Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors

Abstract

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.

Figure 1.

Expression of VEGFR-3 and VEGFR-2 in fetal skin analyzed by in situ hybridization. A, B, and D: Hybridization with the VEGFR-3 antisense probe. The signal originates from a subset of vessels of the dermis (arrows). C: Control hybridization with the VEGFR-3 sense strand. D: In a higher magnification of the area marked in (A) and (B), the autoradiographic grains are shown to be localized to the endothelial cells of a developing vessel. E and F: Hybridization for VEGFR-2. The probe gives signal in developing vessel structures at several levels of the dermis (arrows). B, D, and F: Bright-field photographs of the same sections. Bar: 0.1 mm.

Figure 2.

Radioactive VEGF-C and VEGF binding in human adult skin visualized by autoradiography. A, B, and C: Binding of ¹²⁵I-labeled rh-VEGF-C. The signal is localized to the thin lymphatic vessels of the subpapillary plexus (arrows). D, E, and F: Binding sites are blocked when a 100-fold excess of cold ligand is added to the incubation medium. Some unspecific binding is seen originating from keratin squames in the epidermis (D, arrowhead). G, H, and I: Specific ¹²⁵I-labeled rh-VEGF binding to vascular endothelium, which extends throughout the dermis. (C and I: Higher magnifications (×100) of the marked vessel structures binding the radioactive ligands VEGF-C and VEGF, respectively. Dark-field (A, D, and G) and bright-field (B, C, E, F, H, and I) exposures are shown. Bar: 0.1 mm for A, B, and D to H.

Figure 3.

Comparison of VEGFR-3 expression with two vascular endothelial markers in fetal and adult skin. Arrows indicate vessels in adjacent sections of fetal and adult skin, respectively, which stain for VEGFR-3 (A and B) and CD31 (C and D), but not for PAL-E (E and F), and are therefore presumably lymphatic. Arrowheads identify vessels, which only react with antibodies against the two blood vascular endothelial antigens. In adult skin, the thin VEGFR-3-positive lymphatic vessels could be identified by morphology and lack of a basal lamina (B), but the same vessel could not be traced in the adjacent sections stained for CD31 (D) or PAL-E (F). Bar: 0.17 mm. Insets (A and B, right) illustrate higher magnifications of the lymphatic vessels (arrows) in marked areas of the fetal and adult skin sections, respectively. Bar: 57 μm.

Figure 4.

Comparison of VEGFR-3 expression with double staining for two vascular endothelial markers and with anti-laminin staining. A sample of adult buccal mucosa was stained with the anti-VEGFR-3 mAb (A) and an adjacent section, first with PAL-E (B, red-colored endothelia) and then with anti-CD31 (B, blue-colored endothelia), to distinguish lymphatic structures from blood vessels. PAL-E also decorates the epidermal basal lamina (B, open triangle). Comparison of VEGFR-3 and laminin expression in adult skin is shown in (C) and (D). Arrows indicate lymphatic vessels of adult skin, which stain for VEGFR-3 (C). Arrowheads show blood vessels with basement membranes, which stain for laminin (D), but only very weakly for VEGFR-3. Bars: 35 μm (A and B) and 0.11 mm (C and D).

Figure 5.

Comparison of VEGFR-3 expression with vascular endothelial markers in lymphangiomatosis (A to D) and in capillary cutaneous (E and F) and intramuscular (G and H) hemangiomas. Arrows indicate collapsed, VEGFR-3-positive thin endothelium-lined anastomosing channels dissecting through dermal connective tissue in a lymphangiomatosis sample (A). Negative control was done by blocking anti-VEGFR-3 with the immunogen (B). Adjacent sections were also stained for CD31 (C) and vWF (D). Note that in contrast to the buccal mucosa sample (Figure 4) ▶ , CD31 identifies the endothelial cells lining lymphatic spaces in lymphangiomatosis, whereas the vWF staining is weak or absent. In cutaneous hemangioma, the well-formed capillaries with prominent endothelial cells show very little or no expression of VEGFR-3 (E), but are strongly CD31 positive (F). In a frozen section of intramuscular hemangioma, weak VEGFR-3 staining is detected (G, arrows) in part of the CD31-positive vessels (H). Bars: 65 μm (A to D, 48 μm (E and F), and 140 μm (G and H).