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      Tongxinluo attenuates reperfusion injury in diabetic hearts by angiopoietin-like 4-mediated protection of endothelial barrier integrity via PPAR-α pathway

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          Abstract

          Objective

          Endothelial barrier function in the onset and Tongxinluo (TXL) protection of myocardial ischemia/reperfusion (I/R) injury, and TXL can induce the secretion of Angiopoietin-like 4 (Angptl4) in human cardiac microvascular endothelial cells during hypoxia/reoxygenation. We intend to demonstrate whether TXL can attenuate myocardial I/R injury in diabetes, characterized with microvascular endothelial barrier disruption, by induction of Angptl4-mediated protection of endothelial barrier integrity.

          Methods and results

          I/R injury was created by coronary ligation in ZDF diabetic and non-diabetic control rats. The animals were anesthetized and randomized to sham operation or I/R injury with or without the exposure to insulin, rhAngptl4, TXL, Angptl4 siRNA, and the PPAR-α inhibitor MK886. Tongxinluo, insulin and rhAngptl4 have the similar protective effect on diabetic hearts against I/R injury. In I/R-injured diabetic hearts, TXL treatment remarkably reduced the infarct size, and protected endothelial barrier integrity demonstrated by decreased endothelial cells apoptosis, microvascular permeability, and myocardial hemorrhage, fortified tight junction, and upregulated expression of JAM-A, integrin-α5, and VE-cadherin, and these effects of TXL were as effective as insulin and rhAngptl4. However, Angptl4 knock-down with siRNA interference and inhibition of PPAR-α with MK886 partially diminished these beneficial effects of TXL and rhAngptl4. TXL induced the expression of Angptl4 in I/R-injured diabetic hearts, and was canceled by Angptl4 siRNA and MK886. TXL treatment increased myocardial PPAR-α activity, and was abolished by MK886 but not by Angptl4 siRNA.

          Conclusions

          TXL protects diabetic hearts against I/R injury by activating Angptl4-mediated restoration of endothelial barrier integrity via the PPAR-α pathway.

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          Most cited references41

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          A potential role for angiopoietin 2 in the regulation of the blood-retinal barrier in diabetic retinopathy.

          Although VEGF has been identified as an important mediator of the blood-retinal barrier alteration in diabetic retinopathy, the hypothesis for this study was that that other molecules, including the angiopoietins (Ang-1 and -2), may play a role. The expression of angiopoietins was analyzed in an animal model of diabetic retinopathy, and the role of Ang-2 in the regulation of diabetes-induced alterations of vascular permeability was characterized. Diabetes was induced in rats, and human retinal endothelial cells (HRECs) were grown in media with 5.5 or 30.5 mM glucose. Levels of Ang-1 and -2 mRNA and protein were analyzed. Fluorescence-based assays were used to assess the effect of Ang-2 on vascular permeability in vivo and in vitro. The effect of Ang-2 on VE-cadherin function was assessed by measuring the extent of tyrosine phosphorylation. Ang-2 mRNA and protein increased in the retinal tissues after 8 weeks of diabetes and in high-glucose-treated cells. Intravitreal injection of Ang-2 in rats produced a significant increase in retinal vascular permeability. Ang-2 increased HREC monolayer permeability that was associated with a decrease in VE-cadherin and a change in monolayer morphology. High glucose and Ang-2 produced a significant increase in VE-cadherin phosphorylation. CONCLUSIONS; Ang-2 is upregulated in the retina in an animal model of diabetes, and hyperglycemia induces the expression of Ang-2 in isolated retinal endothelial cells. Increased Ang-2 alters VE-cadherin function, leading to increased vascular permeability. Thus, Ang-2 may play an important role in increased vasopermeability in diabetic retinopathy.
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            Insulin resistance in the vasculature.

            Insulin resistance is typically defined as a reduced ability of insulin to induce glucose uptake by target tissues such as fat and skeletal muscle cells. It accompanies several disease states, including obesity, type 2 diabetes, hepatitis C, and polycystic ovary syndrome, and is a primary feature of metabolic syndrome. Outside of its effects on blood glucose levels, insulin resistance is also associated with a 2- to 3-fold increased risk of cardiovascular mortality. In 1996, Alain Baron, Helmut Steinberg, and colleagues demonstrated that insulin resistance is associated with endothelial dysfunction. This seminal observation led to significant advances in our understanding of insulin's action in health and disease.
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              Protection against myocardial infarction and no-reflow through preservation of vascular integrity by angiopoietin-like 4.

              Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell-cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI. We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4-deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4-deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4-deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits. These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: SoftwareRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: SoftwareRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: ResourcesRole: Software
                Role: ConceptualizationRole: ResourcesRole: SoftwareRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: ResourcesRole: Software
                Role: InvestigationRole: MethodologyRole: ResourcesRole: Software
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                18 June 2018
                2018
                : 13
                : 6
                : e0198403
                Affiliations
                [1 ] State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
                [2 ] Division of Cardiovascular Medicine, University of Texas Health Science Center at Houston, Houston, TX, United States of America
                [3 ] Peking Key Laboratory for Pre-clinical Evaluation of Cardiovascular Implant Material, State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases, Animal Experimental Center, Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
                Virginia Commonwealth University Department of Internal Medicine, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Article
                PONE-D-18-01997
                10.1371/journal.pone.0198403
                6005559
                29912977
                d794db49-5e9e-446a-972e-dc0b7a9d02d8
                © 2018 Qi et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 21 January 2018
                : 20 May 2018
                Page count
                Figures: 7, Tables: 0, Pages: 19
                Funding
                Funded by: National Basic Research Program (973 Program) in China
                Award ID: 2012CB518602
                Award Recipient :
                Funded by: CAMS Innovation Fund for Medical Sciences (CIFMS)
                Award ID: 2016-I2M-1-009
                Award Recipient :
                Funded by: National Science Foundation of China
                Award ID: 81370223
                Award Recipient :
                Funded by: National Science Foundation of China
                Award ID: 81573957
                Award Recipient :
                This study was supported by National Basic Research Program (973 Program) in China (No. 2012CB518602), CAMS Innovation Fund for Medical Sciences (CIFMS) (No. 2016-I2M-1-009), the National Science Foundation of China (No. 81370223 and 81573957) and Postgraduates’ Innovative Foundation of PUMC, China (2013-1002-39).
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                All relevant data are within the paper and its Supporting Information files, and found at: https://doi.org/10.6084/m9.figshare.6455966.v1

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