title: Physiologic ischaemic training induces endothelial progenitor cell mobilization and myocardial angiogenesis via endothelial nitric oxide synthase related pathway in rabbits.
abstract: OBJECTIVE
  Ischaemia-induced angiogenesis promises to improve neovascularization by delivery of angiogenic factors or endothelial progenitor cells (EPCs) to cardiac ischaemic areas. In order to avoid the risk of excessive myocardial ischaemia, therefore, we hypothesized that physiological ischaemic training (PIT) of normal skeletal muscle might contribute to myocardial angiogenesis via nitric oxide mediated mobilization of EPCs from the bone marrow in the established rabbit model of controllable myocardial ischaemia.
  
  
  METHODS
  The rabbits were grouped by sham-operation, myocardial ischaemia without PIT, PIT and PIT with pretreatment with the endothelial nitric oxide synthase (eNOS) inhibitor L-nitroarginine methyl ester (L-NAME). Controlled myocardial ischaemia was modelled by a water balloon constrictor implanted on the left ventricular branch in a rabbit. The PIT procedure included three cycles of 3 min of cuff inflation followed by 5 min of deflation on hind limbs of the rabbits for 4 weeks. At the endpoints, circulating EPCs (CD34/Flk-1) were measured by fluorescence-activated cell sorter; capillary density, by immunohistochemistry; blood flow, by a microsphere technique; endothelial nitric oxide synthase (eNOS) mRNA and protein, by real-time reverse transcriptase (RT)-PCR and Western blotting.
  
  
  RESULTS
  The mRNA levels of eNOS were significantly higher in the PIT and L-NAME groups than in the sham-operation group (P < 0.05). Phospho-eNOS protein expression was higher in the PIT group than in the sham-operation and myocardial ischaemia without PIT groups (P < 0.05), and the effect was inhibited by L-NAME pretreatment (P < 0.05). Compared with sham-operation and myocardial ischaemia without PIT groups, the PIT group had the highest EPC count (P < 0.001), and the increase of capillary density (P < 0.01) and collateral blood flow (P < 0.05) in the ischaemic myocardium was consistent with the finding of EPC count. These effects were also inhibited by pretreatment with the eNOS inhibitor L-NAME. Capillary density and collateral blood flow were highly correlated with the increase of EPC count (r = 0.913 and r = 0.929, respectively, P = 0.000).
  
  
  CONCLUSIONS
  PIT improved EPC mobilization and contributed to compensatory neovascularization via eNOS-related pathway. These results might support the future development of strategies for therapeutic neovascularization.
authors.affiliation: Department of Rehabilitation Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China *Both Mingyue Xiao and Xiao Lu contributed equally to this work.
authors.forename: Mingyue
authors.initials: M
authors.lastname: Xiao
authors.forename: Xiao
authors.initials: X
authors.lastname: Lu
authors.forename: Jianan
authors.initials: J
authors.lastname: Li
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.forename: Yongxue
authors.initials: Y
authors.lastname: Li
mesh_terms: D000818:Animals; D002196:Capillaries / Q000473:pathology; D002452:Cell Count; D002465:Cell Movement / Q000502:physiology; D003326:Coronary Circulation / Q000502:physiology; D003331:Coronary Vessels / Q000473:pathology; D042783:Endothelial Cells / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods*; D019157:Ischemic Preconditioning, Myocardial / Q000379:methods*; D017202:Myocardial Ischemia / Q000097:blood / Q000628:therapy; D018919:Neovascularization, Physiologic / Q000502:physiology*; D009569:Nitric Oxide / Q000097:blood; D052250:Nitric Oxide Synthase Type III / Q000097:blood / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology; D013234:Stem Cells / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000097:blood / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III