PKH26 AND I-125 PKH95 - CHARACTERIZATION AND EFFICACY AS LABELS FOR IN-VITRO AND IN-VIVO ENDOTHELIAL-CELL LOCALIZATION AND TRACKING

PKH26, a fluorescent cell label, and PKH95, a I-125-radioactive cell l abel, are both potentially valuable endothelial cell labels because th ey bind irreversibly within cell membranes. These labels would be part icularly well suited to tract transplanted endothelial cells in vivo. However, no previous studies documenting lack of transfer of the label to unlabeled endothelial cells, as well as the effect of the label on endothelial cell function, have been undertaken. The purpose of this study was to determine the optimal method of endothelial cell (EC) lab eling with PKH26 and PKH95, whether significant EC-to-EC transfer of t he label occurs, the effects of these labels on EC proliferation and m embrane function, and the feasibility of using these labels for long-t erm quantitative EC tracking in vivo. Canine ECs in confluent monolaye rs or in cell suspension were labeled by exposure to 1.0 or 5.0 mu M P KH26 for 1, 3, or 5 min. Cell viability was determined by trypan blue exclusion. The percentage of cells labeled and their fluorescence inte nsity were determined in a fluorescent-activated cell sorter (FACS). E ffect of the label on cell function was assessed by measuring EC proli feration rates as well as intercellular adhesion molecule (ICAM) expre ssion before and after induction with tumor necrosis factor (TNF). To determine if transfer of the label occurs, both labeled and nonlabeled ECs were grown in coculture and subjected to FAGS analysis. For in vi vo cell tracking, doubly labeled ECs were injected into the femoral ar tery of rat hindlimbs, and whole-body tissue analysis was undertaken t o determine labeled-EC distribution at 60 days. Endothelial cells were labeled with equal efficiency as monolayers or in suspension. Labelin g had no effect on EC proliferation rates nor on TNF-induced upregulat ion of ICAM expression. Coculture experiments revealed no significant label transfer to nonlabeled ECs. In vivo cell tracking studies docume nted that 8% of label remained within the skeletal muscle capillaries at 60 days after injection. PKH26 and PKH95 labels incorporate stably into EC membranes, do not alter endothelial cell function, and provide a precise means for quantitative EC tracking and histologic localizat ion both in vitro and in vivo. These labels should prove to be very us eful for studies of endothelial cell biology and transplantation. (C) 1996 Academic Press, Inc.