Abstract
Purpose :
Proteins in the eye lens accumulate chemical modifications during aging. Carbonyl and oxidative stress are among the major underlying drivers of such modifications. Oxidative stress leads to oxidation of glutathione (GSH) and ascorbate and results in the formation of highly reactive dicarbonyls that react with proteins to form advanced glycation end products (AGEs) in lens proteins. AGEs induce crosslinking and aggregation of proteins and contribute to lens aging and cataract formation. To inhibit AGE formation, we have developed a product, that is disulfide linked between a glutathione diester and a mercaptoethylguanidine (MEG) moiety and we named it Carboxitin.
Methods :
Human lens epithelial cells (HLECs) in culture were incubated with Carboxitin for 4 h. Water-soluble protein from human lenses was glycated with ascorbic acid (1 mM) in the presence or absence of Carboxitin (0.5 mM) for 3 and 7 days. Organ cultured mouse lenses (from 5-6 months old mice) were incubated with or without Carboxitin (2 mM) for 24 h and then with erythrulose (0.5 mM) for 72 h at 37°C. Mouse lens stiffness was measured using a compressive mechanical strain. The protein crosslinking levels were assessed by SDS-PAGE on 4-20% gradient gels and western blotting for crystallins. Carboxitin metabolites and AGEs were measured by LC-MS2.
Results :
Carboxitin was taken up and metabolized to GSH esters and MEG in HLECs. Carboxitin inhibited ascorbate-mediated AGE formation in human lens proteins and inhibited formation of 3-deoxythreosone, a major ascorbate-derived AGE precursor. Mouse lenses incubated with Carboxitin showed higher levels of glutathione esters and MEG. Carboxitin inhibited erythrulose-mediated protein crosslinking and AGE formation and the increase in stiffness in organ-cultured mouse lenses.
Conclusions :
Carboxitin enhances GSH ester levels and traps a dicarbonyl and inhibits AGE formation in the lens. These properties of Carboxitin could be exploited to develop a therapy against AGE-mediated lens stiffness during presbyopia.
This is a 2020 ARVO Annual Meeting abstract.