Yeh Jui-Ning, Yip Hon-Kan, Shao Pei-Lin, Chiang John Y, Wu Shun-Cheng, Sung Pei-Hsun, Sheu Jiunn-Jye, Guo Jun
Department of Cardiology, The First Affiliated Hospital, Jinan University Guangzhou 510630, Guangdong, China.
Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine Kaohsiung 83301, Taiwan.
Am J Transl Res. 2024 Sep 15;16(9):5020-5037. doi: 10.62347/OCFT1003. eCollection 2024.
This study tested whether combined shock wave (SW)-facilitated melatonin (Mel) delivered into endothelial progenitor cells (EPCs) (EPC) plus S-nitroso-N-acetyl-DL-penicillamine (SNAP) was superior to merely one modality alone for improving critical limb ischemia (CLI) in rats.
SD rats (n = 50) were equally categorized into group 1 (sham-control), group 2 (CLI), group 3 (CLI + SNAP), group 4 (CLI + EPC), and group 5 (CLI + EPC + SNAP), and ischemia-involved quadriceps were harvested by day 14.
An in vitro study showed that at time points of 24/48/72 h, the cell viability/protein expression of endothelial nitric oxide synthase (eNOS)/and cellular expression of nitric oxide (NO) were highest in EPCs, lowest in EPCs + menadione, and much higher in EPC + Mena than in EPCs + Mena + Mel. Protein levels of oxidative-stress (NOX-1/NOX-2/oxidized protein)/early (AN-V/PI)/late (AN-V/PI) apoptosis and total intracellular/mitochondrial reactive oxygen species ROS exhibited an antithetical trend of cell viability among the groups (all P<0.0001). Matrigel assay of angiogenesis/positively-stained NO cells showed that they were much higher in EPCs + SNAP than in EPCs only (all P<0.0001). Ex vivo angiogenesis/arterial relaxation of carotid-artery rings were highest in left-common-carotid-artery (LCCA) + SNAP, lowest in LCCA + Mena, and notably higher in LCCA than in LCCA + Mena + SNAP (all P<0.0001). Laser Doppler showed ischemic to normal-blood-flow (INBF) ratio was highest in group 1, lowest in group 2, and it progressively increased from groups 3 to 5 (all P<0.0001). The protein levels of oxidative-stress (NOX-1/NOX-4/oxidized protein)/apoptotic [cleaved-caspase-3/cleaved apoptosis/mitochondrial-damage (cytosolic-cytochrome-C/p-DRP-1)]/fibrotic (Smad3/TGF-β)/inflammatory (MMP-9/IL-1β/TNF-α/NF-κB) biomarkers, exhibited an opposite trend, whereas the protein level of endothelial-cell surface markers (CD31/vWF/eNOS) and number of small vessels exhibited an identical pattern of INBF ratio among the groups (all P<0.0001).
Combined EPC and SNAP therapy offered a synergic effect toward rescuing from CLI.
本研究旨在测试将冲击波（SW）促进褪黑素（Mel）输送到内皮祖细胞（EPC）中，并联合使用S-亚硝基-N-乙酰-DL-青霉胺（SNAP），是否比单独使用一种方法更能有效改善大鼠的严重肢体缺血（CLI）。
将50只SD大鼠平均分为1组（假手术对照组）、2组（CLI组）、3组（CLI + SNAP组）、4组（CLI + EPC组）和5组（CLI + EPC + SNAP组），在第14天采集缺血相关的股四头肌。
体外研究表明，在24/48/72小时时间点，EPC中的细胞活力/内皮型一氧化氮合酶（eNOS）的蛋白表达/一氧化氮（NO）的细胞表达最高，EPC + 甲萘醌中的最低，且EPC + 甲萘醌组高于EPC +甲萘醌 + Mel组。氧化应激（NOX-1/NOX-2/氧化蛋白）/早期（An-V/PI）/晚期（An-V/PI）凋亡以及细胞内/线粒体活性氧（ROS）的总蛋白水平在各组间呈现与细胞活力相反的趋势（均P<0.0001）。基质胶血管生成试验/NO阳性染色细胞显示，EPC + SNAP组比仅EPC组高得多（均P<0.0001）。离体血管生成/颈动脉环的动脉舒张在左颈总动脉（LCCA）+ SNAP组最高，LCCA +甲萘醌组最低，LCCA组明显高于LCCA +甲萘醌 + SNAP组（均P<0.0001）。激光多普勒显示缺血与正常血流（INBF）比值在1组最高，2组最低，从3组到5组逐渐升高（均P<0.0001）。氧化应激（NOX-1/NOX-4/氧化蛋白）/凋亡[裂解的半胱天冬酶-3/裂解的凋亡/线粒体损伤（细胞溶质细胞色素C/p-DRP-1）]/纤维化（Smad3/TGF-β）/炎症（MMP-9/IL-1β/TNF-α/NF-κB）生物标志物的蛋白水平呈现相反趋势，而内皮细胞表面标志物（CD31/vWF/eNOS）的蛋白水平和小血管数量在各组间呈现与INBF比值相同的模式（均P<0.0001）。
EPC和SNAP联合治疗对挽救严重肢体缺血具有协同作用。
