title: Establishing 3-nitrotyrosine as a biomarker for the vasculopathy of Fabry disease.
abstract: The endothelial dysfunction of Fabry disease results from α-galactosidase A deficiency leading to the accumulation of globotriaosylceramide. Vasculopathy in the α-galactosidase A null mouse is manifested as oxidant-induced thrombosis, accelerated atherogenesis, and impaired arterial reactivity. To better understand the pathogenesis of Fabry disease in humans, we generated a human cell model by using RNA interference. Hybrid endothelial cells were transiently transfected with small interfering RNA (siRNA) specifically directed against α-galactosidase A. Knockdown of α-galactosidase A was confirmed using immunoblotting and globotriaosylceramide accumulation. Endothelial nitric oxide synthase (eNOS) activity was correspondingly decreased by >60%. Levels of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species and quantified using mass spectrometry, increased by 40- to 120-fold without corresponding changes in other oxidized amino acids, consistent with eNOS-derived reactive nitrogen species as the source of the reactive oxygen species. eNOS uncoupling was confirmed by the observed increase in free plasma and protein-bound aortic 3NT levels in the α-galactosidase A knockout mice. Finally, 3NT levels, assayed in biobanked plasma samples from patients with classical Fabry disease, were over sixfold elevated compared with age- and gender-matched controls. Thus, 3NT may serve as a biomarker for the vascular involvement in Fabry disease.
authors.affiliation: Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Liming
authors.initials: L
authors.lastname: Shu
authors.affiliation: Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Vivekanandan-Giri
authors.affiliation: Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Subramaniam
authors.initials: S
authors.lastname: Pennathur
authors.affiliation: Department of Endocrinology and Metabolism, Academic Medical Centre, Amsterdam, The Netherlands.
authors.forename: Bouwien E
authors.initials: BE
authors.lastname: Smid
authors.affiliation: Department of Medical Biochemistry, Academic Medical Centre, Amsterdam, The Netherlands.
authors.forename: Johannes M F G
authors.initials: JM
authors.lastname: Aerts
authors.affiliation: Department of Endocrinology and Metabolism, Academic Medical Centre, Amsterdam, The Netherlands.
authors.forename: Carla E M
authors.initials: CE
authors.lastname: Hollak
authors.affiliation: Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Shayman
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D016022:Case-Control Studies; D002460:Cell Line; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism; D000795:Fabry Disease / Q000150:complications* / Q000235:genetics / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008875:Middle Aged; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014652:Vascular Diseases / Q000209:etiology* / Q000378:metabolism*; D055815:Young Adult; D000519:alpha-Galactosidase / Q000037:antagonists & inhibitors / Q000235:genetics
chemical_list: D015415:Biomarkers; D034741:RNA, Small Interfering; C002744:3-nitrotyrosine; D014443:Tyrosine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C576330:GLA protein, human; D000519:alpha-Galactosidase