Vascular endothelial cells sense and respond to pressure by molecular mechanism(s) which, to date, remain poorly understood. The present study investigated basic fibroblast growth factor (bFGF) signaling as a putative mechanotransduction pathway involved in the proliferative responses of human umbilical vein endothelia cells (HUVECs) to 60/20 mm Hg cyclic pressure at 1 Hz for 24 h. Under these conditions, the enhanced proliferative response of these HUVECs was not associated with an increased synthesis/release of bFGF, but involved rapid (within 30 min from the onset of exposure to pressure) tyrosine phosphorylation of the bFGF receptor, FGFR-2. Furthermore, monoclonal antibodies to either bFGF or FGFR-2 attenuated the increased proliferation of HUVECs exposed to 60/20 mm Hg cyclic pressure. HUVECs proliferation under 60/20 mm Hg at 1 Hz cyclic pressure is, therefore, dependent upon bFGF and involves FGFR-2 activation.

We have previously utilized a combination of high throughput sequencing and genome-wide microarray profiling analyses to identify novel cell-surface proteins expressed in human umbilical vein endothelial cells. One gene identified by this approach encodes a type I transmembrane receptor that shares sequence homology with the intracellular domain of members of the interleukin-17 (IL-17) receptor family. Real-time quantitative PCR and Northern analyses revealed that this gene is highly expressed in human umbilical vein endothelial cells and in several highly vascularized tissues such as kidney, colon, skeletal muscle, heart, and small intestine. In addition, we also found that it is also highly expressed in the ductal epithelial cells of human salivary glands, seminal vesicles, and the collecting tubules of the kidney by in situ hybridization. This putative receptor, which we have termed human SEF (hSEF), is also expressed in a variety of breast cancer tissues. In co-immunoprecipitation assays, this receptor is capable of forming homomeric complexes and can interact with fibroblast growth factor (FGF) receptor 1. Overexpression of this receptor inhibits FGF induction of an FGF-responsive reporter gene in human 293T cells. This appears to occur as a result of specific inhibition of p42/p44 ERK in the absence of upstream MEK inhibition. This inhibitory effect is dependent upon a functional intracellular domain since deletion mutants missing the IL-17 receptor-like domain lack this inhibitory effect. These findings are consistent with the recent discovery of the zebrafish homologue, Sef (similar expression to fgf genes), which specifically antagonizes FGF signaling when ectopically expressed in zebrafish or Xenopus laevis embryos. Based on sequence and functional similarities, this novel IL-17 receptor homologue represents a potential human SEF and is likely to play critical roles in endothelial or epithelial functions such as proliferation, migration, and angiogenesis.

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 (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.

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.

Although numerous studies have documented the importance of mechanical forces in regulating many endothelial cell functions, the effects of these physical stimuli on endothelial barrier function are not well characterized. The present study used a custom-designed, cyclic pressure system to expose human umbilical vein endothelial cells (HUVECs) to physiologically relevant sinusoidal pressures and demonstrated that exposure to 140/100, but not to 60/20, mm Hg cyclic pressure at 1 Hz for 18 h resulted in a significant (p <.05) reduction in transendothelial permeability to albumin. Moreover, these cyclic pressure-selective changes in HUVEC barrier function occurred concomitantly with redistribution of intracellular tight junction protein zona occludens (ZO)-1 and reorientation of the F-actin cytoskeleton. In contrast, exposure of HUVECs to cyclic pressure had no affect on localization of adherens junctions proteins, vascular endothelial (VE)-cadherin, and beta-catenin. These results, therefore, provide the first evidence that select levels of cyclic pressure, a mechanical force pertinent to the hemodynamic vascular milieu, modulates the endothelial barrier function concomitant with an altered distribution of tight junction component, ZO-1.

DNA microarrays were used to measure the time course of gene expression during skeletal muscle damage and regeneration in mice following femoral artery ligation (FAL). We found 1,289 known sequences were differentially expressed between the FAL and control groups. Gene expression peaked on day 3, and the functional cluster "inflammation" contained the greatest number of genes. Muscle function was depressed for 3 days postligation, but returned to normal by day 7. Decreased muscle function was accompanied by reduced expression of genes involved in mitochondrial energy production, muscle contraction, and calcium handling. The induction of MyoD on day 1 denoted the beginning of muscle regeneration and was followed by the reemergence of the embryonic forms of muscle contractile proteins, which peaked at day 7. Transcriptional analysis indicated that the ischemic skeletal muscle may transition through a functional adaptation stage with recovery of contractile force prior to full regeneration. Several members of the insulin-like growth factor axis were coordinately induced in a time frame consistent with their playing a role in the regenerative process.

Recent advances in gene expression profiling have led to the development of comprehensive databases which can be queried in various manners. In the present report, we have taken a list of genes previously associated with angiogenesis, either in in vivo or in in vitro models, and queried a commercial database established by GeneLogic® to determine the relative expression of these candidate genes in normal kidneys and in renal cell carcinomas (RCC). We identified a number of genes, including CXCR4, matrix metalloproteinase 9, thrombospondin 2, and vascular endothelial growth factor, that were highly expressed in RCC versus normal tissue. One gene, hevin, appears to be selectively upregulated in RCC in contrast to downregulation of this gene in lung and colon tumors. This approach provides a powerful means to identify potential markers of tumor vascularization.

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.

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.

Mechanical forces modulate endothelial cell functions through several mechanisms including regulation of gene transcription. In the present study, gene transcription by human umbilical vein endothelial cells (HUVEC) either maintained under control pressure (that is, standard cell culture conditions equivalent to 0.15 mmHg sustained hydrostatic pressure) or exposed to 60/20 mmHg sinusoidal pressures at 1 Hz were compared using Affymetrix GeneChip microarrays to identify cellular/molecular mechanisms associated with endothelial cell responses to cyclic pressure. Cyclic pressure selectively affected transcription of 14 genes that included a set of mechanosensitive proteins involved in hemostasis (tissue plasminogen activator), cell adhesion (integrin-alpha2), and cell signaling (Rho B, cytosolic phospholipase A2), as well as a unique subset of cyclic pressure-sensitive genes such as vascular endothelial growth factor (VEGF)-C and transforming growth factor (TGF)-beta2. The present study also provided first evidence that VEGF-C, the most highly induced gene under 60/20 mmHg, mediated HUVEC proliferation in response to this cyclic pressure. Cyclic pressure is, therefore, a mechanical force that modulates endothelial cell functions (such as proliferation) by activating a specific transcriptional program.

PAK1 is a protein kinase downstream of the small GTPases Rac and Cdc42 that previous work has implicated in endothelial cell migration via modulation of cell contraction. The first proline-rich region of PAK that binds to an SH3 domain from the adapter protein NCK was responsible for these dominant-negative effects. To test the role of PAK in angiogenesis, we prepared a peptide in which the proline-rich region was fused to the polybasic sequence from the HIV Tat protein to facilitate entry into cells. We show that the short peptide selectively binds NCK, whereas a mutant peptide does not. Treatment of cells with the PAK peptide but not the control peptide disrupts localization of PAK. This peptide specifically inhibited endothelial cell migration and contractility similarly to full-length dominant-negative PAK. In an in vitro tube-forming assay, the PAK peptide specifically blocked formation of multicellular networks. In an in vivo chick chorioallantoic membrane assay, the PAK peptide specifically blocked angiogenesis. These results, therefore, suggest a role for PAK in angiogenesis.

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.

ABSTRACTObjective: Adhesive interactions between tumor cell surface receptors and endothelial cell adhesion molecules are thought to contribute to tumor cell arrest and extravasation during hematogenous metastasis. Recent reports suggest that melanoma cell integrin α4β1 (very late antigen‐4, VLA‐4) interaction with the inducible cell adhesion molecule, vascular cell adhesion molecule‐1 (VCAM‐1), is critical for tumor cell arrest. However, no information is available regarding microvascular VCAM‐1 expression during spontaneous melanoma metastasis. The objectives of this study were to evaluate VCAM‐1 expression in pulmonary and extrapulmonary vascular beds during melanoma progression, and to determine whether there is an organ‐specific profile for VCAM‐1 expression which corresponds with the clinical pattern of melanoma metastasis.Methods: The dual‐radiolabeled monoclonal antibody (mAb) technique for quantification of VCAM‐1 in different vascular beds was applied to a physiological model of melanoma (B16‐BL6) metastasis. Measurements of VCAM‐1 were obtained when primary tumors reached 5 mm in size, and every 7 days following removal of the primary lesion. Histological examinations were performed, and mice were placed into 2 groups, based on the presence (+colonies) or absence (−colonies) of pulmonary metastases. VCAM‐1 measurements obtained from several organ systems were then compared between these 2 groups of mice. Localization of VCAM‐1 was achieved through immunohistochemical staining of tissues. Plasma collected from each experimental animal, as well as melanoma‐conditioned media, was assayed to determine levels of the cytokines tumor necrosis factor‐α (TNF‐α) and interleukin‐1α (IL‐1α).Results: Data collected from the dual‐radiolabeled mAb technique indicate that 3 weeks following removal of the primary lesion, there is a tendency for VCAM‐1 expression to increase in cardiac, hepatic, and cerebral vascular beds. Four weeks following primary resection, when pulmonary metastatic burden was maximal, VCAM‐1 was significantly upregulated in each of these vascular beds. Results obtained from the lung indicate that VCAM‐1 remains unchanged in pulmonary vessels at all time points examined. Immunohistochemical staining of heart and brain support the radiolabeled mAb measurements, and reveals that these organs exhibit an inflammatory phenotype in mice with heavy pulmonary tumor burden. Furthermore, 25% of these mice had histological evidence of melanoma metastases in heart and brain. Transplantation of liver fragments from mice with advanced pulmonary metastases into subcutaneous tissue of donor mice resulted in the formation of melanotic outgrowths. Plasma levels of the cytokines TNF‐α and IL‐1α were negligible in both groups of mice.Conclusions: Our results indicate that upregulation of VCAM‐1 is not a prerequisite for the formation of pulmonary metastases during spontaneous melanoma metastases. However, once lung metastases become well established, organ‐specific increases in VCAM‐1 expression become apparent. Furthermore, these organ‐specific increments in VCAM‐1 expression correspond with documented clinical patterns of melanoma metastasis. The enhanced expression of VCAM‐1 is independent of systemic levels of TNF‐α and IL‐1α, but may be the result of melanoma‐induced alterations at the local level, as we found evidence of melanoma cell occupation in heart, brain, and liver in pulmonary metastases‐bearing mice.

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in both physiological and pathological processes. Hepatocyte growth factor (HGF) is a mesenchyme-derived mitogen that also stimulates cell migration, and branching and/or tubular morphogenesis of epithelial and endothelial cells. In the present study, we tested the hypothesis that simultaneous administration of HGF and VEGF would synergistically promote new blood vessel formation. HGF acted in concert with VEGF to promote human endothelial cell survival and tubulogenesis in 3-D type I collagen gels, a response that did not occur with either growth factor alone. The synergistic effects of VEGF and HGF on endothelial survival correlated with greatly augmented mRNA levels for the anti-apoptotic genes Bcl-2 and A1. Co-culture experiments with human neonatal dermal fibroblasts and human umbilical vein endothelial cells demonstrated that neonatal dermal fibroblasts, in combination with VEGF, stimulated human umbilical vein endothelial cells tubulogenesis through the paracrine secretion of HGF. Finally, in vivo experiments demonstrated that the combination of HGF and VEGF increased neovascularization in the rat corneal assay greater than either growth factor alone. We suggest that combination therapy using HGF and VEGF co-administration may provide a more effective strategy to achieve therapeutic angiogenesis.

Vascular endothelial cell growth factor (VEGF) binds to 2 related receptor tyrosine kinases, known as kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1). The KDR has been shown to mediate VEGF-stimulated endothelial cell mitogenesis, migration, and permeability. The Flt-1 receptor has been suggested to mediate VEGF-stimulated endothelial branching morphogenesis, a process whereby endothelial cells, in the presence of a 3D milieu composed of extracellular matrix components and a mixture of growth factors, undergo a morphological transition into a tubular network with many lumina. In the present study, we have used 2 independent endothelial cell tube formation models and highly selective VEGF mutants for the KDR and Flt-1 receptors. We demonstrate that KDR, not Flt-1, stimulation is responsible for the induction of endothelial tubulogenesis. In addition, we demonstrate a modulatory role for Flt-1 in VEGF-mediated tube formation. We also report that VEGF-driven endothelial tube formation is inhibited by selective inhibitors of mitogen-activated protein kinase activation and p38 protein kinase.