title: Lack of involvement of CEP adducts in TLR activation and in angiogenesis.
abstract: Proteins that are post-translationally adducted with 2-(ω-carboxyethyl)pyrrole (CEP) have been proposed to play a pathogenic role in age-related macular degeneration, by inducing angiogenesis in a Toll Like Receptor 2 (TLR2)-dependent manner. We have investigated the involvement of CEP adducts in angiogenesis and TLR activation, to assess the therapeutic potential of inhibiting CEP adducts and TLR2 for ocular angiogenesis. As tool reagents, several CEP-adducted proteins and peptides were synthetically generated by published methodology and adduction was confirmed by NMR and LC-MS/MS analyses. Structural studies showed significant changes in secondary structure in CEP-adducted proteins but not the untreated proteins. Similar structural changes were also observed in the treated unadducted proteins, which were treated by the same adduction method except for one critical step required to form the CEP group. Thus some structural changes were unrelated to CEP groups and were artificially induced by the synthesis method. In biological studies, the CEP-adducted proteins and peptides failed to activate TLR2 in cell-based assays and in an in vivo TLR2-mediated retinal leukocyte infiltration model. Neither CEP adducts nor TLR agonists were able to induce angiogenesis in a tube formation assay. In vivo, treatment of animals with CEP-adducted protein had no effect on laser-induced choroidal neovascularization. Furthermore, in vivo inactivation of TLR2 by deficiency in Myeloid Differentiation factor 88 (Myd88) had no effect on abrasion-induced corneal neovascularization. Thus the CEP-TLR2 axis, which is implicated in other wound angiogenesis models, does not appear to play a pathological role in a corneal wound angiogenesis model. Collectively, our data do not support the mechanism of action of CEP adducts in TLR2-mediated angiogenesis proposed by others.
authors.affiliation: Analytical Sciences, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Gounarides
authors.affiliation: Analytical Sciences, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Cobb
authors.affiliation: Analytical Sciences, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Analytical Sciences, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Frank
authors.initials: F
authors.lastname: Cook
authors.affiliation: Analytical Sciences, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Yang
authors.affiliation: Analytical Sciences, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: Yin
authors.affiliation: Global Discovery Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Erik
authors.initials: E
authors.lastname: Meredith
authors.affiliation: Analytical Sciences, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Chang
authors.initials: C
authors.lastname: Rao
authors.affiliation: Developmental and Metabolic Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Qian
authors.initials: Q
authors.lastname: Huang
authors.affiliation: Developmental and Metabolic Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: YongYao
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Karen
authors.initials: K
authors.lastname: Anderson
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Andrea
authors.initials: A
authors.lastname: De Erkenez
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Sha-Mei
authors.initials: SM
authors.lastname: Liao
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Maura
authors.initials: M
authors.lastname: Crowley
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Natasha
authors.initials: N
authors.lastname: Buchanan
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Stephen
authors.initials: S
authors.lastname: Poor
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Yubin
authors.initials: Y
authors.lastname: Qiu
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Fassbender
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Siyuan
authors.initials: S
authors.lastname: Shen
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Amber
authors.initials: A
authors.lastname: Woolfenden
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Amy
authors.initials: A
authors.lastname: Jensen
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Cepeda
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Bijan
authors.initials: B
authors.lastname: Etemad-Gilbertson
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Shelby
authors.initials: S
authors.lastname: Giza
authors.affiliation: Global Discovery Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Muneto
authors.initials: M
authors.lastname: Mogi
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Bruce
authors.initials: B
authors.lastname: Jaffee
authors.affiliation: Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America.
authors.forename: Sassan
authors.initials: S
authors.lastname: Azarian
mesh_terms: D000818:Animals; D020256:Choroidal Neovascularization / Q000473:pathology; D004195:Disease Models, Animal; D057809:HEK293 Cells; D006801:Humans; D007834:Lasers; D007962:Leukocytes / Q000378:metabolism; D008810:Mice, Inbred C57BL; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D011758:Pyrroles / Q000378:metabolism*; D012160:Retina / Q000378:metabolism / Q000473:pathology; D051195:Toll-Like Receptor 2 / Q000819:agonists / Q000378:metabolism*
chemical_list: C000602461:2-(omega-carboxyethyl)pyrrole; D011758:Pyrroles; D051195:Toll-Like Receptor 2