Characterization and quantification of porcine circulating endothelial cells.
BACKGROUND
Endothelial damage is a critical step in the development of (xeno) transplantation-related and cardiovascular pathology. In humans, the amount of circulating endothelial cells (CEC) correlates to disease intensity and functions as a valuable damage marker. While (xeno) transplantation and cardiovascular research is regularly performed in porcine models, the paucity of antibodies against porcine endothelium epitopes hinders the use of CEC as damage marker.


OBJECTIVE
This study aimed to develop a method for porcine CEC detection using anti-human antibodies against porcine endothelium epitopes.


METHODS
Human umbilical vein endothelial cells (HUVEC, control) and their swine equivalent (SUVEC) were used to assess the cross-species immunoreactivity of fluorescently labeled anti-human CD31/CD51/CD54/CD62E/CD105/CD106/CD144/CD146/PAL-E/lectin-1/vWF antibodies by isotype-controlled fluorescence-activated cell sorting (FACS) and confocal microscopy. Next, reactivity was ascertained with mature porcine kidney-derived endothelial cells (PKEC), and a FACS-based whole blood CEC quantification method was employed using osmotic erythrolysis and CD105 and CD146 double staining after CD45 exclusion.


RESULTS
Of the 21 assayed antibodies, the MEM-229 clone of CD105 and P1H12 clone of CD146 showed immunoreactivity with SUVEC and PKEC. Double staining showed baseline porcine CEC count of 673.1 ± 551.4 CEC/ml, while the first 7.5 ml of drawn blood (representative of vascular damage) contained 1118 ± 661.4 CEC/ml (n = 14, P = 0.04). A second experiment (n = 5) including CD45 exclusion identified only 14.5 ± 10.8% double-positive CD105-146 events per ml blood.


CONCLUSIONS
Porcine endothelium can be specifically labeled using anti-human CD146 and CD105 antibodies. These antibodies can therefore be used for the identification and quantification of CEC in porcine whole blood by FACS after osmotic erythrolysis.
Department of Surgery (Surgical Laboratory), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Ivo C J H
IC
Post
Robert P
RP
Weenink
Albert C W A
AC
van Wijk
Michal
M
Heger
Anita N
AN
Böing
Robert A
RA
van Hulst
Thomas M
TM
van Gulik
D000818:Animals; D000910:Antibodies, Heterophile / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology; D015478:Antigens, Heterophile / Q000276:immunology; D001773:Blood Cells / Q000166:cytology* / Q000276:immunology*; D051929:CD146 Antigen / Q000276:immunology; D002452:Cell Count / Q000379:methods / Q000662:veterinary; D002478:Cells, Cultured; D003429:Cross Reactions; D000071063:Endoglin; D042783:Endothelial Cells / Q000166:cytology* / Q000276:immunology*; D005434:Flow Cytometry; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D018613:Microscopy, Confocal; D011956:Receptors, Cell Surface / Q000276:immunology; D034421:Sus scrofa / Q000097:blood* / Q000276:immunology*; D014183:Transplantation, Heterologous / Q000009:adverse effects / Q000276:immunology
D000910:Antibodies, Heterophile; D015703:Antigens, CD; D015478:Antigens, Heterophile; D051929:CD146 Antigen; C064206:ENG protein, human; D000071063:Endoglin; C497902:MCAM protein, human; D011956:Receptors, Cell Surface