Effect Of Tongxinluo Protecting Endothelium Barrier On Reducing Myocardial Reperfusion Injury In Diabetes Mellitus And Its Mechanism

Part 1 Cardioprotection of Tongxinluo against acute myocardial infarction/reperfusion injury: reanalysis based on Sigmoid Emax mathematical modelingObjective: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 evaluate the severity of IRI with the formula:IRI(%)=necrosis size(%)-no-reflow size(%) and then investigate the key targets of TXL against I/R injury (IRI) among the cardiac structure-function network.Methods:To evaluate the severity of lethal IRI, a mathematical model was established according to the relationship between myocardial no-reflow size and necrosis size based on the data from 19 pieces of our published paper. A total of 168 mini-swine were employed in subsequent reanalysis. IRI severity among IPC, ischemia postconditioning, simvastatin, TXL, rosuvastatin, nicorandil, carvedilol, adenosine, valsartan, tiroffiban, verapamil and diltiazem interventions was compared. 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.Results: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 (R2=0.92, R2=0.57, P<0.01, respectively). Functional and structural indices correlate positively with NRS (R2=0.64, R2=0.62, P<0.01, respectively). TXL recovers SUR2, iNOS activity, eNOS activity, VE-cadherin, β-catenin, y-catenin and P-selectin with a trend toward the sham group (all P<0.05). The effect of TXL is similar with that of simvastatin (all P>0.05). Moreover, TXL increases PKA activity and eNOS expression with a trend away from the sham group (all P<0.05). The effect of TXL is similar with that of simvastatin (all P>0.05). Among the above nine indices, eNOS activity, eNOS, VE-cadherin, β-catenin and y-catenin expression were significantly up-regulated by TXL compared with IPC (all P>0.05), simvastatin (all P>0.05) or CCB (all P<0.05) and these five microvascular structure and function-related indices may be the key targets of TXL in minimizing IRI.Conclusion:Our study underlines the lethal IRI as one of the causes of myocardial necrosis. Pretreatment with TXL ameliorates myocardial IRI through promoting cardiac microvascular structure and function by simulating IPC. The effect of TXL is similar with that of statin.Part 2 Enhancing endothelial barrier function in vitro:induction of Angptl4 by Tongxinluo via PPARa pathway protects diabetic hearts from reperfusion injuryObjective:Endothelial barrier is disrupted by hypoxia/reoxygenation and high glucose condition. The study in part 2 had revealed that effect of TXL in protecting endothelial barrier function was related to reducing infarct size. However, whether endothelial cell-derived Angptl4 protects reperfused diabetic myocardium in a paracrine way remains unkown. Using knock-down technique in endothelial cells, this study was designed to confirm the core role of Angptl4 in protecting endothelial barrier function in high glucose condition. Futhermore, this study aimed to verify the relationship between different kinds of endothelial junction protein and cardioprotection of TXL.Methods:The 5th passage human cardiac microvascular endothelial cells (CMECs) were cultured in high glucose (18 mM) followed by 2 h of glucose-oxygen-serum deprivation and 2 h of reoxygenation (OGSD/R). CMECs were randomized devided into normal glucose+OGSD/R, high glucose+OGSD/R, insulin(1nM), Angptl4(1μg/ml), TXL(800μg/ml), Angptl4+negative control siRNA、TXL+negative control siRNA、Angptl4+Angptl4 siRNA、TXL+Angptl4 siRNA、Angptl4+MK886(PPARa inhibitor,1 μM)、TXL+MK886、MK886 groups. RNA interfering technique was applied to knockdown Angptl4 in vitro. Endothelial monolayer permeability was measured using fluorescein-isothiocyanate (FITC)-dextran. Cellular skeleton and adhesion junction protein VE-cadherin location and internalization was detected by confocal microscope. Angptl4 in the supernatant and peroxisome proliferator activated receptor-α (PPARa) activity in the nucleus was measured by ELISA. Real time-PCR was used to detect Angptl4 mRNA level. Angptl4, integrin-α5, β-integrin,JAM-A, Occludin, p120-catenin and VE-cadherin expression was measured by Western blot. Furthermore, membrane expression of integrin-a5, JAM-A and VE-cadherin was measured by Western blot.Results:TXL remarkablely reduced endothelial monolayer permeability in the OGSD/R stimulated CMECs under high glucose condition compared with high glucose group (P<0.05). The effect of TXL on endothelial monolayer permeability was similar with those of insulin and Angptl4 (P>0.05).Both Angptl4 knock-down and MK886 could significantly inhibit the effect of TXL (all P<0.05).In the OGSD/R stimulated CMECs under high glucose condition, TXL pretreatment increased PPARa activity and Angptl4 expression remarkablely (all P<0.05). These effects of TXL were similar with those of insulin and Angptl4 (all P>0.05). Both Angptl4 knock-down and MK886 could significantly inhibit the effect of TXL (all P<0.05).In the OGSD/R stimulated CMECs under high glucose condition, TXL increased the expression of JAM-A, VE-cadherin and integrin-a5 (all P<0.05). Both Angptl4 knock-down and MK886 could significantly inhibit the effect of TXL on JAM-A and VE-cadherin expression(all P<0.05).High glucose impaired membrane JAM-A, membrane VE-cadherin and membrane integrin-α5 in CMECs under OGSD/R conditions (all P<0.05). TXL increased the expression of membrane VE-cadherin and membrane integrin-a5 compared with high glucose group (all P<0.05).The protection of TXL was similar with that of insulin and Angptl4 (all P>0.05) but the protection of TXL on membrane integrin-a5 expression was inhibited by Angptl4-siRNA or MK886 (all P<0.05).Conclusion:High glucose worsens hypoxia/reoxygenation injury of CMECs under OGSD/R conditions. The protection of TXL is similar with that of insulin and Angptl4. TXL could decrease endothelial monolayer permeability and apoptosis of CMECs partly by PPARα activation-mediated Angptl4 upregulation.Part 3 Enhancing endothelial barrier function in vivo:induction of Angptl4 by Tongxinluo via PPARa pathway protects diabetic hearts from reperfusion injuryObjective:To investigate whether the cardioprotective effects of Tongxinluo (TXL) against myocardial ischemia/reperfusion injury relates to the PPARa activation-mediated upregulation of Angptl4 in reperfused diabetic hearts.Methods:In a 45minute ischemia and 180-minute reperfusion model,104 ZDF diabetic rats were randomized into diabetes+sham operation, diabetes+myocardial infarction(MI), non-diabetes+MI, insulin(5U/100g body weight), Angptl4(10μg/kg body weight), TXL(5mg/100g body weight), Angptl4+control siRNA, TXL+control siRNA, Angptl4+Angptl4 siRNA, TXL+Angptl4 siRNA, Angptl4+MK886(a PPARa inhibitor,0.4mg/kg body weight), TXL+MK886, MK886 groups(n=8 in each group). RNA interfering technique was applied to knockdown Angptl4 in vivo. Hemodynamics was monitored during the procedure. The area of necrosis (AN) was determined by pathological studies. Microvascular permeability was determined by Miles method of FITC-dextran staining. Myocardial hemorrhage was studied by hematoxylin-eosin (H-E) staining. Myocardial myeloperoxidase (MPO) and p120-catenin expression was detected by immunohistochemistry. Microvascular fine structure was observed by transmission electron microscopy (TEM). Blood glucose and serum activity of creatine kinase (CK) were determined by biochemical methods. Serum cardiac Troponin Ⅰ/(cTnI) and PPARa activity in the myocardial tissue were detected by ELISA. Myocardial apoptosis was detected with double staining with TUNEL and a molecular marker of endothelial cell CD34. Myocardial expression of PPARα, Angptl4, integrin-α5, JAM-A and VE-cadherin were detected by Western blot.Results:Hemodynamics was modified in insulin group, Angptl4 group and TXL group, compared with diabetes+MI group (all P<0.01). SiRNA-mediated Angptl4 konck-down could obviously abolish the effect of TXL (all P<0.01). MK886 worsened the hemodynamics compared with TXL group (P<0.01).TXL pretreatment remarkablely reduced the area of infarction compared with diabetes+MI group. There were no significant difference of the area of infarction among TXL, insulin and Angptl4 groups (all P>0.05). Angptl4 konck-down could obviously abolish the effect of TXL (P<0.01). TXL+MK886 increased the infarct size compared with TXL (P<0.01).The florescein isothiocyanate (FITC)-dextran concentration was also lower in TXL group than in diabetes+MI group (P<0.05). There were no significant difference of FITC-dextran concentration among insulin, Angptl4 and TXL groups (all P>0.05). Angptl4 siRNA knock-down could obviously decrease FITC-dextran concentration (P<0.05).H-E staining revealed that TXL reduced local hemorrhage score from 1.98 to 1.21 (P<0.05). Effect of TXL in reducing local hemorrhage score was similar with those of insulin and Agnptl4 (all P>0.05). Angptl4 knock-down diminished the effect of TXL (P<0.05). MK886 inhibited the effect of TXL (P<0.05).Immunohistochemical examination revealed that TXL reduced MPO expression but increased p120-catenin expression in the area at risk (all P<0.05). The effects of TXL, insulin and Angptl4 were similar in both MPO and p120-catenin expression (all P>0.05). Angptl4 knock-down and MK886 could inhibit the effect of TXL (all P<0.05).Westerrn blot analysis showed that endothelial junction protein expression of JAM-A, VE-cadherin, Integrin-a5 was increased by TXL pretreatment. There was no significant difference for the above junction protein between TXL and Angptl4 groups (all P>0.05) and there was no significant difference for JAM-A, VE-cadherin and p120-catenin expression between TXL and insulin groups(all P>0.05). Angptl4 knock-down and MK886 could inhibit the effect of TXL (all P<0.05).Myocardial PPARa activity was elevated by TXL pretreatment compared with diabetes+MI group (P<0.01). Effect of TXL in increasing PPARa activity was similar with that of insulin (P>0.05). MK886 diminished PPARa activity in contrast to TXL group (P<0.01). TXL pretreatment increased Angptl4 protein expression compared with diabetes+MI group. Effect of TXL in upregulating Angptl4 expression was similar with those of insulin and Angptl4. Angptl4 knock-down could diminish the effect of TXL. The expression of Angptl4 was lower in TXL+MK886 group than in TXL group (P<0.01).Conclusion:Pretreatment with single loading dose of TXL just 1 hour before AMI can attenuate the diabetic hearts against myocardial ischemia/reperfusion injury by reducing cardiac microvascular endothelial cell apoptosis and enhancing endothelial cell barrier function. The cardioprotection of TXL is similar with insulin or Angptl4, and relates to the PPARa activation-mediated upregulation of Angptl4 in reperfused diabetic hearts.