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.
J Cardiovasc Med (Hagerstown). 2014 Apr;15(4):280-7. doi: 10.2459/JCM.0b013e32836009fe.
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.
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.
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).
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.
通过向心脏缺血区域输送血管生成因子或内皮祖细胞（EPCs），缺血诱导的血管生成有望改善新血管生成。为了避免过度心肌缺血的风险，因此，我们假设正常骨骼肌的生理缺血训练（PIT）可能通过一氧化氮介导的 EPC 从骨髓动员到在可控性心肌缺血兔模型中建立的心肌内来促进心肌血管生成。
通过将水气球收缩器植入兔左心室分支来建立心肌缺血模型，将兔子分为假手术组、无 PIT 的心肌缺血组、PIT 组和 PIT 预处理内皮型一氧化氮合酶（eNOS）抑制剂 L-硝基精氨酸甲酯（L-NAME）组。PIT 程序包括三组 3 分钟的袖带充气，然后在兔子的下肢放气 5 分钟，持续 4 周。在终点，通过荧光激活细胞分选术测量循环 EPC（CD34/Flk-1）；通过免疫组织化学测量毛细血管密度；通过微球技术测量血流；通过实时逆转录（RT）-PCR 和 Western 印迹测量内皮型一氧化氮合酶（eNOS）mRNA 和蛋白质。
与假手术组相比，PIT 组和 L-NAME 组的 eNOS mRNA 水平显著升高（P<0.05）。与假手术组和无 PIT 的心肌缺血组相比，PIT 组磷酸化 eNOS 蛋白表达更高（P<0.05），且 L-NAME 预处理抑制了该作用（P<0.05）。与假手术组和无 PIT 的心肌缺血组相比，PIT 组 EPC 计数最高（P<0.001），缺血心肌内毛细血管密度（P<0.01）和侧支血流（P<0.05）增加与 EPC 计数的增加一致。这些作用也被 eNOS 抑制剂 L-NAME 预处理所抑制。毛细血管密度和侧支血流与 EPC 计数的增加高度相关（r=0.913 和 r=0.929，P=0.000）。
PIT 通过与 eNOS 相关途径改善 EPC 动员，促进代偿性新血管生成。这些结果可能支持治疗性血管新生策略的未来发展。
