Real Time High-Resolution Imaging of Porcine Endothelial Glycocalyx Shedding by Human Serum in an in Vitro Microfluidic Model of Pig-to-Human Xenotransplantation

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Abstract

Background

The glycocalyx, a complex layer of membrane-bound proteoglycans, sialic acid-containing glycoproteins and glycolipids is a key element for the maintenance of homeostasis in blood vessels. Pathophysiological conditions such as ischemia/reperfusion injury and vascular diseases lead to damage of the glycocalyx impairing endothelial functions. Increased capillary permeability, adhesion of immune cells, thrombosis and vascular inflammation are typical consequences of glycocalyx shedding. In xenotransplantation, donor graft endothelium interfaces with the recipient blood, resulting in glycocalyx degradation and consequent switch of the graft endothelium to a pro-coagulant and pro-inflammatory phenotype. In the present study a microfluidic perfusion system was used to assess the shedding of heparan sulfate (HS), the main component of the endothelial glycocalyx, after perfusion of porcine aortic endothelial cells (PAEC) with normal human serum (NHS).

Methods

Wild type PAEC were seeded in microchannels of 550 μm diameter with round cross-sections and left to reach confluence overnight under static conditions. A peristaltic pump was then connected and a flow of 600 μl/min, corresponding to a shear stress of 10 dyn/cm2, applied for 48 hours. Flow-adapted PAEC as well as PAEC grown under static conditions were incubated with fluorescent wheat germ agglutinin (WGA), which binds to N-acetyl-D-glucosamine (GlcNAc) and N-acetyl neuraminic acid (NeuAc) components of the glycocalyx, or fluorescence-labeled anti-HS antibody. Subsequently, PAEC microchannels were perfused for 2 hours with 10% NHS or cell culture medium. During NHS perfusion, real-time fluorescence imaging was performed using high-speed confocal microscopy.

Results

PAEC exposed to physiological flow for 48 hours considerably upregulated the expression of HS as compared to static culture conditions (43% ± 3% increase of HS-staining) whereas GlcNAc / NeuAc staining was reduced (61% ± 10% reduction). Perfusion of PAEC-microchannels with 1:10 diluted NHS at 10 dyn/cm2 led to a significant, time-dependent reduction of both HS (38% ± 6% reduction after 10 minutes) and GlcNAc / NeuAc staining (43% ± 9% reduction). Longer perfusion times led to increasing detachment of PAEC, which could be visualized by confocal microscopy.

Conclusion

Our preliminary data confirm that flow and shear stress are important factors affecting the composition of the endothelial glycocalyx in terms of expression of HS, GlcNAc, and NeuAc. The reduced staining of these sugar molecules during NHS perfusion supports the idea that in a pig-to-human xenotransplantation setting the porcine endothelial glycocalyx is shed after contact with human serum. Experiments with several different types of transgenic porcine endothelial cells are currently ongoing to provide insight into the role of the different plasma cascade systems in the shedding of the glycocalyx.

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