Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly.
Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.
Sweeney
Shawn M
SM
Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
DiLullo
Gloria
G
Slater
Simon J
SJ
Martinez
José
J
Iozzo
Renato V
RV
Lauer-Fields
Janelle L
JL
Fields
Gregg B
GB
San Antonio
James D
JD
D001665:Binding Sites / Q000235:genetics; D002196:Capillaries / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D024042:Collagen Type I / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D004789:Enzyme Activation / Q000187:drug effects; D005109:Extracellular Matrix / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents / Q000378:metabolism; D038982:Integrin alpha2beta1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000502:physiology*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014471:Umbilical Veins / Q000166:cytology; D048051:p38 Mitogen-Activated Protein Kinases
D024042:Collagen Type I; D007202:Indicators and Reagents; D038982:Integrin alpha2beta1; D008164:Luminescent Proteins; D010446:Peptide Fragments; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases