Nishida Ryota, Fukui Tomoaki, Sawauchi Kenichi, Kumabe Yohei, Kondo Hyuma, Yamamoto Yuya, Takase Kyohei, Yoshikawa Ryo, Niikura Takahiro, Kuroda Ryosuke, Oe Keisuke
Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.
Department of Orthopaedic Surgery, Hyogo Prefectural Nishinomiya Hospital, Hyogo, Japan.
PLoS One. 2025 Sep 16;20(9):e0332673. doi: 10.1371/journal.pone.0332673. eCollection 2025.
Transcutaneous CO2 application promotes fracture healing and osteogenesis via angiogenesis. However, its molecular mechanism remains unclear. This therapy transiently decreases intra-tissue pH in the affected area to approximately 7.0 for 20 min, followed by recovery. We hypothesized that such intermittent pH changes activate endothelial cells similarly to acidic preconditioning. We aimed to investigate the response of human umbilical vein endothelial cells (HUVECs) to daily intermittent low-pH stimulation. HUVECs were cultured under three conditions: daily 20-min exposure to a low-pH medium (approximately 7.0), followed by a return to a control medium (approximately 7.4) (Change group); continuous exposure to a low-pH medium (Low pH group); constant culturing in a control medium (Control group). Cell proliferation, tube formation, migration, and protein/gene expression were assessed. Tube formation and migration were significantly enhanced in the Change group compared with those in the Control group, and tube formation was also increased in the Low pH group. Western blotting revealed upregulated expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 in the Change group. CD31 gene expression was elevated in the Low pH and Change groups on Days 1 and 7, respectively. Phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (AKT) levels in the Low pH and Change groups were significantly increased compared with those in the Control, except for ERK2 in the Low pH group. The suppression of ERK1/2 or AKT by their inhibitors (U0126 and LY294002, respectively) during low-pH exposure inhibited proliferation and tube formation, indicating that the ERK1/2 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/AKT pathways mediate endothelial cell activation in response to intermittent acidic stimulation. Intermittent low-pH exposure enhanced HUVEC activity, mediated by the ERK1/2 MAPK and PI3K/AKT pathways. Our findings provide insights into the mechanism by which transcutaneous CO2 application promotes angiogenesis and tissue repair.
经皮应用二氧化碳通过血管生成促进骨折愈合和成骨。然而，其分子机制仍不清楚。这种疗法会使受影响区域的组织内pH值短暂降至约7.0并持续20分钟，随后恢复。我们推测这种间歇性pH变化与酸性预处理类似地激活内皮细胞。我们旨在研究人脐静脉内皮细胞（HUVECs）对每日间歇性低pH刺激的反应。HUVECs在三种条件下培养：每日暴露于低pH培养基（约7.0）20分钟，然后恢复到对照培养基（约7.4）（变化组）；持续暴露于低pH培养基（低pH组）；在对照培养基中持续培养（对照组）。评估细胞增殖、管形成、迁移以及蛋白质/基因表达。与对照组相比，变化组的管形成和迁移显著增强，低pH组的管形成也增加。蛋白质印迹显示变化组中血管内皮生长因子（VEGF）和VEGF受体2的表达上调。低pH组和变化组的CD31基因表达分别在第1天和第7天升高。与对照组相比，低pH组和变化组中磷酸化的细胞外信号调节激酶1和2（ERK1/2）以及蛋白激酶B（AKT）水平显著升高，但低pH组中的ERK2除外。在低pH暴露期间用其抑制剂（分别为U0126和LY294002）抑制ERK1/2或AKT会抑制增殖和管形成，表明ERK1/2丝裂原活化蛋白激酶（MAPK）和磷脂酰肌醇-3激酶（PI3K）/AKT途径介导内皮细胞对间歇性酸性刺激的激活。间歇性低pH暴露增强了HUVEC活性，由ERK1/2 MAPK和PI3K/AKT途径介导。我们的研究结果为经皮应用二氧化碳促进血管生成和组织修复的机制提供了见解。
