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Small Molecule Modulation of the Integrated Stress Response Governs the Keratoconic Phenotype In Vitro.
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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 TNF? and IL-1? 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.
SUBMITTER: Soiberman US
PROVIDER: S-EPMC6686743 | biostudies-literature | 2019 Aug
REPOSITORIES: biostudies-literature
Publications
Small Molecule Modulation of the Integrated Stress Response Governs the Keratoconic Phenotype In Vitro.
Investigative ophthalmology & visual science 20190801 10
<h4>Purpose</h4>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 recove ...[more]