TSP1 ameliorates age-related macular degeneration by regulating the STAT3-iNOS signaling pathway

Exp Cell Res. 2020 Mar 1;388(1):111811. doi: 10.1016/j.yexcr.2019.111811. Epub 2019 Dec 30.

Abstract

Age-related macular degeneration is a progressive ocular disease that is the leading cause of vision loss among elderly. AMD usually is divided into two types: wet and dry AMD, which is linked with inflammation. Choroidal Neovascularization (CNV) formation or wet AMD is also associated with oxidative stress. Previously, TSP1 has been shown to have a significant alleviating effect on CNV in TSP1 knockout (TSP1-/-) mice. However, the mechanism by which TSP1 ameliorates CNV remains unclear. Here we report that TSP1 reduces nitric oxide production to prevent cells from forming tubes formation and reduced the levels of vascular endothelial growth factor (VEGF) and lipid peroxides (LPO) during oxidative stress. We measured RF/6A cell viability by CCK-8 assay and apoptosis by flow cytometry. RF/6A cell were transfected with TSP1 and STAT3 overexpression, and then the mRNA and protein levels of TSP1 and also the signal pathways were detected by qRT-PCR and Western blot analysis. Migration assays were performed using a transwell system. Co-Immunoprecipitation was used to analyze the binding relationship between CD47 and SHP-2. The results show that overexpression of TSP1 alleviated the damage of oxidative stress to RF/6A cells including increased cell activity and migration, decreased apoptosis and reduced migration compared to the control group. SHP-2 was activated by TSP1 through its receptor CD47 and STAT3 phosphorylation was reduced by activation of SHP-2, thereby blocking STAT3-iNOS pathway and reducing NO concentration in RF/6A cells ultimately protecting them from oxidative stress. Finally, the CNV mice model confirmed that TSP1 overexpression could protect the mice against CNV in vivo, modified the antioxidants levels and decreased the expression of TNF-α and IL-6 under laser irradiation. These results indicate a potential mechanism of TSP1 to slow down formation of CNV in wet AMD, which may bring hope for new treatment strategies.

Keywords: Age-related macular degeneration; CNV; TSP1; Tubule formation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis
  • CD47 Antigen / metabolism
  • Cell Line
  • Lipid Peroxidation
  • Macaca mulatta
  • Macular Degeneration / metabolism*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase Type II / metabolism*
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / metabolism
  • STAT3 Transcription Factor / metabolism*
  • Signal Transduction
  • Thrombospondin 1 / genetics
  • Thrombospondin 1 / metabolism*
  • Vascular Endothelial Growth Factor A / metabolism

Substances

  • CD47 Antigen
  • STAT3 Transcription Factor
  • Thrombospondin 1
  • Vascular Endothelial Growth Factor A
  • Thbs1 protein, mouse
  • Nitric Oxide
  • Nitric Oxide Synthase Type II
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11
  • Ptpn11 protein, mouse