title: Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling.
abstract: Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is known to be Vegfa dependent, we tested the hypothesis that Crim1 is involved in the regulation of Vegfa signaling. Consistent with this hypothesis, we showed that VEC-specific conditional compound heterozygotes for Crim1 and Vegfa exhibit a phenotype that is more severe than each single heterozygote and indistinguishable from that of the conditional homozygotes. We further showed that human CRIM1 knockdown in cultured VECs results in diminished phosphorylation of VEGFR2, but only when VECs are required to rely on an autocrine source of VEGFA. The effect of CRIM1 knockdown on reducing VEGFR2 phosphorylation was enhanced when VEGFA was also knocked down. Finally, an anti-VEGFA antibody did not enhance the effect of CRIM1 knockdown in reducing VEGFR2 phosphorylation caused by autocrine signaling, but VEGFR2 phosphorylation was completely suppressed by SU5416, a small-molecule VEGFR2 kinase inhibitor. These data are consistent with a model in which Crim1 enhances the autocrine signaling activity of Vegfa in VECs at least in part via Vegfr2.
authors.affiliation: Divisions of Pediatric Ophthalmology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
authors.forename: Jieqing
authors.initials: J
authors.lastname: Fan
authors.forename: Virgilio G
authors.initials: VG
authors.lastname: Ponferrada
authors.forename: Tomohito
authors.initials: T
authors.lastname: Sato
authors.forename: Shruti
authors.initials: S
authors.lastname: Vemaraju
authors.forename: Marcus
authors.initials: M
authors.lastname: Fruttiger
authors.forename: Holger
authors.initials: H
authors.lastname: Gerhardt
authors.forename: Napoleone
authors.initials: N
authors.lastname: Ferrara
authors.forename: Richard A
authors.initials: RA
authors.lastname: Lang
mesh_terms: D000483:Alleles; D000818:Animals; D019898:Autocrine Communication; D052004:Bone Morphogenetic Protein Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006579:Heterozygote; D006720:Homozygote; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D018919:Neovascularization, Physiologic; D020286:Pericytes / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D012171:Retinal Vessels / Q000196:embryology / Q000254:growth & development* / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: C486175:CRIM1 protein, human; C518411:Crim1 protein, mouse; D008565:Membrane Proteins; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D040301:Vascular Endothelial Growth Factor Receptor-2; D052004:Bone Morphogenetic Protein Receptors
keywords: Angiogenesis; Crim1; Endothelial cell; Vegfa