高壓氧處理之人類臍靜脈內皮細胞所誘導之血管內皮細胞生長因子 VEGF 表現的分子機制探討
Mechanisms of The Induction of Vascular Endothelial Growth Factor Expression by Hyperbaric Oxygen in Human Umbilical Vein Endothelial Cells
中文摘要
高壓氧治療可促進血管新生，進而加速和缺氧相關的傷口癒合，而血管內皮細 胞生長因子 (VEGF) 及其接受器系統無論在生理或病理性的血管新生都是最主 要的調控者。然而高壓氧調控血管內皮細胞活化的機制尚未明瞭。本實驗即是在 探討高壓氧活化血管內皮細胞生長所扮演的角色及其可能的調控機制。我們由 Real-time PCR 及北方墨點分析數據證實，高壓氧可誘發血管內皮細胞生長因子
基因的表現，並於4 小時達到最高峰，且高壓氧所誘發 VEGF mRNA 的表現可
被MEK 的抑制劑 PD98059 及 JNK 的抑制劑 SP600125 所抑制。另一方面，西方 墨點分析法顯示，VEGF 蛋白最大表現量在 8 小時，ERK1/2 的磷酸化則在 15 分 鐘最強。此外，VEGF 的 promoter 上有 hypoxia inducible factor-1 (HIF-1) 和 Activator protein-1 (AP-1) 轉錄因子的結合位，由 electrophoretic mobility shift assay (EMSA) 結果顯示，高壓氧處理 1 小時可明顯活化此二轉錄因子，且其 DNA-protein 的結合能力皆會被 PD98059 及 SP600125 所抑制。因此推測高壓氧 可能是透過JNK-mitogenic-activated protein kinase 及 ERK-mitogenic-activated protein kinase 的訊息傳遞路徑，活化 HIF-1 和 AP-1 轉錄因子而啟動 VEGF 基因 的表現，進而促進血管的新生。
英文摘要
One of the biological effects of hyperbaric oxygen (HBO) therapy in enhancing ischemia-related wound healing is the induction of angiogenesis. The vascular
endothelial growth factor VEGF/VEGF receptor system plays a central regulatory role in physiological and pathological angiogenesis. During embryogenesis, the
VEGF/VEGF receptor system is critically involved in the formation of the vascular system by regulating both the growth and the survival of blood vessels. The
mechanism by which hyperbaric oxygen regulates angiogenesis is presently unclear.
The aim of the present study was to investigate the mechanism of the activation of VEGF gene activation in human umbilical vein endothelial cells (HUVECs) exposed to HBO. Preliminary Real-time quantitative PCR studies revealed that the expression of VEGF mRNA was induced after 4 hours of exposure. The induction of VEGF mRNA was further confirmed by Northern blot analysis. Western blot analysis showed that HBO enhanced the VEGF protein level and increased the ERK1/2 activity, with a maximal level after 8 hours and 15 min, respectively. Whereas, JNK
inhibitor SP600125 and MEK inhibitor PD98059 blocked the HBO-induced VEGF mRNA expression. Since VEGF promoter region contains AP-1 and HIF-1 binding sites, stimulation of HUVECs with HBO enhanced AP-1 and HIF-1 binding to their DNA binding sites as demonstrated by electrophoretic mobility shift assay(EMSA).
This effect was also inhibited by SP600125 and PD98059. These data suggested that HBO-induced VEGF expression might be through AP-1 and HIF-1 stimulation, and required the involvement of JNK and ERK-mitogenic-activated protein kinase signaling pathways, which may play an important role in HBO-induced angiogenesis.
