Role Of Thioredoxin-interacting Protein (TXNIP)-Wnt In Diabetic Myocardial Infarction-induced Angiogenetic Suppression

The incidence of diabetes recently has been booming in the world. Patients withDiabetes increase more than7million per year. At present, China has more than94milliondiabetic patients. That is, China has the most diabetic patients in the world. In addition,there are about150million Chinese people who are involved in pre-diabetic state.Meanwhile, Cardiovascular complications are the leading killers of diabetic patients. Therisk of coronary heart disease in diabetic patients increases2-4times. More than75%ofdiabetic patients are hospitalized because of coronary heart disease and more than50%ofdiabetic patients die from coronary heart disease. More importantly, diabetes not onlyincreases the incidence of myocardial infarction, but also increases the incidence of angina,heart failure, death after myocardial infarction. At present, the treatment strategy fordiabetic myocardial tissue damage still can’t fundamentally curb the progression ofdiabetic cardiovascular complications. The incidence and mortality of cardiovascular accidents in patients with diabetes mellitus remain unacceptably high, so prevention andcontrol task is very high.As for myocardial ischemia, establishment of angiogenesis and collateral circulation isvery important for the improvement and maitenance of blood supply of ischemicmyocardium. However, diabetes significantly inhibits the establishment of collateralcirculation in ischemic myocardium, manifested as the density of diabetic capillaries andsmall arteries after myocardial infarction was significantly lower than non-diabetic group,which may be an important approach to increase the coronary heart disease mortality indiabetes, but its internal mechanism remains unclear. Due to the presence of autonomicneuropathy, diabetic patients are often with painless myocardial ischemia and evenpainless myocardial infarction, so they cannot receive timely medical treatment. Therefore,the establishment of compensatory angiogenesis and collateral circulation for theprognosis of patients with diabetes is particularly important to help maintain cardiacfunction, reduce the occurrence of heart failure and sudden death. Clarifying the keymechanisms has important theoretical and clinical value for improving the prognosis ofdiabetes myocardial infarction.Thioredoxin interacting protein (TXNIP), as a hotspot marker in the field of diabetesresearch in recent years, plays a key role in mediating pancreas islet β-cell damage in thedevelopment of diabetes. TXNIP promoter region contains a carbohydrate responseelement ChoRE. The transcription and expression of TXNIP are affected by the regulationof blood glucose. According to the current research progress, We proposed the followingquestions in this study:①TXNIP shows high expression in diabetes, and it is an earlyresponse gene of diabetes and high blood glucose，which eventually leads to oxidativestress, inflammation, cell apoptosis, etc. Whether the effect of diabetes on ischemicmyocardial angiogenesis is also associated with TXNIP?②Wnt/β-catenin signalingpathway regulates cell proliferation, migration and differentiation during embryonicdevelopment. This signaling pathway has played an important role in the process ofangiogenesis, myocardial hypertrophy, atherosclerosis development. Whether TXNIPcould regulate cell functions through Wnt/β-catenin?③Wnt/β-catenin signaling pathway in mice can be activated after myocardial infarction, and β-catenin nucleartranslocation of coronary artery endothelial cells increases in ischemic myocardium.However, what is the expression level of β-catenin of cardiac endothelial cells under thecondition of diabetes after myocardial infarction? All of these problems above need to befurther in-depth studied.【Objective】1. To establish diabetic myocardial infarction model and make clear that the formationdisorder of cardiac angiogenesis under diabetic myocardial infarction is related todiabetes-aggravated myocardial infarction injury.2. To analyze the expression of TXNIP and β-catenin in diabetic myocardial infarctioncondition, and explore the underlying mechanism related to diabetes myocardialinfarction-induced impairment of cardiac angiogenesis and collateral circulation.3. To clarify the status of β-catenin in diabetes myocardial infarction-inducedimpairment of cardiac angiogenesis and collateral circulation, and the ability to partialrecovery of cardiac angiogenesis in diabetes myocardial infarction by correcting Wnt.【Methods】1.8week old C57male mice received intraperitoneal injection of streptozotocin (STZ,50mg/kg,5consecutive days).3consecutive random blood glucose>11.1mmol/Lwas regarded as diabetes successful model. In the basis of diabetic model, diabeticmyocardial infarction model was established by ligation of the left anteriordescending coronary artery.2. The mice were randomly divided into4groups respectively, that was: Control+sham(CS), diabetes+sham (DS), Control+myocardial infarction (CMI), diabetes+myocardial infarction (DMI). To detect cardiac endothelial cell proliferation,apoptosis, TXNIP and β-catenin protein levels, expression of superoxide anion4daysafter myocardial infarction, and to detect the cardiac cardiovascular density, andcardiac function30days after myocardial infarction.3. The diabetic myocardial infarction mice were randomly divided into two groups:diabetic myocardial infarction (DMI) and DMI+lithium chloride (LiCl). To detect levels of endothelial cell proliferation, apoptosis, endothelial cell β-catenin proteinlevel4days after myocardial infarction, and to detect the cardiac cardiovasculardensity, and cardiac function30days after myocardial infarction.4. Using double immunofluorescent labeling with TUNEL and CD31, cardiacendothelial cell apoptosis in mice was observed with a laser confocal microscope.5. Using double immunofluorescent labeling with CD31and Ki67, CD31and TXNIP,CD31and β-catenin, respectively, cardiac endothelial cell proliferation, TXNIP andβ-catenin expression were observed with a laser confocal microscope.6. Using immunohistochemistry to label CD31, cardiac vascular density was captured.7. Small animal ultrasound was used to detect cardiac function in mice.8. Cultivated human umbilical vein endothelial cells (HUVEC) were divided into thefollowing groups:A. Control group (5.5mmol/L glucose), high glucose group (22mmol/L glucose,33mmol/L glucose).B. Control group (5.5mmol/L glucose), high glucose (33mmol/L glucose)+si NCgroup, high glucose (33mmol/L glucose)+si TXNIP.C. Control group (5.5mmol/L glucose), high glucose (33mmol/L glucose)+si NCgroup, high glucose (33mmol/L glucose)+si TXNIP group, high glucose(33mmol/L glucose)+H2O2group,high glucose (33mmol/L glucose)+H2O2+siTXNIP group.D. Control group (5.5mmol/L glucose), Control (5.5mmol/L glucose)+Wnt3a(200ng/ml) group, high glucose group (33mmol/L glucose), high glucose (33mmol/L glucose)+Wnt3a (200ng/ml).E. Control group (5.5mmol/L glucose), Control (5.5mmol/L glucose)+LiCl (200μmol/L) group, high glucose group (33mmol/L glucose), high glucose (33mmol/L glucose)+LiCl (200μmol/L).9. Using luciferase report system to detect β-catenin activity.10. The real time PCR method was used for the transcription levels of TXNIP, Cyclin D1,C-myc. 11. TXNIP and β-catenin protein levels were detected by Western blotting andimmunofluorescence methods.12. Myocardial ROS production was detected by superoxide anion probe (DHE).13. Flow cytometry was used to detect the cell cycle of HUVEC cells.14. HUVEC cell proliferation was detected by MTT assay.15. The scratches and Transwell methods were used to detect the migration of HUVECcells.【Results】1. The cardiac systolic function declined slightly and endothelial cell apoptosis increasedin diabetes group. Compared with non diabetes, cardiac systolic function decreased,cardiac endothelial cell apoptosis increased, cardiac endothelial cell proliferation andcardiac vascular densities decreased after myocardial infarction in diabetic group.2. The expression of cardiac and cardiac endothelial TXNIP significantly increased,nuclear tranlocation of β-catenin decreased in diabetic group. The expression level ofTXNIP significantly increased, luciferase report system readings significantly reduced,and so did nuclear translocation in HUVEC cells cultured in high glucose conditions.The transcription levels of β-catenin downstream genes Cyclin D1and C-myc weresignificantly reduced in a glucose concentration dependent manner. Compared withnon diabetes, diabetes significantly increased the TXNIP expression and reducedβ-catenin activity in the heart and cardiac endothelial cells after myocardial infarction.Compared with non diabetes, diabetes increased ROS production after myocardialinfarction.3. Compared to high glucose group, si TXNIP could induce reading recovery ofluciferase report system. High glucose conditions with ROS scavenger NACrecovered luciferase report system readings. High glucose and si TXNIP conditionswith H2O2downregulated luciferase report system readings.4. Wnt3a (200ng/ml) treatment for36h promoted the expression of β-catenin andnuclear translocation. High glucose decreased β-catenin expression and nucleartranslocation, and adding Wnt3a partially recovered β-catenin expression and nuclear translocation.5. High glucose for36h reduced the proportion of S phase, which was on behalf of theproliferation of HUVEC cells, and cell growth was significantly inhibited. Thesupplement of exogenous Wnt3a can partially restore the percentage of S phaseand the proliferation ability of HUVEC. High glucose inhibited HUVEC cellmigration. Supplement of exogenous Wnt3a can partially restore the migration abilityof HUVEC. High glucose could also inhibit luciferase report system readings andnuclear translocation, and supplementing LiCl (200μmol/L) recovered luciferasereport system readings and nuclear translocation.6. Intraperitoneal injection of LiCl (200mg/kg) could partially restore the diabeticmyocardial infarction-induced β-catenin decrease in cardiac endothelial cells, promotethe proliferation of cardiac endothelial cells, and reduce apoptosis of cardiacendothelial cells.7. Intraperitoneal injection of LiCl (200mg/kg) could partially restore the angiogenicdisorders caused by diabetic myocardial infarction, and improve left ventricularsystolic function.【Conclusion】Our current study demonstrated that diabetic myocardial ischemia reducedcompensatory angiogenesis in the heart. We found that the expression of TXNIPsignificantly increased in the peripheral region of diabetic myocardial infarction inendothelial cells and its effect on angiogenesis for the first time. We also clarified theregulation mechanism of potential inhibiting angiogenesis gene TXNIP to the proliferationrelated gene β-catenin. Recovery of Wnt signal can significantly improve diabeticangiogenesis and cardiac function after myocardial infarction. This study provides a newtreatment/therapeutic target and important theory basis for angiogenesis disorders afterdiabetic myocardial ischemia.