Aucoin, Michelle Marie Butler (1992-04). In vitro studies of coronary venular endothelial cells during growth and stress. Doctoral Dissertation.
Thesis
An endothelial cell line obtained from bovine coronary venules (CVEC) was used to study the in vitro effects of pathophysiologic modulators such as second messengers, receptor-mediated agonists, and stress. One series of experiments evaluated the regulation of gap junctional intercellular communication (GJIC) mediated by connexin43 (Cx43), the putative connexin species present in endothelium. Fluorescence recovery after photobleaching was employed to monitor GJIC between endothelial cells that had been treated with either cyclic adenosine monophosphate (cAMP), tetradecanoylphorbol acetate (TPA), growth factors, hypoxia, or heat stress. The results suggested that endothelial cell intercellular communication, and presumably Cx43, can be regulated by treatment with cAMP, TPA, adenosine, hypoxia, and heat. It is possible that these effects are mediated through phosphorylation events on Cx43. A second set of experiments focused on the induction of heat shock protein 70 (HSP70) by reagents that promote endothelial cell division, and by heat shock itself. For these studies, total RNA and protein were extracted from CVEC following exposure to serum, basic fibroblast growth factor, hypoxia, or heat. As determined by Northern blotting, HSP70 was strongly induced by heat, and less strongly induced by hypoxia and serum. These studies indicate a possible connection between the induction of HSP70 and initiation of DNA synthesis. A final group of experiments was designed to expand the previous study by monitoring HSP70 and intracellular glutathione levels in CVEC exposed to oxidative injury. For these analyses, CVEC were treated with a combination of xanthine oxidase and hypoxanthine, followed by evaluation of HSP70 mRNA and protein, and measurement of relative glutathione (GSH) concentration. GSH was assessed as relative fluorescence intensity by using a fluorescent dye specific for GSH, monochlorobimane. The results of these assays indicated that after oxidative injury, there was a profound reduction in intracellular GSH and a significant increase in HSP70 mRNA and protein. Therefore, CVEC serve as a good in vitro model for evaluating the effects of pathophysiologic regulators and stress on several parameters, including GJIC, HSP70 protein and mRNA dynamics, and intracellular glutathione concentrations.
