PROTEIN MARKERS OF HYPOXIA AND ANGIOGENESIS IN TEAR FLUID OF PATIENTS WITH TRAUMATIC CORNEAL INJURY

Abstract

The aim of our study was to evaluate tear levels of some protein endpoints that can reflect intensities of hypoxia, angiogenesis and tissue remodeling in wounded cornea. Methods. We examined 21 patients (21 eyes) with nonpenetrating corneal injuries. The patients underwent standard ophthalmological examination including previous history and ocular symptoms, visual acuity test, complete anterior and posterior eye segments examination using slit lamp biomicroscopy, evaluation of corneal staining with fluorescein, ophthalmoscopy. Healthy volunteers (n = 10) served as a control. Tear fluid was collected from patients and control volunteers with the use of a disposable tip micropipette. From the lower arch of the conjunctiva without instillation of anesthetic, tears were collected in a sterile plastic Eppendorf tube and frozen at -20 oC before laboratory examination. Proteins of tear fluids were separated by SDS-PAGE (loading 50 µg total protein per track). Then, levels of hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and angiostatins were measured by western blot. Active MMP-9 levels were evaluated by gelatin zymography. The results of blot and zymography assays were processed by densitometric software and then analyzed statistically with the use of Mann-Whitney U-test. Results. Elevated HIF-1α (P<0.001) and angiostatins (P<0.05) levels were revealed by western blot in tear fluid samples collected from patients with injured cornea in comparison with the control group. It is noteworthy that extremely low amounts of VEGF were detected in tear fluid from injured eyes, in spite of abundance of its transcription inducer HIF-1α. Dramatically increased levels of active MMP-9 were found in the tear fluids of patients with corneal wounds, while no significant collagenolytic activity was observed in tears from healthy eyes. There is a strong correlation between extent of corneal lesions and changes in markers expression. Conclusions. Tear levels of HIF-1α and angiostatin as well as MMP-9 activity could represent valuable biomarkers of corneal injury severity in traumatic eye.