Marc G. in many types of cancer, and the basis for surgical and radiation treatment of local lymph nodes. Recent evidence suggests that tumor lymphangiogenesis, the growth of tumor-associated lymphatic vessels, promotes lymphatic metastasis (1C4) and that the inhibition of lymphangiogenesis may provide a new strategy to block lymph node metastasis in cancer therapy (5). Vascular endothelial growth factor (VEGF)-C (6) and VEGF-D (7), which are related to the major angiogenic factor VEGF (8, 9), are distinguished by their capacity to stimulate the growth of lymphatic vessels (18, 19). In the corneal assay, blood vessels must penetrate an avascular space to reach the angiogenesis-inducing stimulus. Dye injection and electron microscopic examination have shown that lymphatic vessels can grow into the cornea after injury, although no lymphatic vessels exist in the normal mammalian cornea (20, 21). Lymphatic vessels currently can be identified in tissues by using VEGFR-3 and two other markers for the lymphatic endothelium. These are LYVE-1 (22, 23), a new homologue RI-2 of the CD44 glycoprotein, a lymphatic endothelial receptor for hyaluronan, and a transmembrane glycoprotein of the mucin class, podoplanin (24). We reasoned that the cornea would be a suitable site to study the effects of lymphangiogenic factors by using these markers. Fibroblast growth factor-2 (FGF-2) is a heparin-binding protein that RI-2 induces cell proliferation or differentiation in a variety of cell types of mesodermal and neuroectodermal origin (25). It is also one of the first factors shown to play an important role in physiological and pathological angiogenesis (26). and purified as described (34). Recombinant human VEGF165 was obtained from R&D Systems, and FGF-2 from Pharmacia UpJohn (Milan, Italy). Rat monoclonal antibodies against mouse VEGFR-3 and rabbit polyclonal antibodies against mouse LYVE-1 were used as described (23, 35). Mouse Corneal Micropocket Assay. The mouse corneal assay was performed according to procedures described (19). Corneal micropockets were created with a modified von Graefe cataract knife in both eyes of each mouse. A micropellet (0.35 0.35 mm) of sucrose aluminum sulfate (Bukh Meditec, Copenhagen, Denmark) coated with hydron polymer type NCC (IFN Science, New Brunswick, NJ) containing 160 ng of VEGF-C or VEGF, or 80 ng of FGF-2, was implanted into each pocket. The pellet was positioned 0.6C0.8 mm from the limbus. After implantation, erythromycin ophthalmic ointment was applied to the eyes. The eyes were examined by a slit-lamp biomicroscope at the indicated days. Vessel length and clock hours of circumferential neovascularization were measured. For the inhibition studies, mice that received corneal implants containing FGF-2 were randomized into two groups and given i.p. injections of neutralizing anti-VEGFR-3 antibodies or nonblocking anti-VEGFR-2 antibodies (35) (600 g per mouse) on postoperative days 0, 2, and RI-2 4. The corneas were photographed on day 5 by a slit-lamp biomicroscope, and the immunohistochemical analysis was performed as described below. Immunohistochemistry. Mice were killed between days 5 and 13 after the implantation of the pellets. Enucleated eyes were fixed in 3% paraformaldehyde, dehydrated, and embedded in paraffin and sectioned radially in parallel to the growing limbal vessels (see Fig. ?Fig.22 and and = 3 each) analyzed by immunostaining as described in and and 450.) (and and ?and55 and and and and assays, models permit the assessment of systemic host factors that influence the growth of the lymphatic vessels. For example, sprouting of lymphatic vessels into tumor stroma may not occur because these vessels lack sufficient pressure to penetrate into the stroma, which has a high interstitial pressure RI-2 (40). Results with isolated lymphatic endothelial cells could also be misleading because many cultured endothelial cells and pericytes/smooth muscle cells located close to the lymphatic vessels express VEGF-C constitutively (13, 41). Endogenous VEGF-C mRNA was not down-regulated in the cultured vascular smooth muscle cells by serum starvation, nor was it stimulated by FGF-2. However, VEGF-C mRNA expression increased after FGF-2 CREB-H stimulation in blood vascular, but not lymphatic endothelial cells. This finding might explain, in part, the phenomenon of concurrent blood vessel.