CD73~+ Human Adipose-derived Mesenchymal Stem Cells Promote Myocardial Repair Through CD73/adenosine Axis

BackgroundMyocardial infarction(MI)can cause a large number of myocardial cell death and ventricular remodeling.Due to the very limited ability of myocardial regeneration,cell transplantation therapy brings a new dawn for the treatment of heart diseases.Currently,mesenchymal stem cells(MSCs)are hot research topics in cell therapy.CD73,CD105,and CD90 are dominant markers of MSCs,but the role of CD73 in cell therapy for myocardial injury is rarely reported.In the treatment of myocardial injury,cell transplantation not only focuses on cell replacement and endocrine mechanisms,but also should attach great importance to the immune regulation of transplanted cells on the myocardial microenvironment.Are CD73~+adiposetissue derived mesenchymal stem cells(ADMSCs)involved in immune regulation in the treatment of myocardial infarction?It is urgent for us to explore and comprehensively analyze the mechanism of CD73~+ADMSCs treatment myocardium injury.It provides theoretical and experimental basis for clinical development and use of CD73+cells.It provides molecular reference standards for screening cells for mesenchymal stem cells in the treatment of myocardial injury and other diseases.Objectives1.To explore whether there is CD73/adenosine axis in the treatment of myocardial injury by human ADMSCs.2.Evaluate the therapeutic effect of CD73~+ADMSCs transplantation on myocardial infarction,and explore whether human ADMSCs play an immunomodulatory role through CD73/adenosine axis to promote infarct myocardial repair.Methods1.Flow cytometry was used to detect the expression of ADMSCs surface markers CD29,CD44,CD73,CD90,CD105 and CD45.2.Construct CD73 siRNA interference vector to transfect ADMSCs,use Western blotting and Real-time PCR to detect the expression of CD73,use CD73 inhibitor adenosine 5'-?,?-methylene diphosphate(adenosine-5'-?,?-methylene diphosphate(APCP)to inhibits CD73 activity.ADMSCs were inoculated into 6-well plates,and added adenosine 5?-monophosphate(AMP)substrate.Experimental grouping:simple media group(Media Only group),blank control group(ADMSCs group),substrate control group(ADMSCs+AMP group),CD73 activity inhibition group(ADMSCs+APCP+AMP group),lentiviral control group(ADMSCs+LV+AMP group)and CD73expression interference group(ADMSCs+CD73siRNA+AMP group).Liquid phase Liquid chromatography mass spectrometry(LC-MS)was used to detect the content of adenosine in the cell supernatant of each group.3.Preparation of rat myocardial infarction model,using doppler ultrasound and electrocardiogram to detect them.The transplanted cells were multi-pointly injected into myocardial infarction area.The experiment was divided into sham-operated group(Sham group),myocardial infarction group(MI group),ADMSCs transplantation group(MI+ADMSCs group),CD73 activity inhibition group(MI+ADMSCs+APCP group),lentiviral control group(MI+ADMSCs+LV group)and CD73 expression interference group(MI+ADMSCs+CD73siRNA group).On the first days and third day,liquid chromatography mass spectrometry and xylenol orange were used to detect the adenosine and hydrogen peroxide content in the myocardial infarction area,respectively.On the 1d,3d,5d,7d,14d,21d,and 28d,oppler ultrasound was used to detect left ventricular ejection fraction(LVEF),left ventricular short-axis shortening rate(LVFS),and left ventricular end-systolic volume(LVESV)of each group.In the 2w and 4w,Real-time PCR was used to detect the expression of transforming growth factor-?1(TGF-?1)and interferon-?(IFN-?)in the myocardial tissue of the cell transplantation area.Results1.ADMSCs highly expressed CD29,CD44,CD73 and CD105,and lowly expressed CD45.2.After CD73siRNA was transfected into ADMSCs,the expression of CD73 protein and gene decreased(P<0.001).The results of adenosine in vitro showed that:the amount of adenosine in the ADMSCs+AMP group,ADMSCs+APCP+AMP group and ADMSCs group decreased in order,comparied between groups(P<0.001);ADMSCs+LV+AMP group was higher than ADMSCs+CD73siRNA+AMP group(P<0.001).3.The results of adenosine in vivo showed that:After 1 day of cells transplantation in each group,the content of adenosine in MI+ADMSCs group was higher than MI group and MI+ADMSCs+APCP group(P<0.01);The content of adenosine in MI+ADMSCs+LV group,MI group and MI+ADMSCs+CD73 siRNA group decreased in order,comparied between groups(P<0.001).After 3 days of cell transplantation in each group,the content of adenosine MI+ADMSCs group was higher than MI group(P<0.001)and MI+ADMSCs+APCP group(P<0.05);The content of adenosine in MI+ADMSCs+LV group was higher than MI group and MI+ADMSCs+CD73siRNA group.4.Compared with Sham group,the ECG of MI showed that ST segment raised upward.After 1d-28d of cells transplantation into the area of myocardial infarction,the results of doppler ultrasound showed that LVEF and LVFS of MI group were lower than Sham group,and LVESV was higher than Sham group(P<0.05).LVEF and LVFS of MI+ADMSCs group and MI+ADMSCs+LV group were higher than MI group(except the third day),and LVESV was lower than MI group(P<0.05).After 5 days,14 days,21 days and 28 days of cell transplantation after MI,the MI+ADMSCs group and MI+ADMSCs+LV group improved heart function better than MI+ADMSCs+APCP group and MI+ADMSCs+CD73siRNA group(P<0.05).5.After 1 day of cells transplantation in each group,the content of hydrogen peroxide in MI group was higher than Sham group(P<0.001)?MI+ADMSCs group(P<0.01)and MI+ADMSCs+LV group(P<0.01);the content of hydrogen peroxide in MI+ADMSCs and MI+ADMSCs+LV group were lower than MI+ADMSCs+APCP group(P<0.05)and MI+ADMSCs+CD73siRNA group(P<0.01),respectively.After3 days of cells transplantation in each group,the content of hydrogen peroxide in MI group was higher than Sham group(P<0.001)?MI+ADMSCs group(P<0.01)and MI+ADMSCs+LV group(P<0.01);the content of hydrogen peroxide in MI+ADMSCs and MI+ADMSCs+LV group were lower than MI+ADMSCs+APCP group(P<0.05)and MI+ADMSCs+CD73siRNA group(P<0.05),respectively.6.After 14d and 28d of cells transplantation in each group,the content of TGF-?in MI+ADMSCs group and MI+ADMSCs+LV group were higher than MI+ADMSCs+APCP group and MI+ADMSCs+CD73siRNA group respectively.The result of IFN-?was contrary to the result of TGF-?(P<0.05).Conclusions1.When transplanting CD73~+ADMSCs to treat myocardial infarction,by inhibiting the activity or expression of CD73,the content of adenosine was down-regulated on 1d and 3d after cell transplantation.It indicates the existence of CD73/ADO axis.2.Transplantation of CD73~+ADMSCs is effective in the treatment of myocardial infarction.When the activity and expression of CD73 were inhibited,the effect of cardiac function at different time points decreased,and the content of hydrogen peroxide increased on 1d and 3d.3.After 14 days and 28 days of cell transplantation,CD73 activity and expression were inhibited,TGF-?expression was down-regulated,and the proinflammatory molecule IFN-?expression was up-regulated.It suggests that human ADMSCs can regulate the immune microenvironment of infarcted myocardial tissue through CD73/adenosine axis to promote infarcted myocardial repair.