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.

With the development of calcium-sensitive fluorescent dyes and videomicroscopic imaging, several investigators have located the changes in intracellular calcium in the cytoplasm, in the perinuclear region, and possibly in the nucleus. However, the presence of calcium in the nucleus is often difficult to ascertain because the fluorescence derived from the perinuclear area interferes with that of the nucleus. We have used confocal microscopy together with two calcium-sensitive dyes [acetoxymethyl esters of fluo 3 (fluo 3-AM) and rhod 2 (rhod 2-AM)] to analyze the cytosolic and nuclear calcium distribution in vascular smooth muscle and endothelial cells studied at rest and after stimulation with receptor-dependent (angiotensin, vasopressin) and receptor-independent (KCl) stimuli. With fluo 3-AM, the baseline fluorescence was located in the cytoplasm but was slightly higher in the nucleus. With all stimuli, the fluorescence intensity increased in both compartments but remained more pronounced within the nucleus. Yet, after calibration, the cytosolic calcium concentration was greater than that of the nucleus at rest and was equally high after stimulation, suggesting different properties of fluo 3 in the cytosol and in the nucleus. With rhod 2-AM, baseline fluorescence was low in the nucleus and high in the cytosol. Cell stimulation caused an initial increase in cytosolic calcium with no change in the nucleus followed by a rise in both compartments. Thus the stimulation of vascular cells is associated with marked increases in cytosolic and nuclear calcium. Fluo 3-AM seems to be a better indicator of nuclear calcium than rhod 2-AM. The increases in nuclear calcium induced by angiotensin II and vasopressin may contribute to their cell proliferative effect.

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