Detection of Proteins Associated with Extracellular Matrix Regulation in the Aqueous Humour of Patients with Primary Glaucoma

ABSTRACT

Purpose

The protein composition of aqueous humour (AH) has held significant relevance and remains to be the prime sample in the discovery of biomarkers in glaucoma. The purpose of this study is to analyze the AH protein concentrations in primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) and further examine the proteome changes compared to cataract control.

Methods

AH was collected from 90 POAG, 72 PACG, 78 cataracts (controls) in this study. The total protein was quantified using Bradford’s assay. Samples were subjected to trypsin digestion followed by liquid chromatography-mass spectrometry (LC-MS) for proteomic studies (n = 3 per group). The extracellular matrix has a major influence on the AH outflow, and the regulator proteins osteopontin (OPN), cathepsin D, and cystatin C detected by mass spectrometry are validated in AH samples by Western blot and turbidimetric immunoassay.

Results

We observed a significant increase in protein levels of POAG (p = .0009); interestingly, a similar increase in PACG compared to cataract (p < .0001) and POAG (p = .02). Proteomics analysis identified 184, 190, and 299 proteins in control, POAG and PACG. OPN was increased in POAG (p = .0319) and PACG (p = .0103) compared to control. The precursor form of cathepsin D was increased in POAG and decreased in PACG, though not significant compared to control. Cystatin C was also increased in both POAG (p = .0310) and PACG (p = .0125) compared to control.

Conclusion

In this study, we report for the first time that PACG cohort had higher total protein compared to controls. A qualitative comparison of proteomes revealed increased numbers of proteins identified in PACG. We assume that elevated levels of OPN and cystatin C in POAG and PACG along with altered cathepsin levels may contribute to ECM aberration in glaucoma.

KEYWORDS:

• Glaucoma
• aqueous humour
• osteopontin
• cystatin C
• cathepsin D

Additional information

Funding

This work was supported by the Indian Council of Medical Research under the grant number 5/4/6/4/oph/12-NCD-II.