探討內生性一氧化氮在缺氧狀態下調控血管新生作用的分子機制

Abstract

細胞偵測氧氣濃度改變的部份能力是由缺氧調控因子 (hypoxia inducible factor, HIF)調控，主要參與缺氧反應的isoform 是HIF-1α。HIF-1α為促進血管新生的轉錄因子，它能啟動並強化血管內皮細胞生長因子 (vascular endothelial growth factor，VEGF) 的基因表現，促使細胞具血管新生能力。已知HIF-1α之蛋白質穩定性受一氧化氮 (NO)調控。此外，也有研究指出，在缺氧情況下外加NO 供應者 (NO donor) 會抑制 VEGF-A 的轉錄及轉譯。但是，內生性NO 是否參與缺氧細胞內的活化血管新生訊息仍未知。我們以小鼠血管內皮細胞 (MS-1) 為研究對像，探討在缺氧情況下，內生性NO 對於HIF-1α穩定性及VEGF-A 蛋白質分泌作用的調控機制。我們觀察到，缺氧30 分鐘的MS-1 細胞有大量內生性NO 產生，此高量的內生性NO 會一直維持到12 小時。在經過16 小時至24 小時的缺氧刺激後，內生性NO 的量便開始下降。此外，缺氧MS-1 細胞之內生性NO 濃度的消長伴隨著HIF-1α 穩定性、VEGF-A 因之轉錄和轉譯以及VEGF-A 蛋白質的分泌作用的變化。值得注意的是，在缺氧刺激12 小時之後，MS-1 細胞之內生性NO 的量下降同時伴隨著VEGF-A 分泌作用的開始。因此，我們提出「翹翹板理論」來說明內生性NO 和VEGF-A 分泌作用在缺氧MS-1 細胞內的關係，並藉由CSNO (NO 提供者)和cPTIO (NO 清除劑) 調控內生性NO 的量來觀察VEGF-A 分泌作用是否因此改變。缺氧的MS-1 細胞在接受外加NO後有較高量的內生性NO，並且分泌較少量的VEGF-A; 反之，同時給予MS-1 細胞缺氧刺激及cPTIO 會提高VEGF-A 的分泌作用。 我們同時也探討由缺氧MS-1 細胞釋放的VEGF-A 是否具功能性及促進血管新生之能力。為此，我們給予人類臍帶靜脈血管內皮細胞 (human umbilical veinendothelial cell, HUVEC) 在缺氧情情況下由缺氧MS-1 細胞、外加CSNO 的缺氧MS-1 細胞及同時受缺氧及cPTIO 處理的MS-1 細胞得到之conditioned medium 來觀察血管新生作用的變化。結果指出，由缺氧MS-1 細胞得到的conditioned medium能活化HUVEC 細胞內血管新生的訊息傳遞。重要的是，由同時受缺氧及cPTIO 處理的MS-1 細胞得到的conditioned medium 更能提高HUVEC 細胞的血管新生作用。反之，由外加CSNO 的缺氧MS-1 細胞得到的conditioned medium 無法有效活化HUVEC細胞內的血管新生訊息。 根據上述實驗結果，我們認為缺氧MS-1 細胞之內生性NO 的含量可決定VEGF-A 分泌作用的程度。因此，經由調控之後的內生性NO 會影響被釋放出來之VEGF-A 蛋白質的多寡，進而造成HUVEC 細胞內之血管新生作用的變化。

Data accumulated indicate that the ability of cells to sense changes in oxygen concentration is partially mediated by the transcriptional regulator hypoxia-inducible factor (HIF). The major isoform involved in hypoxic response is HIF-1α, a pro-angiogenic transcription factor which contributes to the enhancement of VEGF expression and angiogenesis under hypoxia. In previous studies, it has been shown that the stability of HIF-1α may be regulated in a nitric oxide (NO)-dependent manner. In addition, NO supplied by NO donor has been suggested to affect both mRNA expression and protein expression of VEGF-A under hypoxia. However, it is not known whether intrinsic NO participates in the regulation of angiogenic signaling in cells suffered from hypoxia. In the current study, we have investigated if intrinsic NO plays a critical role in HIF- 1α stabilization and VEGF-A protein secretion under hypoxia. Mouse endothelial MS-1 cells were used to delineate the regulatory mechanism involved in this process. We observed that intrinsic NO production was boosted dramatically within 30 minutes of hypoxic treatment in MS-1 cells. In long-term hypoxia after 12 hours hypoxic stimulation, the cellular NO level dropped gradually and the amount of intrinsic NO maintained in a relatively low status within 16 hours to 24 hours. Furthermore, HIF-1α stability, VEGF-A mRNA expression, intracellular VEGF-A protein and VEGF-A protein secretion were regulated in a manner concomitant with the levels of cellular NO in response to hypoxia. Notably, the decrease of intrinsic NO production is matched with the onset of VEGF-A release 12 hours post hypoxia. Therefore, we hypothesized that there is a seesaw relationship between the level of intrinsic NO production and VEGF-A release in MS-1 cells response to hypoxia. Upon observing this interesting phenomenon, we introduced CSNO, the NO donor, and cPTIO, the NO scavenger, into hypoxic MS-1 cell model system to manipulate the level of intrinsic NO, and further checked the effect of manipulated NO on VEGF-A release. In cells treated with 24 hours hypoxia and CSNO, we observed higher level of cellular NO and a significant decrease of VEGF-A release. Whereas in cells cotreated with 16 hours hypoxia and cPTIO, the amount of released VEGF-A is increased. We also investigated whether hypoxia-induced release of VEGF-A from MS-1 cells is functional and could promote angiogenesis in healthy endothelial cells. Moreover, effects of hypoxic-conditioned medium isolated from CSNO or cPTIO-treated MS-1 cells on angiogenesis was also under discussing. For this, we treated Human Umbilical Vein Endothelial Cells (HUVEC) with VEGF-A-contained hypoxic-conditioned medium obtained from MS-1 cells in response to hypoxia or hypoxia plus CSNO/cPTIO. We observed that hypoxic-conditioned medium obtained from MS-1 cells exposed to hypoxia was capable of activating signals leading to angiogenesis. Importantly, hypoxic-conditioned medium obtained from MS-1 cells cotreated with hypoxia and cPTIO further enhanced the angiogenic ability in HUVEC, whereas HUVEC treated with hypoxic conditioned-medium obtained from MS-1 cells in response to hypoxia and CSNO showed reduced angiogenesis. Based on these observations, we proposed that the level of intrinsic NO production is critical to govern the release of VEGF-A in MS-1 cells response to hypoxia. Therefore, the manipulated intrinsic-NO level in hypoxic MS-1 cells may affect the amount of released VEGF-A, thus changing the angiogenic ability in healthy HUVEC.