Biomedical subjects

Stephen D Robinson

Publications and source records attributed to Stephen D Robinson.

4 recordsLinked to original sources

PubMed2006

Generation of genetically modified embryonic stem cells for the development of knockout mouse animal model systems.

The aim of our lab is to understand the contributions made by cell adhesion molecules in the processes of disease. Much of our recent work has focused on the role played by beta3-integrin in mediating pathological angiogenesis. It is fair to state that without the ability to manipulate the mouse genome, and specifically to create knockout mice, the advances we have made in this field would not be nearly as significant as they are. The ability to generate knockout mice depends on the two technological breakthroughs of the ability to isolate and culture mouse embryonic stem (ES) cells and the methods employed for achieving targeted gene replacement in these cells by homologous recombination. Here, we present the methods we have found to be successful, and that we routinely employ to grow and manipulate ES cells, as well as those to screen and identify homologous recombinants.

Animals↗

PubMed2004

Elevated Flk1 (vascular endothelial growth factor receptor 2) signaling mediates enhanced angiogenesis in beta3-integrin-deficient mice.

Tumor growth, tumor angiogenesis, and vascular endothelial growth factor (VEGF)-specific angiogenesis are all enhanced in beta(3)-integrin-null mice. Furthermore, endothelial cells isolated from beta(3)-null mice show elevated levels of Flk1 (VEGF receptor 2) expression, suggesting that beta(3)-integrin can control the amplitude of VEGF responses by controlling Flk1 levels or activity. We now show that Flk1 signaling is required for the enhanced tumor growth and angiogenesis seen in beta(3)-null mice. Moreover, beta(3)-null endothelial cells exhibit enhanced migration and proliferation in response to VEGF in vitro, and this phenotype requires Flk1 signaling. Upon VEGF stimulation, beta(3)-null endothelial cells exhibit higher levels of phosphorylated Flk1 and extracellular-related kinases 1 and 2 than wild-type endothelial cells. Furthermore, signaling via ERK1/2 is required to mediate the elevated responses to VEGF observed in beta(3)-null endothelial cells and aortic rings in vitro. These data confirm that VEGF signaling via Flk1 is enhanced in beta(3)-integrin-deficient mice and suggests that this increase may mediate the enhanced angiogenesis and tumor growth observed in these mice in vivo.

Animals↗

PubMed2004

Beta3-integrin regulates vascular endothelial growth factor-A-dependent permeability.

OBJECTIVE: Beta3-integrin deficiency has been implicated in increasing levels of Flk-1 expression on endothelial cells and enhancing vascular endothelial growth factor (VEGF)-induced angiogenesis. We determined the role of beta3-integrin in mediating VEGF-A-induced blood vessel permeability through Flk-1. METHODS AND RESULTS: Using the Miles assay, we demonstrated that VEGF-A-induced plasma leakage was enhanced in beta3-null mice when compared with wild-type controls. This was not caused by any changes in blood vessel structure (as detected by light or electron microscopy) or by changes in endothelial cell-cell adhesion proteins (as determined by Western blot analysis, flow cytometry, and immunofluorescence). Circulating levels of VEGF, baseline blood vessel leakage, and leakage in response to an acute inflammatory stimulus were identical in wild-type and beta3-null mice. However, VEGF-A-induced leakage was abolished in beta3-null mice by the inhibition of Flk-1, indicating that the elevated levels of Flk-1 on beta3-null endothelial cells enhance VEGF-A-induced permeability. CONCLUSIONS: beta3-integrin-deficiency increases the sensitivity of endothelial cells to VEGF-A by elevating Flk-1 expression and, as a consequence, enhances VEGF-A-mediated permeability.

Animals↗

PubMed2002

Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins.

Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.

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