Abstract Body (Do not enter title and authors here): Background: Heparan sulfate (HS) proteoglycans act as mechanosensors on endothelial cells (ECs), regulating EC morphology and function. HS expression is affected by culture under static or dynamic conditions. HS response to inflammatory stimulus under both conditions is not well characterized. This study investigated HS expression on human aortic ECs (HAECs) under static and arterial flow conditions.
Hypothesis: Inflammation modeled by TNFa significantly decreases HS epitope on HAECs under both static and arterial flow conditions.
Aims: To establish the effect of TNFa on HS expression in HAECs.
Methods: Passages 4 through 8 HAECs (ATCC) were cultured to confluence in endothelial growth medium (Vasculife) in Ibitreat µ-Slide 8 well high chambered coverslip slides or Ibitreat µ-Slide VI 0.4 flow channel slides. Cells were treated with TNFa at 100 ng/mL for 3 hours under static conditions or conditioned with 10 dyn/ cm2 of shear stress for 24 hours and then treated with TNFa at the same concentration added to the circulating media for 3 hours. HAECs were fixed in 2% paraformaldehyde/ 0.1% glutaraldehyde for 30 minutes followed by blocking with 2% goat serum for 30 minutes, both at room temperature. Primary antibody to the 10E4 HS epitope was incubated at 4°C overnight (1:100; 10E4 epitope, AMS Biotechnology, USA) followed by incubation in Alexa Fluor 488 goat anti-mouse secondary antibody (1:300, Molecular Probes, USA) for 1 hour at room temperature. HAECs nuclei were stained using 4′,6-diamidino-2-phenylindole and immersed in phosphate buffered saline for confocal imaging using a laser scanning microscope (Zeiss, LSM 880, 20X).
Results: TNFa significantly (p < 0.05) increased HS expression in HAEC monolayers treated under static conditions compared to untreated control and heparinase III treated HAECs (Figure 1A). HAEC monolayers conditioned under arterial shear stress expressed significantly (p < 0.05, ANOVA with Tukey’s post-hoc) higher HS levels compared to baseline static controls; however, flow conditioned HAECs did not show any difference in HS expression under untreated compared to TNFa conditions (Figure 1B).
Conclusion: These data indicate that fluid shear stress may program endothelial cells to significantly alter their HS expression and response to inflammatory stimuli.