| Summary: | 碩士 === 臺灣大學 === 生化科學研究所 === 98 === 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.
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