title: Nitrite Modification of Extracellular Matrix Alters CD46 Expression and VEGF Release in Human Retinal Pigment Epithelium.
abstract: OBJECTIVE
  Loss of CD46 has recently been implicated in choroidal neovascularization in mice. Herein we investigated the effect of nitrite modification of the extracellular matrix (ECM) as an in vitro model of "aging" and its effect on CD46 expression and vascular endothelial growth factor (VEGF) release in cocultured human retinal pigment epithelium (RPE).
  
  
  METHODS
  ARPE-19 cells were plated onto RPE-derived ECM conditions (untreated; nitrite modified; nitrite modified followed by washing with Triton X-100; or nitrite modified followed by washing with Triton X-100 and coated with extracellular matrix ligands). Cells were cultured for 7 days and CD46 expression was analyzed by immunohistochemistry and Western blot. Additionally, CD46 short interfering RNA (siRNA) was transfected into ARPE-19 cells, and VEGF levels were determined by ELISA. Finally, in the same ECM conditions, ARPE-19 cells were challenged with normal human serum and VEGF levels determined by ELISA.
  
  
  RESULTS
  CD46 is expressed on the basolateral surface of ARPE-19 cells on RPE-derived ECM. Nitrite modification of ECM reduced the expression of CD46 on ARPE-19 cells by 0.5-fold (P = 0.003) and increased VEGF release in ARPE-19 cells by 1.7-fold (P < 0.001). CD46 knockdown also increased release of VEGF on the apical and basal sides of ARPE-19 cells in culture by 1.3- (P = 0.012) and 1.2-fold (P = 0.017), respectively.
  
  
  CONCLUSIONS
  Nitrite modification of the ECM decreased CD46 expression and increased the release of VEGF from ARPE-19 cells. Changes in CD46 expression may lead to changes in VEGF and play a pathologic role in the development of age-related macular degeneration.
authors.affiliation: Department of Ophthalmology Medical University of South Carolina, Charleston, South Carolina, United States 2Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, South Carolina, United States.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Fields
authors.affiliation: Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, New York, United States.
authors.forename: Hui
authors.initials: H
authors.lastname: Cai
authors.affiliation: Department of Ophthalmology Medical University of South Carolina, Charleston, South Carolina, United States.
authors.forename: Hannah E
authors.initials: HE
authors.lastname: Bowrey
authors.affiliation: Department of Ophthalmology Medical University of South Carolina, Charleston, South Carolina, United States.
authors.forename: Ernesto F
authors.initials: EF
authors.lastname: Moreira
authors.affiliation: Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina, United States.
authors.forename: Monika
authors.initials: M
authors.lastname: Beck Gooz
authors.affiliation: Department of Ophthalmology Medical University of South Carolina, Charleston, South Carolina, United States.
authors.forename: Kannan
authors.initials: K
authors.lastname: Kunchithapautham
authors.affiliation: Department of Ophthalmology Medical University of South Carolina, Charleston, South Carolina, United States.
authors.forename: Jie
authors.initials: J
authors.lastname: Gong
authors.affiliation: Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, United States.
authors.forename: Emma
authors.initials: E
authors.lastname: Vought
authors.affiliation: Department of Ophthalmology Medical University of South Carolina, Charleston, South Carolina, United States.
authors.forename: Lucian V
authors.initials: LV
authors.lastname: Del Priore
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004247:DNA / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D051916:Membrane Cofactor Protein / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D009573:Nitrites / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: C497859:Mcp protein, mouse; D051916:Membrane Cofactor Protein; D009573:Nitrites; D042461:Vascular Endothelial Growth Factor A; D004247:DNA