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

Investigative Ophthalmology & Visual Science
Uri Simcha SoibermanJames William Foster

Abstract

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. 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. 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 res...Continue Reading

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Citations

Jan 22, 2021·Current Eye Research·Hsiao-Sang ChuJames Foster
Dec 17, 2020·Asia-Pacific Journal of Ophthalmology·Rohit ShettySwaminathan Sethu
Apr 10, 2020·Progress in Retinal and Eye Research·Marina S GorbatyukOleg S Gorbatyuk
Aug 10, 2021·Experimental Eye Research·C PetersonJ Foster

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Methods Mentioned

BETA
scraping
zymography
PCR
ELISA

Software Mentioned

GraphPad Prism
Zen
Axiovision

Related Concepts

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Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis