Retinal pigment epithelium cells produce VEGF in response to oxidized phospholipids through mechanisms involving ATF4 and protein kinase CK2

Exp Eye Res. 2013 Nov:116:177-84. doi: 10.1016/j.exer.2013.08.021. Epub 2013 Sep 7.

Abstract

Oxidized phospholipids (OxPLs) are pleiotropic lipid mediators known to induce proangiogenic and proinflammatory cellular effects that are increasingly recognized to be involved in a number of physiologic and pathologic processes in the retina. Immunohistochemical studies have detected OxPLs in retinal structures, such as retinal pigment epithelium (RPE) or photoreceptor cells. This study analyzed whether OxPLs could play a role in upregulation of VEGF, which is a cause of pathological neovascularization characteristic of eye diseases such as age-related macular degeneration. We confirmed accumulation of OxPLs in the eye using reversed-phase liquid chromatography coupled to mass spectrometry. Multiple species of oxidized phosphatidylcholines (OxPCs) were detected in human vitreous, including biologically active fragmented species POVPC, PGPC, PONPC and PAzPC. In in vitro experiments human fetal RPE and primary RPE cells were stimulated with OxPLs. Primary RPE cells were transfected with small interfering RNAs targeting ATF4. mRNA levels of VEGF in fetal and primary RPE cells were determined by real-time quantitative PCR. VEGF protein concentrations were measured in culture medium by ELISA. We found that OxPCs and other classes of OxPLs upregulated the expression of VEGF in fetal and primary RPE cells, which critically depended on ATF4. In addition, upregulation of VEGF in primary RPE cells was blocked by a chemical inhibitor of protein kinase CK2 known to suppress induction of ATF4 and VEGF by OxPLs. Our data show that different species of OxPLs, which are present in the human eye are capable of stimulating expression of VEGF in fetal and primary RPE cells via ATF4-dependent mechanisms.

Keywords: RPE; angiogenesis; oxidized phospholipids; protein kinase CK2; unfolded protein response.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activating Transcription Factor 4 / biosynthesis
  • Activating Transcription Factor 4 / genetics*
  • Blotting, Western
  • Casein Kinase II / biosynthesis
  • Casein Kinase II / genetics*
  • Cells, Cultured
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Macular Degeneration / genetics
  • Macular Degeneration / metabolism
  • Macular Degeneration / pathology
  • Mass Spectrometry
  • Oxidation-Reduction
  • Phospholipids / metabolism*
  • RNA, Messenger / genetics*
  • Real-Time Polymerase Chain Reaction
  • Retinal Pigment Epithelium / metabolism*
  • Retinal Pigment Epithelium / pathology
  • Up-Regulation*
  • Vascular Endothelial Growth Factor A / biosynthesis
  • Vascular Endothelial Growth Factor A / genetics*

Substances

  • ATF4 protein, human
  • Phospholipids
  • RNA, Messenger
  • Vascular Endothelial Growth Factor A
  • Activating Transcription Factor 4
  • Casein Kinase II