Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas

Abstract

Background: Lymphangiomas are neoplasias of childhood. Their etiology is unknown and a causal therapy does not exist. The recent discovery of highly specific markers for lymphatic endothelial cells (LECs) has permitted their isolation and characterization, but expression levels and stability of molecular markers on LECs from healthy and lymphangioma tissues have not been studied yet. We addressed this problem by profiling LECs from normal dermis and two children suffering from lymphangioma, and also compared them with blood endothelial cells (BECs) from umbilical vein, aorta and myometrial microvessels.

Methods: Lymphangioma tissue samples were obtained from two young patients suffering from lymphangioma in the axillary and upper arm region. Initially isolated with anti-CD31 (PECAM-1) antibodies, the cells were separated by FACS sorting and magnetic beads using anti-podoplanin and/or LYVE-1 antibodies. Characterization was performed by FACS analysis, immunofluorescence staining, ELISA and micro-array gene analysis.

Results: LECs from foreskin and lymphangioma had an almost identical pattern of lymphendothelial markers such as podoplanin, Prox1, reelin, cMaf and integrin-alpha1 and -alpha9. However, LYVE-1 was down-regulated and VEGFR-2 and R-3 were up-regulated in lymphangiomas. Prox1 was constantly expressed in LECs but not in any of the BECs.

Conclusion: LECs from different sources express slightly variable molecular markers, but can always be distinguished from BECs by their Prox1 expression. High levels of VEGFR-3 and -2 seem to contribute to the etiology of lymphangiomas.

Figure 1

Expression of blood and lymphatic markers on large vessel endothelial cells. Typical profiles obtained by FACS analysis with the panendothelial markers CD31/PECAM-1, Tie2 and CD105/endoglin on primary HUVECs and HAECs (passage 3–5). Profiles obtained with lymphatic markers podoplanin and LYVE-1 on both of the control cell types are also indicated. The mean fluorescence intensities were normalized to the background fluorescence of the secondary antibody alone (grey).

Figure 2

Expression of von Willebrand factor (vWF), CD31/PECAM-1 and Prox1 in large vessel control cells. Granular intracellular localization of vWF, membranous localization of CD31 and nuclear localization of Prox1 was detected by immunocytology in HUVEC and HAEC. Nuclei were counter-stained with DAPI. One representative staining out of several independent experiments is shown. Magnification, × 200.

Figure 3

Characterization of LECs isolated from dermal microvascular endothelial cells. FACS sorting of HDMECs with the two antibodies against podoplanin and LYVE-1. Only podoplanin⁺ cells are found in gate R3 and podoplanin⁺/LYVE-1⁺ cells are found in gate R4. A total number of 9,6 × 10⁵ cells from passage 6 have been used for cell sorting.

Figure 4

FACS analysis of blood markers on dermal microvascular endothelial cells before and after podoplanin magneto-bead sorting. Typical profiles obtained with the panendothelial markers CD31/PECAM-1, Tie2 and CD105/endoglin on primary HDMECs and on the same cells (passage 3–5) after podoplanin sorting with magneto beads.

Figure 5

FACS analysis of lymphatic markers on dermal microvascular endothelial cells before and after podoplanin magneto-bead sorting. Typical profiles obtained with lymphatic markers podoplanin, LYVE-1 and VEGFR-3 before and after podoplanin-mediated cell sorting with a FACS sorter

Figure 6

Expression of CD31/PECAM-1, LYVE-1 and Prox1 in dermal microvascular endothelial cells before and after LYVE-1 sorting. Membranous localization of CD31, LYVE-1 and podoplanin, and nuclear localization of Prox1 was detected by immunocytology in HDMECs. Nuclei were counter-stained with DAPI in some specimens. One representative staining out of several independent experiments is shown. Magnification, × 200.

Figure 7

Expression of lymphatic markers on UtMVECmyo. A: Typical FACS analysis profiles obtained with the panendothelial marker CD31/PECAM-1 and the lymphendothelial markers LYVE-1 and podoplanin. B: Immunostaining for CD31, Prox1, podoplanin and LYVE-1. Nuclei were counter-stained with DAPI. One representative staining out of several independent experiments is shown. Magnification, × 200.

Figure 8

Isolation and characterization of LECs from lymphangiomas. Lymphendothelial cells (LECs) from lymphangioma explants isolated by „cell sweeping” were photographed under phase contrast microscopy. a) The cells from patient-A show the typical cobblestone morphology and are contact inhibited (passage 7, magnification, × 40.). Expression of CD31/PECAM-1 together with Prox1 in LECs (b). Membranous localization of CD31 and nuclear localization of Prox1 was detected by immunocytology. Magnification, × 200. c) Same specimen as in b) showing CD31 expression and nuclear counter-staining with DAPI. Magnification, × 200. d) Podoplanin staining. Nuclei were counter-stained with DAPI. One representative staining out of several independent experiments is shown.

Figure 9

Isolation and characterization of LECs from lymphangiomas. LECs from lymphangioma explants (patient-B) isolated by CD31 microbead sorting shown by phase contrast microscopy. The culture was contaminated with stromal cells (a). Isolation of LECs with CD31 micro beads. Indicated are cell numbers after each step.(b). Cells are further cultivated and displayed the typical cobblestone morphology (c). d) Same specimen as in c) stained for CD31 and Prox1. Magnification: × 40 in (a) and × 100 in (c) and (d).

Figure 10

FACS analysis of blood vessel markers on LECs from lymphangioma patients. Expression of blood endothelial markers. Typical profiles obtained with the panendothelial markers CD31/PECAM-1, Tie1, Tie2 and VEGFR-2 on primary LECs from the two patients A and B (passage 4–6). All of the markers are expressed.

Figure 11

FACS analysis of lymph vessel markers on LECs from lymphangioma patients. Expression of lymph endothelial markers on LECs from lymphangioma patients. Typical profiles obtained with the markers podoplanin (three different antibodies for LEC-B), LYVE-1 and VEGFR-3 on primary LECs from the two patients A and B (passage 4–6).