The aim of this research is to investigate the role of local blood flow as an initializing and localizing factor for thrombosis and vessel wall thickening. This research will use cultured human endothelial cells exposed to various regimes of physiological fluid shear stress which include steady, pulsatile, reversing, and reattaching flows. Three major areas of investigation are proposed which emphasize (i) signal transduction, (ii) thrombosis and (iii) hyperplasia. The first area involves the use of gel shift assays to measure the intracellular concentration of the transcriptional regulator c-fos/c-jun complex. Shear-activation of protein kinase C will be evaluated with these studies to help relate rapid signaling events with longer term changes that occur in endothelial cells. This approach will help identify the genetic outcomes which are predicted by the growing data base of secondary messengers involved in transduction of shear stress signals. The second area is the study of the endothelial coagulant state during exposure to fluid shear stresses. These studies will use epi-fluorescence video imaging to measure dynamic changes in thrombomodulin and protein S concentrations on endothelial cells surfaces exposed to hemodynamic forces. Additionally, measurements of tPA and PAI-1 expression and expression of the adhesion mediators ICAM, GMP-140, and PECAM will add to this evaluation of the surface character of endothelial cells exposed to various flows. In light of our previous demonstration of large changes in endothelial expression of tPA by steady arterial shear stresses, these studies are of particular importance to the pharmacology of anticoagulation and fibrinolytic therapy where hemodynamic forces are an important parameter. The third area of study will expand our demonstrated technique of measuring changes in endothelial cell mRNA levels using polymerase chain reaction technology. Measurement of endothelial mRNA levels for PDGF A and B chain, TGFbeta, and basic FGF in endothelial cells exposed to shear stress has great importance to issues of intimal hyperplasia, a pathology known to be localized by low and reversing flows. It is hypothesized that reversing flows and flow reattachment zones may be particularly strong inducers of growth factor expression while steady flows may not induce growth factors and may even suppress their expression. Understanding the mechanisms by which hemodynamic forces regulate endothelial cell gene expression may provide targets for intervention of vascular disorders which occur with aging, bypass grafts, or angioplasty. Also, the success of seeding vessels with genetically engineered endothelial cells for human gene therapy must take into account endothelial function in hemodynamic environments.
| Yamaguchi, M; Diamond, S; Watanabe, H et al. (1993) Heparin and dibutyryl cAMP modulate gene expression in stimulated human saphenous vein smooth muscle cells. In Vitro Cell Dev Biol Anim 29A:867-72 |
