Endothelial Cell Migratory Response to Simple and Spatial Gradients in Wall Shear Stress

AIChE: Annual Meeting, Salt Lake City, UT. November 8-13, 2015
Session: Bio-Fluid Dynamics
Talk Title: Endothelial cell migratory response to simple and spatial gradients in wall shear stress
Keywords: Endothelial cell, cell migration, wall shear stress, fluid flow
Vinay N. Surya, Maggie A. Ostrowski, Eleftheria Michalaki, Alexander R. Dunn, Gerald G. Fuller

Endothelial cells (ECs) line the inner surface of blood and lymphatic vessels and are highly sensitive to fluid flow as part of their physiological function. EC orientation with flow, migration and vessel development are profoundly influenced by hydrodynamic stresses, affecting the ability to develop capillaries through angiogenesis and formation of valves through valvulogenesis. How ECs sense fluid flow is a central and unanswered question in cardiovascular research with important implications in cardiovascular disease and tumor metastasis.

Previously, we developed a high-throughput flow chamber which applies low Reynolds number impinging flow to a monolayer of ECs. This flow profile is physiologically relevant to vessel bifurcations, where the spatial gradient in wall shear stress may mechanically trigger angiogenic and valvulogenic cues (Figure 1). Using this device, we previously found that human lymphatic microvascular endothelial cells migrate against the flow direction, towards regions of higher shear stress. The gradient in wall shear stress also triggers the same physiological response as was observed in in vivo mouse embryonic studies, including increased expression of the two transcription factors involved in valve formation,  prospero-homeobox protein 1 and forkhead box protein C2, indicating a plausible connection between mechanically triggered cell migration and vessel structure formation.

We have used the high-throughput flow chamber to probe how endothelial cells from different regions of the human vasculature respond to spatial gradients in wall shear stress. We have examined how coronary artery, aortic, umbilical vein, and blood and lymphatic microvascular ECs respond to both simple shear and gradients in shear stress in an effort to determine similarities and differences in the migratory responses (Figure 2). We have found that while the lymphatic microvascular ECs uniquely migrate against the flow, large and small blood vessel ECs display very different migratory responses to the same shear stress gradient. Large vessel blood ECs align and move with the flow direction, while blood microvascular ECs adopt an azimuthal orientation to the flow direction with no preference for migration direction.

Ongoing work aims to develop microfluidic channels with physiologically relevant geometries that mimic the complex flows found in vivo. We are currently pursuing constricting geometries such as those found at the sites of valve formation in the venous and lymphatic systems, and U-shaped geometries, which model the low and high curvature geometries characteristic of Dean flows in the descending aorta. In both cases, we hypothesize that the cellular response to fluid flow will depend on the absolute magnitude and spatial gradient in wall shear stress, a topic which to our knowledge is relatively unexplored despite its physiological significance.

Figure 1. A conceptual schematic of the relevant vessel fluid flows experienced at a vessel bifurcation, which can be broken down into two simple flow profiles: Poiseuille and impinging flow. A monolayer of endothelial cells (red) are seeded on a gelatin-coated coverslip, submerged in cell culture medium (light blue) and experience fluid flow of the cell culture medium. Brightfield and fluorescent microscopy are used to capture migratory response over time.

Figure 2. (A) The impinging flow device uses a submerged nozzle to flow media (light blue) over ECs (red) seeded on a glass substrate (dark blue). Six identical jets provide ease of use with standard 6-well tissue culture plates. (B) Finite element simulation (COMSOL) shows the cross-sectional fluid velocity profile. Fluid streamline arrows (red) indicate that the cells experience a spatially varying, axisymmetric flow. (C) An impinging flow chamber produces a spatially conserved, highly tunable wall shear stress profile (controlled by pump flow rate) to produce physiologically relevant gradients in wall shear stress. Wall shear stress (blue) increases to a maximum at ~400 microns from the stagnation point, then decays to zero. (D) Endothelial cells from different vessel regions display widely varying responses to the same spatial gradient in wall shear stress after 20 hours of exposure to impinging flow. The total distance a cell travels while exposed to impinging flow is plotted against its radial displacement, i.e. radial movement towards the jet center (negative value) or movement away from the jet center (positive value). N = 100 cells per cell type. Of the cell types tested, only lymphatic endothelial cells collectively migrate upstream, against the direction of fluid flow.