Key Laboratory of Medical Electrophysiology of Ministry of Education and Medical Electrophysiological Key Lab of Sichuan Province, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, Luzhou, China.
Am J Physiol Cell Physiol. 2023 Nov 1;325(5):C1228-C1243. doi: 10.1152/ajpcell.00048.2023. Epub 2023 Sep 18.
Extracellular adenosine triphosphate (ATP) is one of the most abundant biochemical constitutes within the stem cell microenvironment and is postulated to play critical roles in cell migration. However, it is unclear whether ATP regulates the cell migration of CD34 vascular wall-resident stem/progenitor cells (VW-SCs) and participates in angiogenesis. Therefore, the biological mechanisms of cell migration mediated by ATP was determined by in vivo subcutaneous matrigel plug assay, ex vivo aortic ring assay, in vitro transwell migration assay, and other molecular methods. In the present study, ATP dose-dependently promoted CD34 VW-SCs migration, which was more obviously attenuated by inhibiting or knocking down P2Y2 than P2Y6. Furthermore, it was confirmed that ATP potently promoted the migration of resident CD34 cells from cultured aortic artery rings and differentiation into endothelial cells in matrigel plugs by using inducible lineage tracing -CreER; R26-tdTomato mice, whereas P2Y2 and P2Y6 blocker greatly inhibited the effect of ATP. In addition, ATP enhanced the protein expression of stromal interaction molecule 1 (STIM1) on cell membrane, blocking the calcium release-activated calcium (CRAC) channel with shSTIM1 or BTP2 apparently inhibited ATP-evoked intracellular Ca elevation and channel opening, thereby suppressing ATP-driven cell migration. Moreover, extracellular signal-regulated protein kinase (ERK) inhibitor PD98059 and p38 inhibitor SB203580 remarkably inhibited ERK and p38 phosphorylation, cytoskeleton rearrangement, and subsequent cell migration. Unexpectedly, it was found that knocking down STIM1 greatly inhibited ATP-triggered ERK/p38 activation. Taken together, it was suggested that P2Y2 signaled through the CRAC channel mediated Ca influx and ERK/p38 pathway to reorganize the cytoskeleton and promoted the migration of CD34 VW-SCs. In this study, we observed that the purinergic receptor P2Y2 is critical in the regulation of vascular wall-resident CD34 cells' migration. ATP could activate STIM1-mediated extracellular Ca entry by triggering STIM1 translocation to the plasma membrane, and knockdown of STIM1 prevented ERK/p38 activation-mediated cytoskeleton rearrangement and cell migration.
细胞外三磷酸腺苷 (ATP) 是干细胞微环境中最丰富的生化成分之一，据推测在细胞迁移中发挥关键作用。然而，尚不清楚 ATP 是否调节 CD34 血管壁驻留干细胞/祖细胞 (VW-SC) 的细胞迁移并参与血管生成。因此，通过体内皮下基质胶塞测定、体外主动脉环测定、体外 Transwell 迁移测定和其他分子方法确定了由 ATP 介导的细胞迁移的生物学机制。在本研究中，ATP 剂量依赖性地促进 CD34 VW-SC 的迁移，而用 P2Y2 抑制剂或敲低 P2Y2 比 P2Y6 更明显地减弱了这种作用。此外，通过使用诱导谱系追踪 -CreER;R26-tdTomato 小鼠，证实 ATP 可强力促进培养的主动脉环中驻留 CD34 细胞的迁移，并在基质胶塞中分化为内皮细胞，而 P2Y2 和 P2Y6 阻滞剂则极大地抑制了 ATP 的作用。此外，ATP 增强了细胞膜上基质相互作用分子 1 (STIM1) 的蛋白表达，用 shSTIM1 或 BTP2 阻断钙释放激活钙 (CRAC) 通道明显抑制了 ATP 诱导的细胞内 Ca 升高和通道开放，从而抑制了 ATP 驱动的细胞迁移。此外，细胞外信号调节蛋白激酶 (ERK) 抑制剂 PD98059 和 p38 抑制剂 SB203580 显著抑制了 ERK 和 p38 的磷酸化、细胞骨架重排和随后的细胞迁移。出乎意料的是，发现敲低 STIM1 大大抑制了 ATP 触发的 ERK/p38 激活。总之，这表明 P2Y2 通过 CRAC 通道介导的 Ca 内流和 ERK/p38 途径信号转导来重排细胞骨架并促进 CD34 VW-SC 的迁移。在本研究中，我们观察到嘌呤能受体 P2Y2 在调节血管壁驻留 CD34 细胞的迁移中起着关键作用。ATP 可以通过触发 STIM1 向质膜易位来激活 STIM1 介导的细胞外 Ca 内流，并且敲低 STIM1 可防止 ERK/p38 激活介导的细胞骨架重排和细胞迁移。
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