Effect of laminar shear stress on gene regulation, protein synthesis, and protein secretion by cultured human endothelial cells
Diamond, Scott Lee
Matthews, Kathleen S.
Doctor of Philosophy
To test the hypothesis that wall shear stress generated by blood flow may regulate endothelial cell expression of blood clot dissolving proteins or vasoactive proteins, an in vitro perfusion system was used to expose human umbilical vein endothelial cell monolayers to well defined, laminar fluid flow. Protein production studies utilized immunoassays, while semi-quantitative studies of messenger RNA levels in a small numbers of cells required a reverse transcription/polymerase chain reaction technique. Secretion by endothelial cells of the two main regulators of the fibrinolytic (ie blood clot dissolving) system, tissue plasminogen activator (tPA) and plasminogen activator inhibitor, type 1 (PAI-1) were not affected by exposure to venous levels of shear stress (4 dynes/cm$\sp2$). However, at arterial shear stresses of 15 and 25 dynes/cm$\sp2$, the tPA secretion rate was 2.1 and 3.0 times greater, respectively, than the basal tPA secretion rate. PAI-1 secretion was unstimulated by shear stress over the entire physiological range. The tPA mRNA level was many fold higher ($>$10 fold) in endothelial cells sheared for 24 hours than in stationary controls. The mRNA level of the common house-keeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was found to be the same in control and sheared cells. The fact that GAPDH was unregulated indicates selectively in cellular response to shear stress in addition to validating the PCR technique. Endothelin (ET), a 21-amino acid peptide secreted by endothelial cells, has vasoconstrictor and mitogenic activity for vascular smooth muscle cells. Fluid shear stress of 25 dynes/cm$\sp2$ caused a rapid and sustained drop in endothelin production after only 2 hours exposure to shear stress. Endothelin secretion was not affected by venous shear stress of 4 dynes/cm$\sp2$. The mRNA level for endothelin in cells exposed to shear stress was almost undetectable, indicating that the drop in protein secretion is due to a drop in transcription of the message RNA for endothelin. The mRNA level of basic fibroblast growth factor (bFGF) was found to be the same in cells sheared for 24 hours as in controls. Enhancement of the fibrinolytic potential of endothelial cells in response to hemodynamic forces involves transcriptional events and could explain the deposition of fibrin in low shear zones near arterial bifurcations. Flow-regulated ET expression may explain the inverse correlation of fluid shear stress with: (1) the localization of atherosclerotic lesions near vessel bifurcations and; (2) the severity of intimal hyperplasia in surgical vein bypass grafts and vessel anastomotic sites.