Abstract
Purpose :
Post-mitotic corneal endothelial cells are subject to external and internal stressors with aging. To identify the mechanisms contributing to the age-associated decline in corneal endothelial functions, we undertook ex-vivo and in-vitro analysis.
Methods :
For in-vitro studies, primary bovine corneal endothelial cells (BCEC) were cultured with passage 1 considered as early passage and passage 4 as late passage. We analyzed changes in cell viability and mitochondrial functions using flow cytometry analysis of Annexin V and JC-1, respectively. We measured oxidative stress using DCFDA assay and immunofluorescence for Nitrotyrosine. Changes in protein clearance pathways were assessed using autophagy flux (DALGreen assay), autophagosome formation (DAPGreen assay), and proteasomal chymotrypsin activities. Jess immunoassay analysis was carried out for mitochondrial, autophagy, proteasome-related proteins, and antioxidant proteins. For ex-vivo assays, we used young (5-week-old) and aged (30-week-old) C57BL/6J mice. All the analyses mentioned above were also carried out in the animal studies.
Results :
Both in-vitro and ex-vivo studies showed an increase in oxidative stress, antioxidant protein expressions, autophagy flux, and proteasomal activities with an increase in passages and age, respectively. With the increase in passage numbers, BCEC showed an upregulation of autophagy-related proteins - Beclin1 (Passage 4- 1.6-fold), LAMP2A (Passage 4- 2-fold), proteasome activity regulator- αPA28 (Passage 4- 1.4-fold), and antioxidant protein- catalase (Passage 4- 1.3-fold) compared to Passage 1. Similar findings were observed in the corneal endothelium of older mice.
Conclusions :
Our preliminary data indicates an interplay between oxidative stress protein clearance pathways and age. Future studies will identify the precise mechanisms that are behind these changes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.