Macrorheology and adaptive microrheology of endothelial cells subjected to fluid shear stress

Abstract

Vascular endothelial cells (ECs) respond to temporal and spatial characteristics of hemodynamic forces by alterations in their adhesiveness to leukocytes, secretion of vasodilators, and permeability to blood-borne constituents. These physiological and pathophysiological changes are tied to adaptation of cell mechanics and mechanotransduction, the process by which cells convert forces to intracellular biochemical signals. The exact time scales of these mechanical adaptations, however, remain unknown. We used particle-tracking microrheology to study adaptive changes in intracellular mechanics in response to a step change in fluid shear stress, which simulates both rapid temporal and steady features of hemodynamic forces. Results indicate that ECs become significantly more compliant as early as 30 s after a step change in shear stress from 0 to 10 dyn/cm(2) followed by recovery of viscoelastic parameters within 4 min of shearing, even though shear stress was maintained. After ECs were sheared for 5 min, return of shear stress to 0 dyn/cm(2) in a stepwise manner did not result in any further rheological adaptation. Average vesicle displacements were used to determine time-dependent cell deformation and macrorheological parameters by fitting creep function to a linear viscoelastic liquid model. Characteristic time and magnitude for shear-induced deformation were 3 s and 50 nm, respectively. We conclude that ECs rapidly adapt their mechanical properties in response to shear stress, and we provide the first macrorheological parameters for time-dependent deformations of ECs to a physiological forcing function. Such studies provide insight into pathologies such as atherosclerosis, which may find their origins in EC mechanics.

Fig. 1

High-resolution imaging and tracking of endogenous vesicles in endothelial cells. A: bovine aortic endothelial cell (EC) imaged under high-resolution differential interference contrast microscopy. Endogenous vesicles (highlighted by white boxes) were tracked in a focal plane 2–4 μm from the base of the cell. Arrow indicates direction of fluid flow. Two-dimensional trajectories of endogenous vesicles calculated before step flow (preshear) was imposed (B), 30 s (C) and 4 min (D) after the onset of step flow (during shear), and 30 s after step flow was turned off (postshear; E).

Fig. 2

EC mechanics in response to step change in shear stress from 0 to 10 dyn/cm². A: ensemble-averaged mean square displacement (MSD) plotted against increasing time lags exhibits power-law scaling. α < 1 indicates subdiffusive behavior of tracked endogenous vesicles. B: creep compliance curves of ECs for all experimental conditions. A twofold increase in creep compliance compared with preshear values was observed at very early time points (30 s) after onset of step shear stress. No change in creep compliance was observed when compared with initial static conditions, after shearing cells for 4 min, and after shear stress was removed in a stepwise manner. Data are means ± SE (n = 6). *P < 0.05 for 30 s step shear vs. static control preshear. Error bars for 4 min shear and 5 min postshear are not shown for clarity.

Fig. 3

Shear-induced adaptive microrheology. Frequency-dependent elastic (A) and viscous (B) moduli decreased rapidly after exposure to step shear of 0 to 10 dyn/cm². After 4 min of shearing was completed, values returned to preshear levels. No change in rheology was seen after shear stress was removed. 1Pa = 10 dyn/cm². Data are means ± SE; n = 6. *Significant differences between preshear and 30 s shear rheological moduli (P < 0.05) at all frequencies (A) and for indicated frequencies (B). Error bars for 4 min shear and 5 min postshear are not shown for clarity. C: frequency-dependent viscoelastic phase angles indicate that ECs cross over from the viscous to the elastic regime at frequency of 1 s⁻¹. Imposition of step shear stress increases the crossover frequency to 3 s⁻¹.

Fig. 4

Macrorheology of endothelial cells. Heterogeneous global cell deformation measured from average vesicle displacements due to step change in shear stress. A: ECs exhibited time-dependent deformation when step shear stress was turned on (0 to 10 dyn/cm²). #Significant displacements compared with 0 displacement using 95% confidence intervals. B: negligible cell deformation was seen upon removal of shear stress in a stepwise manner (10 to 0 dyn/cm²). Data are means ± SD (n = 6). C: curve-fitting average creep response to a phenomenological model shows that ECs behave like viscoelastic liquids with a relaxation time of 3 s. Inset: Voigt-Maxwell model used to describe the creep response of ECs.