Cui Xiaodong, Li Xiaoxia, He Yanting, Yu Jie, Fu Jie, Song Bo, Zhao Robert Chunhua
Department of Basic Medicine, Institute of Stem Cell and Regenerative Medicine, Qingdao University Medical College, Qingdao University, Qingdao, P.R. China.
School of Basic Medical Sciences, Weifang Medical University, Weifang, P.R. China.
Stem Cells Dev. 2021 Jul 1;30(13):671-682. doi: 10.1089/scd.2021.0062. Epub 2021 Jun 7.
It has been shown that the KCa3.1 channel-specific blocker, TRAM34, is a promising antiatherosclerosis (AS) agent, but its side effects restrict its clinical application. Notably, its effect on endothelial progenitor cells (EPCs) is unclear. We aim to unravel the effect of TRAM34 on EPCs and identify the underlying mechanism. Rats were injected intraperitoneally with TRAM34, and EPCs were isolated from bone marrow. The gene and protein levels of corresponding factors were detected by real-time PCR, enzyme-linked immunosorbent assay, western blotting, and fluorescence-activated cell sorting. Liquid chromatography-tandem mass spectrometry (LC-MS) was applied to detect metabolite differences. We showed that when rats were treated with TRAM34 in vivo, colony formation and proliferation of early EPCs were reduced, but their senescence and apoptosis were enhanced. Moreover, TRAM34 enhanced NOX activity, promoted an increase in intracellular ROS levels, increased PKC expression, and subsequently promoted EPC senescence, which is unfavorable for EPC angiogenesis in vivo and in vitro. Combining these results with LC-MS data, we found that TRAM34 significantly promoted pyrimidine and purine metabolism, leading to cellular senescence. Furthermore, the NOX inhibitor, Setanaxib, enhanced antioxidant metabolic pathways, especially S-adenosylmethioninamine (SAM) metabolism, to exert an antisenescence effect. Finally, we confirmed that SAM alleviates TRAM34-induced cellular senescence, suggesting an efficient approach to improve the quality of endogenous EPCs. This study reveals the mechanism of TRAM34-induced EPC senescence, providing a solution for the extended application of KCa3.1 inhibitor in AS.
已有研究表明，KCa3.1通道特异性阻滞剂TRAM34是一种很有前景的抗动脉粥样硬化（AS）药物，但其副作用限制了其临床应用。值得注意的是，其对内皮祖细胞（EPCs）的影响尚不清楚。我们旨在阐明TRAM34对EPCs的影响并确定其潜在机制。给大鼠腹腔注射TRAM34，然后从骨髓中分离EPCs。通过实时PCR、酶联免疫吸附测定、蛋白质印迹和荧光激活细胞分选检测相应因子的基因和蛋白质水平。应用液相色谱 - 串联质谱（LC - MS）检测代谢物差异。我们发现，当在体内用TRAM34处理大鼠时，早期EPCs的集落形成和增殖减少，但其衰老和凋亡增强。此外，TRAM34增强了NOX活性，促进细胞内ROS水平升高，增加PKC表达，随后促进EPC衰老，这对体内外EPC血管生成不利。将这些结果与LC - MS数据相结合，我们发现TRAM34显著促进嘧啶和嘌呤代谢，导致细胞衰老。此外，NOX抑制剂Setanaxib增强了抗氧化代谢途径，特别是S - 腺苷甲硫胺（SAM）代谢，以发挥抗衰老作用。最后，我们证实SAM可减轻TRAM34诱导的细胞衰老，提示这是一种提高内源性EPCs质量的有效方法。本研究揭示了TRAM34诱导EPC衰老的机制，为KCa3.1抑制剂在AS中的扩展应用提供了解决方案。
