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.

中文翻译：

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结合 NOX/ROS/PKC 信号通路和代谢组学分析揭示了 TRAM34 诱导的内皮祖细胞衰老的机制

已经表明，KCa3.1 通道特异性阻断剂 TRAM34 是一种很有前途的抗动脉粥样硬化 (AS) 药物，但其副​​作用限制了其临床应用。值得注意的是，它对内皮祖细胞 (EPC) 的影响尚不清楚。我们旨在揭示 TRAM34 对 EPC 的影响并确定其潜在机制。大鼠腹腔注射 TRAM34，并从骨髓中分离 EPC。采用实时荧光定量PCR、酶联免疫吸附法、蛋白质印迹法和荧光激活细胞分选法检测相应因子的基因和蛋白水平。液相色谱-串联质谱 (LC-MS) 用于检测代谢物差异。我们发现，当大鼠在体内接受 TRAM34 治疗时，早期 EPC 的集落形成和增殖减少，但它们的衰老和凋亡增强。此外，TRAM34 增强 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，这对体内和体外 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 显着促进了嘧啶和嘌呤代谢，导致细胞衰老。此外，NOX 抑制剂 Setanaxib 可增强抗氧化代谢途径，尤其是 S-腺苷甲硫胺 (SAM) 代谢，从而发挥抗衰老作用。最后，我们证实 SAM 减轻了 TRAM34 诱导的细胞衰老，这表明了一种提高内源性 EPC 质量的有效方法。本研究揭示了 TRAM34 诱导 EPC 衰老的机制，