Effects And Mechanism Of TXL-improved Angiogenesis And Cardiac Function In Ischemic Heart

BackgroundMyocardial infarction (MI) is a major cause of morbidity and mortality. It remains responsible for about one-third of heart failure cases in our world, although there are lots of therapeutic approaches. In fact, sudden occlusion of a major coronary artery can result in acute myocardial ischemia and rapid apoptosis of cardiomyocytes, which leads to progressive fibrous replacement of myocardium and left ventricular (LV) dilatation. The progressive deterioration LV remodeling contributes to post-MI heart failure, and the prognosis of patients with heart failure is still poor. Therefore, targeting promotion of new vessels to increase blood flow to ischemic tissues is a promising option to treat ischemic heart disease.Tongxinluo (TXL) is a traditional Chinese compound prescription and has been approved by the State Food and Drug Administration of China in1996for the treatment of angina pectoris and ischemic stroke, as we previously described. Increasing evidence has indicated that the traditional Chinese medicine TXL has cardioprotective functions. Treatment with TXL is effective in lowering serum lipid levels, inhibiting plaque inflammation, and enhancing stability of vulnerable plaques. It can also reduce myocardial no-reflow and ischemia-reperfusion injury and modulate vascular endothelial function.However, it remains unclear whether TXL has an effect on cardiac function and heart remodeling after myocardial infarction. Therefore, we designed this experiment to investigate the potential role and mechanism of TXL in mice model of MI by ligation of anterior descending branch (LAD).Object1. To establish a mice model of myocardial infarction and to observe the effects of TXL on postischemic cardiac dysfunction and cardiac remodeling.2. To elucidate the possible underlying mechanisms of protective effects of TXL in MI.Methods1. Animal groupingTen-week-old male C57BL/6background wild-type mice were randomly divided into five groups:(1) sham group (Sham);(2) MI-control group (Control), where the mice were treated with vehicle (physiological saline) alone(10mL/kg/day);(3) MI-low dose TXL group (TXL-L), where mice were treated with0.38g/kg/day TXL;(4) MI-medium dose TXL (TXL-H) group, where mice were treated with1.5g/kg/day TXL.(5) MI-high dose TXL (TXL-H) group, where mice were treated with1.5g/kg/day TXL.2. Surgical ProceduresAll animal surgeries were carried out under isoflurane (2%) to minimize the pain. Mice were orally intubated and artificially ventilated by using a rodent respirator. Hearts were then exposed through the left lateral thoracotomy. MI was created by permanent LAD ligation with a7-0suture line. The occlusion of coronary was confirmed by pallor and regional wall motion abnormality of the left ventricle. The sham group underwent the same time matched surgical procedure without ligation. The mice were either subjected to left anterior descending coronary artery ligation (LAD) or the same time-matched surgical procedure without ligation.3. Echocardiographic AnalysisTransthoracic echocardiography was performed in isoflurane anesthetized mice30days after MI surgical intervention to evaluate left ventricle function. Cardiac dimensional and functional parameters were analyzed by using a high resolution echocardiography system (Vevo770) with a35MHz linear array transducer. M-mode echocardiography of the left ventricle at the papillary muscle level was performed. Diastolic left ventricular internal diameter (LVIDd) and systolic left ventricular internal diameter (LVIDs) were measured. The left ventricular fractional shortening (%FS) and ejection fraction (%EF) were automatically calculated by the echocardiographic system.4. Histological AnalysisHistological analysis was assessed in perfusion/fixed hearts collected from mice at7days or30days after surgery. The hearts were harvested and fixed in4%formalin for24hours. The formalin-fixed tissues were embedded in paraffin wax and cut into5μm sections for hematoxylin-eosin and Masson’s trichrome staining. Infarct size was expressed as the sum of the epicardial and endocardial scar length divided by the sum of the LV epicardial and endocardial circumferences. Masson’s trichrome staining was used to detect interstitial fibrosis in the border zones. The morphologic parameters and CVF were measured using Image-Pro Plus6.0, by two blinded observers.5. TUNEL Assay.The apoptosis of cardiomyocytes was assessed by using In Situ Cell Death Detection Kit. Cells were defined as apoptotic if the whole nuclear area of the cell was labeled positively.6. Capillary DensityFor the measurement of capillary density (counts/mm2), we performed immunohistochemical analysis of CD31/Platelet Endothelial Cell Adhesion Molecule-1.7. Western Blot AnalysisProtein in the border zone of heart tissues was extracted at7days after surgery. Protein of equal mounts was separated on10%-15%SDS-PAGE and electrotransferred onto nitrocellulose membrane. Blots were incubated with primary antibodies at4℃room overnight and with secondary antibody for2h at room temperature. Signals were detected by enhanced chemiluminescence (Millipore) and analyzed by the use of Image-ProPlus6.0.8. Electrophoretic Mobility Shift Assay (EMSA) AnalysisEMSA was performed by using Light Shift Chemiluminescent EMSA Kit according to the manufacturer’s instructions. Nuclear protein in the border zone of heart tissues was collected and extracted by Nuclear Extraction Kit.9. Statistical Analysis.The data are expressed as mean±SEM. SPSS for Windows v.18.0was used for statistical analysis. Intergroup comparisons involved one-way ANOVA followed by LSD’s test (with equal variances assumed) or Dunnett’sT3test (with equal variances not assumed). Probabilities of0.05or less were considered to be statistically significant.Results1. TXL attenuated LAD induced mortality in miceTreatment of TXL reduced LAD-induced mortality in mice.2. Echocardiographic AnalysisTXL treatment improved post-MI cardiac dysfunction. Left ventricular function was preserved in TXL treatment groups at30days after coronary ligation.3. Infarct sizeTXL Therapy Attenuated Infarction Size after MI. Compared with the sham group, LAD ligation induced myocardial infarction, while TXL treatment could significantly reduce the size of infarction at a dose-dependently manner.4. Myocardium interstitial fibrosisMasson’s trichrome staining was performed to show the fibrosis in the border zone. TXL treatment could significantly reduce the fibrosis.5. Tunnel assayTXL therapy reduced MI-induced cardiomyocyte apoptosis.6. Capillary Density. Compared with the control group, increased capillary was observed in the TXL treatment groups.7. Western blotTXL treatment increased the expression of PI3K, and VEGF and elevated phosphorylation of AKT, ERK, and eNOS.8. EMSAThe DNA binding activity of HIF-la was significantly increased in TXL treatment groups compared with control group.Conclusions:1. Compared with the control group, TXL treatment restored cardiac function; increased revascularization, attenuated cardiomyocyte apoptosis, and reduced interstitial fibrosis.2. TXL may improve cardiac function and ameliorate cardiac remodeling by increasing neovascularization through enhancing the phosphorylation of Akt and ERK, the expression and activity of HIF-la, and the protein level of VEGF and p-eNOS. Background:Myocardial infarction (MI) is a common presentation of ischemic heart disease defined as interruption of the blood flow to the heart because of the occlusion of a coronary artery. The World Health Organization’s World Health Statistics2013report indicates that ischemic heart disease is on the rise and remains the most prevalent cause of death globally, constituting>14%of all deaths. Neovascularization, however, is impaired in myocardial infarction, which leads to incomplete blood flow restoration and significant residual tissue ischemia. Myocardial ischemia causes cardiomyocyte death, adverse ventricular remodeling, and ventricular dysfunction. Uninterrupted progression of the remodeling process may eventually lead to heart failure, the severity of which is an important determinant of prognosis.The ability of the organism to develop new blood vessels (neovascularization) constitutes an important adaptive response to vascular occlusive diseases. However, increasing evidence suggests that postnatal neovascularization relies not exclusively on the sprouting of pre-existing vessels, but also the contribution of bone marrow-derived circulating endothelial progenitor cells (EPCs). Endothelial progenitor cells (EPCs) home to sites of ischemia and contribute to neovascularization in ischemic tissue. Experimental and clinical studies demonstrate that treatment of acute myocardial infarction with EPCs results in a reduction in infarct size. Insufficient number of EPCs after MI may contribute to insufficient angiogenesis and exacerbation of MI. The Stromal cell-derived factor-la/CXC chemokine receptor4system is pivotally involved in the mobilization and recruitment of stem and progenitor cells to the heart after MI. The bone marrow-derived cells, which express CXCR4, have been shown to act as vascular stem/progenitor cells (including EPCs) and promote angiogenic processes after MI.Based on our experiment part I, we have proved TXL treatment restored cardiac function, and increased revascularization. However, whether TXL can modulate EPC mobilization and functions in the setting of AMI is not known. Hence, the aim of this experiment is to demonstrate the importance and mechanism of TXL on EPCs mobilization and functions.In the present study, we hypothesized that after an AMI, the numbers circulating EPCs will be heightened with tongxinluo treatment in mice and formation of tube-like structures of EPCs could be enhanced though up-regulated a SDF-la/CXCR4System.Object:1. In vivo, observe the tongxinluo mobilization ability of EPCs;2. In vivo, observe the expression of SDF-la in ischemia heart;3. In vitro, observe the ability of tonginluo enhancing formation of tube-like structures of EP Cs.4. Explore the mechanism of tongxinluo activating EPCs.Methods1. Animal groupingA total of75ten-week-old male C57BL/6background wild-type mice were randomly divided into five groups:(1) sham group(Sham);(2) MI-control group (Control), where the mice were treated with vehicle (physiological saline) alone(10mL/kg/day);(3) MI-low dose TXL group (TXL-L), where mice were treated with0.38g/kg/day TXL;(4) MI-medium dose TXL (TXL-M) group, where mice were treated with0.75g/kg/day TXL.(5) MI-high dose TXL (TXL-H) group, where mice were treated with1.5g/kg/day TXL. 2. Surgical Procedures.All animal surgeries were carried out under isoflurane (2%) to minimize the pain. Mice were orally intubated and artificially ventilated by using a rodent respirator. Hearts were then exposed through the left lateral thoracotomy. MI was created by permanent LAD ligation with a7-0suture line. The occlusion of coronary was confirmed by pallor and regional wall motion abnormality of the left ventricle. The sham group underwent the same time matched surgical procedure without ligation.3. Quantification of circulating endothelial progenitor cells (EPCs)Blood was harvest from heart at3days post MI. The peripheral blood mononuclear cells were incubated for90min at room temperature with FITC conjugated anti-mouse CD34and APC-conjugated anti-mouse KDR. After incubation, cells were washed with PBS twice. The number of CD34+/KDR+cells within the monocytic cell population was counted for quantification of EPCs,4. Isolation、culture and identification of EPCsEPCs were obtained form marrow cavity by density gradient centrifugation15min at2000×g. EPCs were cultured with endothelial cell growth medium-2(EGM-2) in0.1%mouse vitronectin coated dish. The phenotype of Endothelial progenitor cells was confirmed by using dual binding of DiI-ac-LDL and FITC-UEA-1lectin.5. MTT assay50μg/mL、100μg/mL、200μg/mL、400μg/mL、800μg/mL、2000μg/mL of tongxinluo were used to stimulate EPCs for48h, then the cell viability of EPCs was measured by MTT.6. Optimal concentration screening testsAfter cultured with serum-free EGM-2medium for12h, EPCs were stimulated by EGM-2containing tongxinluo at0μg/mL、50μg/mL、100μg/mL、200μg/mL for24hours seperatedly. Then the EPCs were collected and the expression of CXCR4was evaluated.7. The optimum action time tests After cultured with serum-free EGM-2medium for12h, EPCs were stimulated by EGM-2containing200ug/mL tongxinluo for0h、6h、12h、18h、24h、36h seperatedly. Then collected the EPCs to the expression of CXCR4.8. In vitro Matrigel tube formation assayBD Matrigel TM basement membrane matrix was melted at4℃overnight, placed50μL matrigel in96-well plates and allowed to solidify at37℃for30min. After incubated by different treats, EPCs (1×104) were resuspended in50μL of EGM-2and plated on Matrigel. After8h, the mean tube length was calculated.9. Immunohistochemical analysisImmunohistochemical analysis was assessed in perfusion/fixed hearts collected from mice at3days after surgery. The hearts were harvested and fixed in4%formalin for24hours. The formalin-fixed tissues were embedded in paraffin wax and cut into5μm sections for immunohistochemical staining. Paraffin sections were incubated in3%H2O2to inhibit the endogenous peroxidase activity, blocked with5%BSA for30min, and incubated with the antibodies at4℃overnight. Then, the sections were incubated with secondary antibody and visualized by using a DAB kit.10. Western Blot AnalysisTotal proteins were obtained from myocardial tissue and EPCs. Protein extracts were subjected to SDS-PAGE, transferred to PVDF membranes.11. RT-PCR AnalysisTotal RNA was obtained from myocardial tissue and EPCs with the mRNA abstraction kit.. RT-PCR was carried out by the routine two-step method. The mRNA exression of CXCR4and GAPDH was analyzed.12. Statistical Analysis.The data are expressed as mean±SEM. SPSS for Windows v.18.0was used for statistical analysis. Intergroup comparisons involved one-way ANOVA followed by LSD’s test (with equal variances assumed) or Dunnett’sT3test (with equal variances not assumed). Probabilities of0.05or less were considered to be statistically significant. Results:1. Tongxinluo elevated the quantity of EPCsThe quantity of EPCs in peripheral blood was increased in the mice of MI groups. Tongxinluo further elevated the quantity of EPCs.2. Immunohistochemical stainingCompared with the sham and MI group, tongxinluo could increased the expression of SDF-la in the heart.3. Isolation culture and identification of EPCsWe have shown the EPCs at1day^4day and7day. The phenotype of endothelial progenitor cells was confirmed by using dual binding of DiI-ac-LDL and FITC-UEA-1lectin.4. The effects of tongxinluo on cell viability50μg/mL、100μg/mL、200μg/mL、400μg/mL of tongxinluo could promote the proliferation and viability of EPCs,yet the cell viability was suppressed in the concentration in800μg/mL and1000μg/mL. Therefore,50ug/mL、100μg/mL、200μg/mL、400μg/mL of tongxinluo were used in the following studies.5. Optimal concentration and optimal timeThe expression of CXCR4of EPCs increased to the top when stimulated by tongxinluo at200μg/mL for24hours.6. Matrigel tube formation assayTongxinluo enhanced formation of tube-like structures of EPCs on Matrigel. Cotreatment with AMD3100significantly blocked tongxinluo’s effect.Conclusion:1. Tongxinluo could elevate the quantity of EPCs in peripheral blood;2. Tongxinluo could increase the expression of SDF-la in the heart;3. The expression of CXCR4of EPCs increased to the top when stimulated by tongxinluo at200μg/mL for24hours; 4. Tongxinluo enhanced formation of tube-like structures of EPCs on Matrigel. Cotreatment with CXCR4antagonist AMD3100significantly blocked tongxinluo’s effect;...