title: Flow cytometric analysis of circulating microvesicles derived from myocardial Ischemic preconditioning and cardioprotection of Ischemia/reperfusion Injury in rats.
abstract: OBJECTIVE
  To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles (MVs) from myocardial ischemic preconditioning (IPC) treated rats (IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion (I/R) injury in rats.
  
  
  METHODS
  Myocardial IPC was elicited by three.cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending (LAD) coronary artery. Platelet-free plasma (PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFR PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 µm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure (MAP), heart rate (HR) and ST-segment of electro-cardiogram (ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin (HE) staining. The activity of plasma lactate dehydrogenase (LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining.
  
  
  RESULTS
  Total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs (LMVs) and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (<1 Vm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats (P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR (P<0.01), decreased ST-segment and LDH activity (P < 0.05, P < 0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment (P < 0.01).
  
  
  CONCLUSIONS
  The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.
authors.forename: Miao
authors.initials: M
authors.lastname: Liu
authors.forename: Yi-lu
authors.initials: YL
authors.lastname: Wang
authors.forename: Man
authors.initials: M
authors.lastname: Shang
authors.forename: Yao
authors.initials: Y
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.forename: Shao-xun
authors.initials: SX
authors.lastname: Wang
authors.forename: Su
authors.initials: S
authors.lastname: Wei
authors.forename: Kun- wei
authors.initials: K
authors.lastname: Zhang
authors.forename: Chao
authors.initials: C
authors.lastname: Liu
authors.forename: Yan-na
authors.initials: YN
authors.lastname: Wu
authors.forename: Ming-lin
authors.initials: ML
authors.lastname: Liu
authors.forename: Jun-qiu
authors.initials: JQ
authors.lastname: Song
authors.forename: Yan-xia
authors.initials: YX
authors.lastname: Liu
mesh_terms: D000818:Animals; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D003331:Coronary Vessels / Q000473:pathology; D005434:Flow Cytometry; D006339:Heart Rate; D019157:Ischemic Preconditioning, Myocardial; D009203:Myocardial Infarction / Q000503:physiopathology*; D015428:Myocardial Reperfusion Injury / Q000503:physiopathology*; D009206:Myocardium / Q000473:pathology; D010641:Phenotype; D051381:Rats