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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 7, 2002 as doi:10.1096/fj.01-1010fje. |
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* Anatomisches Institut,
Abteilung Plastische und Handchirurgie,
Zentrum für Kinderheilkunde und Jugendmedizin, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany;
Harris Birthright Research Centre for Fetal Medicine, King‘s College Hospital, London, UK;
Universitäts-Frauenklinik, Kiel, Germany;
|| Molecular and Cancer Biology Laboratory, University of Helsinki, Helsinki, Finland; and
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institute of Health, Bethesda, Maryland, USA
2Correspondence: Anatomisches Institut der Albert-Ludwigs-Universität Freiburg, Albertstrasse 17, 79104 Freiburg, Germany. E-mail: Joerg.Wilting{at}anat.uni-freiburg.de
SPECIFIC AIMS
The identification of lymphatic vessels and lymphatic endothelium has always been problematic. In histological sections, lymphatic endothelial cells (LECs) can hardly be identified. The homeobox-containing transcription factor Prox1, the mammalian homologue of the Drosophila gene prospero, has been shown to be expressed in LECs but not in blood vascular endothelial cells (BECs) of murine and avian embryos. We have investigated the expression of Prox1, vascular endothelial growth factor receptor 3 (VEGFR-3), CD31, CD34, and PAL-E in human tissues by immunofluorescence. We studied cryosections from human fetuses, skin from healthy adults and lymphedema patients, as well as hemangiomas and lymphangiomas from children between 6 days and 63 months of age.
PRINCIPAL FINDINGS
1. Prox1 is coexpressed with VEGFR-3 in lymphatic endothelium
We studied the expression of Prox1 protein with double immunofluorescence staining of fetal tissues. It is well known that the mesenteric blood vessels are accompanied by numerous lymphatics. In 19-wk-old human fetuses, the mesenteric lymphatic collectors are positive for both VEGFR-3 (flt4) and Prox1 (Figure 1
A, B). VEGFR-3 is located on the surface of the LECs whereas Prox 1 is found in the nucleus. We observed double positivity for VEGFR-3 and Prox1 in LECs in all parts of the intestine. This also holds true for the initial lymphatic capillaries in the center of the intestinal villi (Fig. 1C, D
). Therefore, like in mouse and chick embryos, Prox1 marks the lymphatics in human fetuses. This is supported by double staining with Prox1 and PAL-E antibodies. PAL-E has often been used as a marker of blood vessels, as in our studies. The two markers are mutually exclusive (Fig. 1E, F
). PAL-E is expressed in microvessels and veins but not in lymphatics, which are Prox1 positive. Besides lymphatics, we observed Prox1 expression in a small number of intestinal epithelial cells probably representing enteroendocrine cells. To investigate whether Prox1 is expressed in tissues of adults, we studied buccal skin of healthy adults and scrotal skin of a 25-year-old lymphedema patient who underwent resection surgery. We observed Prox1- and VEGFR-3-positive lymphatics in the dermis; again, expression of PAL-E and Prox1 was mutually exclusive. In the hypodermis of the lymphedema patient we found extremely dilated, Prox1-positive/PAL-E-negative lymphatics.
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2. In contrast to VEGFR-3, Prox1 retains its lymphatic specificity in hemangiomas
We then studied the expression of Prox1 in combination with other endothelial markers in hemangiomas (6 specimens). The dense plexus of blood capillaries is Prox1-negative/CD31-positive. Lymphatics, which are occasionally located in the vicinity of the hemangiomas, express CD31 weakly and are Prox1-positive/CD31 positive. They also express VEGFR-3. However, in contrast to Prox1, VEGFR-3 is also expressed in a considerable number of BECs of the hemangiomas. The BECs of hemangiomas are strongly positive for CD34. Double staining with Prox1 and CD34 revealed that the blood capillaries are Prox1-negative/CD34-positive, whereas lymphatic capillaries in the dermis adjacent to the hemangioma are Prox1-positive/CD34-negative. This staining pattern is also found in the lymphatic trunks. In one patient, the hemangioma was located in the dermis of the medial side of the thigh and contained superficial lymphatic trunks that accompany the great saphenous vein. The LECs of these trunks are Prox1- positive/CD34-negative.
3. Prox1 is a constitutive marker of lymphangioma endothelial cells
Lymphangiomas are characterized by multiple endothelial lined cysts containing serous fluid. Our immunohistological studies show that the LECs in lymphangiomas are Prox1-positive/CD31-positive. The CD31 staining appears to be stronger than in LECs located in the periphery of the hemangiomas. We observed that LECs of lymphangiomas usually are Prox-positive/CD34-negative and only a very small subset of them is CD34-positive. However, the situation is complicated by the fact that elongated CD34+ cells are present in the stroma of lymphangiomas and often are intimately attached to the LECs. Furthermore, LECs of lymphangiomas express VEGFR-3, which has been observed earlier.
The results are summarized in Table 1
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CONCLUSIONS
Morphological characteristics of endothelial cells in vascular malformations and tumors do not allow a clear distinction between the BECs and LECs. Vascular malformations often appear as combined malformations involving blood vessels and the lymphatics. In ‘lymphedema’ patients, a broad spectrum from lymphatic aplasia to hyperplasia can be observed. A precise diagnosis of such diseases is only possible with the help of molecular markers that distinguish between BECs and LECs. Our studies show that Prox1 is a highly specific marker for LECs in normal and diseased tissues. In this respect, Prox1 is better than VEGFR-3, which is also expressed in early embryonic blood vessels and hemangiomas. In LECs, Prox1 is coexpressed with CD31 and VEGFR-3 but not with PAL-E and generally not with CD34. However, CD34-positive LECs can occasionally be observed in lymphangiomas. We have previously shown that Prox1 is a reliable marker of LECs in experimental melanomas expressing high levels of VEGF-C and containing lymphatic vessels, whereas VEGFR-3 seems to lose its lymphatic specificity in tumors because of its up-regulation in blood vessels. VEGFR-3 and CD34 are less reliable markers for LECs and BECs, respectively, because exceptions from their normal expression patterns are found in pathological tissues. We therefore recommend the use of Prox1 as a LEC marker in histological sections. Since Prox1 is expressed in some nonendothelial cell types such as hepatocytes and pancreatic epithelial cells, double staining with Prox1 and CD31 is the most reliable method to characterize LECs. Coexpression of Prox1 with other LEC markers such as LYVE-1 and podoplanin needs further study.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-1010fje; to cite this article, use FASEB J. (June 7, 2002) 10.1096/fj.01-1010fje 
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