Small Molecule Modulation of the Integrated Stress Response Governs the Keratoconic Phenotype In Vitro

Invest Ophthalmol Vis Sci. 2019 Aug 1;60(10):3422-3431. doi: 10.1167/iovs.19-27151.

Abstract

Purpose: The degenerative corneal disease keratoconus is a leading indicator for corneal transplant with an unknown etiology. We recently identified the activation of the integrated stress response (ISR) in ex vivo human corneas and in vitro cell culture. Utilizing small molecules to modulate the ISR we sought to investigate the effects of stimulating the ISR in healthy cells to recapitulate aspects of the in vitro keratoconic phenotype and whether relieving the ISR signaling would recover the disease phenotype.

Methods: Corneal fibroblasts were extracted from patients undergoing corneal transplant or unaffected cadaverous donor limbal rings. Cells were exposed to the DNA damage-inducible protein (GADD34) inhibitor SAL003 to stimulate the ISR, or Trans-ISRIB to relieve ISR signaling pathway. Collagen production was assessed through hydroxyproline production, Sirius Red incorporation, or quantitative (q)PCR. Western blotting, hydroxyproline, and qPCR were used to assess components of the ISR pathway and collagen production.

Results: ISR stimulation through SAL003 resulted in significant decrease of hydroxyproline and COL1A1 transcription and eventual apoptosis in normal fibroblasts. Patient (KC) fibroblast production of hydroxyproline was increased in response to ISRIB, while matrix metalloproteinase (MMP)9 production was lowered. The prospective biomarker of keratoconus prolactin-inducible factor was also upregulated in KC fibroblast cultures in response to ISRIB. Inflammatory markers TNFα and IL-1β were unaffected.

Conclusions: Activation of the ISR is sufficient to recapitulate many key aspects of the KC phenotype in unaffected cells in vitro. Inhibition of the ISR also relieves many of the hallmarks of KC in affected cells. Therefore, targeting of the ISR through small molecules is a potential therapeutic path for small molecule treatment of keratoconus.

Publication types

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

MeSH terms

  • Acetamides / pharmacology*
  • Blotting, Western
  • Cell Count
  • Cells, Cultured
  • Collagen Type I / genetics
  • Corneal Keratocytes / drug effects*
  • Corneal Keratocytes / metabolism
  • Cyclohexylamines / pharmacology*
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Hydroxyproline / metabolism
  • Keratoconus / pathology*
  • Matrix Metalloproteinase 2 / genetics
  • Matrix Metalloproteinase 9 / genetics
  • Phenotype
  • Prospective Studies
  • Protein Phosphatase 1 / antagonists & inhibitors
  • Real-Time Polymerase Chain Reaction
  • Stress, Physiological / drug effects*

Substances

  • 2-(4-chlorophenoxy)-N-(4-(2-(4-chlorophenoxy)acetamido)cyclohexyl)acetamide
  • Acetamides
  • Collagen Type I
  • Cyclohexylamines
  • Enzyme Inhibitors
  • collagen type I, alpha 1 chain
  • PPP1R15A protein, human
  • Protein Phosphatase 1
  • MMP2 protein, human
  • Matrix Metalloproteinase 2
  • Matrix Metalloproteinase 9
  • Hydroxyproline