Purpose: We investigated the role of Gal 1-3αGal, aGPIb, and sialoadhesin carbohydrates (CHOs) in the interaction of human PMNs with wild-type (WT), GalTKO, and 10GE porcine aortic endothelial cells (EC) under physiologic microvascular shear stress and temperature, using human coronary endothelial cells (HCE) as a negative control.
Methods: Microfluidic channels were seeded with WT, GalTKO, 10GE, and human coronary ECs. ECs were stimulated with 10 n/µl rh-TNFα for 3 hours (to induce IRI-mediated E-selectin upregulation) and perfused with fresh human whole blood stained with calcein to detect non-RBC cells. 1F1 was perfused over EC while a-GP1b was added to blood for 30 mins, both before the perfusion over 60 minutes under microvascular flow conditions (5 dynes/cm² shear stress) at 37°C. A total of 181 images were captured during each channel perfusion, and stacked images were then processed using ImageJ software. Applying signal threshold, size of the objects, and circularity, neutrophils were distinguished and analyzed for PMN rolling and movement (mean speed [pixels/frame] and track duration [Frames]). HCE was used as a negative control for PMN interactions. Results from multiple experimental conditions were analyzed using one-way ANOVA with post-hoc tests.
Results: Absence of Gal 1-3αGal significantly decreased PMN rolling and its duration compared to WT. Sialoadhesin blockade (mAb 1F1) and GPIb inhibition further reduced the duration of PMN tracking and increased the mean speed of migration on EC (P < 0.005 for all experimental groups except WT+GP1b vs Gal+GP1b). PMN rolling closely correlated with diffuse PLT adhesion, primarily occurring at EC-EC junctions among all cell lines.
Conclusion: PMN rolling on and migration over porcine EC under IRI conditions is reduced by GalTKO gene modification and further attenuated by sialoadhesin blockade and GPIb inhibition. These findings support that inhibition of carbohydrate-driven pro-inflammatory interactions mediated by PMNs and other blood elements reduces inflammation and improves xenograft outcomes. PMN localized rolling and migration in association with adherent PLTs implicates coagulation pathway activation in the residual sequestration observed with ECs treated with 1F1 and GPIb blockade. These results suggest that genetic and pharmacologic strategies targeting thrombodysregulation may further mitigate inflammation in xenografts. We will evaluate the effect of these agents on multi-GE ECs next