Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan.
Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 83301, Taiwan.
Int J Mol Sci. 2020 Oct 23;21(21):7887. doi: 10.3390/ijms21217887.
This study tested whether circulatory endothelial progenitor cells (EPCs) derived from peripheral arterial occlusive disease (PAOD) patients after receiving combined autologous CD34+ cell and hyperbaric oxygen (HBO) therapy (defined as rejuvenated EPCs) would salvage nude mouse limbs against critical limb ischemia (CLI). Adult-male nude mice ( = 40) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 (CLI-EPCs (6 × 10) derived from PAOD patient's circulatory blood prior to CD34 cell and HBO treatment (EPC) by intramuscular injection at 3 h after CLI induction) and group 4 (CLI-EPCs (6 × 10) derived from PAOD patient's circulatory blood after CD34 cell and HBO treatment (EPC) by the identical injection method). By 2, 7 and 14 days after the CLI procedure, the ischemic to normal blood flow (INBF) ratio was highest in group 1, lowest in group 2 and significantly lower in group 4 than in group 3 ( < 0.0001). The protein levels of endothelial functional integrity (CD31/von Willebrand factor (vWF)/endothelial nitric-oxide synthase (eNOS)) expressed a similar pattern to that of INBF. In contrast, apoptotic/mitochondrial-damaged (mitochondrial-Bax/caspase-3/PARP/cytosolic-cytochrome-C) biomarkers and fibrosis (Smad3/TGF-ß) exhibited an opposite pattern, whereas the protein expressions of anti-fibrosis (Smad1/5 and BMP-2) and mitochondrial integrity (mitochondrial-cytochrome-C) showed an identical pattern of INBF (all < 0.0001). The protein expressions of angiogenesis biomarkers (VEGF/SDF-1α/HIF-1α) were progressively increased from groups 1 to 3 (all < 0.0010). The number of small vessels and endothelial cell surface markers (CD31/vWF) in the CLI area displayed an identical pattern of INBF (all < 0.0001). CLI automatic amputation was higher in group 2 than in other groups (all < 0.001). In conclusion, EPCs from HBO-C34+ cell therapy significantly restored the blood flow and salvaged the CLI in nude mice.
这项研究旨在测试接受联合自体 CD34+细胞和高压氧（HBO）治疗后从外周动脉闭塞性疾病（PAOD）患者循环中获得的内皮祖细胞（EPC）（定义为再生 EPC）是否能挽救裸鼠肢体免受严重肢体缺血（CLI）的影响。成年雄性裸鼠（n=40）平均分为 4 组：1 组（假手术对照）、2 组（CLI）、3 组（CLI-EPCs（6×10），来源于 PAOD 患者在接受 CD34 细胞和 HBO 治疗前的循环血液（EPC），在 CLI 诱导后 3 小时通过肌肉内注射）和 4 组（CLI-EPCs（6×10），来源于 PAOD 患者在接受 CD34 细胞和 HBO 治疗后的循环血液（EPC），通过相同的注射方法）。在 CLI 手术后 2、7 和 14 天，1 组的缺血与正常血流（INBF）比值最高，2 组最低，4 组明显低于 3 组（<0.0001）。内皮功能完整性（CD31/血管性血友病因子（vWF）/内皮型一氧化氮合酶（eNOS））的蛋白水平表达与 INBF 相似。相反，凋亡/线粒体损伤（线粒体-Bax/半胱天冬酶-3/PARP/胞质细胞色素-C）和纤维化（Smad3/TGF-β）标志物表现出相反的模式，而抗纤维化（Smad1/5 和 BMP-2）和线粒体完整性（线粒体细胞色素-C）的蛋白表达与 INBF 表现出相同的模式（均<0.0001）。血管生成生物标志物（VEGF/SDF-1α/HIF-1α）的蛋白表达从 1 组到 3 组逐渐增加（均<0.0010）。CLI 区域的小血管数量和内皮细胞表面标志物（CD31/vWF）与 INBF 表现出相同的模式（均<0.0001）。2 组的 CLI 自动截肢率高于其他组（均<0.001）。综上所述，HBO-C34+细胞治疗的 EPC 显著恢复了血流，挽救了裸鼠的 CLI。
