Comparison of hypoxic effects induced by chemical and physical hypoxia on cardiomyocytes.
Zhao Ru-Zhou,Jiang Shuai,Ru Ning-Yu,Jiao Bo,Yu Zhi-Bin
Canadian journal of physiology and pharmacology
The degree and duration of chemical hypoxia induced by sodium dithionite (NaSO) have not been reported. It is not yet clear how much reduction in the O concentration (physical hypoxia) can lead to hypoxia in cultured cardiomyocytes. In this study, oxygen microelectrodes were used to measure changes in the O concentration in media containing different concentrations of NaSO. Then, hypoxic effects of 0.8, 1.0, and 2.0 mM NaSO or 1%, 3%, and 5% O in cultured cardiomyocytes from neonatal rats were observed and compared. The results showed that the O concentration failed to remain constant by NaSO treatment during the 180-minute observation period. Only the 2.0 mM NaSO group significantly increased the expression of hypoxia-inducible factor 1α (HIF-1α) and hypoxic responses. Notably, 3% O only significantly increased the expression of HIF-1α in cardiomyocytes, while 1% O not only increased the expression of HIF-1α but also increased the apoptotic rate in cardiomyocytes. These results suggest that NaSO is not suitable for establishing a hypoxic model in cultured neonatal rat cardiomyocytes, and neonatal rat cardiomyocytes cultured at or below 1% O induced significant hypoxic effects, which can be used as a starting O concentration for establishing a hypoxic cell model.
Improvement of tube formation model of cell: Application for acute hypoxia in in vitro study of angiogenesis.
Li Fengyang,Gou Xue,Xu Dan,Han Dan,Hou Kai,Fang Weirong,Li Yunman
Angiogenesis caused by acute vascular occlusion occurs in various ischemic diseases. The in vitro tube formation assay by endothelial cells is a rapid, quantitative method for drug discovery on angiogenesis. Tube formation assay on Matrigel has been widely used to identify the angiogenesis, however, there are some problems to limit its application. In this study, we found for the first time that sodium dithionite (SD) could induce endothelial cell tube formation without Matrigel under hypoxia condition. To further verify our findings, the angiogenesis related proteins and mRNA at different time points after tube formation were measured both in primary human large-vessel endothelial cell (HUVECs) and murine microvascular endothelial cell line (Bend.3). In conclusion, compared with traditional tube formation on Matrigel, the novel model exhibits the following advantages: (1) Combination oxygen glucose deprivation with sodium dithionite (OGD-SD) model is operated more easily than traditional tube formation. (2) OGD-SD can be used for not only cell imaging, but also immunofluorescence, protein extraction and gene analysis. (3) OGD-SD is more applicable to acute hypoxia model of endothelial cell in vitro. (4) OGD-SD may be more suitable to identify molecular mechanism of compound that intervenes processes of pro-tube formation, tube formation and tube disconnection.