Endothelial progenitor cells from infantile hemangioma and umbilical cord blood display unique cellular responses to endostatin

Abstract

Infantile hemangiomas are composed of endothelial cells (ECs), endothelial progenitor cells (EPCs), as well as perivascular and hematopoietic cells. Our hypothesis is that hemangioma-derived EPCs (HemEPCs) differentiate into the mature ECs that comprise the major compartment of the tumor. To test this, we isolated EPCs (CD133(+)/Ulex europeus- I(+)) and mature ECs (CD133(-)/Ulex europeus-I(+)) from proliferating hemangiomas and used a previously described property of hemangioma-derived ECs (HemECs), enhanced migratory activity in response to the angiogenesis inhibitor endostatin, to determine if HemEPCs share this abnormal behavior. Umbilical cord blood-derived EPCs (cbEPCs) were analyzed in parallel as a normal control. Our results show that HemEPCs, HemECs, and cbEPCs exhibit increased adhesion, migration, and proliferation in response to endostatin. This angiogenic response to endostatin was consistently expressed by HemEPCs over several weeks in culture, whereas HemECs and cbEPCs shifted toward the mature endothelial response to endostatin. Similar mRNA-expression patterns among HemEPCs, HemECs, and cbEPCs, revealed by microarray analyses, provided further indication of an EPC phenotype. This is the first demonstration that human EPCs, isolated from blood or from a proliferating hemangioma, are stimulated by an angiogenesis inhibitor. These findings suggest that EPCs respond differently from mature ECs when exposed to angiogenic or antiangiogenic signals.

Figure 1.

EC marker expression and characterization. (A) Cytometric analysis of ECs and hematopoietic markers in cultured HemEPCs, HemECs, cbEPCs, and HDMECs. Solid gray histograms represent cells stained with fluorescence-conjugated antibodies. Black lines show cells stained with fluorescence-conjugated isotype control antibodies. (B) Indirect immunofluorescent staining of cells, grown in monolayers, with CD31, VE-cadherin, and VWF antibodies showing positive staining for EC markers (all cells were passage 8). Images were taken with a Nikon Eclipse TE300 (Nikon, Melville, NY) using Spot Advanced 3.5.9 software (Diagnostic Instruments, Sterling Heights, MI) and a 20×/0.45 objective lens.

Figure 2.

ES-induced adhesion, migration, and proliferation of EPCs. Increased adhesion (A), migration (B), and proliferation (C) of HemEPCs, HemECs, and cbEPCs in response to ES. (For the adhesion and the proliferation assay, data are presented relative to control. *P < .05 compared with control; †P < .05 compared with VEGF treatment group; n = 3/treatment; all cells were passage 9). Error bars represent SEM.

Figure 3.

Effect of long-term culture on the ES response. (A) Analysis of the ES- and VEGF-induced migration of the cells at passages 6, 12, 18, and 24. (*P < .05 compared with passage 6 and 12; n = 3/treatment.) (B) RT-PCR analysis of EC markers at passage 9 and passage 25. HDMECs served as a positive control.

Figure 4.

E-selectin expression in EPCs. Flow cytometric analysis of E-selectin expression in HemEPCs, cbEPCs, and HDMECs; HemEPCs and cbEPCs were assayed at passage 9 and passage 25. Cells were treated with media alone, TNF-α (10 ng/mL), or ES (10 ng/mL) for 5 hours prior to harvesting for analysis.

Figure 5.

Gene expression-profiling. Linear regression analysis of mRNA levels in HDMECs/HemECs (A), HDMECs/cbEPCs (B), HDMECs/HemEPCs (C), HemECs/cbEPCs (D), HemECs/HemEPCs (E), and HemEPCs/cbEPCs (F). (A-F) Mean and 2 SD limits are indicated by gray lines. The array contained 113 genes encoding matrix proteins and proteins involved in cell-cell and cell-matrix interactions.