Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts

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.

Figure 1

Fold change and p value data from microarray experiments. Encircled points represent transcripts for ADH1B (Probe sets 209612_s_at and 209613_s_at). Dashed line marks that p=0.05 and statistical significance.

Figure 2

Protein expression of 80 kDa dimer and 40 kDa monomer subunits of alcohol dehydrogenase in cultured corneal fibroblasts. C1-C5 are normal corneal fibroblast cell lines, and K1–K3 are keratoconus corneal fibroblast cell lines.

Figure 3

Alcohol dehydrogenase immunoreactivity in a normal cornea, Fuchs’ dystrophy cornea, and keratoconus cornea. Diaminobenzidine (DAB) brown staining of the keratocytes of normal corneas and Fuchs’ dystrophy corneas indicates presence of alcohol dehydrogenase in contrast to keratoconus keratocytes.

Figure 4

Alcohol dehydrogenase is a dimeric zinc metalloenzyme that catalyzes the reversible oxidation of alcohols to aldehydes.