The development of atherosclerosis is thought to be initiated by a dysfunctional state of the vascular endothelium. The proposal that mechanical forces play a role in the localization of this disease has led researchers to develop in vitro models to assess their effects on cultured endothelial cells. The arterial endothelium is exposed simultaneously to circumferential hoop stretch and wall shear stress, yet previous investigations have focused on the isolated effects of either cyclic stretch or shear stress. The influence of physiological levels of combined shear stress and hoop stretch on the morphology and F-actin organization of bovine aortic endothelial cells was investigated. Cells subjected for 24 hours to shear stresses higher than 2 dyne/cm2 or to hoop stretch greater than 2% elongated significantly compared with unstressed controls and oriented along the direction of flow and perpendicular to the direction of stretch. Exposure to more than 4% stretch significantly enhanced the responses to shear stress. Both shear stress and hoop stretch induced formation of stress fibers that were aligned with the cells' long axes. Simultaneous exposure to both stimuli appeared to enhance stress fiber size and alignment. These results indicate that shear stress and hoop stretch synergistically induce morphological changes in endothelial cells, which suggests that circumferential strain might modulate sensitivity of endothelial cells towards shear stress.

The proposal of the role of mechanical forces as a localizing factor of atherosclerosis has led many researchers to investigate their effects on vascular endothelial cells. Most previous efforts have concentrated on either the fluid shear stress, which results from the flow of blood, or the circumferential "hoop" stretch, which results from the expansion of the artery during the cardiac cycle. In fact, arterial endothelial cells are subjected to both fluid shear stress and cyclic hoop stretch in vivo. Therefore, a more complete investigation of mechanical phenomena on endothelial cell behavior should include both kinds of mechanical stimuli. This study was undertaken to design an experimental apparatus that could subject cultured vascular endothelial cells to simultaneous physiologic levels of both shear stress and cyclic hoop stretch. The experimental apparatus consists of four cylindrical elastic tubes so that the following conditions may be studied: (a) static conditions: (b) shear stress only; (c) hoop stretch only; and (d) shear stress and hoop stretch. In order to establish the functional capabilities of the apparatus, bovine pulmonary artery endothelial cells were cultured in the tubes, and their morphology and f-actin structure were observed with confocal microscopy. The cells remained healthy and attached to the walls throughout the 24 hr experiment. Preliminary results indicated that the alignment of endothelial cells subjected to shear stress was significantly enhanced by the addition of hoop strain.

Cultured vascular endothelial cells undergo significant morphological changes when subjected to sustained fluid shear stress. The cells elongate and align in the direction of applied flow. Accompanying this shape change is a reorganization at the intracellular level. The cytoskeletal actin filaments reorient in the direction of the cells' long axis. How this external stimulus is transmitted to the endothelial cytoskeleton still remains unclear. In this article, we present a theoretical model accounting for the cytoskeletal reorganization under the influence of fluid shear stress. We develop a system of integro-partial-differential equations describing the dynamics of actin filaments, the actin-binding proteins, and the drift of transmembrane proteins due to the fluid shear forces applied on the plasma membrane. Numerical simulations of the equations show that under certain conditions, initially randomly oriented cytoskeletal actin filaments reorient in structures parallel to the externally applied fluid shear forces. Thus, the model suggests a mechanism by which shear forces acting on the cell membrane can be transmitted to the entire cytoskeleton via molecular interactions alone.

Hemodynamic forces have been shown to modulate the expression of endothelin (ET-1) and endothelin-converting enzyme (ECE-1) in endothelial cells. We have subjected E.A. hy 926 cells in culture to steady fluid shear stress with and without flow-induced pressure. The effect of combining these two mechanical forces on the expression of genes in the ET system was studied and the changes were compared to the mRNA levels in static culture. Analysis of total RNA by Northern blot analysis and RNAse protection showed that steady shear stress induced ET-1 gene expression three- to fourfold in this system. The same condition had little to no effect on altering expression of ECE-1 isoforms. A range of flow-induced pressure (80-160 mm Hg) was not able to further augment ET-1 or ECE-1 gene expression. Overall, with the mechanical environment studied, we have been able to detect a predominant contribution of shear stress to altering the ET-1 gene in our system. Furthermore, this induction was independent of an alteration of ECE-1 gene levels, suggesting that these two genes have a different pattern of regulation by the same stimuli in this cell type.

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EINLEITUNG: Die Übertragung von äußeren Kräften auf Endothelzellen führt zu signifikanten biochemischen und morphologischen Veränderungen [1]. Untersuchungen in-vivo und in-vitro demonstrieren im besonderen den Einfluß von fluß-induzierten Scherspannungen auf die Funktion und Erscheinungsform der Zellen [2]. Die Zellen verlängern und reorientieren sich in der Richtung der auf sie einwirkenden Blutströmung. Dieses Phänomen steht u.a. im Verdacht, in der Lokalisierung von arterioskeloritischen Plaques im Blutgefa'ßsystem involviert zu sein [3]. Die Änderung der äußeren morphologischen Form wird begleitet von einer Reorganisation des Zellskelettes im allgemeinen, und des F-Actin-Zytoskelettes im besonderen. Die einzelnen Actin-Filamente des grobmaschigen isotropen Netz-werkes, sowie vor allem die dominanten Actin-Stressfasern (siress fibers) reorientieren sich parallel zur langen Hauptachse der Zellen, und somit parallel zur Flußrichtung [4]. Die ursächlichen Mechanismen dieses Reorientierungsprozesses sind aber noch unklar und umstritten [5] und daher Gegenstand dieser Studie.

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