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A microfluidic oxygen gradient demonstrates differential activation of the hypoxia-regulated transcription factors HIF-1α and HIF-2α
Integrative Biology ( IF 2.5 ) Pub Date : 2017-08-18 00:00:00 , DOI: 10.1039/c7ib00099e
Megan L. Rexius-Hall 1, 2, 3, 4 , Jalees Rehman 3, 4, 5, 6, 7 , David T. Eddington 1, 2, 3, 4
Affiliation  

Gas-perfused microchannels generated a linear oxygen gradient via diffusion across a 100 μm polydimethylsiloxane (PDMS) membrane. The device enabled exposure of a single monolayer of cells sharing culture media to a heterogeneous oxygen landscape, thus reflecting the oxygen gradients found at the microscale in the physiological setting and allowing for the real-time exchange of paracrine factors and metabolites between cells exposed to varying oxygen levels. By tuning the distance between two gas supply channels, the slope of the oxygen gradient was controlled. We studied the hypoxic activation of the transcription factors HIF-1α and HIF-2α in human endothelial cells within a spatial linear gradient of oxygen. Quantification of the nuclear to cytosolic ratio of HIF immunofluorescent staining demonstrated that the threshold for HIF-1α activation was below 2.5% O2 while HIF-2α was activated throughout the entire linear gradient. We show for the first time HIF-2α is subject to hyproxya, hypoxia by proxy, wherein hypoxic cells activate HIF in close-proximity normoxic cells. These results underscore the differences between HIF-1α and HIF-2α regulation and suggest that a microfluidic oxygen gradient is a novel tool for identifying distinct hypoxic signaling activation and interactions between differentially oxygenated cells.

中文翻译:

微流氧梯度表明缺氧调节的转录因子HIF-1α和HIF-2α的差异激活

气体灌注的微通道中产生的线性氧含量梯度经由扩散穿过100μm的聚二甲基硅氧烷(PDMS)膜。该设备能够使共享培养基的单层细胞暴露于异质氧环境,从而反映出在生理环境中在微观尺度上发现的氧梯度,并允许暴露于变化的细胞之间实时交换旁分泌因子和代谢产物。氧气含量。通过调节两个气体供应通道之间的距离,可以控制氧气梯度的斜率。我们研究了在氧的空间线性梯度内人内皮细胞中转录因子HIF-1α和HIF-2α的低氧激活。HIF免疫荧光染色的核质比定量表明,HIF-1α激活的阈值低于2.5%O 2而HIF-2α在整个线性梯度中均被激活。我们首次显示HIF-2α受到代理人的低氧,缺氧的影响,其中低氧细胞激活近距离常氧细胞中的HIF。这些结果强调了HIF-1α和HIF-2α调节之间的差异,并表明微流氧梯度是一种新的工具,可用于识别不同的低氧信号激活和差异化氧合的细胞之间的相互作用。
更新日期:2017-09-18
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