BENEFICIAL EFFECTS OF ANGIOSTATIN K1-3 AND LACTOFERRIN IN ALKALI-BURNED RABBIT CORNEA: A COMPARATIVE STUDY
Authors:   Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv, Bilous V. 
Published: August 2024

Corneal injury is associated with hypoxia-induced neovascularization, which interferes optical transparency of the cornea, resulting in vision loss and blindness. The treatment of corneal damage remains a ignificant unmet medical need. Lactoferrin (Lf) and angiostatins (AS) are naturally occurring antiinflammatory and antiangiogenic proteins, which play important roles in eye physiology and can be used as protectors against corneal diseases. The aim of the study was to evaluate and compare the effects of plasminogen fragment AS K1-3 and Lf (as a referent preparation) on the levels of the principal markers of angiogenesis, apoptosis, and autophagy in the burn-injured cornea. Additionally, effects of AS on the metabolic activity of macrophages have been studied. Materials and methods. Experimental model of corneal burn was induced by NaOH application to rabbit’s eye surface. One of the group of animals with injured cornea topically received AS, which were applied as eye drops (1 μM solution) daily for 14 days after injury, another one was treated by the equimolar solution of Lf. The levels of protein markers of angiogenesis (VEGF), apoptosis (caspase-3), and autophagy (beclin-1) were evaluated in corneal lysates by western blot. Histological analysis was performed by hematoxylin/eosin staining of corneal slices followed by light microscopy. Effects of AS in the range of concentrations 10-200 nM on the activity of phagocytic cells were assessed in murine macrophage cell line J744.2 by the test with nitroblue tetrazolium (NBT). The quantitative results were analysed with the use of Mann-Whitney U-test. The difference between group mean values was considered significant at P < 0.05. Results. Dramatically increased levels of protein markers related to angiogenesis, apoptosis, and utophagy were found in the corneas with burn-induced injury. AS or Lf-based treatment reduced burninduced overexpression of VEGF, caspase-3, and beclin-1. It is important to note that AS, along with anti-VEGF activity, appeared to more effectively suppress apoptosis and autophagy by decreasing tested markers near the control level, as compared with Lf. Histochemical examination revealed typical signs of fibrotic and necrotic changes in the injured corneas, which were associated with excessive inflammatory infiltration and intense neovascularization. Treatment of burn-induced injury with AS alleviated histopathological changes in cornea, which is evidenced by improving epithelial regeneration, reducing neovascularization, nd moderating leucocyte infiltration. These observations are in agreement with the ata of NBT test suggesting that AS in the concentrations >50 nM is able to moderately inhibit metabolic activity of macrophages up to 30% as compared with intact cells (P<0.05). Conclusions. Obtained results indicate that AS may serve as an effective treatment option to relief alkali-induced corneal injury with the efficacy comparable or even higher than that of firmly established eye protective protein Lf.