(379c) Endothelial Cells Migrate Upstream and Align Against the Shear Stress Field Created By Impinging Flow

Endothelial Cells
Migrate Upstream and Align Against the Shear Stress Field Created by Impinging
Flow

Spatial gradients in wall shear stress have emerged
as an important factor for cardiovascular development and disease. High shear
and vortical flow were required for normal heart development in zebrafish,
where occluded flow results in an abnormal third chamber.¹ Large
shear stress gradients likewise exist in the heart tube of the developing quail
embryo, where high localized shear stresses in the outflow tract coincide with
the future locations of aortic and pulmonary valve formation.^2,3 ECs
lining the aortic valve leaflets, where high shear stress gradients occur,
align perpendicular to flow and may reflect a response to the high shear stress
present in the left ventricle.⁴ Importantly, the mechanisms
underlying ECs response to spatial gradients in wall shear stress remain essentially
unknown. In order to study the influence of shear stress gradients on
endothelial cell response, we built an impinging flow cell that exposes
endothelial cells to a gradient of shear stress whose slope can be tuned by
changing the flow rate. We investigated the response of endothelial cells to shear
stress gradients that ranged from 0 to a peak shear stress of between 9-220
dynes/cm².

Materials and
Methods

We
built an impinging flow cell that exposes endothelial cells to a gradient of
shear stress. The impinging flow cell device is used to produce gradients in
shear stress while imaging the cellular migration and reorientation over a 21-hour
period. A photo of the device is shown in Fig. 1a. The device was fabricated
out of acrylic in order to enable live-cell imaging by allowing light to pass
through the transparent device and illuminate the cells while tracking their
response to impinging flow. The device is mounted on an x-y-z translation stage
suspended at a fixed height of 1 mm over a cell culture dish seeded with ECs.
The impinging flow exit orifice is oriented normal to the EC surface and
dispenses fluid (cell culture media) onto the EC monolayer seeded on a cell
culture dish The orifice radius is 0.35 mm. A pump is
used to recirculate cell culture media onto the cell surface.

(a)                                                           (b)                                                                 (c)             

Figure 1. Device design and
modeling of impinging flow cell. (a) Impinging flow cell device is used to
produce gradients in shear stress while imaging the cellular migration and
reorientation. The fluid flow enters the impinging flow chamber shown at the
left and impinges onto the cell culture dish normal to the cell surface. Fluid
leaves the dish at the same rate through a tube located at the edge of the dish
via recirculation by a peristaltic pump  (b) Velocity flow field from finite element
analysis performed in Comsol shown in vertical cross section for flow through
an impinging flow cell. Flow rate of 1.5 mL/min for an orifice with a 0.35 mm
radius located 1 mm above the cell surface is modeled. Fluid velocity magnitude
is represented with color, fluid direction with arrows and flow streamlines
with black lines. The conditions show a low flow region (depicted in blue) at r = 0 and far from the orifice. The
inlet flow rate corresponds to jet Reynolds numbers of 67 and demonstrates that
the flow is in the laminar regime. (c) Wall shear stress on the cell culture dish
is plotted as a function of radius from the impinging flow. Shear stress ranges
from zero (at r = 0) directly under
the exit of the impinging flow cell orifice (the stagnation point) and
increasing symmetrically and radially outward to a maximum at a distance of
0.350 mm. The region of maximum in shear stress is radially symmetric and
corresponds to the radius of the impinging exit orifice. Shear stress values
from the analysis are plotted for flow rates of 1.5 mL/min and 4.5 mL/min.

Results

At high confluency, endothelial cells migrate against the direction of fluid flow for
all flow rates tested and concentrate in the region of maximum wall shear
stress, while sub-confluent endothelial cells migrate with the flow direction. In addition, endothelial cells align
parallel to the flow at low wall shear stresses but orient perpendicular to the
flow direction and in a densely packed arrangement above a critical threshold
in local wall shear stress as shown in Fig. 2b, within region 2.

Figure 2. Orientation analysis of HMVEC's exposed to flow in the impinging flow
cell for 21 hours. The peak location in shear stress is shown as a yellow
dashed ring. Cells have been colored to clearly observe their long axis with
respect to the fluid flow direction. Blue indicates EC alignment
parallel with flow, red is perpendicular to flow, and green is
intermediate orientation. Blank lines indicate concentric rings with a spacing
of 0.185 mm to show axisymmetric cell response.  (a) Image of HMVECs exposed to 1.5mL/min flow
producing peak WSS of 9 dynes/cm². Cells beyond the shear stress
maximum are primarily oriented with flow (regions 3, 4 and 5), while cells in
the stagnation region (region 1) are predominantly unaligned. Gray region with
'*' represents an area where valid EC images were not obtained because of poor
contrast. (b) HMVECs exposed to
impinging flow rate of 4.5 mL/min with maximum shear stress of 72 dynes/cm².
Cells in the vicinity of the wall shear stress maximum (region 2) are
predominantly oriented perpendicular to flow (red), while cells beyond this
region are oriented parallel (blue) to the flow. (c) HMVECs exposed to 9 mL/min
flow with peak WSS of 220 dynes/cm². Cells in regions 1, 2, and much
of 3 were ablated due to high shear stress. Cells in regions 3-5 oriented
perpendicular to flow. Scale bars equal 100 µm.

Our observations suggest that endothelial cells are
exquisitely sensitive to both the magnitude and spatial gradients in wall shear
stress. The perpendicular reorientation of ECs at high shear stress values
agrees with in vivo data where ECs
are observed to orient perpendicular to flow at the valve
leaflet where shear stress is high. Their resulting migration and polarization
may play presently unrecognized roles both during cardiovascular development
and disease, particularly in regions of complex flow. The mechanisms underlying
these responses may additionally play important roles during development, where
high shear and large gradients exist transiently during heart development.

1.     
Hove, J. R. et al. Intracardiac fluid forces are an
essential epigenetic factor for embryonic cardiogenesis. Nature (2003).

2.     
Peterson, L. M. et al. 4D shear stress maps of the
developing heart using Doppler optical coherence tomography. Biomed Opt Express 3, 3022-3032 (2012).

3.     
Garita, B. et al. Blood flow dynamics of one
cardiac cycle and relationship to mechanotransduction and trabeculation during
heart looping. Am J Physiol Heart Circ
Physiol 300 (2011).

4.      Deck, J. D. Endothelial cell orientation on aortic
valve leaflets. Cardiovasc Res 20, 760-767 (1986).