Origins of circulating endothelial cells and endothelial outgrowth from blood

Abstract

Normal adults have a small number of circulating endothelial cells (CEC) in peripheral blood, and endothelial outgrowth has been observed from cultures of blood. In this study we seek insight into the origins of CEC and endothelial outgrowth from cultures of blood. Fluorescence in situ hybridization analysis of blood samples from bone marrow transplant recipients who had received gender-mismatched transplants 5-20 months earlier showed that most CEC in fresh blood had recipient genotype. Endothelial outgrowth from the same blood samples after 9 days in culture (5-fold expansion) was still predominantly of the recipient genotype. In contrast, endothelial outgrowth after approximately 1 month (102-fold expansion) was mostly of donor genotype. Thus, recipient-genotype endothelial cells expanded only approximately 20-fold over this period, whereas donor-genotype endothelial cells expanded approximately 1000-fold. These data suggest that most CEC in fresh blood originate from vessel walls and have limited growth capability. Conversely, the data indicate that outgrowth of endothelial cells from cultures of blood is mostly derived from transplantable marrow-derived cells. Because these cells have more delayed outgrowth but a greater proliferative rate, our data suggest that they are derived from circulating angioblasts.

Figure 1

Endothelial expansion from buffy coat mononuclear cells of normal blood. On day 2 and for subsequent passages (indicated by arrows), the number of endothelial cells was confirmed by staining for P1H12 and vWF, and that number was consistent with the cell count by morphology. All data points plotted as mean ± SD (n = 5 for culture up to passage 6; n = 4 for subsequent passages).

Figure 2

Outgrowth endothelial cell morphology and phenotype. Outgrowth cells have typical endothelial morphology (a). The remaining parts (b–e) show, as labeled at the bottom of each, constitutive and activated phenotype by immunofluorescent staining. Outgrowth endothelial cells incorporated acetylated LDL and were positive for vWF. They were negative for VCAM-1 but expressed it upon stimulation.

Figure 3

Flow cytometry analysis of outgrowth endothelial cell phenotype. In each graph, the black line outlines the region of fluorescent intensity for cells labeled with negative control antibody. The filled region identifies cells labeled with antibody for the expression marker indicated above each graph. Outgrowth cells are negative for CD14 (monocyte marker) and positive for flk-1, vWF, CD36, and the endothelial-specific marker VE-cadherin.

Figure 4

FISH for endothelial genotype. Cells were double-labeled for endothelial phenotype and gender genotype. Cell surface staining for P1H12, shown here in blue, identified the cells as endothelial (9). A rhodamine-conjugated probe was used to identify Y chromosomes; a FITC-conjugated probe was used to identify X chromosomes. This illustration, using blood from subject D, shows 1 endothelial cell in the upper-right corner with male genotype and 1 endothelial cell in the bottom-left corner with female genotype. Cytospin and fixation procedure causes some distortion of cell morphology and loss of cytoplasm. There was no morphologic difference between donor-genotype and recipient-genotype endothelial cells.

Figure 5

Endothelial outgrowth from peripheral blood of gender-mismatched bone marrow transplant recipients showing: (a) the total number of cells in culture at the 3 time points (mean ± SD; n = 4); (b) the percentage of cells at the 3 time points having recipient or donor genotype; (c) the overall fold expansion, as well as the fold expansion of cells of the 2 separate genotypes. (n = 4, mean ± SD for day 0 and 27 ± 4; n = 2, mean for day 9.)