Analysis of the effect of disturbed flow on monocytic adhesion to endothelial cells
Introduction
Adhesion of circulating monocytes to vascular endothelial cells (ECs) is an initial step in the sequence of events leading to atherosclerosis (Ross, 1993). The early atherosclerotic lesions preferentially localize at branches and curvatures of the arterial system, where the local flow is often disturbed (e.g., flow separation, recirculation, flow reattachment, and nonuniform shear stress distribution) (Ku et al., 1985), suggesting that hemodynamic factors play a role in the initiation and progression of the disease. There is considerable evidence that spatial variations of shear stress in the disturbed flow region exert significant influences on ECs (DePaola et al., 1992; Truskey et al., 1995; Tardy et al., 1997; Chiu et al., 1998; Gabriels and Paul, 1998; DePaola et al., 1999; Davies et al., 1999; Nagel et al., 1999; Davies, 2000; Phelps and DePaola, 2000; Hsu et al., 2001). In vivo, the localization of monocyte binding may be affected by the local fluid dynamics of the arterial flow (Margolin et al., 1995). The flow disturbance may cause an increased mass transfer, thereby enhancing the interactions between circulating monocytes and vascular ECs that ultimately contribute to the observed pathology of the arterial wall (Barber et al., 1998).
The effects of disturbed flow on interactions between blood cells and surfaces have been studied in vitro (Karino and Goldsmith, 1979; Pritchard et al., 1995; Barber et al., 1998; Hinds et al., 2001; Skilbeck et al., 2001). Investigating the adhesion of human platelets to collagen-coated surfaces in, and downstream of, an annular vortex distal to a tubular sudden expansion, Karino and Goldsmith (1979) demonstrated a peak platelet adhesion within the vortex, while the adhesion was minimal at the reattachment point. In addition, they found that the presence of erythrocytes caused a marked increase in adhesion of platelets in the vortex. Determining the rolling velocity and adhesive rate of monocytes on a chemotactic peptide-coated silicone surface in the recirculation flow distal to a expansion, Pritchard et al. (1995) demonstrated an increase in adhesion downstream of the expansion site. Barber et al. (1998) reported that the arrest of monocytes on ECs occurs preferentially in the vicinity of the reattachment point in recirculation flow. Hinds et al. (2001) found that flow patterns and biological activities of the surface are the major factors responsible for leukocyte adhesion, with pulsatile flow causing a lesser cell adhesion to E-selectin-coated surface than steady flow. Recently, Skilbeck et al. (2001) investigated the adhesive behavior of neutrophils to P-selectin-coated surface under disturbed flow by microscopic observation and computational simulation.
Despite the large number of studies on the effects of disturbed flow on cell adhesion to biological or synthetic surfaces, the detailed mechanisms underlying the effects of such flow conditions on regional monocyte attachment to ECs remain unclear. The distributions of cell adhesion to ECs under disturbed flow are believed to rely on the local collision frequencies between the flowing cells and the endothelial surface (Munn et al., 1994). The hemodynamic factors that may affect the collision frequencies include local shear stress distribution, cell concentration, residence time near the endothelial surface, and velocity components of flowing cells perpendicular to the wall (Karino and Goldsmith, 1979; Munn et al., 1994). It is also likely that disturbed flow may alter the adhesive properties of ECs by modulating the surface expression of adhesive proteins (Walpola et al., 1995). In order to understand the adhesion behavior of monocytes in the complex flow environment that exists in arterial branches and bends, it is necessary to develop a method to characterize the flow dynamics and to quantitatively determine the factors contributing to the monocyte adhesion under disturbed flow.
In the present study, we applied the micron-resolution particle image velocimetry (μPIV) technique to analyze the relevant hemodynamic factors that contribute to the monocyte adhesion to ECs under disturbed flow, e.g., the shear stress distribution, local cell concentration near the wall, and residence time, using our developed vertical-step flow (VSF) chamber (Chiu et al., 1998). Computational simulations of the flow fields and cell trajectories were performed with conditions identical to those used for the experiments to allow proper comparison. The distribution of monocyte adhesion to ECs under disturbed flow was investigated in the VSF with the μPIV system. The long-term effects of disturbed flow on the adhesive properties of ECs were studied by examining the expressions of various adhesive proteins on ECs exposed to disturbed flow for 24 h. To the best of our knowledge, this study may be the first report of its kind in utilizing the sophisticated μPIV technique to quantitatively determine relevant hemodynamic factors contributing to monocyte adhesion to ECs under disturbed flow and in identifying the effect of prolonged disturbed flow on the adhesive properties of ECs. The aims of the present research are to elucidate the effects of disturbed flow on monocyte adhesion and the mechanism of monocyte recruitments under such complex flow environments.
Section snippets
Endothelial cell culture
ECs were isolated from fresh human umbilical cords by using the collagenase perfusion technique (Gimbrone, 1976). The cell pellet was re-suspended in medium M199 (Gibco, Grand Island, New York) supplemented with 20% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). ECs were grown in Petri dishes for three days and then seeded onto glass slides (75 mm by 38 mm, Corning, New York) pre-coated with fibronection (Sigma). Secondary cultures were used for the experiments within two
Characterization of flow fields in the VSF
The variations of reattachment lengths in the VSF with Reynolds numbers (ranging from 3 to 180) were determined by the μPIV technique (Fig. 3A). The results computed at various Reynolds numbers are quantitatively in agreement with our present experimental data, as well as the experimental data of Armaly et al. (1983), in which a large-scale backward-facing step flow was studied with identical experimental conditions using laser Doppler anemometry (LDA). The near-wall velocity components and
Discussion
This is the first report that utilizes the newly developed μPIV technique to determine the contribution of hemodynamic factors to monocytic adhesion to ECs under disturbed flow and analyzes the long-term effects of disturbed flow on the adhesive properties of ECs. The μPIV studies on the motions of flowing THP-1 cells under disturbed flow have demonstrated the existence of a higher near-wall cell concentration and a longer residence time in the regions near the step and the reattachment point.
Acknowledgements
This work was supported in part by grants ME-090-PP-I3 and ME-091-PP-I3 from the National Health Research Institutes, Taiwan, ROC. The authors are indebted to Dr. Ned H.C. Hwang for helpful advice and discussion, and to Mr. Chien Yuan Chen for computational work.
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