Electrical impedance of cultured endothelium under fluid flow.

The morphological and functional status of organs, tissues, and cells can be assessed by evaluating their electrical impedance. Fluid shear stress regulates the morphology and function of endothelial cells in vitro. In this study, an electrical biosensor was used to investigate the dynamics of flow-induced alterations in endothelial cell morphology in vitro. Quantitative, real-time changes in the electrical impedance of endothelial monolayers were evaluated using a modified electric cell-substrate impedance sensing (ECIS) system. This ECIS/Flow system allows for a continuous evaluation of the cell monolayer impedance upon exposure to physiological fluid shear stress forces. Bovine aortic endothelial cells grown to confluence on thin film gold electrodes were exposed to fluid shear stress of 10 dynes/cm2 for a single uninterrupted 5 h time period or for two consecutive 30 min time periods separated by a 2 h no-flow interval. At the onset of flow, the monolayer electrical resistance sharply increased reaching 1.2 to 1.3 times the baseline in about 15 min followed by a sustained decrease in resistance to 1.1 and 0.85 times the baseline value after 30 min and 5 h of flow, respectively. The capacitance decreased at the onset of flow, started to recover after 15 min and after slightly overshooting the baseline values, decreased again with a prolonged exposure to flow. Measured changes in capacitance were in the order of 5% of the baseline values. The observed changes in endothelial impedance were reversible upon flow removal with a recovery rate that varied with the duration of the preceding flow exposure. These results demonstrate that the impedance of endothelial monolayers changes dynamically with flow indicating morphological and/or functional changes in the cell layer. This in vitro model system (ECIS/Flow) may be a very useful tool in the quantitative evaluation of flow-induced dynamic changes in cultured cells when used in conjunction with biological or biochemical assays able to determine the nature and mechanisms of the observed changes.

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.

Spatial variations in endothelial barrier function in disturbed flows in vitro.

Hindered barrier function has been implicated in the initiation and progression of atherosclerosis, a disease of focal nature associated with altered hemodynamics. In this study, endothelial permeability to macromolecules and endothelial electrical resistance were investigated in vitro in monolayers exposed to disturbed flow fields that model spatial variations in fluid shear stress found at arterial bifurcations. After 5 h of flow, areas of high shear stress gradients showed a 5.5-fold increase in transendothelial transport of dextran (molecular weight 70,000) compared with no-flow controls. Areas of undisturbed fully developed flow, within the same monolayer, showed a 2.9-fold increase. Monolayer electrical resistance decreased with exposure to flow. The resistance measured during flow and the rate of change in monolayer resistance after removal of flow were lowest in the vicinity of flow reattachment (highest shear stress gradients). These results demonstrate that endothelial barrier function and permeability to macromolecules are regulated by spatial variations in shear stress forces in vitro.

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.

Mechanical forces alter growth factor release by pleural mesothelial cells.

The mesothelial cells that form the visceral pleura of the lung are subjected to physical forces such as stretch due to lung expansion and fluid shear stress due to the sliding motion of the lung against the chest wall. In this study, the effect of mechanical forces on the production of growth factors by mesothelial cells was investigated. Rat visceral pleura mesothelial (RVPM) cells were exposed to fluid shear stress by perfusing a column of cell-covered beads. RVPM cells grown on a silicone elastomer were subjected to cyclic strain by applying an oscillating vacuum to the bottom of the wells using the Flexercell apparatus. Fluid shear stress (5.2-15.7 dyn/cm2) stimulated the release of endothelin-1 (ET-1) by RVPM cells two- to fivefold over static cells. ET-1 secretion by RVPM cells was also stimulated approximately twofold by cyclic stretch (20% maximum strain, 30 cycles/min). RVPM cells released significant levels of platelet-derived growth factor (PDGF), but there was no effect of either shear stress or cyclic strain on PDGF release. These results suggest that the production of growth factors by pleural mesothelial cells is regulated in part by physical forces.

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.

Shear stress alters pleural mesothelial cell permeability in culture.

The sliding motion of the lung against the chest wall creates a shear stress in the pleural space, which can be as high as 60 dyn/cm2, depending on the respiration rate. Such shear stresses may affect the mesothelial cells that line the pleural space on the lung (visceral pleura) and chest wall (parietal pleura). When exposed to shear stress (17 dyn/cm2) in a parallel-plate flow chamber for 22 h, rat visceral pleura mesothelial cells were not altered morphologically and did not align in the direction of flow, in contrast to the shape changes observed for bovine aortic endothelial cells. By using mesothelial cells cultured on porous microcarrier beads, we measured the permeability of the cells at different flows in a cell-column chromatography assay. The permeabilities to sodium fluorescein and cyanocobalamin increased from 8.2 +/- 1.0 and 7.8 +/- 0.7 x 10(-5) cm/s to 22.5 +/- 1.2 and 21.8 +/- 3.0 x 10(-5) cm/s, respectively, when the flow was increased from 0.9 to 3.5 ml/min (corresponding to average shear stresses of 4.7-18.4 dyn/cm2). The permeabilities returned to baseline values when the flow was reduced. Cytochalasin D stimulated an increase in permeability that was not augmented by a subsequent increase in shear stress. These results suggest that the barrier function of mesothelial cells is responsive to changes in fluid shear stress.

Adsorption of proteins out of plasma onto glass from a separated flow.

Perturbations in the adsorption of plasma proteins caused by flow separation were studied quantitatively. An instrument was constructed that causes flow to separate over approximately half the width of a standard microscope slide and the pattern of protein deposition in and near the separated flow was observed by staining the slide with black iron oxide. The slide was mounted at the edge of a Couette flow field established between two concentric cylinders, the outer of which was rotating. The slide was located on the stationary, inner cylinder just downstream of a rectangular bar that causes the flow to separate. After exposure to dilute plasma injected upstream of the bar, the slide was removed and stained with oxide suspension. The resulting, visible pattern was scanned through a video camera and analyzed to yield relative values of stain density that could be quantified. The oxide patterns suggest that proteins were deposited onto the slide less rapidly in and just downstream of the separated flow region than farther downstream. At a shear rate of 6.61 s-1, corresponding to a velocity of 1.32 cm s-1 0.2 cm above the point of flow separation, overall amounts of adsorbed proteins increased with exposure time in the range 3-30 min with the exception of a period from 10 to 11 min when all data show a temporary decrease. In calibration experiments, oxide failed to adhere to slides exposed to purified albumin but adhered copiously to slides exposed to purified fibrinogen. These results suggest that the oxide patterns following plasma exposure are attributable primarily to fibrinogen and that the temporary decrease in the separated flow experiments is attributable to the displacement of fibrinogen by a less stainable protein, conjecturally high molecular weight kininogen and factor XII. This study yields quantitative information confirming earlier findings that were less controlled and non-quantitative. It confirms the hypothesis that the sequence of protein deposition from dilute plasma to glass surfaces is delayed in regions of separated flow.

Vascular endothelium responds to fluid shear stress gradients.

In vitro investigations of the responses of vascular endothelium to fluid shear stress have typically been conducted under conditions where the time-mean shear stress is uniform. In contrast, the in vitro experiments reported here have re-created the large gradients in surface fluid shear stress found near arterial branches in vivo; specifically, we have produced a disturbed-flow region that includes both flow separation and reattachment. Near reattachment regions, shear stress is small but its gradient is large. Cells migrate away from this region, predominantly in the downstream direction. Those that remain divide at a rate that is high compared with that of cells subjected to uniform shear. We speculate that large shear stress gradients can induce morphological and functional changes in the endothelium in regions of disturbed flow in vivo and thus may contribute to the formation of atherosclerotic lesions.