title: The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell lumen formation.
abstract: A role for fibroblasts in physiological and pathological angiogenesis is now well recognized; however, the precise mechanisms underlying their action have not been determined. Using an in vitro angiogenesis model in combination with a candidate gene approach, column chromatography, and mass spectrometry, we identify two classes of fibroblast-derived factors--one that supports vessel sprouting but not lumen formation, and one that promotes lumen formation. In the absence of fibroblasts a combination of angiopoietin-1, angiogenin, hepatocyte growth factor, transforming growth factor-α, and tumor necrosis factor drives robust endothelial cell (EC) sprouting; however, lumens fail to form. Subsequent addition of fibroblast-conditioned medium restores lumenogenesis. Using small interfering RNA-mediated knockdown, we show that five genes expressed in fibroblasts--collagen I, procollagen C endopeptidase enhancer 1, secreted protein acidic and rich in cysteine, transforming growth factor-β-induced protein ig-h3, and insulin growth factor-binding protein 7--are necessary for lumen formation. Moreover, lumen formation can be rescued by addition of purified protein to knockdown cultures. Finally, using rheology, we demonstrate that the presence of these matricellular proteins results in significantly stiffer gels, which correlates with enhanced lumen formation. These findings highlight the critical role that fibroblast-derived extracellular matrix components play in EC lumen formation and provide potential insight into the role of fibroblasts in the tumor microenvironment.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Newman
authors.forename: Martin N
authors.initials: MN
authors.lastname: Nakatsu
authors.forename: Wayne
authors.initials: W
authors.lastname: Chou
authors.forename: Paul D
authors.initials: PD
authors.lastname: Gershon
authors.forename: Christopher C W
authors.initials: CC
authors.lastname: Hughes
mesh_terms: D042501:Angiogenic Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002845:Chromatography; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000378:metabolism / Q000494:pharmacology; D005109:Extracellular Matrix / Q000737:chemistry* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D000071816:Glycodelin; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D009389:Neovascularization, Pathologic; D018919:Neovascularization, Physiologic; D011257:Pregnancy Proteins / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D012212:Rheology; D015398:Signal Transduction / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D000099222:betaIG-H3 Protein
chemical_list: D042501:Angiogenic Proteins; D002352:Carrier Proteins; D003094:Collagen; D017077:Culture Media, Conditioned; D016326:Extracellular Matrix Proteins; D000071816:Glycodelin; D006023:Glycoproteins; D047908:Intracellular Signaling Peptides and Proteins; D011257:Pregnancy Proteins; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein; C045246:PAEP protein, human; C491555:PCOLCE protein, human