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Hyperplasia of Lymphatic Vessels in VEGF-C Transgenic Mice

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Science  30 May 1997:
Vol. 276, Issue 5317, pp. 1423-1425
DOI: 10.1126/science.276.5317.1423

Abstract

No growth factors specific for the lymphatic vascular system have yet been described. Vascular endothelial growth factor (VEGF) regulates vascular permeability and angiogenesis, but does not promote lymphangiogenesis. Overexpression of VEGF-C, a ligand of the VEGF receptors VEGFR-3 and VEGFR-2, in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement. Thus, VEGF-C induces selective hyperplasia of the lymphatic vasculature, which is involved in the draining of interstitial fluid and in immune function, inflammation, and tumor metastasis. VEGF-C may play a role in disorders involving the lymphatic system and may be of potential use in therapeutic lymphangiogenesis.

The four known members of the VEGF family, VEGF (1), VEGF-B (2), VEGF-C (3, 4), and platelet-derived growth factor (5) have different roles as regulatory factors of endothelia (6). In order to clarify the function of VEGF-C in vivo, its cDNA was cloned between the human keratin 14 (K14) promoter and polyadenylation signal for expression in transgenic mice (7). The K14 promoter has been shown to target gene expression to the basal cells of stratified squamous epithelia (8). Of the 27 mice analyzed at 3 weeks of age, three were transgene-positive, having approximately 40 to 50, 20, and 4 to 6 copies, respectively, of the transgene in their genome. The latter two mice transmitted the gene to two of 40 and six of 11 pups, respectively.

The transgenic mice were small and had slightly swollen eyelids and poorly developed fur. Histological examination showed that the epidermis was hyperplastic and the number of hair follicles was reduced; these effects were considered secondary to other phenotypic changes (9). The dermis was atrophic, and its connective tissue was replaced by large, dilated vessels devoid of red cells but lined with a thin endothelial cell layer (Fig. 1, A and B). Such abnormal vessels were confined to the dermis and resembled the dysfunctional, dilated spaces characteristic of hyperplastic lymphatic vessels (10). In addition, the ultrastructural features were reminiscent of lymphatic vessels, which differ from blood vessels in that they have overlapping endothelial junctions, anchoring filaments in the vessel wall, and a discontinuous or even partially absent basement membrane (Fig. 1C) (11). Antibodies to collagen types IV and XVIII (12), and laminin gave little or no staining of the vessels, whereas the basement membrane staining of other vessels was prominent (Fig. 1D) (13). The endothelium was also characterized by positive staining with monoclonal antibodies to desmoplakins I and II, expressed in lymphatic, but not in vascular endothelial cells (14). Collectively, these findings suggest that the abnormal vessels are of lymphatic origin.

Figure 1

Analysis of the skin of transgenic mice. (A) Hematoxylin-eosin stained section of the skin of a 2-month-old transgenic mouse. Hyperkeratotic epidermis (e) showed underlying vessel spaces lined with endothelium (arrows) but devoid of red cells (compare with the dermal vein shown with an arrowhead). The dermis (d) is atrophic compared with the control littermate skin (B) (45% versus 65% of the dermal thickness, respectively), and the muscle layer (m) is also reduced. (C) Electron microscopy shows the endothelial junctions of an abnormal vessel (arrow) (25); l, lumen; m, mesenchyme. In (D), the basal lamina is stained for type XVIII collagen in veins (arrowheads) but not in the lymphatic endothelium (arrow) (26). Scale bars for (A) and (B), 250 μm; (C), 100 nm; and (D), 25 μm.

Abundant VEGF-C mRNA was detected in the epidermis and hair follicles of the transgenic mice (Fig. 2, A and B), whereas mRNAs encoding its receptors VEGFR-3 (15) and VEGFR-2 (16), as well as the Tie-1 endothelial receptor tyrosine kinase (17), were expressed in endothelial cells lining the abnormal vessels (Fig. 2, C and E) (13). In the skin of littermate control animals, VEGFR-3 was detected only in the superficial subpapillary layer of lymphatic vessels, whereas VEGFR-2 was observed in all endothelia (Fig. 2, D and F), in agreement with earlier findings (18, 19).

Figure 2

In situ hybridization analysis of the skin of K14–VEGF-C transgenic mice. (A) and (B) show hybridization of transgenic skin with VEGF-C antisense and sense probes. (C) and (D) and (E) and (F) show in situ hybridization for VEGFR-3 and VEGFR-2, respectively, in the skin of transgenic and littermate control animals (27). Arrows in (A) indicate basal keratinocytes; in (C) through (E), they point out the lymphatic endothelium; and in (F), arrows show endothelial cells. Scale bar, 20 μm.

The lymphatic endothelium has a great capacity to distend in order to adapt to functional requirements. To ascertain whether vessel dilation was due to endothelial distension or proliferation, we carried out in vitro proliferation assays. The VEGF-C receptor interaction in the transgenic mice apparently transduces a mitogenic signal, because in contrast to littermate controls, the lymphatic endothelium of the skin from young K14–VEGF-C mice showed increased DNA synthesis, as demonstrated by bromodeoxyuridine (BrdU) incorporation followed by staining with antibodies to BrdU (Fig. 3, A and B).

Figure 3

Immunohistochemical analysis of endothelial proliferation and intravital fluorescence microlymphography and microvessel angiography. (A) and (B) show staining of cells in the S phase of the cell cycle, through BrdU incorporation into DNA and its immunohistochemical detection (28). In 2-week-old transgenic mice, the nuclear staining was observed in many endothelial cells of the lymphatic vessels (lv) as well as in keratinocytes [red arrows in (A)]. In nontransgenic littermates, mainly nuclei of keratinocytes of epidermis and some dermal cells are stained [red arrows in (B)]; unstained nuclei were observed in both cases (green arrows). Ø marks artefactual detachment of the epidermis during sample preparation. (C) and (D) illustrate the lymphatic vessels of transgenic and control skin, respectively, through fluorescence microscopy after intradermal injection of FITC-dextran (21, 29). The measured parameters are diameter (d) [in (C) and (D)] and horizontal (h) and vertical (v) mesh sizes [in (D) only]. Blood vessels of the ear after injection of FITC-dextran into the tail vein of transgenic and control mice are shown in (E) and (F), respectively (24). Scale bars for (A) and (B), 5 μm; (C) and (D), 250 μm; (E) and (F), 1 mm.

Angiogenesis is a multistep process that includes endothelial sprouting, migration, and proliferation (20). To estimate the contribution of such processes to the transgenic phenotype, we analyzed the morphology and function of the lymphatic vessels, using fluorescent microlymphography (21). A typical honeycomb-like network with similar mesh sizes was detected in both control and transgenic mice (Fig. 3, C and D), but the diameter of the vessels in transgenic mice was approximately twice that of controls (Table1). Some dysfunction of the abnormal vessels was indicated by the fact that it took longer for the dextran to completely fill the abnormal vessels. Injection of fluorescein isothiocyanate (FITC)–dextran into the tail vein, followed by fluorescence microscopy of the ear showed that the blood vascular morphology was unaltered and leukocyte rolling and adherence appeared normal (Fig. 3, E and F) (13). Thus, the endothelial proliferation induced by VEGF-C leads to hyperplasia of the superficial lymphatic network, but does not induce the sprouting of new vessels.

Table 1

Structural parameters of lymphatic and blood vessel networks for transgenic and control mice. Mesh size describes vessel density. Diameters and mesh sizes are in micrometers (mean ± SD).

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These effects of VEGF-C overexpression are unexpectedly specific, particularly as VEGF-C is also capable of binding to and activating VEGFR-2, which is the major mitogenic receptor of blood vessel endothelial cells (16). In culture, high concentrations of VEGF-C stimulate the growth and migration of bovine capillary endothelial cells that express VEGFR-2, but not significant amounts of VEGFR-3 (3). In addition, VEGF-C induces vascular permeability in the Miles assay, presumably by its effect on VEGFR-2 (22). In vivo, the specific effects of VEGF-C on lymphatic endothelial cells may reflect a requirement for the formation of VEGFR-3 × VEGFR-2 heterodimers for endothelial cell proliferation at physiological concentrations of the growth factor. Such possible heterodimers may help to explain how three homologous VEGFs exert partially redundant, yet strikingly specific, biological effects.

In summary, VEGF-C appears to induce specific lymphatic endothelial proliferation and hyperplasia of the lymphatic vasculature in vivo. So far, there is no evidence that tumors can stimulate the growth of new lymphatic vessels (23), but further studies should establish the role of VEGF-C in lymphangiomas and in tumor metastasis via the lymphatic vasculature as well as in various other disorders involving the lymphatic system and their treatment.

  • * These authors contributed equally to this work.

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