Integrated Stress Response and Decreased ECM in Cultured Stromal Cells From Keratoconus Corneas

Investigative Ophthalmology & Visual Science
James W FosterShukti Chakravarti

Abstract

Keratoconus (KC) is a multifactorial disease where progressive thinning and weakening of the cornea leads to loss of visual acuity. Although the underlying etiology is poorly understood, a major endpoint is a dysfunctional stromal connective tissue matrix. Using multiple individual KC corneas, we determined that matrix production by keratocytes is severely impeded due to an altered stress response program. KC and donor (DN) stromal keratocytes were cultured in low glucose serum-free medium containing insulin, selenium and transferrin. Fibronectin, collagens and proteins related to their chaperone, processing and export, matrix metalloproteinase, and stress response related proteins were investigated by immunoblotting, immunocytochemistry, hydroxyproline quantification, and gelatin zymography. Multiplexed mass spectrometry was used for global proteomic profiling of 5 individual DN and KC cell culture. Transcription of selected proteins was assayed by qPCR. DN and KC cells showed comparable survival and growth. However, immunoblotting of selected ECM proteins and global proteomics showed decreased fibronectin, collagens, PCOLCE, ADAMTS2, BMP1, HSP47, other structural and cytoskeletal proteins in KC. Phosphorylated (p) eIF2α, a tr...Continue Reading

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Citations

Aug 8, 2019·Investigative Ophthalmology & Visual Science·Uri Simcha SoibermanJames William Foster
Aug 31, 2019·Scientific Reports·Rabab SharifDimitrios Karamichos
Oct 17, 2019·Cells·Tina B McKayDimitrios Karamichos
Jun 9, 2020·Journal of Current Ophthalmology·Siamak Zarei-GhanavatiPardis Eghbali
Sep 10, 2020·Frontiers in Cell and Developmental Biology·Sudan PuriVivien J Coulson-Thomas
Oct 28, 2019·Omics : a Journal of Integrative Biology·Vishal ShindeShukti Chakravarti
Jan 22, 2021·Current Eye Research·Hsiao-Sang ChuJames Foster
May 23, 2020·Experimental Eye Research·Gary H F YamVishal Jhanji
Feb 21, 2021·Stem Cells and Development·Jan KosslVladimir Holan
Aug 13, 2021·Scientific Reports·Ann J LigockiCarmelo Romano
Aug 10, 2021·Experimental Eye Research·C PetersonJ Foster

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Methods Mentioned

BETA
PCR
zymography

Software Mentioned

Cellsense
Zeiss
Proteome Discoverer
cell
ImageJ
SEQUEST
sense

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