Immunohistochemical Analysis of Cellular Prion Protein Expression on Vascular Endothelial Cells.
Authors: Sivakumaran Muttuswamy , Frost Christine 
Published: April 2020

Abstract
The majority of the cellular prion protein (PrPc), the normal counterpart of the putative infectious agent of variant Creutzfeldt-Jakob (vCJD) disease, present in human blood is associated with the plasma compartment. But the source of this ‘soluble’ prion protein is unclear. Based on the results of in-vitro experiments using cultured umbilical cord or adult vascular endothelial cells, some investigators have suggested that vascular endothelial cells may be an important source of plasma PrPc. Our previous immunohistochemical studies using tissue specimens obtained from healthy individuals and a monoclonal antibody to PrP (3F4 recognising an epitope between aminoacids 109–111 of the protein), however, have shown that normal vascular endothelial cells exhibit minimal or no expression of this protein. Our finding has raised an important question whether the lack of expression seen with the above antibody may be a result of truncation of PrPc in tissues resulting in removal of 3F4-binding site. In order to establish the true nature of prion protein expression on normal, unstimulated, unmanipulated endothelial cells, we have extended the study using seven antibodies that recognise different domains, spanning the whole human PrP protein molecule. Antibodies against epitopes 23–85 (FH11 & BG4), 63–99 (BE12), 79–97 (AHP498T, CD230), 109–112 (3F4), 140–180 (KG9 & DF7) were chosen for the study. Institutional and Local Research Ethics Committee approvals were obtained for this study. Formalin fixed, paraffin- embedded tissue blocks from normal human umbilical cord (12), aorta (4), saphenous vein (4), kidney (1) and cerebrum (1) were selected from the tissue bank at Peterborough District Hospital, United Kingdom. Cerebral tissue and a platelet ‘clot’ prepared from pooled normal platelet concentrates were used as positive controls for PrPc expression. Sections of normal kidney were included as positive control for antibody QBEND10 which was used to demonstrate endothelial cells. Tissue sections from all cases were studied with haematoxylin and eosin (H&E) and immunohistochemistry (avidin-biotin horseradish peroxidase technique) using above antibodies. All the sections were examined by light microscopy to assess qualitatively the presence or absence of immunostaining. The intensity of labeling was evaluated by a scoring system: W - weak immunostaining, S- strong immunostaining, and N- negative immunostaining. The results show that the monoclonal antibody QBEND10 clearly identifies and delineates vascular endothelial cells in umbilical cord and adult blood vessels. All anti-PrPc antibodies show positive reaction, albeit to varying intensities, with cerebrum and platelets. None of the seven anti-PrP antibodies used in this study show positive reactivity with endothelial cells of umbilical cord, aorta or saphenous vein. Based on these results we conclude that normal, uncultured, unstimulated vascular endothelial cells do not express cellular prion protein in detectable quantities and, therefore, it is unlikely that vascular endothelial cell is a major source of PrPc in plasma in healthy individuals.