Journal of Biological Chemistry
Volume 290, Issue 6, 6 February 2015, Pages 3405-3417
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Cell Biology
Targeted Identification of Sialoglycoproteins in Hypoxic Endothelial Cells and Validation in Zebrafish Reveal Roles for Proteins in Angiogenesis

https://doi.org/10.1074/jbc.M114.618611Get rights and content
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The formation of new vessels in the tumor, termed angiogenesis, is essential for primary tumor growth and facilitates tumor invasion and metastasis. Hypoxia has been described as one trigger of angiogenesis. Indeed, hypoxia, which is characterized by areas of low oxygen levels, is a hallmark of solid tumors arising from an imbalance between oxygen delivery and consumption. Hypoxic conditions have profound effects on the different components of the tumoral environment. For example, hypoxia is able to activate endothelial cells, leading to angiogenesis but also thereby initiating a cascade of reactions involving neutrophils, smooth muscle cells, and fibroblasts. In addition, hypoxia directly regulates the expression of many genes for which the role and the importance in the tumoral environment remain to be completely elucidated. In this study, we used a method to selectively label sialoglycoproteins to identify new membrane and secreted proteins involved in the adaptative process of endothelial cells by mass spectrometry-based proteomics. We used an in vitro assay under hypoxic condition to observe an increase of protein expression or modifications of glycosylation. Then the function of the identified proteins was assessed in a vasculogenesis assay in vivo by using a morpholino strategy in zebrafish. First, our approach was validated by the identification of sialoglycoproteins such as CD105, neuropilin-1, and CLEC14A, which have already been described as playing key roles in angiogenesis. Second, we identified several new proteins regulated by hypoxia and demonstrated for the first time the pivotal role of GLUT-1, TMEM16F, and SDF4 in angiogenesis.

Background: Target identification on tumor microvascularization is an opportunity for cancer therapy.

Results: Membrane and secreted glycoproteins were identified on endothelial cells under hypoxia, and their function was assessed in zebrafish.

Conclusion: Three novel hypoxia-regulated glycoproteins involved in angiogenesis have been identified and validated.

Significance: We describe an approach that can be used to rapidly identify novel angiogenesis-related genes and their protein products.

Angiogenesis
Endothelial Cell
Hypoxia
Vascular Biology
Zebrafish
Adhesion Receptor

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