996-14 Hypoxia-induced Alterations in Gene Expression in Cardiac and Endothelial Cells

Altered gene expression patterns accompany episodes of hypoxia both with and without reoxygenation. We are using cultured cardiac myocyte and endothelial cell models to identify genes that are differentially regulated byoxygen levels. This study employs a PCR-based RNA fingerprinting technique, differential display, that allows the sampling of the effects of hypoxia on gene expression over multiple time points with and without reoxygenation. Differential display was performed using total RNA isolated from cardiac AT-l cells or endothelial cells. Cells were exposed to two main experimental protocols: anoxia for six hours with or without a four hour reoxygenation, or 24 hours of hypoxia (1% oxygen). Oxygen tension in the culture media was monitored using a silver-platinum electrode. Total RNA was isolated and used as the template for reverse transcriptase reactions and subsequent differential display PCR reactions. Three candidate differentially expressed genes have been identified in the initial set of experiments in AT-1 cells. These results suggest that this fingerprinting methodology will enable the identification of oxygen-regulated genes and will increase our understanding of the molecular basis of cellular responses to hypoxia.