Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
Am J Physiol Cell Physiol. 2010 Jun;298(6):C1527-37. doi: 10.1152/ajpcell.00484.2009. Epub 2010 Feb 24.
Hypoxia plays an important role in vascular development through hypoxia-inducible factor-1alpha (HIF-1alpha) accumulation and downstream pathway activation. We sought to explore the in vitro response of cultures of human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), human endothelial progenitor cells (hEPCs), and human umbilical cord vein endothelial cells (HUVECs) to normoxic and hypoxic oxygen tensions. We first measured dissolved oxygen (DO) in the media of adherent cultures in atmospheric (21% O(2)), physiological (5% O(2)), and hypoxic oxygen conditions (1% O(2)). In cultures of both hEPCs and HUVECs, lower oxygen consumption was observed when cultured in 1% O(2). At each oxygen tension, feeder-free cultured hESCs and iPSCs were found to consume comparable amounts of oxygen. Transport analysis revealed that the oxygen uptake rate (OUR) of hESCs and iPSCs decreased distinctly as DO availability decreased, whereas the OUR of all cell types was found to be low when cultured in 1% O(2), demonstrating cell adaptation to lower oxygen tensions by limiting oxygen consumption. Next, we examined HIF-1alpha accumulation and the expression of target genes, including VEGF and angiopoietins (ANGPT; angiogenic response), GLUT-1 (glucose transport), BNIP3, and BNIP3L (autophagy and apoptosis). Accumulations of HIF-1alpha were detected in all four cell lines cultured in 1% O(2). Corresponding upregulation of VEGF, ANGPT2, and GLUT-1 was observed in response to HIF-1alpha accumulation, whereas upregulation of ANGPT1 was detected only in hESCs and iPSCs. Upregulation of BNIP3 and BNIP3L was detected in all cells after 24-h culture in hypoxic conditions, whereas apoptosis was not detectable using flow cytometry analysis, suggesting that BNIP3 and BNIP3L can lead to cell autophagy rather than apoptosis. These results demonstrate adaptation of all cell types to hypoxia but different cellular responses, suggesting that continuous measurements and control over oxygen environments will enable us to guide cellular responses.
缺氧通过缺氧诱导因子-1α（HIF-1α）的积累和下游途径的激活，在血管发育中起着重要作用。我们试图探索体外培养的人胚胎干细胞（hESC）、诱导多能干细胞（iPSC）、人内皮祖细胞（hEPC）和人脐静脉内皮细胞（HUVEC）对常氧和低氧氧张力的反应。我们首先测量了贴壁培养物在大气（21% O2）、生理（5% O2）和低氧氧条件下（1% O2）培养基中的溶解氧（DO）。在 1% O2 培养时，观察到 hEPC 和 HUVEC 的耗氧量较低。在每种氧张力下，无饲养层培养的 hESC 和 iPSC 被发现消耗相当数量的氧气。转运分析表明，hESC 和 iPSC 的耗氧率（OUR）随着 DO 可用性的降低而明显降低，而当在 1% O2 中培养时，所有细胞类型的 OUR 均较低，表明细胞通过限制耗氧量来适应较低的氧张力。接下来，我们检查了 HIF-1α的积累和靶基因的表达，包括 VEGF 和血管生成素（ANGPT；血管生成反应）、GLUT-1（葡萄糖转运）、BNIP3 和 BNIP3L（自噬和细胞凋亡）。在 1% O2 中培养的所有四种细胞系中均检测到 HIF-1α的积累。相应地，在 HIF-1α积累的情况下，检测到 VEGF、ANGPT2 和 GLUT-1 的上调，而仅在 hESC 和 iPSC 中检测到 ANGPT1 的上调。在 24 小时低氧培养后，所有细胞中均检测到 BNIP3 和 BNIP3L 的上调，而使用流式细胞术分析未检测到细胞凋亡，表明 BNIP3 和 BNIP3L 可导致细胞自噬而不是凋亡。这些结果表明所有细胞类型都能适应缺氧，但细胞反应不同，这表明对氧环境的连续测量和控制将使我们能够指导细胞反应。
