Department of Plastic and Reconstructive Surgery, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 123, Dapi Road, Niaosung Dist., Kaohsiung City, 833253, Taiwan.
Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, 833253, Taiwan.
Stem Cell Res Ther. 2022 Sep 2;13(1):447. doi: 10.1186/s13287-022-03119-0.
This study tested the hypothesis that overexpression of cellular prion protein in endothelial progenitor cells (PrPc-EPCs), defined as "rejuvenated EPCs," was superior to EPCs for salvaging the critical limb ischemia (CLI) induced after 28-day chronic kidney disease (CKD) induction in rat.
Cell viability and flow cytometric analyses of early/late apoptosis/total-intracellular ROS/cell cycle (sub-G1, G2/M phase) were significantly higher in EPCs + HO than in EPCs that were significantly reversed in PrPc-EPCs + HO (all p < 0.001). The protein expressions of inflammation (IL-1ß/IL-6/MMP-9/p-NF-κB) were significantly increased in EPC + TNF-α than in EPCs that were significantly reversed in PrPc-EPCs + TNF-α (all p < 0.001). Adult-male SD rats (n = 8/each group) were categorized into group 1 (sham-operated control), group 2 (CKD + CLI), group 3 [CKD + CLI + EPCs by intravenous (0.6 × 10)/intra-muscular (0.6 × 10) injections at 3 h after CLI induction], group 4 (CKD + CLI + PrPc-EPCs/dose-administration as group 3) and group 5 (CKD + CLI + siPrnp-EPCs/dose-administration as group 3). By day 14 after CLI induction, the ratio of ischemia to normal blood flow (INBF) in CLI area was highest in group 1/lowest in group 2/significantly higher in group 4 than in groups 3/5 and significantly higher in group 3 than in group 5 (all p < 0.0001). Histopathology demonstrated that the angiogenesis (number of small vessels/CD31 + cells) exhibited a similar trend, whereas the fibrosis/kidney injury score exhibited an opposite pattern of INBF among the groups (all p < 0.0001). The protein expressions of angiogenesis (SDF-1α/VEGF/CXCR4)/cell-stress signaling (p-PI3K/p-Akt/p-m-TOR) were significantly and progressively increased from groups 1-4 that were reversed in group 5 (all p < 0.0001). The protein expressions of fibrotic (p-Smad3/TGF-ß)/oxidative-stress (NOX-1/NOX-2/oxidized-protein)/apoptotic (mitochondrial-Bax/cleaved caspase3/cleaved PARP)/mitochondrial-damaged (cytosolic-cytochrome-C) biomarkers displayed an opposite pattern of INBF among the groups (all p < 0.0001).
PrPc-EPCs were superior to EPCs only therapy for salvaging the CLI.
本研究旨在验证这样一个假设，即在经过 28 天慢性肾病（CKD）诱导后发生的大鼠临界肢体缺血（CLI）中，内皮祖细胞（EPC）中细胞朊蛋白的过度表达（定义为“再生 EPC”）优于 EPC 用于挽救。
细胞活力和早期/晚期凋亡/总细胞内 ROS/细胞周期（亚 G1、G2/M 期）的流式细胞术分析在 EPC+HO 中显著高于 EPC，在 PrPc-EPC+HO 中则显著逆转（均 p<0.001）。在 EPC+TNF-α中，炎症（IL-1β/IL-6/MMP-9/p-NF-κB）的蛋白表达显著增加，而在 PrPc-EPC+TNF-α中则显著逆转（均 p<0.001）。成年雄性 SD 大鼠（每组 8 只）分为 1 组（假手术对照）、2 组（CKD+CLI）、3 组[CKD+CLI+静脉（0.6×10）/肌内（0.6×10）注射 EPCs，在 CLI 诱导后 3 h 给药]、4 组（CKD+CLI+PrPc-EPCs/剂量给药如第 3 组）和 5 组（CKD+CLI+siPrnp-EPCs/剂量给药如第 3 组）。在 CLI 诱导后 14 天，CLI 区域的缺血与正常血流（INBF）比值在第 1 组中最高，在第 2 组中最低，在第 4 组中显著高于第 3 组/第 5 组，在第 3 组中显著高于第 5 组（均 p<0.0001）。组织病理学显示，血管生成（小血管/CD31+细胞的数量）呈相似趋势，而纤维化/肾脏损伤评分则呈相反的 INBF 模式（均 p<0.0001）。血管生成（SDF-1α/VEGF/CXCR4）/细胞应激信号（p-PI3K/p-Akt/p-m-TOR）的蛋白表达水平从第 1 组到第 4 组逐渐升高，而第 5 组则逆转（均 p<0.0001）。纤维化（p-Smad3/TGF-β）/氧化应激（NOX-1/NOX-2/氧化蛋白）/细胞凋亡（线粒体-Bax/cleaved caspase3/cleaved PARP）/线粒体损伤（细胞质-cytochrome-C）生物标志物的蛋白表达在各组中呈相反的 INBF 模式（均 p<0.0001）。
PrPc-EPC 优于单独 EPC 治疗，可挽救 CLI。
