Blocking neuropilin-2 function inhibits tumor cell metastasis.

Metastasis, which commonly uses lymphatics, accounts for much of the mortality associated with cancer. The vascular endothelial growth factor (VEGF)-C coreceptor, neuropilin-2 (Nrp2), modulates but is not necessary for developmental lymphangiogenesis, and its significance for metastasis is unknown. An antibody to Nrp2 that blocks VEGFC binding disrupts VEGFC-induced lymphatic endothelial cell migration, but not proliferation, in part independently of VEGF receptor activation. It does not affect established lymphatics in normal adult mice but reduces tumoral lymphangiogenesis and, importantly, functional lymphatics associated with tumors. It also reduces metastasis to sentinel lymph nodes and distant organs, apparently by delaying the departure of tumor cells from the primary tumor. Our results demonstrate that Nrp2, which was originally identified as an axon-guidance receptor, is an attractive target for modulating metastasis.

Bv8 and endocrine gland-derived vascular endothelial growth factor stimulate hematopoiesis and hematopoietic cell mobilization.

Bv8 and endocrine-gland-derived VEGF (EG-VEGF), or prokineticins, are two highly related, secreted proteins that we previously described as selective angiogenic mitogens. Here we describe the expression and functional characterization of Bv8 in peripheral blood cells, notably monocytes, neutrophils, and dendritic cells, and in the bone marrow. In human and mouse, the two Bv8 G protein-coupled receptors are expressed in hematopoietic stem cells and specific mature blood cells, including lymphocytes. Bv8 is highly expressed by neutrophils at sites of inflammation and can stimulate migration of monocytes, in a pertussis toxin-sensitive manner. Bv8, or EG-VEGF that shares the same receptors, increased numbers of colony-forming units granulocytic and monocytic in cultures of human or mouse hematopoietic stem cells. Systemic in vivo exposure to Bv8 or EG-VEGF resulted in significant increases in total leukocyte, neutrophil, and monocyte counts. Additionally, adenovirus (Av)Bv8 or AvEG-VEGF delivered just before 5-fluorouracil injury promoted the survival of hematopoietic cells and enhanced progenitor mobilization. In conclusion, Bv8 can promote survival and differentiation of the granulocytic and monocytic lineages. Bv8 potentially modulates growth, survival, and function of cells of the innate and adaptive immune systems, possibly through autocrine or paracrine signaling mechanisms.

Using gene expression profiling to identify the molecular basis of the synergistic actions of hepatocyte growth factor and vascular endothelial growth factor in human endothelial cells.

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) are two potent endothelial mitogens with demonstrated angiogenic activities in animal models of therapeutic angiogenesis. Several recent studies suggest that these growth factors may act synergistically, although the mechanism of this interaction is not understood. Changes in the gene expression profile of human umbilical vein endothelial cells treated with HGF, VEGF or the combination of the two were analyzed with high-density oligonucleotide arrays, representing approximately 22000 genes. Notably, the genes significantly up- and downregulated by VEGF versus HGF exhibited very little overlap, indicating distinct signal transduction pathways. The combination of HGF and VEGF markedly increased the number of significantly up- and downregulated genes. At 4 h, the combination of the two growth factors induced a number of chemokine and cytokines and their receptors (IL-8, IL-6, IL-11, CCR6, CXCR1,CXC1 and IL17RC), numerous genes involved in growth factor signal transduction (egr-1, fosB, grb10, grb14,MAP2K3,MAP3K8, MAPKAP2,MPK3, DUSP4 and DUSP6), as well as a number of other growth factors (PDGFA, BMP2, Hb-EGF, FGF16, heuregulin beta 1, c-kit ligand, angiopoietin 2 and angiopoietin 4 and VEGFC). In addition, the VEGF receptors neuropilin-1 and flt-1 were also upregulated. At 24 h, a clear 'cell cycle' signature is noted, with the upregulated expression of various cell cycle control proteins and gene involved in the regulation of mitosis and mitotic spindle assembly. The receptor for HGF, c-met, is also upregulated. These data are consistent with the hypothesis that the combination of HGF and VEGF results in the cooperative upregulation of a number of different molecular pathways leading to a more robust proliferative response, that is, growth factor(s), receptors, molecules involved in growth factor signal transduction, as well as, at later time points, upregulation of the necessary cellular proteins required for cells to escape cell cycle arrest and enter the cell cycle.

Stanniocalcin 1 is an autocrine modulator of endothelial angiogenic responses to hepatocyte growth factor.

Stanniocalcin 1 (STC1) is a secreted glycoprotein originally described as a hormone involved in calcium and phosphate homeostasis in bony fishes. We recently identified the mammalian homolog of this molecule to be highly up-regulated in an in vitro model of angiogenesis, as well as focally and intensely expressed at sites of pathological angiogenesis (e.g. tumor vasculature). In the present study, we report that STC1 is a selective modulator of hepatocyte growth factor (HGF)-induced endothelial migration and morphogenesis, but not proliferation. STC1 did not inhibit proliferative or migratory responses to vascular endothelial growth factor or basic fibroblast growth factor. The mechanism of STC1 inhibitory effects on HGF-induced endothelial migration seem to occur secondary to receptor activation because STC1 did not inhibit HGF-induced c-met receptor phosphorylation, but did block HGF-induced focal adhesion kinase activation. In the mouse femoral artery ligation model of angiogenesis, STC1 expression closely paralleled that of the endothelial marker CD31, and the peak level of STC1 expression occurred after an increase in HGF expression. We propose that STC1 may play a selective modulatory role in angiogenesis, possibly serving as a "stop signal" or stabilizing factor contributing to the maturation of newly formed blood vessels. HGF is a mesenchyme-derived pleiotropic factor with mitogenic, motogenic, and morphogenic activities on a number of different cell types. HGF effects are mediated through a specific tyrosine kinase, c-met, and aberrant HGF and c-met expression are frequently observed in a variety of tumors. Recent studies have shown HGF to be a potent growth factor implicated in wound healing, tissue regeneration, and angiogenesis.

Branching out: a molecular fingerprint of endothelial differentiation into tube-like structures generated by Affymetrix oligonucleotide arrays.

The process of endothelial differentiation into a network of tube-like structures with patent lumens requires an integrated program of gene expression. To identify genes upregulated in endothelial cells during the process of tube formation, RNA was prepared from several different time points (0, 4, 8, 24, 40, and 48 hours) and from three different experimental models of human endothelial tube formation: in collagen gels and fibrin gels driven by the combination of PMA (80), bFGF (40 ng/ml) and bFGF (40 ng/ml) or in collagen gels driven by the combination of HGF (40 ng/ml) and VEGF (40 ng/ml). Gene expression was evaluated using Affymetrix Gene Chip oligonucleotide arrays. Over 1000 common genes were upregulated greater than twofold over baseline at one or more time points in the three different models. In the present study, we discuss the identified genes that could be assigned to major functional classes: apoptosis, cytoskeleton, proteases, matrix, and matrix turnover, pumps and transporters, membrane lipid turnover, and junctional molecules or adhesion proteins.

Vascular endothelial growth factor-induced genes in human umbilical vein endothelial cells: relative roles of KDR and Flt-1 receptors.

OBJECTIVE: This study evaluated the relative roles of the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 in the mediation of altered gene expression elicited by VEGF. METHODS AND RESULTS: We used mutants of VEGF selective for the KDR and Flt-1 receptors to differentiate gene expression patterns mediated by wild-type VEGF (VEGFwt) in human umbilical vein endothelial cells. RNA was extracted from cells treated for 24 hours with 1 nmol/L of each ligand, and gene expression was monitored by using oligonucleotide arrays (Affymetrix U95A). We report that activation of KDR was sufficient to upregulate all the genes induced by VEGFwt. In contrast, there were no genes selectively upregulated by the Flt-selective mutant. However, high concentrations of the Flt-selective mutant could augment the expression of some genes induced by submaximal concentrations of VEGFwt but not the KDR-selective mutant. CONCLUSIONS: The binding of VEGF to its receptor, KDR, is necessary and sufficient to induce the gene expression profile induced by this growth factor. Furthermore, in human umbilical vein endothelial cells, the Flt-1 receptor appears to act as a decoy receptor, tempering the response to lower concentrations of VEGF.

Stanniocalcin 1 alters muscle and bone structure and function in transgenic mice.

Fish stanniocalcin (STC) inhibits uptake of calcium and stimulates phosphate reabsorption. To determine the role of the highly homologous mammalian protein, STC-1, we created and characterized transgenic mice that express STC-1 under control of a muscle-specific promoter. STC-1 transgenic mice were smaller than wild-type littermates and had normal growth plate cartilage morphology but increased cartilage matrix synthesis. In STC-1 mice, the rate of bone formation, but not bone mineralization, was decreased. Increased cortical bone thickness and changes in trabeculae number, density, and thickness in STC-1 mice indicated a concomitant suppression of osteoclast activity, which was supported by microcomputed tomography analyses and histochemistry. Skeletal muscles were disproportionately small and showed altered function and response to injury in STC-1 mice. Electron microscopy indicated that muscle mitochondria were dramatically enlarged in STC-1 mice. These changes in STC-1 mice could not be explained by deficits in blood vessel formation, as vascularity in organs and skeletal tissues was increased as was induction of vascularity in response to femoral artery ligation. Our results indicate that STC-1 can affect calcium homeostasis, bone and muscle mass and structure, and angiogenesis through effects on osteoblasts, osteoclasts, myoblasts/myocytes, and endothelial cells.

In silico data filtering to identify new angiogenesis targets from a large in vitro gene profiling data set.

The objective of this study was to use gene expression data from well-defined cell culture models, in combination with expression data from diagnostic samples of human diseased tissues, to identify potential therapeutic targets and markers of disease. Using Affymetrix oligonucleotide array technology, we identified a common profile of genes upregulated during endothelial morphogenesis into tubelike structures in three in vitro models of angiogenesis. Rigorous data selection criteria were used to identify a list of over 1,000 genes whose expression was increased more than twofold over baseline at either 4, 8, 24, 40 or 50 h. To further refine and prioritize this list, we used standard bioinformatic algorithms to identify potential transmembrane and secreted proteins. We then overlapped this gene set with genes upregulated in colon tumors vs. normal colon, resulting in a subset of 128 genes in common with our endothelial list. We removed from this list those genes expressed in 6 different colon tumor lines, resulting in a list of 24 putative, vascular-specific angiogenesis-associated genes. Three genes, gp34, stanniocalcin-1 (STC-1), and GA733-1, were expressed at levels 10-fold or more in colon tumors compared with normal mucosa. We validated the vascular-specific expression of one of these genes, STC-1, by in situ hybridization. The ability to combine in vitro and in vivo data sets should permit one to identify putative angiogenesis target genes in various tumors, chronic inflammation, and other disorders where therapeutic manipulation of angiogenesis is a desirable treatment modality.