Hemodynamics and the focal origin of atherosclerosis: a spatial approach to endothelial structure, gene expression, and function.

Atherosclerosis originates at predictable focal and regional sites that are associated with complex flow disturbances and flow separations in large arteries. The spatial relationships associated with hemodynamic shear stress forces acting on the endothelial monolayer are considered in experiments that model regions susceptible to atherosclerosis (flow disturbance) and resistant to atherosclerosis (undisturbed flow). Flow disturbance in vitro induced differential expression at the single gene level as illustrated for the intercellular communication gene and protein, connexin 43. Transcription profiles of individual endothelial cells isolated from both disturbed and undisturbed flow regions exhibited more expression heterogeneity in disturbed than in undisturbed flow. We propose that within highly heterogeneous populations of endothelial cells located in disturbed flow regions, proatherosclerotic gene expression may occur within the range of expression profiles induced by the local hemodynamics. These may be sites of initiation of focal atherosclerosis. Mechanisms are proposed to account for heterogeneous endothelial responses to shear stress by reference to the decentralized model of endothelial mechanotransduction. Length scales ranging from centimeters to nanometers are useful in describing regional, single cell, and intracellular mechanotransduction mechanisms.

A spatial approach to transcriptional profiling: mechanotransduction and the focal origin of atherosclerosis.

The initiation and progression of focal atherosclerotic lesions has long been known to be associated with regions of disturbed blood flow. Improved precision in experimental models of spatially defined flow has recently been combined with regional and single-cell gene-expression profiling to investigate the relationships linking haemodynamics to vessel-wall pathobiology.

Spatial and temporal regulation of gap junction connexin43 in vascular endothelial cells exposed to controlled disturbed flows in vitro.

Hemodynamic regulation of the endothelial gap junction protein connexin43 (Cx43) was studied in a model of controlled disturbed flows in vitro. Cx43 mRNA, protein expression, and intercellular communication were mapped to spatial variations in fluid forces. Hemodynamic features of atherosclerotic lesion-prone regions of the vasculature (flow separation and recirculation) were created for periods of 5, 16, and 30 h, with laminar shear stresses ranging between 0 and 13.5 dynes/cm2. Within 5 h, endothelial Cx43 mRNA expression was increased in all cells when compared with no-flow controls, with highest levels (up to 6- to 8-fold) expressed in regions of flow recirculation corresponding to high shear stress gradients. At 16 h, Cx43 mRNA expression remained elevated in regions of flow disturbance, whereas in areas of fully developed, undisturbed laminar flow, Cx43 expression returned to control levels. In all flow regions, typical punctate Cx43 immunofluorescence at cell borders was disrupted by 5 h. After 30 h of flow, disruption of gap junctions persisted in cells subjected to flow separation and recirculation, whereas regions of undisturbed flow were substantially restored to normal. These expression differences were reflected in sustained inhibition of intercellular communication (dye transfer) throughout the zone of disturbed flow (84.2 and 68.4% inhibition at 5 and 30 h, respectively); in contrast, communication was fully reestablished by 30 h in cells exposed to undisturbed flow. Up-regulation of Cx43 transcripts, sustained disorganization of Cx43 protein, and impaired communication suggest that shear stress gradients in regions of disturbed flow regulate intercellular communication through the expression and function of Cx43.

Spatial relationships in early signaling events of flow-mediated endothelial mechanotransduction.

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.

Effects of wall shear stress and fluid recirculation on the localization of circulating monocytes in a three-dimensional flow model.

There is a correlation between the location of early atherosclerotic lesions and the hemodynamic characteristics at those sites. Circulating monocytes are key cells in the pathogenesis of atherosclerotic plaques and localize at sites of atherogenesis. The hypothesis that the distribution of monocyte adhesion to the vascular wall is determined in part by hemodynamic factors was addressed by studying monocyte adhesion in an in vitro flow model in the absence of any biological activity in the model wall. Suspensions of U937 cells were perfused (Re = 200) through an axisymmetric silicone flow model with a stenosis followed by a reverse step. The model provided spatially varying wall shear stress, flow separation and reattachment, and a three-dimensional flow pattern. The cell rolling velocity and adhesion rates were determined by analysis of videomicrographs. Wall shear stress was obtained by numerical solution of the equations of fluid motion. Cell adhesion patterns were also studied in the presence of chemotactic peptide gradients. The cell rolling velocity varied linearly with wall shear stress. The adhesion rate tended to decrease with increasing local wall shear stress, but was also affected by the radial component of velocity and the dynamics of the recirculation region and flow reattachment. Adhesion was increased in the vicinity of chemotactic peptide sources downstream of the expansion site. Results with human monocytes were qualitatively similar to the U937 experiments. Differences in the adhesion rates of U937 cells occurring solely as a function of the fluid dynamic properties of the flow field were clearly demonstrated in the absence of any biological activity in the model wall.

Hemodynamic forces and vascular cell communication in arteries.

As the interface between the blood and the rest of the vessel wall, the endothelium is directly affected by hemodynamic shear stress (frictional) forces that locally regulate vascular tone and are implicated in the localization of atherosclerosis. There are many diverse responses of endothelial cells to hemodynamically related mechanical stresses ranging from ion channel activation to gene regulatory events. The processes of force transmission from the blood to the cell, and force transduction within the endothelium to electrophysiologic, biochemical, and transcriptional responses are poorly understood. This article reviews the principal mechanisms currently thought to be involved and outlines the signal pathways from the endothelium to underlying smooth-muscle cells.