Rap1 GTPase Inhibits Tumor Necrosis Factor-α-Induced Choroidal Endothelial Migration via NADPH Oxidase- And NF-κB-Dependent Activation of Rac1

Am J Pathol. 2015 Dec;185(12):3316-25. doi: 10.1016/j.ajpath.2015.08.017. Epub 2015 Oct 23.

Abstract

Macrophage-derived tumor necrosis factor (TNF)-α has been found in choroidal neovascularization (CNV) surgically removed from patients with age-related macular degeneration. However, the role of TNF-α in CNV development remains unclear. In a murine laser-induced CNV model, compared with un-lasered controls, TNF-α mRNA was increased in retinal pigment epithelial and choroidal tissue, and TNF-α colocalized with lectin-stained migrating choroidal endothelial cells (CECs). Inhibition of TNF-α with a neutralizing antibody reduced CNV volume and reactive oxygen species (ROS) level around CNV. In CECs, pretreatment with the antioxidant apocynin or knockdown of p22phox, a subunit of NADPH oxidase, inhibited TNF-α-induced ROS generation. Apocynin reduced TNF-α-induced NF-κB and Rac1 activation, and inhibited TNF-α-induced CEC migration. TNF-α-induced Rac1 activation and CEC migration were inhibited by NF-κB inhibitor Bay11-7082. Overexpression of Rap1a prevented TNF-α-induced ROS generation and reduced NF-κB and Rac1 activation. Activation of Rap1 by 8-(4-chlorophenylthio)adenosine-2'-O-Me-cAMP prevented TNF-α-induced CEC migration and reduced laser-induced CNV volume, ROS generation, and activation of NF-κB and Rac1. These findings provide evidence that active Rap1a inhibits TNF-α-induced CEC migration by inhibiting NADPH oxidase-dependent NF-κB and Rac1 activation and suggests that Rap1a de-escalates CNV development by interfering with ROS-dependent signaling in several steps of the pathogenic process.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Movement / physiology
  • Cells, Cultured
  • Choroid / metabolism
  • Choroidal Neovascularization / genetics
  • Choroidal Neovascularization / metabolism*
  • Choroidal Neovascularization / pathology
  • Endothelial Cells / physiology
  • Enzyme Activation / physiology
  • Female
  • Mice, Inbred C57BL
  • NADPH Oxidases / physiology*
  • NF-kappa B / physiology*
  • Neuropeptides / metabolism*
  • Neuropeptides / physiology
  • Reactive Oxygen Species / metabolism
  • Signal Transduction / physiology
  • Tumor Necrosis Factor-alpha / biosynthesis
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / physiology*
  • Up-Regulation / physiology
  • rac1 GTP-Binding Protein / metabolism*
  • rac1 GTP-Binding Protein / physiology

Substances

  • NF-kappa B
  • Neuropeptides
  • Rac1 protein, mouse
  • Reactive Oxygen Species
  • Tumor Necrosis Factor-alpha
  • NADPH Oxidases
  • rac1 GTP-Binding Protein