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

<div><p>Objective</p><p>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.</p><p>Methods and results</p><p>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.</p><p>Conclusions</p><p>TXL protects diabetic hearts against I/R injury by activating Angptl4-mediated restoration of endothelial barrier integrity via the PPAR-α pathway.</p></div

Identification of endothelial cell apoptosis in the I/R-injured hearts treated with or without TXL by confocal microscopy.

<p>Endothelial cells were identified by red fluorescence (CD34), total cell number was detected by blue fluorescence (DAPI DNA staining), and apoptosis was detected by green fluorescence (TUNEL). Apoptotic endothelial cells were detected and counted by colocalized red and green (displayed as yellow). I/R injury induced significant ECs apoptosis in both MI control and diabetic MI rats (n = 8 in each group). Treatment with insulin, rhAngptl4 or TXL ameliorated ECs apoptosis compared with the diabetic MI controls. Whereas, co-treatment with Angptl4 siRNA partially blocked the beneficial effect of TXL. Administration of the PPARα inhibitor MK886 also reversed the inhibition effect of TXL on ECs apoptosis, but not reduce ECs apoptosis in the rhAngptl4-treated animals. Red arrows indicate endothelial cells and white arrows show apoptotic cells. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198403#pone.0198403.g001" target="_blank">Fig 1</a>.</p

Expression levels of JAM-A, VE-cadherin, and integrin-α5 in the I/R-injured hearts with or without TXL treatment.

<p>I/R injury decreased the expression levels of JAM-A (A), VE-cadherin (B), and Integrin-α5 (C). Pre-treatment with insulin, rhAngptl4, or TXL up-regulated expression levels of JAM-A (A), VE-cadherin (B), and Integrin-α5 (C). Addition of Angptl4 siRNA canceled the effects of TXL-induced up-regulation of JAM-A (A) and VE-cadherin (B), but not Integrin-α5 (C). Co-treatment with MK886 abolished the TXL-upregulated expression of JAM-A (A), VE-cadherin (B), and Integrin-α5 (C). Compared with DB-sham group, *<i>P</i><0.05, ** <i>P</i><0.01; Compared with the DB-MI group, ^†<i>P</i><0.05, ^††<i>P</i><0.01; Compared with the TXL group, ^‡<i>P</i><0.05, ^‡‡<i>P</i><0.01; Compared with the rhAngptl4+siR group, ^§§<i>P</i><0.01. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198403#pone.0198403.g001" target="_blank">Fig 1</a>.</p

Evaluation of intramyocardiac hemorrhage in the infarcted hearts treated with or without TXL.

<p>Sections of the hearts were stained with hematoxylin-eosin (n = 8 in each group). DB-sham group has no obvious extravasation of red blood cells in the interstitial space (A). I/R injury induced apparent extravasation of red blood cells into the interstitial space in both DB-MI (B) and non-DB-MI (C) groups. Treatment with insulin (D), rhAngptl4 (E) or TXL (F) greatly decreased extravasation of red blood cells. Combination with angptl4 siRNA canceled the effects of rhAngptl4 (I) and TXL (J). However, combination with MK886 abolished the effect of TXL (L) but not rhAngptl4 (K). Images were taken under a Leica microscope with 40×objective. Black arrows indicate intra-myocardiac hemorrhage. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198403#pone.0198403.g001" target="_blank">Fig 1</a>.</p

Histopathologic assessments of the area at risk and necrosis in the infarcted hearts treated with or without TXL in the presence or absence of signal regulators.

<p>The area at risk and necrosis was respectively examined by Evans blue and triphenyltetrazolium chloride (TTC) staining (n = 8 in each group). The health myocardium was stained blue by Evans blue, the area at risk (AAR) was not stained by Evans blue. TTC-unstained white myocardium was identified as the area of necrosis (AN). Abbreviations: DB-sham = Diabetic sham; DB-MI = Diabetic MI control; non-DB-MI = non-diabetic MI control; rhAngptl4 = recombinant human Angptl4; rhAngptl4+siCtrl = rhAngptl4+control siRNA; TXL+siCtrl = TXL+control siRNA; rhAngptl4+siR = rhAngptl4+Angptl4 siRNA; TXL+siR = TXL+Angptl4 siRNA.</p

Expression levels of Angptl4 and analysis of PPAR-α activity in the I/R-injured diabetic hearts treated with or without TXL in the presence or absence of signal regulators.

<p>(A) Angptl4 expression was decreased by I/R injury, and pre-treatment with insulin, rhAngptl4, or TXL reverted this effect. Whereas, Angptl4 siRNA and MK886 abolished the TXL-induced upregulation of Angptl4. (B) I/R injury decreased the PPAR-α activity in the I/R-injured myocardium, and even worse in diabetic hearts. Pre-treatment of insulin, rhAngptl4 and TXL increased the PPAR-α activity. Addition of MK886 but not Angptl4 siRNA abolished the TXL-stimulated PPAR-α activation. Compared with the DB-sham group, *<i>P</i><0.05, **<i>P</i><0.01; Compared with the DB-MI group, ^†<i>P</i><0.05, ^††<i>P</i><0.01; Compared with the TXL group, ^‡<i>P</i><0.05, ^‡‡<i>P</i><0.01; Compared with the rhAngptl4+siR group, ^§§<i>P</i><0.01. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198403#pone.0198403.g001" target="_blank">Fig 1</a>.</p

Cardiac Microvascular Barrier Function Mediates the Protection of Tongxinluo against Myocardial Ischemia/Reperfusion Injury

<div><p>Objective</p><p>Tongxinluo (TXL) has been shown to decrease myocardial necrosis after ischemia/reperfusion (I/R) by simulating ischemia preconditioning (IPC). However, the core mechanism of TXL remains unclear. This study was designed to investigate the key targets of TXL against I/R injury (IRI) among the cardiac structure-function network.</p><p>Materials and Methods</p><p>To evaluate the severity of lethal IRI, a mathematical model was established according to the relationship between myocardial no-reflow size and necrosis size. A total of 168 mini-swine were employed in myocardial I/R experiment. IRI severity among different interventions was compared and IPC and CCB groups were identified as the mildest and severest groups, respectively. Principal component analysis was applied to further determine 9 key targets of IPC in cardioprotection. Then, the key targets of TXL in cardioprotection were confirmed.</p><p>Results</p><p>Necrosis size and no-reflow size fit well with the Sigmoid Emax model. Necrosis reduction space (NRS) positively correlates with I/R injury severity and necrosis size (<i>R²</i>=0.92, <i>R²</i>=0.57, <i>P</i><0.01, respectively). Functional and structural indices correlate positively with NRS (<i>R²</i>=0.64, <i>R²</i>=0.62, <i>P</i><0.01, respectively). TXL recovers SUR2, iNOS activity, eNOS activity, VE-cadherin, β-catenin, γ-catenin and P-selectin with a trend toward the sham group. Moreover, TXL increases PKA activity and eNOS expression with a trend away from the sham group. Among the above nine indices, eNOS activity, eNOS, VE-cadherin, β-catenin and γ-catenin expression were significantly up-regulated by TXL compared with IPC (P>0.05) or CCB (P<0.05) and these five microvascular barrier-related indices may be the key targets of TXL in minimizing IRI.</p><p>Conclusions</p><p>Our study underlines the lethal IRI as one of the causes of myocardial necrosis. Pretreatment with TXL ameliorates myocardial IRI through promoting cardiac microvascular endothelial barrier function by simulating IPC.</p></div

Relationship between NRS and functional or structural indices.

<p><b>A</b>: Association between NRS and functional indices. <b>B</b>: Association between NRS and structural indices. Abbreviations: NRS = necrosis reduction space; PC1 = Principal Component 1.</p

Comparison of lethal IRI under all interventions.

<p><b>A</b>: Illustration for lethal IRI zone. The no-reflow zone was unstained using thioflavin S, and the necrotic zone was unstained using TTC. The surviving myocardium lies outside the dotted line, and the necrotic myocardium lies inside of the dotted line. <b>B</b>: The positive correlation between NRS value and IRI value (<i>R</i>^<i>2</i> = 0.92, P<0.01) indicates NRS is a valuable surrogate marker for lethal IRI severity. <b>C-D</b>: Necrosis size is positively correlated with NRS (<i>R</i>^<i>2</i> = 0.57, P<0.01) or IRI value (<i>R</i>^<i>2</i> = 0.52, P<0.01), underlining the linear relationship between IRI and necrosis size. <b>E</b>: Necrosis size has weak linear relationship with no-reflow size (<i>R</i>^<i>2</i> = 0.10, P<0.01). <b>F-G</b>: Observational values of NRS and IRI were expressed as the mean±SE; the error bars were the standard deviations. The significance of differences between groups was analyzed using the one way ANOVA, *<i>P</i><0.05, **<i>P</i>< 0.01 vs IPC group. Abbreviations: NRS = necrosis reduction space; IRI = ischemia/reperfusion injury.</p

Data sources for mathematical modeling.

<p>Abbreviations:</p><p>BW = body weight;</p><p>MAP = mean aortic pressure;</p><p>HR = heart rates;</p><p>IPC = ischemia preconditioning.</p><p>Data sources for mathematical modeling.</p