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Research Papers

The Response of Human Aortic Endothelial Cells in a Stenotic Hemodynamic Environment: Effect of Duration, Magnitude, and Spatial Gradients in Wall Shear Stress

[+] Author and Article Information
Leonie Rouleau, Joanna Rossi

Department of Chemical Engineering, McGill University, 3610 University, Montreal, QC Canada, H3A 2B2; Montreal Heart Institute, 5000, rue Bélanger, Montreal, QC, Canada, H1T 1C8

Richard L. Leask1

Department of Chemical Engineering, McGill University, 3610 University, Montreal, QC Canada, H3A 2B2; Montreal Heart Institute, 5000, rue Bélanger, Montreal, QC, Canada, H1T 1C8richard.leask@mcgill.ca

1

Corresponding author.

J Biomech Eng 132(7), 071015 (Jun 04, 2010) (11 pages) doi:10.1115/1.4001217 History: Received November 10, 2009; Revised January 21, 2010; Posted February 09, 2010; Published June 04, 2010; Online June 04, 2010

Inflammation plays a key role in the development and stability of coronary plaques. Endothelial cells alter their expression in response to wall shear stress (WSS). Straight/tubular and asymmetric stenosis models were designed to study the localized expression of atheroprone molecules and inflammatory markers due to the presence of the spatial wall shear stress gradients created by an eccentric plaque. The effects of steady wall shear stress duration (0–24 h) and magnitude (4.518dynes/cm2) were analyzed in human abdominal aortic endothelial cells through quantitative real-time polymerase chain reaction (PCR) and immunofluorescence analysis in straight/tubular models. Regional expression was assessed by immunofluorescence and confocal microscopy in stenosis models. Under steady fully developed flow, endothelial cells exhibited a sustained increase in levels of atheroprotective genes with WSS duration and magnitude. The local response in the stenosis model showed that expression of endothelial nitric oxide synthase and Kruppel-like factor 2 is magnitude rather than gradient dependent. A WSS magnitude dependent transient increase in translocation of transcription factor nuclear factor κB was observed. Intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin exhibited a sustained increase in protein expression with time. The mRNA levels of these molecules were transiently upregulated and this was followed by a decrease in expression to levels lower than static controls. Regionally, increased inflammatory marker expression was observed in regions of WSS gradients both proximal and distal to the stenosis when compared with the uniform flow regions, whereas the atheroprotective markers were expressed to a greater extent in regions of elevated WSS magnitudes. The results from the straight/tubular model cannot explain the regional variation seen in the stenosis models. This may help explain the localization of inflammatory cells at the shoulders of plaques in vivo.

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Copyright © 2010 by American Society of Mechanical Engineers
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Figures

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Figure 1

Straight/tubular and asymmetric stenosis in vitro models (a). Perfusion flow loop diagram (b). Normalized wall shear stress on the stenosis side for different Reynolds numbers (c).

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Figure 2

Wall shear stress magnitude and duration effect on eNOS expression assessed by confocal microscopy (a) and quantitative real-time PCR ((b) and (c)). (b) Effect of time on expression ( ∗P<0.05,  ∗∗P<0.01, and  ∗∗∗P<0.001 with respect to the 0 h levels; ǂǂP<0.01 and ǂǂǂP<0.001 with respect to the 6 h levels; ¤P<0.05, ¤¤P<0.01, and ¤¤¤P<0.001 with respect to the 12 h levels) and (c) of wall shear stress magnitudes after 24 h of perfusion ( ∗∗∗P<0.001 compared with static controls and ǂǂP<0.01 compared with the 4.5 dynes/cm2 condition) (n=3, mean±SEM).

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Figure 3

Wall shear stress magnitude and duration effect on KLF2 expression assessed by confocal microscopy (a) and quantitative real-time PCR ((b) and (c)). (b) Time had a significant impact on expression, with a maximum at 12 h after perfusion ( ∗P<0.05,  ∗∗P<0.01, and  ∗∗∗P<0.001 with respect to the 0 h levels, and ǂP<0.05 with respect to the 6 h levels. (c) After 24 h of perfusion, differences were noted at higher wall shear stress magnitudes ( ∗∗∗P<0.001 compared with static controls, ǂǂP<0.01 compared with the 4.5 dynes/cm2 condition, and ¤P<0.05 compared with the 9 dynes/cm2) (n=3, mean±SEM).

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Figure 4

Qualitative regional expression of eNOS and KLF2 for a steady flow resulting in a mean entrance wall shear stress of 18 dynes/cm2 and after 24 h of perfusion (a). The intensities in the inlet, acceleration, and deceleration regions were quantified (b) ( ∗∗P<0.01 with respect to the inlet, ǂǂP<0.01, and ǂǂǂP<0.001 with respect to the acceleration region) (n=5, mean±SEM).

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Figure 5

Effect of time and wall shear stress magnitude on NF-κB expression as assessed using confocal microscopy

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Figure 6

Effect of wall shear stress magnitude and duration on ICAM-1 expression as assessed by confocal microscopy (a) and quantitative real-time PCR ((b) and (c)). (b) Time dependent variations ( ∗P<0.05,  ∗∗P<0.01, and  ∗∗∗P<0.001 with respect to the 0 h levels, and ǂP<0.05 and ǂǂP<0.01 with respect to the 6 h levels) and (c) long term magnitude effect ( ∗P<0.05 and  ∗∗P<0.01 with respect to the static controls) (n=3, mean±SEM).

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Figure 7

Effect of wall shear stress magnitude and duration on VCAM-1 expression as assessed by confocal microscopy (a) and quantitative real-time PCR ((b) and (c)). (b) Time dependent variations ( ∗P<0.05 and  ∗∗∗P<0.001 with respect to the 0 h levels, and ǂP<0.05 and ǂǂP<0.01 with respect to the 6 h levels) and (c) long term magnitude effect ( ∗P<0.05 with respect to the static controls) (n=3, mean±SEM).

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Figure 8

Effect of wall shear stress magnitude and duration of exposure on E-selectin expression assessed by confocal microscopy (a) and quantitative real-time PCR ((b) and (c)). (b) Time dependent variations ( ∗∗P<0.01 and  ∗∗∗P<0.001 with respect to the 0 h levels, ǂP<0.05 with respect to the 6 h levels, and ¤¤P<0.01 with respect to the 12 h levels) and (c) long term magnitude effect (ǂP<0.05 with respect to the 4.5 dynes/cm2) (n=3, mean±SEM).

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Figure 9

Regional inflammatory marker expression. (a) Expression of NF-κB after 4 h of a perfusion at 4.5 dynes/cm2. (b) ICAM-1, VCAM-1, and E-selectin expression in the inlet, acceleration, and deceleration regions after 24 h of perfusion at 18 dynes/cm2. (c) Ratio relative to the inlet of the number of cells that expressed the cell adhesion molecule on the total number of cells within each field (n=5, mean±SEM,  ∗P<0.05).

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