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 CD 31/ CD 51/ CD 54/ CD 62E/ CD 105/ CD 106/ CD 144/ CD 146/ 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 CD 105 and CD 146 double staining after CD 45 exclusion. Results Of the 21 assayed antibodies, the MEM ‐229 clone of CD 105 and P1H12 clone of CD 146 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 CD 45 exclusion identified only 14.5 ± 10.8% double‐positive CD 105‐146 events per ml blood. Conclusion Porcine endothelium can be specifically labeled using anti‐human CD 146 and CD 105 antibodies. These antibodies can therefore be used for the identification and quantification of CEC in porcine whole blood by FACS after osmotic erythrolysis.