Expression profiling in hypoxia-treated human microvascular endothelial cells

Purpose/Objective: There is accumulating evidence that hypoxia is an independent factor for negative clinical outcome in tumor patients. One hypothesis is that hypoxia leads to increased tumor angiogenesis and increased local invasion due to transcriptional changes. Because the endothelial cell compartment is crucial at least for tumor angiogenesis we have used RNA and protein expression profiling of human microvascular cells treated in vivo with hypoxia to better understand hypoxia-induced pathways in endothelial cells. Materials/Methods: We used primary isolated human lung microvascular, dermal microvascular, and umbilical vein endothelial cells treated 4, 8 and 12h under 0.1% O2. Total RNA and protein extracts were analyzed. Using a large DNA-Chip we have been able to get expression profiles from complete human Unigene II clusters which contain approximately 76,000 elements covering almost 95% of the human genome, in a single experiment, providing clues to the functional role of many genes, including potentially important genes involved in different pathways like angiogenesis and apoptosis. We further used SELDI ProteinChip Arrays to investigate hypoxia-induced proteins using four different surface chemistries (WCX, SAX, H4 and IMAC). Results: We report on the identification of a series of genes and proteins whose expressions varied with different exposure times under hypoxic conditions in vitro. Among the highly significantly up- or down-regulated genes were genes known to be related to apoptosis and cell cycle (11 genes), proteases (13), angiogenesis (12), suppressor genes (13), cancer in a general sense (>40). Upregulated gene examples include NF-kB, HIF1a, AP1, COX2, JUN, VEGF, VEGFR, IGFBP3, downregulated genes were CYCLINB, AP2, BAX, and others. The protein chips revealed more than 10 differentially-expressed proteins from the endothelial cells. Further, the DNA chip technology and protein chip arrays give us the opportunity to investigate a huge amount of expressed sequence tags (ESTs) and uncharacterized genes. Conclusions: Our findings demonstrate that hypoxia can affect the expression pattern of microvascular endothelial cells, and that several genes and proteins are acting in concert in response to this stress. In addition to identifying unexpected genes and proteins, these technologies can facilitate the development of new hypotheses concerning how cells respond to hypoxia and the identification of co-regulated pathways in response to hypoxia microenvironment alterations.