Abstract
Purpose: :
Lysyl oxidase (LOX a copper-dependent amine oxidase), is a collagen cross-linking enzyme, that catalyzes a critical post- translational modification essential for biogenesis of connective tissue matrices. Immature collagen crosslinking and increased levels of matrix metalloproteases (MMP) have been reported in proliferative diabetic retinopathy (PDR). The level of LOX has not been studied in the pathogenesis of PDR and hence the purpose of this study was to estimate the LOX levels in the vitreous of PDR and also in ARPE-19 cultures exposed to high glucose environment.
Methods: :
Undiluted human vitreous specimen was obtained during vitreoretinal surgery, from PDR (n= 10) patients. Vitreous specimens from donor eyeballs were used as control (n=15). Lysyl oxidase activity (+/- LOX inhibitor Beta aminopropionitrile at 500 µM concentration) was estimated by Amplex red dye method using fluorescent ELISA reader (Ex550 Em580). Collagen content in terms of hydroxyproline was measured spectrophotometrically. ARPE-19 cultures were grown in 25cm2 flask to 90 % confluence and exposed to high glucose concentrations (10,20 and 30mM) for 72 hours and the specific activity of LOX was estimated in medium. Total RNA was extracted from cell pellet. TaqMan gene expression assay for LOX was done by relative quantification.
Results: :
The LOX activity was significantly decreased in vitreous of PDR (p=0.014) compared to controls. However the hydroxyproline content was increased significantly in PDR (p=0.012). In ARPE 19 cells exposed to 30mM glucose the specific activity of LOX was significantly reduced (p=0.025) at 72 hours when compared to controls. The LOX mRNA expression also showed a decreasing trend with increasing glucose concentration.
Conclusions: :
These results are suggestive of increased collagen turnover in PDR with a decreased LOX activity, which plays an important role in extracellular matrix remodeling. A similar decrease in LOX activity was observed in ARPE-19 cells exposed to high glucose.
Keywords: retinal pigment epithelium • vitreous • extracellular matrix