Targeted identification of sialoglycoproteins in hypoxic endothelial cells and validation in zebrafish reveal roles for proteins in angiogenesis.
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.
From Sanofi Research and Development, 195 route d'Espagne, 31000 Toulouse, France, Centre de recherche en Cancérologie de Toulouse, INSERM UMR1037, Université de Toulouse, 20-24 rue du pont Saint-Pierre, 31057 Toulouse, France.
Nicolas
N
Delcourt
Biobide, S. L., Paseo Mikeletegi 58, 20009 San Sebastián-Donostia, Spain, and BBD-BioPhenix S. L.-Bionaturis group, Paseo Mikeletegi 56, 20009 San Sebastián-Donostia, Spain.
Celia
C
Quevedo
From Sanofi Research and Development, 195 route d'Espagne, 31000 Toulouse, France.
Christelle
C
Nonne
From Sanofi Research and Development, 195 route d'Espagne, 31000 Toulouse, France.
Pierre
P
Fons
Donogh O'Brien BioConsulting, Les Poirioux, 18310 St. Outrille, France.
Donogh
D
O'Brien
From Sanofi Research and Development, 195 route d'Espagne, 31000 Toulouse, France.
Denis
D
Loyaux
Biobide, S. L., Paseo Mikeletegi 58, 20009 San Sebastián-Donostia, Spain, and.
Maria
M
Diez
From Sanofi Research and Development, 195 route d'Espagne, 31000 Toulouse, France.
François
F
Autelitano
From Sanofi Research and Development, 195 route d'Espagne, 31000 Toulouse, France.
Jean-Claude
JC
Guillemot
From Sanofi Research and Development, 195 route d'Espagne, 31000 Toulouse, France.
Pascual
P
Ferrara
BBD-BioPhenix S. L.-Bionaturis group, Paseo Mikeletegi 56, 20009 San Sebastián-Donostia, Spain.
Arantza
A
Muriana
Biobide, S. L., Paseo Mikeletegi 58, 20009 San Sebastián-Donostia, Spain, and.
Carlos
C
Callol
From Sanofi Research and Development, 195 route d'Espagne, 31000 Toulouse, France.
Jean-Pascal
JP
Hérault
From Sanofi Research and Development, 195 route d'Espagne, 31000 Toulouse, France.
Jean-Marc
JM
Herbert
Centre de recherche en Cancérologie de Toulouse, INSERM UMR1037, Université de Toulouse, 20-24 rue du pont Saint-Pierre, 31057 Toulouse, France.
Gilles
G
Favre
From Sanofi Research and Development, 195 route d'Espagne, 31000 Toulouse, France, francoise.bono@sanofi.com.
Françoise
F
Bono
D000222:Adaptation, Physiological; D000818:Animals; D000075368:Anoctamins; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia; D000071063:Endoglin; D051272:Glucose Transporter Type 1 / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D061307:Human Umbilical Vein Endothelial Cells; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism; D018919:Neovascularization, Physiologic; D039942:Neuropilin-1 / Q000235:genetics / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish
C555078:ANO6 protein, human; D000075368:Anoctamins; D015703:Antigens, CD; C570898:CLEC14A protein, human; D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; C064206:ENG protein, human; D000071063:Endoglin; D051272:Glucose Transporter Type 1; D006023:Glycoproteins; D037181:Lectins, C-Type; D049294:Phospholipid Transfer Proteins; D020543:Proteome; D011956:Receptors, Cell Surface; D012795:Sialoglycoproteins; D039942:Neuropilin-1; C099100:SDF4 protein, human
Adhesion Receptor; Angiogenesis; Endothelial Cell; Hypoxia; Vascular Biology; Zebrafish