Focal adhesion sites were observed in cultured endothelial cells by tandem scanning confocal microscopy and digitized image analysis, techniques that provide real-time images of adhesion site area and topography in living cells. Image subtraction demonstrated that in the presence of unidirectional steady laminar flow (shear stress IrI = 10 dyn/cm2) a substantial fraction of focal adhesion sites remodeled in the direction of flow. In contrast, focal adhesions of control (no flow) cells remodeled without preferred direction. In confluent monolayers subjected to shear stresses of 10 dyn/cm2, cells began to realign in the direction of flow after 7-9 h. This was accompanied by redistribution of intracellular stress fibers, alignment of individual focal adhesion sites, and the coalescence of smaller sites resulting in fewer, but larger, focal adhesions per cell. Cell adhesion, repeatedly calculated in the same cells as a function ofthe areas of focal contact and the separation distances between membrane and substratum, varied by < 10% during both short (30 min), or prolonged (< 24 h), periods of exposure to flow. Consistent with these measurements, the gains and losses of focal adhesion area as each site remodeled were approximately equivalent. When the glass substratum was coated with gelatin, rates of remodeling were inhibited by 47% during flow (r = 10 dyn/ cm2). These studies: (a) reveal the dynamic nature of focal adhesions; (b) demonstrate that these sites at the ablumenal endothelial membrane are both acutely and chronically responsive to frictional shear stress forces applied to the opposite (lumenal) cell surface; and (c) suggest that components of the focal adhesion complex may be mechanically responsive elements coupled to the cytoskeleton. (J. Clin. Invest. 1994.

Blood flow interactions with the vascular endothelium represent a specialized example of mechanical regulation of cell function that has important physiological and pathological cardiovascular consequences. The endothelial monolayer in vivo acts as a signal transduction interface for forces associated with flowing blood (hemodynamic forces) in the acute regulation of artery tone and chronic structural remodeling of arteries, including the pathology of atherosclerosis. Mechanisms related to spatial relationships at the cell surfaces and throughout the cell that influence flow-mediated endothelial mechanotransduction are discussed. In particular, flow-mediated ion channel activation and cytoskeletal dynamics are considered in relation to topographic analyses of the luminal and abluminal surfaces of living endothelial cells.

Real time measurements of cell-substratum adhesion in endothelial cells were obtained by tandem scanning confocal microscopy of sites of focal contact (focal adhesions) at the abluminal cell surface. Focal contact sites were sharply defined (low radiance levels) in the living cell such that the images could be enhanced, digitized, and isolated from other cellular detail. Sites of focal contact are the principal determinant of cell-substratum adhesion. Measurements of (a) the focal contact area and (b) the closeness of contact (inverse radiance) were used to nominally define the adhesion of a single cell or field of cells, and to record spontaneous and induced changes of cell adhesion in real time.The topography of focal contacts was estimated by calculating separation distances from radiance values using a calibration technique based on interference ring optics. While slightly closer contact was noted between the cell membrane and substratum at or near the center of each focal contact, separation distances throughout the adhesion regions were always < 50 nm.Subtraction ofconsecutive images revealed continuous spontaneous remodeling of individual focal adhesions in unperturbed cells during periods of < 1 min. Despite extensive remodeling of focal contact sites, however, cell adhesion calculated for an entire cell over extended periods varied by < 10%. When cytoskeletal stability was impaired by exposure to cytochalasin or when cells were exposed to proteolytic enzyme, endothelial adhesion declined rapidly. Such changes were recorded at the level of single cells, groups of cells, and at single focal adhesions. In both unperturbed and manipulated cells, the dynamics of remodeling and cell adhesion characteristics varied greatly between individual sites within the same cell; disappearance of existing sites and appearance of new ones often occurred within minutes while adjacent sites underwent minimal remodelling.Tandem scanning confocal microscopy image analysis of living cells in real time provides repetitive spatial, temporal, and quantitative information about cell adhesion. Such an approach should allow more precise quantitative analyses to be A preliminary report ofparts ofthis work has appeared in abstract form (1991. FASEB (Fed. Am. Soc. Exp. Biol.) J. 5:1603A).

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