Vascular progenitors derived from murine bone marrow stromal cells are regulated by fibroblast growth factor and are avidly recruited by vascularizing tumors.

Bone marrow-derived stromal cells (BMSC) possess a population of vascular progenitor cells that enable them to acquire a histology and immunophenotype coherent with endothelial cells (EC). Recent evidence indicates that a hypoxic environment such as that encountered in tumor masses regulates BMSC angiogenic properties by pathways that remain to be defined. It is also unclear as to what extent these marrow-derived precursor cells could contribute to the growth of endothelium-lined vessels at the vicinity of tumor masses. In this study, we found that BMSC exhibited the ability to generate three-dimensional capillary-like networks on Matrigel, and that this property was up-regulated by growth factors-enriched conditioned media isolated from several tumor-derived cell lines. In particular, basic fibroblast growth factor, a key mediator of angiogenesis, was found to be the most potent growth factor for inducing BMSC proliferation, migration, and tubulogenesis. The setup of a new two-dimensional in vitro co-culture assay further showed that BMSC were massively recruited when cultured in the presence of either cancerous or differentiated EC lines. In vivo, subcutaneous co-injection of BMSC with U-87 glioma cells in nude mice resulted in the formation of highly vascularized tumors, where BMSC differentiated into CD31-positive cells and localized at the lumen of vascular structures. Our data suggest that BMSC could be recruited at the sites of active tumor neovascularization through paracrine regulation of their angiogenic properties. These observations may have crucial implications in the development of novel therapies using BMSC engineered to secrete anti-cancerous agents and to antagonize tumor progression.

Radiation induced-tubulogenesis in endothelial cells is antagonized by the antiangiogenic properties of green tea polyphenol (-) epigallocatechin-3-gallate.

Radiation therapy is a widely-used option for the treatment of a variety of solid tumors. Although effective, ionizing radiation (IR) may give rise to various side effects, including secondary tumors. In agreement with this, recent reports have demonstrated increased invasive potential in different tumor-derived cell lines following radiation treatment. Many of the molecular effects of IR specifically on the endothelial cells involved in tumor neo-vascularization remain unknown. In this study, we found that low sublethal single doses of IR applied to human umbilical vein endothelial cells stimulated cell migration and in vitro tubulogenesis. This correlated with an increase in membrane type-1 matrix metalloproteinase (MT1-MMP) protein expression, a crucial enzyme that promotes endothelial cell migration and tube formation, and of caveolin-1, a protein that regulates tube formation. Cell adhesion was also promoted by IR, reflected in increased gene expression levels of cell surface beta(3) integrin. Pretreatment of the cells with epigallocatechin-3-gallate (EGCg), a green tea catechin that possesses anti-angiogenic properties, prevented most of the IR-induced cellular and molecular events. These observations suggest that current protocols involving radiation therapy for the treatment of cancer can paradoxically promote angiogenesis, but can be improved by combination with anti-angiogenic molecules such as EGCg to target those tumor-derived endothelial cells that escaped IR-induced apoptosis.

Matrix metalloproteinase regulation of sphingosine-1-phosphate-induced angiogenic properties of bone marrow stromal cells.

OBJECTIVE: Bone marrow-derived stromal cells (MSC) are able to acquire histological and immunophenotypic characteristics consistent with endothelial cells (EC). In this study we examined the effect of sphingosine-1-phosphate (S1P), a platelet-derived bioactive lysophospholipid that is believed to specifically stimulate EC migration and tube formation, on the angiogenic properties of MSC. METHODS: MSC were isolated from murine bone marrow and cultured in the presence of diverse angiogenic growth factors. Using a chemotaxis chamber and Matrigel tubulogenesis assay, we measured the extent of MSC migration and capillary-like structure formation. Western blots and zymography were used to assess the levels and activation states of soluble and membrane-bound matrix metalloproteinase (MMP). RESULTS: We found that S1P strongly induced MSC migration and in vitro capillary-like structure formation. Ilomastat, a broad-spectrum MMP inhibitor, antagonized several angiogenic and S1P-mediated events in MSC. These included 1) the inhibition of S1P-induced tube formation, 2) the inhibition of concanavalin-A (Con-A)-mediated proMMP-2 activation, and 3) the inhibition of S1P- and Con-A-induced caspase-3 activity. Moreover, S1P induced membrane type-1 (MT1)-MMP mRNA and protein expression, but paradoxically antagonized its cell surface proteolytic processing. In addition, anti-angiogenic agents such as Ilomastat, Neovastat, and green tea polyphenol epigallocatechin-3-gallate antagonized the S1P-induced migration of MSC as well as that of transfected COS-7 cells overexpressing the recombinant receptor for S1P, EDG-1. CONCLUSION: Collectively, our results indicate a crucial role for S1P/EDG-1-mediated angiogenic and survival events in the regulation of microvascular network remodeling by MSC, and may provide a new molecular link between hemostasis and angiogenesis processes.

Hypoxia promotes murine bone-marrow-derived stromal cell migration and tube formation.

Recent evidence indicates that bone-marrow-derived stromal cells (MSCs) have a histology coherent with endothelial cells that may enable them to contribute to tumor angiogenesis through yet undefined mechanisms. In this work, we investigated the angiogenic properties of murine MSCs involved in extracellular matrix degradation and in neovascularization that could take place in a hypoxic environment such as that encountered in tumor masses. MSCs were cultured in normoxia (95% air and 5% CO(2)) or in hypoxia (1% oxygen, 5% CO(2), and 94% nitrogen). We found that hypoxic culture conditions rapidly induced MSC migration and three-dimensional capillary-like structure formation on Matrigel. In vitro, MSC migration was induced by growth-factor- and cytokine-enriched conditioned media isolated from U-87 glioma cells as well as from MSCs cultured in hypoxic conditions, suggesting both paracrine and autocrine regulatory mechanisms. Although greater vascular endothelial growth factor levels were secreted by MSCs in hypoxic conditions, this growth factor alone could not explain their greater migration. Interestingly, matrix metalloproteinase (MMP)-2 mRNA expression and protein secretion were downregulated, while those of membrane-type (MT)1-MMP were strongly induced by hypoxia. Functional inhibition of MT1-MMP by a blocking antibody strongly suppressed MSC ability to migrate and generate capillary-like structures. Collectively, these data suggest that MSCs may have the capacity to participate in tumor angiogenesis through regulation of their angiogenic properties under an atmosphere of low oxygen that closely approximates the tumor microenvironment.