rutao Wang, Fu Yi, Chao Gao, Chao Xin, Di Zhu, Yan Li, Wenjun Yan, Haichang Wang & Ling Tao

  2. Department of Anesthesiology, Xijing Hospital, The Fourth Military Medical University, 15 Changle West Road, Xi’an, 710032, China

    Lize Xiong

  3. The Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA

    Erhe Gao

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

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395_2014_415_MOESM1_ESM.tif

Supplement Fig1 NLRP3 siRNA had no effect on the expression of Txnip in CMECs. CMECs were transfected with NLRP3 siRNA for 48 hours, and Txnip expression was determined using Western blots (n = 5 per group). Data were expressed as mean ± SEM (TIFF 324 kb)

395_2014_415_MOESM2_ESM.tif

Supplement Fig 2 NLRP3 siRNA decreased the E-selectin and ICAM-1 expression in CMECs subjected to SI/R. CMECs were transfected with NLRP3 siRNA for 48 hours and then subjected to SI/R. E-selectin (A) and ICAM-1 (B) expression express was determined by western-blot (n = 5 per group). Data were expressed as mean ± SEM. *P<0.05 vs. Sham group; # P<0.05 vs. Vehicle (TIFF 1,627 kb)

395_2014_415_MOESM3_ESM.tif

Supplement Fig3 Txnip overexpression exacerbated NLRP3 inflammasome activation and SI/R injury in CMECs. A) CMECs were transfected with Txnip-GFP for 24 hours, and Txnip expression was determined using Western blots (n = 5 per group). B) CMECs were transfected with Txnip-GFP for 24 hours and then were subjected to SI/R injury followed by 24 hours of LPS treatment, and then were subjected to SI/R injury. IL-1β content was determined using ELISA (n = 6 per group).C and D) CMECs were treated with Txnip-GFP for 24 hours and then subjected to SI/R injury. LDH release and caspase-3 activity were determined using enzyme activity assay kit (n = 6 per group). Data were expressed as mean ± SEM. $$ P<0.01 vs. Control group, *P<0.05, **P<0.01 vs. Sham group; # P<0.05 vs. Vehicle group (TIFF 3,671 kb)

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Liu, Y., Lian, K., Zhang, L. et al. TXNIP mediates NLRP3 inflammasome activation in cardiac microvascular endothelial cells as a novel mechanism in myocardial ischemia/reperfusion injury. Basic Res Cardiol 109, 415 (2014). https://doi.org/10.1007/s00395-014-0415-z

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