Friday September 24, 2021 - 23:35 to 00:50
Differences in the glycocalyx of venous and arterial porcine endothelial cells in a 3D microfluidic model
Anastasia Milusev1,2, Melle Holwerda1, Nicoletta Sorvillo1, Robert Rieben1.
1Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland; 2Graduate school for cellular and biomedical sciences, University of Bern, Bern, Switzerland
Introduction: Vascular endothelial cells of the transplanted organ represent the first point of interaction between donor and recipient and are key players in xenorejection. While several modifications of surface-bound regulatory proteins on porcine endothelial cells have already been introduced, little is known about the role of the porcine endothelial glycocalyx in xenorejection. Endothelial cells are covered by a layer of sugars called the glycocalyx, which is crucial for regulating vascular integrity and hemostasis. Shedding of the endothelial glycocalyx is correlated to activation of the endothelium leading to a pro-inflammatory and pro-coagulant phenotype. Here we aim to characterize the glycocalyx composition of porcine endothelial cells isolated from various anatomical origins and determine whether the heterogeneity of the glycocalyx could play a role in xenorejection.
Methods: Differences in the glycocalyx distribution and composition were investigated by immunofluorescence staining and quantitative real-time PCR of endothelial cells grown both under static conditions and pulsatile flow. We used a 3D microfluidic flow system developed in our laboratory to study the cells in circular, vessel-like microfluidic channels.
Results: Differences in the distribution of sulfated sugars that compose the glycocalyx were observed by immunofluorescence (Fig.1). N-sulfated N-Acetylglucosamine was located at the cell junction on endothelial cells from the thoracic aorta and aortic arch whereas on cells form the vena cava N-sulfated N-Acetylglucosamine was present on the entire cell surface (Fig. 1B), suggesting that the venous and arterial glycocalyx are different. qPCR analysis confirmed that the arterial and venous glycocalyx are differently composed (Fig. 2) as seen by differences in the expression of proteoglycans (Fig. 2A) and enzymes responsible for sulfation (Fig. 2B). In fact, the core proteoglycan perlecan and the N-deacetylase-N-sulfotransferase are upregulated in venous cell whereas the core proteoglycans syndecan-2 and -4 as well as the 3-O sulfotransferase are downregulated compared to arterial endothelial cells.
Conclusion: The glycocalyx of porcine arterial and venous endothelial cells is significantly different which could mean these cells play different roles in xenotransplantation and -rejection. Additional experiments are currently being carried out to determine whether the observed differences in glycocalyx composition also have an effect on the function of the respective endothelial cells, for example with respect to recruitment of regulatory molecules like C1-inhibitor, antithrombin III or superoxide dismutase, which are known to bind to the glycocalyx.