There has been persistent controversy regarding whether hypoxia, an important stimulus for angiogenesis and vascular remodeling, imposes an oxidant stress on target cells. This hypothesis has been difficult to prove using conventional approaches to detect reactive oxygen species (ROS). As an alternate strategy, we used """"""""endonuclease fingerprinting"""""""" to determine if hypoxia caused ROS-dependent modifications in mitochondrial DNA and in selected nuclear genes in rat cultured pulmonary arterial smooth muscle cells (PASMCs). Mitochondrial DNA was spared, but hypoxia caused an unusual pattern of oxidative modifications in nuclear genes that differed from lesions associated with ROS-dependent toxicity; strand breaks were rare, and the density of oxidatively-modified bases was elevated. Against this background, the long-term goal of the proposed research is to define the significance of oxidative DNA modifications which occur in the context of physiological signal transduction. How ROS contribute to hypoxic cell responses is unclear, but our findings that the VEGF promoter region was a prominent site of hypoxia-induced base oxidation and that an antioxidant prevents both DNA modifications and hypoxia-induced accumulation of VEGF mRNA suggests that oxidative base modifications could be linked to expression of hypoxia-responsive genes. The proposed research will test key aspects of the working hypothesis that ROS-dependent modifications in specific DNA sequences contribute to regulation of gene expression in hypoxia. Studies in rat PASMCs will: (1) Test the hypothesis that transcriptionally-active genes which are induced (VEGF) or repressed (ornithine decarboxylase: ODC) by hypoxia exhibit hypoxia-inducted oxidative DNA modifications that differ from those in unresponsive (B-actin) and non-transcribed (insulin) genes; (2) Determine whether hypoxia-induced changes in oxidative DNA modifications are temporally and functionally associated with altered expression of the affected genes; and, (3) Map at single-nucleotide resolution the location of hypoxia-induced oxidative modifications in functionally-distinct sequences in the VEGF gene and determine if the presence of oxidized bases is associated with altered transactivating factor binding. Resolution of these Specific Aims will provide insight into the biological importance of oxidative DNA modifications occurring in the context of hypoxic signal transduction. Our hypothesis, if valid, also could uncover a new paradigm for ROS regulation of gene expression wherein oxidative modifications in critical DNA sequences affects interactions between transcription factors and their responsive elements.
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