(18g) Differential Effect of Shear Stress On Endothelial Cells Response to TNF-Alpha and IL1-Beta Stimulation Is Linked to Cell Shear History

Introduction: The ability to discriminate between
healthy and diseased endothelial cells (ECs) via cell adhesion molecule (CAM)
expression will have significant impact on the design of vascular-targeted
imaging and drug delivery vehicles for treatment of several diseases. However, EC
behavior under chronic inflammation has yet to be fully understood due to the
use of complex in vitro models that
fail to simulate actual physiological inflammation conditions – wherein
the combined effects of chemical and mechanical stimulus are simultaneously
present. Work done by this group showed previously unreported patterns of E-selectin CAM expression on EC exposed to
shear-interleukin-1β (IL-1β) co-stimulation; in this report, human
umbilical vein EC (HUVEC) exposure to combined fluid shear stress and tumor
necrosis factor α (TNFα) is investigated.

Materials and Methods: HUVEC sources were harvested from fresh
cords and subcultured for no more than two passages.
HUVEC monolayers were prepared and subjected to 0 or 12 hrs
of shear stress imparted by fluid flow in a parallel plate flow chamber prior
to simultaneous exposure to shear-cytokine stimulation. This pre-shearing
period preconditions the mechanically-sensitive cells
to a phenotypic state mimicking physiological profiles. IL-1β and TNFα
were used in the subsequent stimulations (individual and combined) to simulate
EC exposure to inflammatory chemicals. To determine HUVEC response, E-selectin (E-sel) – the
hallmark CAM expressed during inflammation – was measured via immunofluorescence
microscopy.

Results and Discussion: We found that like activation with
IL-1β, the response of HUVECs to co-stimulation with fluid shear and
TNFα is dependent on the shear history and the time frame of the following
shear-cytokine activation. The presented data (Fig. 1) suggests that fluid
shear differently affects IL-1β and TNFα signaling –prolonged
exposure to high laminar shear prior (preconditioning) to cytokine-shear
stimulation elicits muted E-sel response for
TNFα activation (clear bars in C versus A) whereas response to IL-1β (filled
bars) is enhanced (Fig. 1B) or muted (Fig. 1C) depending on the length of
shear-cytokine stimulation post-preconditioning.

The observed data for dual-stimulation of
HUVECs with both TNFα/IL-1β, seems to
correlate with E-sel expression for shear-IL-1β
activated monolayers (Fig. 1C). This indicates that the observed E-sel expression trend for shear-IL-1β-only activated
monolayers likely remains relevant even under more complex activation conditions.

Figure 1. E-sel site density of combined and individual TNFα or
IL-1β activation. HUVEC monolayers were treated with 10 ng/mL TNFα (clear), 0.1 ng/mL
IL-1β (filled), or simultaneously with both cytokines at the same
concentrations (grey) for up to 12 hrs under (A)
static-cytokine 
and shear-cytokine (10 dyn/cm2)
activation of (B) na?ve cells and (C) 12 hrs
preconditioned monolayers.

Conclusions: This study explores longer
term activation of HUVECs relevant to time frames encountered during physiological
chronic inflammation and observes previously unreported patterns of
shear-TNFα-induced E-sel expression. However, we
also report that shear-cytokine stimulation of HUVECs is also
cytokine-specific: TNFα activated monolayers elicit an increasingly muted
E-sel expression response relative to IL-1β
activated cells with increasing exposure to fluid
shear stimulus. This indicates that fluid shear plays a significant role in
modulating the pathways for TNFα and IL-1β signaling cascades and
should be further explored to determine additional specific endothelial
responses for potential application in developing targeted therapeutic
treatments.