3-nitrotyrosine and Retinitis

The authors' previous studies showed that the Wnt signaling pathway is activated in the retinas and retinal pigment epithelia of animal models of age-related macular degeneration (AMD) and diabetic retinopathy (DR). The purpose of this study was to investigate the role of the canonical Wnt pathway in pathogenesis of these diseases.. The Wnt pathway was activated using the Wnt3a-conditioned medium and adenovirus expressing a constitutively active mutant of beta-catenin (Ad-S37A) in ARPE19, a cell line derived from human RPE. Ad-S37A was injected into the vitreous of normal rats to activate the Wnt pathway in the retina. Accumulation of beta-catenin was determined by Western blot analysis, and its nuclear translocation was revealed by immunocytochemistry. Inflammatory factors were quantified by Western blot analysis and ELISA. Oxidative stress was determined by measuring intracellular reactive oxygen species (ROS) generation and nitrotyrosine levels.. The Wnt3a-conditioned medium and Ad-S37A both increased beta-catenin levels and its nuclear translocation in ARPE19 cells, suggesting activation of the canonical Wnt pathway. Activation of the Wnt pathway significantly upregulated the expression of VEGF, NF-kappaB, and TNF-alpha. Further, Ad-S37A induced ROS generation in a dose-dependent manner. Wnt3a also induced a twofold increase of ROS generation. Intravitreal injection of Ad-S37A upregulated the expression of VEGF, ICAM-1, NF-kappaB, and TNF-alpha and increased protein nitrotyrosine levels in the retinas of normal rats.. Activation of the canonical Wnt pathway is sufficient to induce retinal inflammation and oxidative stress and plays a pathogenic role in AMD and DR.

Topics: Active Transport, Cell Nucleus; Adenoviridae; Animals; beta Catenin; Cell Line; Culture Media, Conditioned; Humans; Injections, Intraocular; Macular Degeneration; Mice; Oxidative Stress; Rats; Rats, Inbred BN; Reactive Oxygen Species; Retinal Pigment Epithelium; Retinitis; Signal Transduction; Transfection; Tyrosine; Up-Regulation; Vitreous Body; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

Macrophages infiltrating an inflamed or injured tissue undergo development of coordinated sets of properties that contribute to tissue damage, repair, and remodeling. The purpose of this study was to determine whether macrophages isolated from normal or inflamed retina are programmed to a distinct set of properties and to examine whether the development of experimental autoimmune uveoretinitis (EAU) affects macrophage function.. EAU was induced in Lewis rats, and a retina-derived macrophage-enriched population was generated by density centrifugation during the prepeak, peak, and resolution phases of the disease. Cell surface phenotype was assessed by two- and three-color flow cytometry, and function was determined in vitro by nitric oxide (NO) production, with or without further cytokine stimulation or by immunohistochemistry to determine expression of beta-glucuronidase, nitric oxide synthase (NOS)-2, and nitrotyrosine.. Myeloid-derived cells from normal retina were programmed similar to TGF-beta-stimulated uncommitted bone-marrow-derived macrophages (BMDMs). Contrary to BMDM behavior, retina-isolated macrophages displayed distinct properties and phenotype at different phases of the disease course and remained resistant throughout, to further cytokine challenge in vitro. During peak disease, retina-isolated macrophages had characteristics of IFN-gamma/TNF-alpha primed cells (nitrotyrosine positive and NO producing). Despite equivalent numbers of macrophages during resolution, their function reverted to characteristics of TGF-beta primed cells (beta-glucuronidase positive).. Resident retinal myeloid-derived cells are primed and are resistant to further cytokine stimulation, and, similar to macrophages derived during EAU recovery, behave operationally as though TGF-beta primed. During peak inflammation, infiltrating macrophages adapt to concurrent hierarchical Th1 T-cell response (IFN-gamma/TNF-alpha), generating NO. The results provide evidence of in vivo programming of macrophages within the retina.

Topics: Animals; Autoimmune Diseases; Cytokines; Female; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Glucuronidase; Immunoenzyme Techniques; Immunophenotyping; Macrophage Activation; Macrophages; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Inbred Lew; Retina; Retinitis; Tyrosine; Uveitis