Monocyte recruitment to endothelial cells in response to oscillatory shear stress

Abstract

Leukocyte recruitment to endothelial cells is a critical event in inflammatory responses. The spatial, temporal gradients of shear stress, topology, and outcome of cellular interactions that underlie these responses have so far been inferred from static imaging of tissue sections or studies of statically cultured cells. In this report, we developed micro-electromechanical systems (MEMS) sensors, comparable to a single endothelial cell (EC) in size, to link real-time shear stress with monocyte/EC binding kinetics in a complex flow environment, simulating the moving and unsteady separation point at the arterial bifurcation with high spatial and temporal resolution. In response to oscillatory shear stress (tau) at +/- 2.6 dyn/cm2 at a time-averaged shear stress (tau(ave))=0 and 0.5 Hz, individual monocytes displayed unique to-and-fro trajectories undergoing rolling, binding, and dissociation with other monocyte, followed by solid adhesion on EC. Our study quantified individual monocyte/EC binding kinetics in terms of displacement and velocity profiles. Oscillatory flow induces up-regulation of adhesion molecules and cytokines to mediate monocyte/EC interactions over a dynamic range of shear stress +/- 2.6 dyn/cm2 (P=0.50, n=10).

Figure 1

Principle of thermal shear stress sensors. a) Threelayer model. For heat transfer “q_c” refers to convective heat transfer. b) The thermal element resides within a velocity boundary layer. The rate of heat loss from a heated resistive element to the fluid flow is dependent on the velocity profile in the boundary layer. A linear relation is obtained as V²/R∝ τ^1/3 based on heat transfer principles.

Figure 2

Fabrication steps of the microthermal shear stress sensor. a) Thermal oxidation and Si₃N₄ deposition; b) polysilicon deposition and patterning; c) thermal oxidation and Si₃N₄ deposition; d) opening etching holes; e) removing the sacrificial poly-Si layer; f) blocking the etching holes; g) poly-Si deposition, ion implantation with boron and patterning; h) Al deposition and patterning for electrodes and SiO₂ deposition for waterproof.

Figure 3

a) Test channel with the flush-mounted microther- mal shear stress sensor array to the upper wall of the pulsatile flow channel. Confluent BAEC monolayers were seeded on the bottom. b) A photograph of individual shear stress sensors illustrate the polysilicon as an sensing element. The diaphragm (see Fig. 2f) was bent down by the external atmospheric pressure, giving rise to an optical interference patterns referred as Newton rings.

Figure 4

Cell tracking velocimetry: a) Intensity contours of monocytes obtained from digitized video images; b) representative cross-correlation for determining the monocyte displacement.

Figure 5

Real-time shear stress measurement by the microthermal shear stress sensors. a) Pulsatile flow; b) oscillatory flow to simulate the outer wall of arterial bifurcations at the reattachment point.

Figure 6

a) Captured images of monocyte attachment under oscillatory flow. Note that monocyte 2 established a firm attachment to EC at 4.53 s while monocyte 1 continued to undergo to-and-fro locomotion. b) Displacement tracings of monocytes 1 and 2 corresponding to the captured images using cell tracking velocimetry.

Figure 7

Captured images of cell–cell interactions under oscillatory shear stress. a) Monocyte 1 was undergoing tethering while monocyte 2 was in to-and-for locomotion; b) monocytes 1 and 2 established binding; c) monocytes 1 and 2 started to separate; d) monocytes 1 and 2 were apart; e) monocyte 2 resumed to-and-fro locomotion while monocyte 1 remained attached to EC. f) The dark blue profile reflects the real-time oscillatory shear stress. The trajectories of monocytes 1 and 2 from the captured images (a–e) are superimposed with the dotted blue trajectory of the nonviable monocyte (control). g) Velocity profiles of monocytes 1 and 2 are compared with that of the nonviable monocyte (control) in relation to the velocity of oscillating flow field.

Figure 8

Range of shear stress over which 5 distinct cell–cell interactions occurred: 1) monocyte-monocyte binding or 2) separation, 3) monocyte tethering or 4) adhesion to EC monolayers, and 5) monocyte detachment from EC monolayers. The difference among the mean shear stress (open circles with error bars) was statistically insignificant (n= 10 for each event, P= 0.50) for the individual events.

Figure 9

a) Fluorescence signal vs. cycle number for Pselectin and ICAM-1 normalized with GAPDH. b) Bar graphs show relative mRNA expression for P-selectin and ICAM-1 in response to pulsatile vs. oscillatory flow conditions. Values are expressed as mean ± se. *P<0.05 P-selectin vs. control; **P<0.05 ICAM-1 vs. control.

Figure 10

Images of monocyte solid adhesion to EC were captured in response to a) pulsatile flow and b) oscillatory flow. c) Real- time RT-PCR: mRNA expression of MCP-1 and GAPDH in response to control (static condition), oscillatory, pulsatile flow at 4 h; d) Relative changes in MCP-1 mRNA expression. Values are expressed as mean ± se.*P < 0.05 P-selectin vs. control.