Endothelial cell responses to atheroprone flow are driven by two separate flow components: low time-average shear stress and fluid flow reversal

Abstract

To simulate the effects of shear stress in regions of the vasculature prone to developing atherosclerosis, we subjected human umbilical vein endothelial cells to reversing shear stress to mimic the hemodynamic conditions at the wall of the carotid sinus, a site of complex, reversing blood flow and commonly observed atherosclerosis. We compared the effects of reversing shear stress (time-average: 1 dyn/cm(2), maximum: +11 dyn/cm(2), minimum: -11 dyn/cm(2), 1 Hz), arterial steady shear stress (15 dyn/cm(2)), and low steady shear stress (1 dyn/cm(2)) on gene expression, cell proliferation, and monocyte adhesiveness. Microarray analysis revealed that most differentially expressed genes were similarly regulated by all three shear stress regimens compared with static culture. Comparisons of the three shear stress regimens to each other identified 138 genes regulated by low average shear stress and 22 genes regulated by fluid reversal. Low average shear stress induced increased cell proliferation compared with high shear stress. Only reversing shear stress exposure induced monocyte adhesion. The adhesion of monocytes was partially inhibited by the incubation of endothelial cells with ICAM-1 blocking antibody. Increased heparan sulfate proteoglycan expression was observed on the surface of cells exposed to reversing shear stress. Heparinase III treatment significantly reduced monocyte adhesion. Our results suggest that low steady shear stress is the major impetus for differential gene expression and cell proliferation, whereas reversing flow regulates monocyte adhesion.

Fig. 1.

Reversing flow (RF) system validation. A: fluid oscillations in the RF system are driven by a 1-ml syringe attached to a linear motor. The average shear stress of −1 dyn/cm² was generated by an alternating dual-syringe pump in which one syringe fills the CO₂-perfused media reservoir while the other syringe draws media from the media reservoir and across the flow chamber. B: the RF system produces wall shear stresses (solid line), which closely approximate computer simulations of the wall shear stress at the carotid sinus (shaded line). Wall shear stresses produced by the reversing flow system were obtained from high-speed video microscopy used to track polystyrene beads within the flow chamber.

Fig. 2.

Variations in fluid flow patterns cause altered endothelial cell (EC) morphology. A: phase-contrast images of ECs exposed to 15 dyn/cm² of shear stress for 24 h produce elongated cells that are aligned with the direction of shear stress. B–D: ECs exposed to 1 dyn/cm² (B) and ECs exposed to RF (C) are not aligned and are similar to the morphology of cells grown under static conditions (D). Arrows indicate the direction of flow. Scale bar = 100 μm.

Fig. 3.

Venn diagrams providing insight into the mechanisms regulating differential gene expression. A: differentially expressed genes in a comparison of high shear stress (HSS), low shear stress (LSS), or RF to static conditions. Approximately 57% of the differentially expressed genes are found in the central white area of the diagram, indicating that these genes were differentially expressed by all three flow conditions compared with static conditions. B: differentially expressed genes in a comparison of the three flow conditions to each other. The 138 genes found in the magenta region of the diagram are regulated by LSS. The 22 genes found in the yellow region of the diagram are regulated by RF. The five genes found in the blue-green region of the diagram are regulated by HSS.

Fig. 4.

Hierarchical clusters of functional groups. A: functional groups upregulated by LSS. B: functional groups upregulated by RF. Flow conditions and genes associated with functional groups identified by DAVID were clustered according to expression patterns. The color range shown is indicative of the normalized natural log-transformed signal values for each gene according to the normalization procedure described in methods.

Fig. 5.

Cell proliferation is inhibited by HSS compared with LSS. ECs were exposed to shear stress in the presence of 10 μM bromodeoxyuridine (BrdU). Cells were fixed and stained for BrdU incorporation using an anti-BrdU antibody and nuclear DNA using Hoechst-33258. Exposure to all three forms of shear stress caused a reduction in BrdU incorporation compared with static culture (*P < 0.001) Exposure to HSS caused the greatest reduction in BrdU incorporation compared with the other two shear stress conditions (†P < 0.05). There were no significant differences between steady LSS and reversing shear stress. The percentage of nuclei that stained positively for BrdU was determined by counting nuclei in 5 experiments with 8 frames/experiment.

Fig. 6.

THP-1 cells have greater adhesion to ECs exposed to reversing flow. A: THP-1 cells were perfused across ECs pretreated for 24 h with the indicated shear stress conditions. Flow was stopped for 30 s, allowing the THP-1 cells to interact, and, after a 5-min rinse, adherent cells were counted in 10 frames in each of 5–6 separate experiments. *P < 0.001 between RF and all three other conditions. B: ICAM-1 surface expression was significantly upregulated in ECs exposed to 15 dyn/cm² of shear stress or RF for 24 h compared with ECs exposed to 1 dyn/cm² of shear stress or static fluid (*P < 0.05). Data represent flow cytometry data of ICAM-1 fluorescent intensities normalized to the average ICAM-1 fluorescent intensity of ECs exposed to static conditions. C: VCAM-1 surface expression was not significantly changed under any shear stress condition. D: functional blocking of ICAM-1 significantly reduced monocyte adhesion in cells pretreated with RF compared with ECs blocked with either mouse control IgG or VCAM-1 antibody (*P < 0.05). E: surface heparan sulfate proteoglycan expression was elevated in cells exposed to reversing shear stress compared with the three other conditions. Scale bar = 50 μm. F: treatment of cells exposed to reversing shear stress with heparinase III significantly attenuated monocyte adhesion (*P < 0.05).