A New In Vitro Model to Evaluate Differential Responses of Endothelial Cells to Simulated Arterial Shear Stress Waveforms

[+] Author and Article Information
Brett R. Blackman, Guillermo Garcı́a-Cardeña, Michael A. Gimbrone

Center for Excellence in Vascular Biology, and Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115

J Biomech Eng 124(4), 397-407 (Jul 30, 2002) (11 pages) doi:10.1115/1.1486468 History: Received December 01, 2001; Revised April 01, 2002; Online July 30, 2002
Copyright © 2002 by ASME
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Shear stress induced regulation of eNOS protein and mRNA expression in HUVEC. (a) Western blot for eNOS protein following 6 and 24 hours of LSS and ART flow. Static samples represent no flow time-matched controls for each experimental condition. Samples were equally loaded with 50μg of protein, and blots are representative of 3 independent experiments per condition (Densitometry analysis normalized to static experiment: LSS 6h 0.83±0.40 vs. ART6h 1.41±0.09 arbitrary units, p=0.23; LSS 24h 4.03±0.60 vs. ART 24h 1.35±0.46 arbitrary units, p<0.05). (b) Relative mRNA levels of eNOS following 6 and 24 hours of LSS and ART were measured by real-time PCR (Taqman). Measurements were made in duplicate from three independent experiments. Results were normalized using β2 microglobulin, a gene not regulated by these stimuli. Bars represent mean ±SE (LSS: 6h, n=3, 24h, n=3; ART: 6h, n=3, 24h, n=4). *p<0.05.
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Schematic of the Dynamic Flow System and plot of motor-generated arterial waveforms. (a) The DFS is a cone and plate device that mounts on to a microscope stage and permits direct visualization of the cells adhered to the plate surface. The rotation of the cone is controlled via a timing belt connection to the programmable stepper motor. (b) An arterial waveform, developed from phase-encoding MRI of a previously characterized human abdominal aorta 22, was programmed into DFS and used to assess the biological response of ECs for all experiments in this study. Measurements from the DFS are represented for multiple cardiac cycles. Characteristics of this waveform include, a peak shear stress of 20 dyn/cm2 during systole, flow reversal during diastole to a maximum of −3 dyn/cm2, an equivalent time-average of 7.5 dyn/cm2 (depicted by the solid horizontal line), and a pulsation frequency of 1Hz. (c) A blood flow waveform acquired by ultrasound from the brachial artery of a normal human subject (left panel) was programmed into and measured from the DFS (right panel). Plots represent multiple cardiac cycles of the waveform, and the programmed waveform is modeled at a frequency of 1Hz. For the measured waveforms, (b) and (c, right), values of the angular velocity (rev/sec) were measured directly from the motor, and values of shear stress (dyn/cm2) were computed at the plate surface.
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Shear stress induced morphological changes in endo–thelial cells. (a) Images of HUVEC were acquired at the start of the experiment (t=0) and following 24 hours of LSS and ART. The arrow points in the direction of net flow. Images are representative of 3 independent experiments per condition. (b) Time-lapse video and image analysis techniques (see Methods) were used to evaluate changes in EC aspect ratio as a function of time (every 6 minutes) following 24 hours of exposure to LSS or ART. Curves were generated from the mean aspect ratio for each time point (LSS, n=3; ART, n=3). Standard error bars are plotted every 2 hours between 12 and 24 hours to illustrate non-significant differences among the LSS (black) and ART (gray) curves (p>0.05).
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Migration patterns and characteristics of HUVEC exposed to steady and arterial flow. (a) Migratory patterns of four individual ECs (per plot) were tracked for 24 hours under static (no flow), LSS, and ART conditions. Traces of the cell’s spatial movements are representative of the general migratory behaviors observed for that defined condition and were assessed from 3 independent experiments per condition. All distances were computed relative to a given cell’s original centroid (at t=0), which is defined here as the origin. The arrow (right side) points in the direction of net forward flow for LSS and ART. (b) Intrinsic migratory characteristics, RMS cell speed (left) and directional persistence (right), were computed for 24 hours of static, LSS, and ART conditions from the equation describing the mean square displacements. Calculations were made from a total of 30 cells pooled from three independent experiments per condition. Bars represent the mean ±SE. **p<0.005,***p<0.0001, n.s.=not significant.
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Remodeling of cytoskeletal-associated structures following 24 hours of arterial and steady shear stress. Confocal immunofluorescence micrographs show the distribution of (a) actin and plectin in the apical (supranuclear) compartment, and (b) vinculin, in basal compartment, in HUVEC after exposure to static (left), LSS (middle), and ART (right) for 24 hours. Actin stress fibers (top) are stained green and the nuclei counterstained with SYTOX (red). Images are representative of four independent experiments per condition.




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