Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.

Vascular endothelial cells undergo morphogenesis into capillary networks in response to angiogenic factors. We show here that sphingosine-1-phosphate (SPP), a platelet-derived bioactive lipid, activates the EDG-1 and -3 subtypes of G protein-coupled receptors on endothelial cells to regulate angiogenesis. SPP induces the Gi/mitogen-activated protein kinase/cell survival pathway and the small GTPase Rho- and Raccoupled adherens junction assembly. Both EDG-1-and EDG-3-regulated signaling pathways are required for endothelial cell morphogenesis into capillary-like networks. Indeed, SPP synergized with polypeptide angiogenic growth factors in the formation of mature neovessels in vivo. These data define SPP as a novel regulator of angiogenesis.

SummaryPsoriatic skin is characterized by microvascular hyperpermeability and angioproliferation, but the mechanisms responsible are unknown. We report here that the hyperplastic epidermis of psoriatic skin expresses strikingly increased amounts of vascular permeability factor (VPF; vascular endothelial growth factor), a selective endothelial cell mitogen that enhances microvascular permeability. Moreover, two VPF receptors, kdr and fit-l, are overexpressed by papillary dermal microvascular endothelial cells. Transforming growth factor ot (TGF-o 0, a cytokine that is also overexpressed in psoriatic epidermis, induced VPF gene expression by cultured epidermal keratinocytes. VPF secreted by TGF-c~-stimulated keratinocytes was bioactive, as demonstrated by its mitogenic effect on dermal microvascular endothelial cells in vitro. Together, these findings suggest that TGF-ot regulates VPF expression in psoriasis by an autocrine mechanism, leading to vascular hyperpermeability and angiogenesis. Similar mechanisms may operate in tumors and in healing skin wounds which also commonly express both VPF and TGF-ol. p! soriasis is a common, chronic skin disease characterized by recurrent erythematous skin plaques that exhibit epidermal hyperplasia, a variable inflammatory cell infiltrate, and abnormalities of the papillary dermal vasculature (1-4). Microvessels in the papillary dermis of psoriatic plaques are elongate, dilated, and hyperpermeable (5, 6) and more closely resemble postcapillary venules than the capillary loops of normal skin (7,8). Whereas vascular changes may precede inflammatory cell infiltration in developing psoriatic plaques, and may reappear before clinical relapse (9-11), there is increasing evidence that epidermal alterations precede capillary leakiness and vascular anomalies in the development of psoriatic skin lesions (12). Moreover, a previous study demonstrated that the angiogenic properties of psoriatic skin were associated with the epidermis, not the dermis (13).Vascular permeability factor (VPF) is a 32-42-kD glycosylated protein that is overexpressed by many human and animal tumors (14-17) and by the epidermis of healing wounds (18), conditions that, like psoriasis, are associated with enhanced microvascular permeability and angiogenesis. Two tyrosine kinase receptors for VPF, kdr and fit-l, are also overexpressed in the microvessels of tumors that overexpress VPF (19)(20)(21). In vivo, VPF enhances microvascular permeability with a potency some 50,000 times that of histamine and induces angiogenesis (22)(23)(24)(25). In vitro, VPF is a selective mitogen for cultured endothelial cells, hence its alternate name, vascular endothelial growth factor (26, 27). We hypothesized that a cytokine with these properties might play an important role in the pathogenesis of psoriasis.In this report, we demonstrate increased expression of VPF mRNA by the hyperplastic epidermis of lesional psoriatic skin and increased expression of two VPF receptors in psoriatic dermal microvessels. In vitro investigations showe...

Vascular endothelial growth factor (VEGF) has been implicated in the pathologic angiogenesis observed in psoriasis and other chronic inflammatory skin diseases that are characterized by enhanced expression of VEGF by epidermal keratinocytes and of VEGF receptors by tortuous microvessels in the upper dermis. To investigate the functional importance of chronic VEGF overexpression in vivo, we used a keratin 14 promoter expression cassette containing the gene for murine VEGF164 to selectively target VEGF expression to basal epidermal keratinocytes in transgenic mice. These mice demonstrated an increased density of tortuous cutaneous blood capillaries with elevated expression levels of the high affinity VEGF receptors, VEGFR-1 and VEGFR-2, most prominently during the neonatal period. In contrast, no abnormalities of lymphatic vessels were detected. In addition, the number of mast cells in the upper dermis was significantly increased in transgenic skin. Intravital fluorescence microscopy revealed highly increased leukocyte rolling and adhesion in postcapillary skin venules that were both inhibited after injection of blocking antibodies against E- and P-selectin. Combined blocking antibodies against intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 were without effect, whereas an anti-vascular cell adhesion molecule-1/VLA-4 antibody combination almost completely normalized the enhanced leukocyte adhesion in transgenic mice. This study reveals VEGF as a growth factor specific for blood vessels, but not lymphatic vessels, and demonstrates that chronic orthotopic overexpression of VEGF in the epidermis is sufficient to induce cardinal features of chronic skin inflammation, providing a molecular link between angiogenesis, mast cell accumulation, and leukocyte recruitment to sites of inflammation.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5-to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors-the ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins-through induction of mRNAs encoding the ␣ 1 and ␣ 2 subunits. In contrast, VEGF did not induce increased expression of the ␣ 3 ␤ 1 integrin, which also has been implicated in collagen binding. Integrin ␣ 1 -blocking and ␣ 2 -blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and ␣ 1 -blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of ␣ 1 ␤ 1 and ␣ 2 ␤ 1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of ␣ 1 -blocking and ␣ 2 -blocking Abs. In vivo, a combination of ␣ 1 -blocking and ␣ 2 -blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of ␣ 1 ␤ 1 and ␣ 2 ␤ 1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that ␣ 1 ␤ 1 and ␣ 2 ␤ 1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.

FTY720, a potent immunosuppressive agent, is phosphorylated in vivo into FTY720-P, a high affinity agonist for sphingosine 1-phosphate (S1P) receptors. The effects of FTY720 on vascular cells, a major target of S1P action, have not been addressed. We now report the metabolic activation of FTY720 by sphingosine kinase-2 and potent activation of vascular endothelial cell functions in vitro and in vivo by phosphorylated FTY720 (FTY720-P). Incubation of endothelial cells with FTY720 resulted in phosphorylation by sphingosine kinase activity and formation of FTY720-P. Sphingosine kinase-2 effectively phosphorylated FTY720 in the human embryonic kidney 293T heterologous expression system. FTY720-P treatment of endothelial cells stimulated extracellular signal-activated kinase and Akt phosphorylation and adherens junction assembly and promoted cell survival. The effects of FTY720-P were inhibited by pertussis toxin, suggesting the requirement for G i -coupled S1P receptors. Indeed, transmonolayer permeability induced by vascular endothelial cell growth factor was potently reversed by FTY720-P. Furthermore, oral FTY720 administration in mice potently blocked VEGF-induced vascular permeability in vivo. These findings suggest that FTY720 or its analogs may find utility in the therapeutic regulation of vascular permeability, an important process in angiogenesis, inflammation, and pathological conditions such as sepsis, hypoxia, and solid tumor growth.

No abstract

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.