Abstract
Purpose :
Age and intraocular pressure elevation due to increased conventional outflow resistance are the two primary risk factors for glaucoma. However, the effects of aging on conventional outflow tissue homeostasis is unknown. The aim of this study was to determine age-related changes in the proteome of conventional outflow tissues.
Methods :
In accordance with an approved animal protocol (A226-21-11), full thickness limbal strips containing conventional outflow tissues were carefully dissected from eyes of two age groups of male mice (3 month vs. 30 month old, n=4 mice/group). Protein digests (20 µg) were prepared and isolated using a paramagnetic bead protocol and analyzed using quantitative LC/MS/MS. A rigorous quality control protocol was executed before protein data were analyzed for statistical differences in protein expression between groups using two-tailed heteroscedastic t-test on log2-transformed data. The differentially expressed protein dataset was then analyzed for affected cellular pathways by ReactomeGSA software. Sagittal sections of irideo-corneal tissues were immunostained with Iba1 antibodies and imaged using confocal microscopy. Iba1 protein levels in limbal strips containing outflow tissues were quantified by Western blot.
Results :
Analysis of proteomic data showed that 102 proteins were significantly upregulated and 50 proteins significantly downregulated in limbal tissues of elderly mice (1.5-fold change, ANOVA p value <0.05). Pathway analysis of differentially expressed proteins demonstrated that proteins involved in the immune system (20 proteins) and metabolic pathways (19 proteins) were primarily upregulated with age. In contrast, multiple pathways were downregulated with age (e.g. signal transduction, transcription, developmental biology, etc.) and represented by fewer proteins (4-10 proteins). Upregulation of immune system proteins can be partially explained by increased numbers of macrophages in the limbal tissues as indicated by increased Iba1 expression in limbal tissues from elderly eyes (1.3-fold, p=0.01, n=3) and a qualitative increase in Iba1 positive cells, particularly in the trabecular meshwork of elderly eyes.
Conclusions :
While a number of proteins were downregulated with age, the primary finding was the upregulation of proteins involved in cell metabolism and the immune system in conventional outflow tissues with age.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.