Role of 6-tioguanine in a mouse model of diabetic retinopathy

Abstract

Purpose : Diabetic retinopathy (DR) is the most severe ocular complication of diabetes mellitus and is the leading cause of legal blindness among working-age individuals in industrialized countries. Proliferative DR, the most severe form, is characterized by the formation of new vessels. Interestingly, the purine analogue 6-thioguanine (6-TG) has recently demonstrated a remarkable anti-angiogenic activity in acute myeloid leukemia. Therefore, we investigated the putative protective and anti-angiogenic effects of 6-TG in a mouse model of DR. Moreover, an in-silico analysis was performed to assess a possible action of 6-TG trough melanocortin receptors 1 and 5 (MCR1 and MCR5), two receptors with a protective role against DR.

Methods : 6-TG (50 nM to 500 µM) was tested on HUVEC viability and vascularization both innormal glucose (NG) or high glucose (HG). Diabetes was induced in C57BL6J male mice (7-10 weeks) with a single intraperitoneal injection of streptozocin (STZ) 65 mg per kg. Animals received intravitreal injections (5 µl) of 6-TG (0.5-1.5-2.5 mg/kg) every 4 weeks until week 12. Fluorangiography (FA) and immunohistology analysis were performed at baseline (T0) and after 12 weeks (T12). Moreover, 6TG and MCR1 and MCR5 antagonists (AGRP14.3 µM and PG20N 130 nM, respectively) were co-administered both in vitro that in vivo.

Results : 6-TG did not reduce HUVEC cell viability in both NG and HG, while it was able to significantly reduce dose-dependently the branches/field in NG and HG conditions (P < 0.05 vs CTRL), showing a specific anti-angiogenic activity. This was confirmed in STZ-mice, showing a significant reduction in vascular abnormalities at T12 when treated with 1.5 and 2.5 mg/kg 6-TG. These were paralleled by a significant reduction (P < 0.01) of retinal CD34 staining, a marker of formation of new vessels. Moreover, the in-silico analysis highlighted MCR1 and MCR5 as putative targets of 6-TG. These was confirmed by the co-administration of 6-TG and AGRP or PG20N, which abolished the antiangiogenic effects showed by 6-TG alone.

Conclusions : 6-TG showed a specific anti-angiogenic activity in a mouse model of DR. Its actions seem to be partially due to and interactions with MCR1 and MCR5 retinal receptors. These data pave the way to the identification of new biochemical and molecular targets modified by the action of 6-TG in an unusual context compared to chemotherapy, in which 6-TG is widely used.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.