Maria Papadaki

Effects of Fluid Shear Stress on Gene Regulation of Vascular Cells

Hemodynamic forces such as fluid shear stress play an active role in many physiological and pathophysiological processes of the cardiovascular system. Shear stress resulting from blood flow and transmural plasma flux alters the function of vascular cell (primarily endothelial cells), leading to both rapid and slower adaptive tissue responses. Transmission of the shear stress signal throughout the vascular cell involves a complex interplay between cytoskeletal and biochemical elements and results in changes in structure, metabolism, and gene expression. Herein we review current knowledge on flow‐induced mechanotransduction in the vascular endothelial cell and the molecular mechanisms believed responsible for shear‐induced endothelial and smooth muscle cell gene regulation with an emphasis on signal transduction.

Differential Regulation of Protease Activated Receptor-1 and Tissue Plasminogen Activator Expression by Shear Stress in Vascular Smooth Muscle Cells

Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

Effects of shear stress on the growth kinetics of human aortic smooth muscle cells in vitro.

After cardiovascular intervention, smooth muscle cells (SMC) are directly exposed to blood flow and thus their behavior might be affected by fluid hemodynamic forces. The aim of this study was to determine the effect of fluid shear stress on the growth rate of SMC. Human aortic smooth muscle cells (hASMC) were seeded on fibronectin‐coated glass slides and were exposed to different levels of shear stress using parallel plate flow chambers. After 24 h, cell numbers in the stationary and sheared cultures were measured by a Coulter counter. Results demonstrated that increasing shear stress significantly reduces the proliferation rate of hASMC (P < 0.05). Comparable lactate dehydrogenase levels in the media of stationary and flow cultures provided evidence that the reduction of cell number was not due to cell injury. Proliferating cell nuclear antigen (PCNA) immunofluorescence studies indicated that the cell cultures were not growth arrested 24 h after exposure to shear stress, and that the differences in PCNA staining between stationary control and flow cultures were comparable to the cell counts.

Effect of Flow on Gene Regulation in Smooth Muscle Cells and Macromolecular Transport Across Endothelial Cell Monolayers

Endothelial cells line all of the vessels of the circulatory system, providing a non-thrombogenic conduit for blood flow; they regulate many complex functions in the vasculature, such as coagulation, fibrinolysis, platelet aggregation, vessel tone and growth, and leukocyte traffic; and they form the principal barrier to transport of substances between the blood and the surrounding tissue space. The permeability of endothelial cell changes with environmental stimuli; shear stress, in particular, applied either in vivo, or in vitro, induces changes in protein expression and secretion of vasoactive factors by endothelial cells. The ability to study the effects of shear on the macromolecular permeability of the cerebral vasculature is particularly important, since in no other place is the barrier function of the endothelium more important than in the brain. The endothelial cells of this organ have developed special barrier properties that keep the cerebral system from experiencing any drastic change in composition; together with glial cells, they form the blood brain barrier (BBB). We have studied the effect of flow on bovine BBB using flow chambers and tissue culture systems.

Quantitative measurement of shear-stress effects on endothelial cells.

Over the past 20 yr, great strides have been made toward understanding the role of fluid hemodynamic forces in the vascular wall homeostasis at the molecular level. In vivo studies have demonstrated that blood vessels are adaptive to physiological changes in blood flow, with vessels tending to enlarge in areas of high flow and tending to reduce their lumen diameter in low-flow regimes (1,2). Furthermore, altered hemodynamics have been implicated in the pathogenesis of many cardiovascular disorders, such as thrombosis, atherosclerosis, and vessel wall injury. Vascular endothelial cells serve as a barrier between perfused tissues and flowing blood, and they are believed to act as a sensor of the local biomechanical environment. The hemodynamic forces generated in the vasculature include frictional wall shear-stress, cyclic strain, and hydrostatic pressure (3). For the purpose of this chapter, we will focus on methods for examining the link between fluid wall shear-stress and endothelial cell function. Advances in our understanding of the effects of shear-stress on endothelial cell function require that cell populations be exposed to controlled, well-defined, flow-induced shear-stress environments. Since in vivo studies have the inherent problem that they cannot quantitatively define the shearing forces or separate their effects from the other components of the hemodynamic system, in vitro flow studies using cultured cells are extensively used.