Abstract
Purpose :
The obese, type 2 diabetic db/db mouse has been established as suitable model for the pathogenesis of human diabetic retinopathy (DR), but the role of arginase in this model is unknown. Given the association of arginase isoforms, A1 and A2, in several models of retinopathy, we hypothesize that A1 and A2 are associated with the pathophysiology of DR in the db/db mouse. A1/A2 compete with nitric oxide synthase (NOS) for L-arginine and increased A1/A2 decreases nitric oxide (NO) levels. NO is imperative for tissue perfusion in many pathologic disease states, including stroke, DR, and cardiovascular disease (CVD). Increased NO has been shown to be protective in murine models of stroke, CVD, and DR, while A2-/- mice were protected against western diet-induced DR.
Methods :
Whole eyes, retinas, and plasma were collected from 16 (n=8) and 23 week-old (n=6) female db/db mice and their DB/db (control) littermates (#000642). Globes were sectioned and immunofluorescence (IF) was performed for A1, A2, markers of inflammation, and neuroglial activation, including GFAP and IL-6. Retinas were used to western blot for proteins in the NLRP3 inflammasome cascade, products of reactive oxygen species, and hypoxia, including A1, A2, GFAP, HIF-1α, and IL-1β. Western blot signals were quantified by densitometry using ImageJ software and normalized to loading controls. Plasma was used for A1 ELISA analysis, reported in ng/mL. GraphPad Prism 9 was used to implement unpaired t-test analyses. Values of p<0.05 were considered significant.
Results :
A1 levels were markedly increased in both the plasma (16 weeks p=0.011, 23 weeks p<0.0001) and retina lysates (p=0.005) of the db/db mice compared to controls. Retinal A2 was also significantly increased (p=0.0332). Markers of inflammasome activation were significantly increased in db/db retinas (Eg: IL-1β p=0.008). Hypoxia marker HIF-1α, a VEGF transcription factor, was also markedly elevated in db/db retinas (p=0.027). IF of GFAP, an indicator of neurodegeneration, showed increased expression in db/db retinas compared to controls.
Conclusions :
These results are consistent with our hypothesis that A1 and A2 are linked to the pathophysiology of DR in the db/db mouse, as elevated A1 and A2 correlate with increased markers of hypoxia, inflammation, neurodegeneration in db/db mouse retina compare to controls. Further investigation is needed to elucidate the mechanistic effects of A1/A2 on the pathogenesis of db/db DR.
This is a 2021 ARVO Annual Meeting abstract.