TY - JOUR
T1 - Increased endoplasmic reticulum stress in human glaucomatous trabecular meshwork cells and tissues
AU - Peters, Joseph C.
AU - Bhattacharya, Sanjoy
AU - Clark, Abbot F.
AU - Zode, Gulab S.
N1 - Publisher Copyright:
© 2015 The Association for Research in Vision and Ophthalmology, Inc.
PY - 2015
Y1 - 2015
N2 - 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 was to determine whether ER stress is also increased in human glaucomatous TM cells and tissues. Methods. Endoplasmic reticulum stress markers including GRP78, GRP94, and C/EBP homologous protein (CHOP) were examined by immunohistochemistry in the TM of age-matched normal (n = 18) and open-angle glaucoma donors (n = 18). GRP78, GRP94, activating transcription factor (ATF)-4, endoplasmic oxidoreductin-1alpha (ERO-1α), phosphorylated eukaryotic translation initiation factor 2α (EIF-2α), and CHOP were examined by Western blot analysis in TM tissue lysates from age-matched normal (n = 4) and POAG donors (n = 5). In addition, ER stress markers were examined in primary TM cells isolated from normal (n = 4 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 minimum CHOP immunostaining observed in normal TM tissues. However, there was a 3-fold increase in CHOP levels in the glaucomatous TM (P < 0.0001; n = 18), indicating the presence of chronic ER stress in the glaucomatous 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, ERO-1α, and CHOP 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 markers including increased GRP78, GRP94, ATF-4, ERO-1α, and CHOP compared to normal TM cells. However, glaucomatous TM cells did not show splicing of XBP-1, a marker of unfolded protein response pathway. Conclusions. These studies indicate the presence of chronic ER stress in human glaucomatous TM tissues and cells and further suggest that ER stress pathway may provide a novel target for developing disease-modifying glaucoma treatments.
AB - 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 was to determine whether ER stress is also increased in human glaucomatous TM cells and tissues. Methods. Endoplasmic reticulum stress markers including GRP78, GRP94, and C/EBP homologous protein (CHOP) were examined by immunohistochemistry in the TM of age-matched normal (n = 18) and open-angle glaucoma donors (n = 18). GRP78, GRP94, activating transcription factor (ATF)-4, endoplasmic oxidoreductin-1alpha (ERO-1α), phosphorylated eukaryotic translation initiation factor 2α (EIF-2α), and CHOP were examined by Western blot analysis in TM tissue lysates from age-matched normal (n = 4) and POAG donors (n = 5). In addition, ER stress markers were examined in primary TM cells isolated from normal (n = 4 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 minimum CHOP immunostaining observed in normal TM tissues. However, there was a 3-fold increase in CHOP levels in the glaucomatous TM (P < 0.0001; n = 18), indicating the presence of chronic ER stress in the glaucomatous 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, ERO-1α, and CHOP 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 markers including increased GRP78, GRP94, ATF-4, ERO-1α, and CHOP compared to normal TM cells. However, glaucomatous TM cells did not show splicing of XBP-1, a marker of unfolded protein response pathway. Conclusions. These studies indicate the presence of chronic ER stress in human glaucomatous TM tissues and cells and further suggest that ER stress pathway may provide a novel target for developing disease-modifying glaucoma treatments.
KW - CHOP
KW - ER stress
KW - Glaucoma
KW - Trabecular meshwork
UR - http://www.scopus.com/inward/record.url?scp=84939839013&partnerID=8YFLogxK
U2 - 10.1167/iovs.14-16220
DO - 10.1167/iovs.14-16220
M3 - Article
C2 - 26066753
AN - SCOPUS:84939839013
SN - 0146-0404
VL - 56
SP - 3860
EP - 3868
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 6
ER -