Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts

Venkateswara Mootha, J. M. Kanoff, J. Shankardas, S. Dimitrijevich

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Purpose: To identify differentially expressed genes in keratoconus (KC) corneal fibroblasts. Methods: Stromal keratocytes (having a fibroblast morphology) from KC keratoplasty specimens and eye bank donor corneas were isolated and expanded using a serum containing medium. RNA was isolated from three KC fibroblast cultures and five eye bank donor cornea fibroblast cultures. The targets from the cultured fibroblasts were hybridized to the Affymetrix U133 Plus 2.0 microarrays. Western blot analyses of cell lysates were performed to examine protein levels of interest in the two groups. Protein levels of select differentially expressed genes were further examined by immunohistochemistry. Keratocyte staining of archived KC keratoplasty specimens were graded using a 0 to 3+ scale and compared to five archived whole globes having normal corneas as well as to 10 Fuchs' dystrophy keratoplasty specimens. Results: Microarray analysis revealed up to a 212 fold reduction in the mRNA levels of alcohol dehydrogenase (class 1) beta polypeptide (ADH1B) in KC fibroblasts (p=0.04). Decreased alcohol dehydrogenase in KC fibroblasts was confirmed by western blot analysis of early passage primary keratocyte cell lysates. Immunohistochemistry using a monoclonal mouse immunoglobulin G (IgG) against human liver alcohol dehydrogenase revealed a dramatic difference in protein staining in the keratocytes of the KC group compared to the normal cornea group. Immunohistochemistry also showed decreased immunostaining against alcohol dehydrogenase in the KC stromal sections compared to those obtained from Fuchs' endothelial corneal dystrophy samples. Conclusions: Decreased alcohol dehydrogenase in KC corneal fibroblasts represents a strong marker and possible mediator of keratoconus.

Original languageEnglish (US)
Pages (from-to)706-712
Number of pages7
JournalMolecular Vision
Volume15
StatePublished - Apr 10 2009

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Keratoconus
Alcohol Dehydrogenase
Fibroblasts
Cornea
Corneal Transplantation
Eye Banks
Immunohistochemistry
Western Blotting
Fuchs' Endothelial Dystrophy
Staining and Labeling
Proteins
Microarray Analysis
Genes
Immunoglobulin G
RNA

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Mootha, V., Kanoff, J. M., Shankardas, J., & Dimitrijevich, S. (2009). Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts. Molecular Vision, 15, 706-712.

Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts. / Mootha, Venkateswara; Kanoff, J. M.; Shankardas, J.; Dimitrijevich, S.

In: Molecular Vision, Vol. 15, 10.04.2009, p. 706-712.

Research output: Contribution to journalArticle

Mootha, V, Kanoff, JM, Shankardas, J & Dimitrijevich, S 2009, 'Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts', Molecular Vision, vol. 15, pp. 706-712.
Mootha V, Kanoff JM, Shankardas J, Dimitrijevich S. Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts. Molecular Vision. 2009 Apr 10;15:706-712.
Mootha, Venkateswara ; Kanoff, J. M. ; Shankardas, J. ; Dimitrijevich, S. / Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts. In: Molecular Vision. 2009 ; Vol. 15. pp. 706-712.
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