title: Matrix metalloproteinase activity creates pro-angiogenic environment in primary human retinal pigment epithelial cells exposed to complement.
abstract: OBJECTIVE
  Mechanistic studies have shown that inflammation, complement activation, extracellular matrix (ECM) turnover, growth factor imbalance, and oxidative stress are fundamental components of age-related macular degeneration (AMD). Matrix metalloproteinases (MMPs) mediate ECM turnover but also process various bioactive molecules. Here, we tested whether complement attack on RPE monolayers changes MMP secretion and activation, thereby altering the availability of growth factors in the extracellular space.
  
  
  METHODS
  Human embryonic RPE monolayers with stable transepithelial resistance (TER) were established. Complement activation was induced with H₂O₂ and normal human serum. MMP-2/9, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) protein, and mRNA levels were analyzed by Western blotting, ELISA, and real-time PCR; activity of MMP-2/9 by gelatin zymography.
  
  
  RESULTS
  Complement activation resulted in a loss of TER, which required transient membrane attack complex formation, activation of the alternative pathway, and VEGF secretion and signaling. Despite the generation of reactive oxygen species, cellular integrity or intracellular adenosine triphosphate (ATP) levels were unaffected. However, expression of MMP-2/9 and their protease activity was elevated. Inhibition of MMP-2/9 activity increased PEDF and decreased VEGF levels in the apical and basal supernatants but had no effect on their expression levels. VEGF levels in the supernatant correlated with the level TER reduction.
  
  
  CONCLUSIONS
  These studies suggest that complement activation, by altering the expression and activation of MMPs, has the ability to generate a proangiogenic environment by altering the balance between VEGF and PEDF. Our findings link reported results that have been associated with AMD pathogenesis; oxidative stress; complement activation; VEGF/PEDF ratio; and MMP activity.
authors.affiliation: Department of Ophthalmology, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA.
authors.forename: Mausumi
authors.initials: M
authors.lastname: Bandyopadhyay
authors.forename: Bärbel
authors.initials: B
authors.lastname: Rohrer
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D003167:Complement Activation; D003165:Complement System Proteins / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D009024:Morphogenesis; D018384:Oxidative Stress; D060888:Real-Time Polymerase Chain Reaction; D015861:Retinal Neovascularization / Q000196:embryology / Q000201:enzymology* / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000196:embryology / Q000201:enzymology* / Q000473:pathology
chemical_list: D007155:Immunologic Factors; D003165:Complement System Proteins; D020782:Matrix Metalloproteinases