280184 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.
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division - See also TI: Comprehensive Quality by Design in Pharmaceutical Development and Manufacture