The dissertation is devoted to the study of molecular mechanisms of the development of pathological conditions of the cornea, induced by various factors, the influence of which is associated with the development of hypoxia, inflammation, and neovascularization. Since corneal avascularity is a threatening condition that leads to deterioration of vision or its complete loss, today the search for new effective and safe modulators of angiogenesis is an urgent problem of modern biochemistry, biomedicine and clinical ophthalmology.
The object of research of the dissertation is the molecular mechanisms of the development of neovascular diseases of the cornea caused by alkaline burns and chronic alcoholism, as well as the mechanisms of the protective action of angiostatins and thiamine. Therefore, the aim of the work was formulated as follows: to find out the molecular mechanisms of the development of pathological conditions of the cornea and to develop approaches to their correction. Modern biochemical and immunochemical methods were used to fulfill the tasks set in the dissertation, namely: protein gel electrophoresis, immunoblotting, immunoenzymatic analysis, affinity chromatography, enzyme phoresis (gelatin zymography), spectrophotometric methods, histological methods, as well as methods of eukaryotic cultivation cells, immunization of animals and obtaining polyclonal antibodies, determination of the degree of neovascularization according to the Efron scale, methods of statistical processing of results.
The work investigated the effects of physiological inhibitors of neovascularization, angiostatins (K1-3, K5), which suppress angiogenesis through specific inhibition of proangiogenic signaling in endothelial cells and have anti-inflammatory properties. In the presented work, it was established for the first time that angiostatins exhibit a wide range of protective effects in the cornea. In particular, it was shown for the first time that angiostatins in a dose-dependent manner contribute to a decrease in the levels of marker proteins associated with hypoxia (HIF-1α), angiogenesis (VEGF), tissue remodeling and fibrosis (MMP-9), autophagy (Beclin-1), intercellular tight contacts (ZO-1), as well as endoplasmic reticulum stress (GRP-78) as the main links of the pathological process. The protective effects of angiostatins on satellite glia cells in the injured cornea have also been demonstrated. For the first time, angiostatins were shown to regulate the level of the ACE2 protein, which serves as a receptor for the SARS-CoV-2 virus, and to reduce the level of the receptor in corneal damage. For the first time, the absence of cytotoxic effects of angiostatins on cells of the retinal pigment epithelium in the therapeutic range of concentrations (2–100 nM) was proven, which was shown in experiments on RPE cell culture. For the first time, it was shown that administration of thiamine (25 mg/kg body weight) to rats with chronic alcohol intoxication has a corrective effect, reducing the consequences of the toxic effects of ethanol in the cornea. It was established for the first time that vitamin B1 contributes to an increase in the ratio of BclxL/Bax - an indicator that indicates the inhibition of apoptosis and the strengthening of the viability of cells in the tissue of the cornea under the conditions of the toxic effect of ethanol. New are the results that prove the neuroprotective properties of thiamine in the cornea of rats under the conditions of long-term consumption of ethyl alcohol, which is manifested in the normalization of the content of neuronal markers (NF-H, τ-protein and Neu-N), as well as a decrease in the level of activation of satellite glia and the expression of the corresponding GFAP marker.
The obtained results regarding the antiangiogenic effect of angiostatins and their pleiotropic effects in the damaged cornea may serve as a basis for creating a new drugs for treatment of ocular diseases associated with inflammation and neovascularization. The use of angiostatins can be a promising tool for normalizing the condition of the corneal tissue during keratoplasty. The results proving the anti-apoptotic and neuroprotective properties of thiamine may be useful for the development of new vitamin preparations as effective means of correcting the ophthalmic manifestations of chronic alcohol intoxication. The use of polyclonal antibodies to lactoferrin obtained in the work can become the basis for the development of new non-invasive diagnostic methods based on immunochemical detection of the level of lactoferrin in tear fluid, which will make the treatment of patients with eye injuries more personalized. The obtained data create a theoretical basis for the use of a number of protein-biomarkers to assess the degree of destructive changes in the cornea due to its trauma as a basis for non-invasive diagnostics using tear fluid.
