Physiological Ischemic Training Induces Endothelial Progenitor Cell Mobilization And Myocardial Angiogenesis Via A Vascular Endothelial Growth Factor/endothelial Nitric Oxide Synthase–related Pathway

Objective: Ischemia-induced angiogenesis have been promising which aims toimprove neovascularization by delivery of angiogenic factors or endothelialprogenitor cells (EPCs) to cardiac ischemic area. In order to avoid the risk ofexcessive myocardial ischemia, therefore, we hypothesized that physiologicalischemic training (PIT) of normal skeletal muscle might contribute to myocardialangiogenesis via mobilization of EPCs from the bone marrow in the establishedrabbit model of controllable myocardial ischemia.Methods: The rabbits were grouped by sham-operation (Sham), myocardialischemia without PIT (MI), PIT, PIT with the pretreatment with the endothelial nitricoxide synthase (eNOS) inhibitor L-nitroarginine methyl ester (L-NAME), PIT withanti-vascular endothelial growth factor antibody (Anti-VEGF), or both L-NAME andAnti-VEGF antibody (L-NAME+Anti-VEGF). Controlled myocardial ischemia wasmodeled by a water balloon constrictor implanted on their left ventricular branch PITprocedure included three cycles of3minutes of cuff inflation followed by5minutesof deflation on hind limbs of the rabbits for4weeks. At the endpoints, circulatingEPCs (CD34+/Flk-1+) were measured by fluorescence-activated cell sorter; capillarydensity, by immunohistochemistry; blood flow, by a microspheres technique; leftventricular ejection fraction, by echocardiogram; plasma VEGF and nitric oxide, by enzyme linked immunosorbent assay (ELISA) and nitrate reductase method; themRNA and protein expression of VEGF and eNOS, by real-time RT (reversetranscription)–PCR and Western blotting respectively.Results: Compared with Sham and MI groups, the PIT group had the highest EPCcount (P <0.001), and the increase of capillary density (P <0.01) and collateralblood flow (P <0.05) in the ischemic myocardium were consistent with the findingof EPC count. Blockade of VEGF or eNOS prevented all such PIT–induced effectsand caused the decrease of left ventricular ejection fraction (P <0.05). The mRNAlevels of eNOS and VEGF were significantly higher in the groups received PITcompared with the Sham group (P <0.05). VEGF protein expression was higher inthe groups received PIT than the Sham and MI groups (P <0.05), and the effects wasinhibited by Anti-VEGF pretreatment (P <0.05). Phospho-eNOS protein expressionwas higher in the PIT group than the Sham and MI groups (P <0.05), and the effectwas inhibited by L-NAME and Anti-VEGF antibody pretreatment (P <0.05). Theplasma VEGF and nitric oxide were consistent with the myocardial VEGF and eNOSprotein levels respectively. The capillary density and collateral blood flow werehighly correlated with the increase of EPC count (r=0.893and r=0.877,respectively, P=0.000).Conclusion: PIT improved EPC mobilization and myocardial angiogenesis in vivothough VEGF/eNOS-related pathway. Consequently, these results might support thefuture development of strategies for therapeutic neovascularization.