The effect of the environment on plasminogen activators and inhibitors

The aims of this project were to develop a reliable assay for the investigation of small changes in mRNA expression of tissue plasminogen activator (tPA) and Plasminogen Activator Inhibitor (PAI-1) in endothelial cell as a response to changes in environmental conditions such as shear stress and cyclic stretch.;An apparatus was constructed to stimulate shear stress in a vessel by flowing a continuous stream of culture medium across endothelial cell monolayers. This has been used to demonstrate endothelial cell elongation and orientation into the direction of flow and is capable of reproducing shear stresses between 0.2Pa and 4.7Pa.;An RNase Protection assay has been developed for the detection of small changes in mRNA expression of tissue plasminogen activators and inhibitors from Human Umbilical Vein Endothelial Cells. It is possible to assay for tPA, PAI-1 and the internal control house-keeping gene Glyceraldehyde-3-Phosphate Dehydrogenase (G3PDH) in one sample tube. This eliminates the problems of tube to tube variations experienced when using other quantitative methods of mRNA detection. The use of known amounts of control RNAs in the assay would provide a means of quantification of these changes. This assay has been used successfully to show alterations in tPA and PAI-1 mRNA expression in response to shear stress, cyclic stretch and oxidative damage.;There is considerable interest in the nature of the protein factors and DNA sequences that regulate Plasminogen Activator and Plasminogen Activator Inhibitor gene expression, not only because this information may provide new clues as to how the genes are regulated but also because it may identify drugs which chronically lower PAI-1 (or increase tPA). These drugs may represent new thrombolytic agents. Accurate analysis of tPA and PAI-1 mRNA expression would be necessary for the investigation into the involvement of various different factors in the transcriptional control of tPA and PAI-1.