EndothelialCdkn1a(p21) Overexpression and Accelerated Senescence in a Mouse Model of Fuchs Endothelial Corneal Dystrophy

Abstract

Stress of the endoplasmic reticulum and oxidative stress play critical roles in the pathogenesis of Fuchs Endothelial Corneal Dystrophy (FECD). In the normal aging cornea, cellular stress has been associated with a loss in proliferative capacity (premature senescence) of corneal endothelial cells (CECs). The present study used a transgenic Col8a2(Q455K/Q455K) knock-in mouse model of early-onset FECD to identify the endothelial expression profile of specific cellular stress response-related targets, which may be relevant to late-onset FECD. The differential endothelial mRNA levels of cellular stress response-related genes were determined in 12-month-old homozygous Col8a2(Q455K/Q455K) mutant and wild-type mice using customized PCR arrays. Result validation and analysis of additional senescence-related transcripts was performed by real-time PCR. Expression of p53 and p21 was assessed by immunofluorescence. Senescence-associated β-galactosidase (SA-β-Gal) activity was investigated by histochemical labeling. Human FECD samples and normal controls were examined for p21 expression by immunohistochemistry. PCR-array analysis showed greater than 2-fold and/or significantly altered endothelial regulation of 19 cellular stress response-related transcripts in Col8a2(Q455K/Q455K) mutant mice; real-time PCR documented statistically significant upregulation of senescence-associated targets Cdkn1a (p21), Serpine1 (PAI-1), Tagln (Sm22), Fn1 and Clu (ApoJ). Immunofluorescence revealed increased expression of nuclear p53 and p21 in mutant animals. SA-β-Gal staining detected increased proportions of senescent CECs in mutant mice. Human FECD endothelium exhibited increased levels of nuclear p21 protein. Our results identify endothelial Cdkn1a (p21) upregulation in a mouse model of early-onset FECD, confirm overexpression of p21 in late-onset human FECD endothelium, and suggest a role for premature senescence in FECD.