The molecular mechanism of tissue-specific metastasis in tumors endogenously expressing members of the vascular endothelial growth factor (VEGF) family is not yet clear. Here we demonstrate that MMP9 is specifically induced in premetastatic lung endothelial cells and macrophages by distant primary tumors via VEGFR-1/Flt-1 tyrosine kinase (TK) and that it significantly promotes lung metastasis. In a genetic approach using mice, suppression of MMP9 induction by deletion of either VEGFR-1TK or MMP9 markedly reduced lung metastasis. Furthermore, the MMP9 levels in endothelial cells of normal lung lobes from patients carrying distant tumors were significantly elevated as compared with those from patients without tumors. Thus, a block of MMP9 induction via VEGFR-1 inhibition could be useful for the prevention of tumor metastasis in lung.

Flt-1, also known as vascular endothelial growth factor receptor 1 (VEGFR-1), is a high-affinity tyrosine kinase receptor for VEGF and is expressed almost exclusively on vascular endothelial cells. As an exception, Flt-1 transcript was recently found to be expressed in human peripheral blood monocytes. However, the protein of the Flt-1 receptor on the cell surface of monocytes is yet to be identified, and whether the Flt-1 protein is expressed during the differentiation of monocyte-macrophage lineage cells has not been examined. Using monoclonal antibodies against 2 different antigenic epitopes on the Flt-1 extracellular domain, this study found that the major population of the monocyte-marker CD97 ؉ cells in human peripheral blood express Flt-1 as a cell surface molecule. VEGFR-2 (KDR/Flk-1) was not expressed at detectable levels in these cells. An Flt-1 neutralizing monoclonal antibody significantly suppressed VEGF-induced migration of the monocytes, suggesting an important role for Flt-1 in the biologic function of monocytes. Furthermore, CD34 ؉ cells in human cord blood, originally negative for the Flt-1 expression, differentiated into Flt-1 ؉ cells in association with the appearance of monocyte-macrophage markers after a 2-week culture in the presence of hematopoietic cytokines. In addition, the Flt-1 ؉ CD11b ؉ cell fraction from CD34 ؉ cells was found to efficiently differentiate into multinuclear osteoclasts in the presence of macrophage colony-stimulating factor and osteoclast differentiation factor. These results strongly suggest that Flt-1 is a novel cell surface marker as well as a biologically functional molecule for monocyte-macrophage lineages in humans. IntroductionVascular endothelial growth factor (VEGF) and its receptors Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2) are well established as the essential regulatory system for blood vessel formation in embryogenesis. [1][2][3][4] Other VEGF family members such as VEGF-C and VEGF-D and their specific receptor Flt-4 (VEGFR-3) are also important for angiogenesis as well as lymphoangiogenesis in the embryo. 5 The specificity of VEGF and its receptor system for vascular endothelial cells appears to be based on at least 2 factors, the endothelial cell-specific expression of Flt-1 and KDR/Flk-1 6,7 and a unique signaling pathway from the receptors. We have recently shown that VEGF-dependent DNA synthesis in primary endothelial cells is mainly mediated via the phospholipase C␥-protein kinase C-mitogen-activated protein (MAP) kinase pathway, different from the signal transduction of the representative receptor tyrosine kinases such as EGF receptor. 8,9 Most of the signals for VEGF-induced cell proliferation and vascular permeability are considered to be mediated by KDR/ Flk-1, which has strong tyrosine kinase activity. [10][11][12] On the other hand, the Flt-1 bears an affinity for VEGF that is about 10-fold higher than KDR, but contains much weaker tyrosine kinase activity, suggesting a regulatory function in the VEGF signaling in endothelial cells. [13][14][...

Vascular endothelial growth factor (VEGF) and VEGF receptor-1 (VEGFR-1/Flt-1) were shown to be involved in pathological angiogenesis, particularly rheumatoid arthritis (RA). However, the molecular basis of their actions is not fully understood. Here we report that in a murine model of RA, deletion of the tyrosine kinase (TK) domain of VEGFR-1 decreased the incidence and clinical symptoms of RA. Pathological symptoms, such as synovial hyperplasia, inflammatory infiltrates, pannus formation, and cartilage/bone destruction, became milder in Vegfr-1 tk ؊/؊ mice compared with wild-type (Wt) mice in the human T-cell leukemia virus-1 (HTLV-1) pX-induced chronic models. VEGFR-1 TK-deficient bone marrow cells showed a suppression of multilineage colony formation. Furthermore, macrophages induced to differentiate in vitro showed a decrease in immunologic reactions such as phagocytosis and the secretion of interleukin-6 (IL-6) and VEGF-A. IntroductionVascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), including VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4), form a regulatory system crucial for normal development and pathological angiogenesis. [1][2][3][4] VEGFRs are structurally related to the Fms/Kit/PDGFR family and contain an extracellular domain carrying 7 immunoglobulin (Ig)-like repeats and a cytoplasmic tyrosine kinase (TK) domain. 5,6 VEGFR-1 and VEGFR-2 are highly expressed on vascular endothelial cells. [7][8][9][10] We and others have recently shown that VEGFR-1 is expressed not only in vascular endothelial cells but also in monocytes/ macrophages, 11,12 and its signaling is involved in the migration of macrophages toward VEGF-A. 13 In addition to VEGF-A, VEGF-B and PlGF are also ligands for VEGFR-1; thus, they could play a role in this signaling under both physiological and pathological conditions. Other nonendothelial cells, including smooth muscle cells, trophoblasts, and osteoblasts, were reported to express VEGFR-1. 14 Mice lacking Vegfr-2 die in the embryonic stage due to a severe deficiency of vascular development. 15 In contrast, mice lacking Vegfr-1 die due to overgrowth and disorganization of the vascular system. 16 Interestingly, however, mouse embryos lacking the TK domain of Vegfr-1 survive without significant defects, 17 suggesting that VEGFR-1 functions as a negative regulator of vascular development by trapping VEGF-A via its ligand-binding domain. 14 Recently, various studies including ours indicated that the expression of VEGFs and VEGFRs is up-regulated in various diseases. 2,4,18-20 VEGFR-1-mediated signaling was shown to play a significant role in a variety of pathological conditions such as carcinogenesis and inflammatory diseases. 19,21,22 VEGFR-1 signaling facilitates tumor angiogenesis and spontaneous lung metastases by inducing the expression of matrix metalloproteinase 9 (MMP-9). 22-24 A recent study also showed that VEGFR-1 is important for the reconstitution of hematopoiesis in bone marrow (BM) after irradiation. 25 Rheumatoid arthritis (RA) 26 is a...

VEGF and its receptor family including VEGFR-1(Flt-1) are well known to be a crucial regulatory system for normal development and pathological angiogenesis. Rheumatoid arthritis(RA) is a chronic systemic disease characterized by an inflammatory erosive synovicitis, which show marked neovascularization, inflammatory cell infiltration and synovial hyperplasia, then produce a pannus of inflammatory vascular tissue and lead to irreversible cartilage and bone destruction. We have already shown VEGFR-1 is expressed not only in vascular endothelial cells but also in inflammatory cells, especially in monocyte/macrophage. A recent report suggests the involvement of VEGFR-1 in RA by using collagen induced RA mouse model. To examine whether the signaling from VEGFR-1 is important for the pathological process of RA, we used VEGFR-1 tyrosine kinase(−/−) mice which cannot generate the signaling from this receptor, and an arthritis mouse model system carrying Human T-cell leukemia virus(HTLV-1) pX transgene. VEGFR-1 TK(−/−) mice with pX gene clearly showed a reduction in the incidence and the degree of clinical symptom of arthritis. Furthermore, the heterozygote VEGFR-1 TK(+/−) with pX transgene showed a partial decrease in the degree of clinical as well as pathological scores. To explain the reason of reduction of clinical symptoms, we investigated involvement of VEGFR-1 TK signal in lineage of bone marrow hematopoietic stem cell(HSC) to monocyte/macrophage proliferation and differentiation and their immunity. VEGFR-1 TK activities are not associated in number of HSC in bone marrow. However, VEGFR-1 TK(−/−) HSC toward multi-lineage proliferation is suppressed in colony-formation. In addition, failures of monocyte/macrophage faculties are observed in immunological reaction, phagocytosis, cytokine secretion(IL-6, VEGF) and migration. Furthermore, expressions of hematopoiesis and inflammation related genes in VEGFR-1 TK(−/−) macrophage are downregulated by microarray analysis. Next we treated with small molecule inhibitors of VEGF receptor(VEGFR-1 and VEGFR-2) of tyrosine kinase, KRN951, in RA model(pX transgenic model and type II collagen Ab cocktail model) for treatment. Treatment with KRN951 strongly attenuated the disease symptom through inhibiting recruitment of BM hematopoietic cells into peripheral inflammatory cells. These observations indicate that VEGFR-1 signals play an important role in both RA mouse model. The tyrosine kinase activity and the signaling of VEGFR-1 enhances hematopoiesis, proliferation/differentiation and immunity of monocyte/macrophage from bone marrow HSC, and promotes rheumatoid arthritis, which may be a new possibilities for the treatment of RA in humans. Figure Figure

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