A novel method for segmenting growth of cells in sheared endothelial culture reveals the secretion of an anti-inflammatory mediator

Background: Effects of shear stress on endothelium are important for the normal physiology of blood vessels and are implicated in the pathogenesis of atherosclerosis. They have been extensively studied in vitro. In one paradigm, endothelial cells are cultured in devices that produce spatially varying shear stress profiles, and the local profile is compared with the properties of cells at the same position. A flaw in this class of experiments is that cells exposed to a certain shear profile in one location may release mediators into the medium that alter the behaviour of cells at another location, experiencing different shear, thus obscuring or corrupting the true relation between shear and cell properties. Methods: Surface coating methods were developed for attaching cells only to some areas of culture-ware and preventing them from spreading into other regions even during prolonged culture. Results: Segmenting the growth of cells had no effect on cell shape, alignment and number per unit area compared to culturing cells in the whole well, but there were differences in tumour-necrosis-factor-alpha (TNF-alpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and monocyte adherence to the monolayer. Conclusions: The results are consistent with the release of a mediator from cells exposed to high-magnitude uniaxial shear stress that has anti-inflammatory effects on activated endothelium; the mediator may be of importance in atherogenesis. Hence the new methods revealed an important property that would not have been observed without growth segmentation, suggesting that they could find more widespread application.