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Evaluation of corneal epithelial lysyl oxidase (LOX) mRNA expression in keratoconus with disease severity and correlation to MMP9 and collagen gene expression

Poster Details

First Author: N.Pahuja INDIA

Co Author(s):    R. Shetty   V. Arora   P. Srivatsa   A. Ghosh   B. Shetty   A. Ghosh

Abstract Details



Purpose:

Since collagen forms an integral part of the cornea and is most affected in the ectasia that characterizes Keratoconus, it is important to understand the factors that regulate different forms of collagen. We therefore designed this study in primary patient epithelium to understand the role of corneal epithelium in the collagen regulation process. We further correlated the gene expression data with different grades of Keratoconus to elucidate any potential association of these genes with disease severity.

Setting:

A Tertiary eye care facility and teaching institute in South India

Methods:

Corneal epithelium of 83 Keratoconus and 59 control (normal) subjects was collected after prior written consent. Pre- operative and post-operative retinoscopy, slit lamp biomicroscopy and topography using Pentacam (Oculus) and Orbscan was done. Patients were divided into normal and keratoconus and were further classified into 4 different grades of keratoconus as per Amsler-Krumeich classification. Epithelial cells were collected from the keratoconus patients under topical anesthesia before corneal collagen crosslinking or topography guided photorefractive keratectomy (T-PRK) and controls undergoing Photorefractive keratectomy. Epithelial tissue was homogenized in TRIZOL reagent and total mRNA was extracted for gene expression analysis. Total levels of LOX, MMP9, IL6, COL1A1 and COL4A1 were estimated by quantitative real-time PCR for each sample after normalization to actin.

Results:

We observed that LOX transcript levels were reduced in keratoconus patients (1.65+0.23) compared with that of controls (3.02+0.45). Furthermore, the transcript data demonstrated decreasing trend of LOX expression across grades I (2.0+0.36), II (1.25+0.31) and III+IV (1.26+0.58) as per the Amsler-Krumeich grades. However, MMP9 levels were highly up regulated in Keratoconus patients (7.82+1.44) relative to controls (3.12+0.95). Collagen I and collagen IV transcript levels were 14.8+3.4 and 5.0+1.3 in Keratoconus respectively while they were 21.9+5.5 and 3.9+1.5 in controls. IL6 levels did not show any significant trends at the transcript level.

Conclusions:

The data supports the hypothesis that Lysyl oxidase levels are reduced in Keratoconus patients. Up-regulation of MMP9 levels as expected based on previous data was confirmed in these patients. Interestingly, collagen I levels were reduced, while collagen IV levels were slightly increased. The reduction in collagen I, the primary collagen type in cornea may have a functional role in the ectatic process. Type IV collagen is often associated with healing processes and its up regulation may be an attempt by the corneal epithelium of KC patients to counteract the disease. In summary, the reduction in levels of LOX is suggestive of a pathological role of the enzyme in the ectasia associated with Keratoconus and may represent a possible biomarker for disease staging and therapeutic target for treatment in the future. FINANCIAL INTEREST: NONE

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