The Role And Mechanism Of CD44 In Pulsed Electromagnetic Fields(PEMFs) In Promoting Angiogenesis After Myocardial Infarction In Mice

ObjectiveIn our previous study,we found that pulsed electromagnetic fields(PEMFs)of 30 Hz,3m T could improve cardiac function that may be contributed by increased angiogenesis in mice with myocardial infarction.However,its mechanism needs to be elucidated.In addition,previous studies have found that CD44 was low or not expressed in microvascular endothelial cells in mouse normal myocardium,but was highly expressed and a large number of new vessels were formed in the infarct area within 1 to 2 weeks after acute myocardial infarction.Therefore,the purposes of this study were: 1)to investigate the expression pattern of CD44 in the ischemic myocardium of MI mice after PEMFs(30 Hz,3m T)intervention,2)If the expression of CD44 in ischemic myocardium changes after PEMFs intervention,the role of CD44 protein in PEMFs promoting angiogenesis in ischemic myocardium should be further clarified,and the possible mechanism should be explored.Materials and MethodsThe mouse MI model was prepared by left anterior descending coronary artery ligation.The successfully prepared model was randomly divided into the control group and the PEMFs group which received PEMFs intervention(30Hz,3m T,45min/day for 7/14 days).Cardiac function is evaluated by echocardiography.The expression of CD44 in ischemic myocardium was detected by RT-q PCR(Experiment 1).If the CD44 expression is increased in ischemic myocardium after PEMFs intervention,CD44 knockout homozygous mouse(CD44KO)will be paired with local CD44 wild-type(CD44WT)mouse to breed homozygous CD44 WT and CD44 KO mice with similar genetic background for the following experiments(Experiment 2).The successfully prepared MI models were randomly divided into four groups according to their genotypes: WT+Control,WT+PEMFs,KO+Control and KO+PEMFs group.WT+PEMFs and KO+PEMFs group received the same PEMFs intervention as mentioned in experiment 1.Cardiac function,MI size and angiogenesis were evaluated,and the protein or m RNA expression levels of CD44 and related genes were detected(Experiment 3).At the same time,the effect of CD44 on angiogenesis under PEMFs intervention will be further studied using the matrigel angiogenesis model in mice(Experiment 4)and in-vitro mouse aortic ring angiogenesis model(Experiment 5).ResultsIn Experiment 1,echocardiography showed that PEMFs intervention for 1 or2 weeks significantly improved the cardiac function of MI mice.CD44 m RNA expression was increased in the myocardial peri-infarcted area after 2 weeks of PEMFs intervention.In Experiment 2,CD44 wild-type and knockout homozygous mice were successfully bred and be genotyped for subsequent experiments.In Experiment 3,we found that PEMFs intervention for 1 week had improved cardiac function both in WT+PEMFs mice and KO+PEMFs mice,and the improvement in cardiac function in WT+PEMFs mice was more significant than that in KO+PEMFs mice.After 2 weeks of PEMFs intervention,only WT+PEMFs mice had sustained improvement in cardiac function after myocardial infarction,while KO+PEMFs mice no longer had improvement in cardiac function.There was no significant difference in the myocardial infarct size among the four groups after 1 week PEMFs intervention.Comparing to the other three groups,left ventricular infarct size was significantly reduced in the WT+PEMFs group after 2 weeks of intervention.At 1 week after intervention,comparing to WT+Control group,angiogenesis in the myocardial peri-infarcted area was significantly decreased in KO+Control group;comparing to WT+PEMFs group,peri-infarcted area angiogenesis was significantly decreased in KO+PEMFs group.After 2 weeks of PEMFs intervention,Comparing to the other three groups,the capillary density in the myocardial peri-infarcted area in WT+PEMFs group was significantly increased.With the detection of protein and m RNA levels,we found that: 1)CD44/AKT/VEGF signalings pathways were activated in the myocardial peri-infarcted area after 1 week of PEMFs intervention.2)CD44/PKC-α/VEGF/TGF-β1/HIF-1α signalings pathways were activated after 2 weeks of PEMFs intervention.3)CD44 gene deletion upregulates the expression of MMP-2 in the myocardial peri-infarcted area both after 1 and 2 weeks of PEMFs intervention.In experiments 4 and 5,we found that CD44 knockout inhibited in vivo matrix plug angiogenesis and in vitro aortic ring angiogenesis.ConclusionCD44 plays an important role in promoting ischemic myocardial angiogenesis,preventing the enlargement of myocardial infarction area and improving cardiac function in MI mice treated with PEMFs.These effects may be contributed by PEMFs-induced activation of CD44/AKT/VEGF,CD44/PKC-α/VEGF/TGF-β1/HIF-1α and CD44/ MMP-2 related signaling pathways that may have protective effects and pro-angiogenic effects on cells.