Increased endoplasmic reticulum stress in human glaucomatous trabecular meshwork cells and tissues

Abstract

 

Purpose

 

Primary open angle glaucoma (POAG) is the most common form of glaucoma and is accompanied by elevated intraocular pressure (IOP) resulting from increased aqueous humor outflow resistance through the trabecular meshwork (TM). The pathological mechanisms underlying increased outflow resistance have not been fully delineated. We recently demonstrated that chronic endoplasmic reticulum (ER) stress in the TM is associated with ocular hypertension in mouse models of glaucoma. The purpose of this study is to determine whether ER stress is also increased in human glaucomatous TM cells and tissues.

 

Methods

 

ER stress markers (GRP78, GRP94, ATF-4, ERO-1α, phosphorylated elF-2α and CHOP) were examined by immunohistochemistry and Western blot analysis in TM tissues from age-matched normal (n=22) and glaucoma donors (n=23). In addition, ER stress markers were examined in primary TM cells isolated from normal (n = 3 NTM) and glaucoma (n =4 GTM) human donors.

 

Results

 

Immunohistochemical analysis demonstrated a significant increase in GRP78 and GRP94 in the glaucomatous TM (n=18) compared to normal TM (p<0.0001, n=18,). Interestingly, there was little or no detectable level of CHOP observed in normal TM tissues. However, there was a 2-fold increase in CHOP levels in the glaucomatous TM (p<0.0001; n=18), indicating the presence of chronic ER stress in the glaucomatous human TM. Western blot analysis of TM tissue lysates also demonstrated increased ER stress markers in the glaucomatous TM tissues including GRP78, GRP94, ATF-4, ERO-1α and CHOP. Densitometric analysis of Western blots showed a significant increase in ATF-4 (p=0.0247), ERO-1α (p=0.0129) and CHOP (p=0.0294) expression in the glaucomatous TM (n=5) compared to age-matched normal TM (n=4). In addition, primary TM cells obtained from glaucoma donors demonstrated increased ER stress compared to normal TM cells. We further demonstrate that Chop is increased in TM of Tg-MYOC^Y437H mice and deletion of Chop prevented IOP elevation in Tg-MYOC^Y437H mice.

 

Conclusions

 

These studies indicate the presence of chronic ER stress in human glaucomatous TM tissues and cells. These studies further support the pathological role of ER stress in IOP elevation and possible new treatment options via targeting this pathway for reduction of elevated IOP in glaucoma.  

 

Immunostaining for KDEL and CHOP in human TM tissues.