Expression profile of the matricellular protein osteopontin in primary open-angle glaucoma and the normal human eye

Abstract

PURPOSE. To characterize the role of osteopontin (OPN) in primary open-angle glaucoma (POAG) and normal eyes. METHODS. OPN quantification was performed by enzyme-linked immunosorbent assay in aqueous humor (AH) obtained from human donor eyes (POAG and normal) and surgical samples (POAG and elective cataract removal). OPN expression and localization in whole eye tissue sections and primary normal human trabecular meshwork (NTM) cells were studied by Western blot and immunohistochemistry. Latanoprost-free acid (LFA)-treated NTM cells were analyzed for OPN gene and protein expression. Intraocular pressure was measured by tonometry, and central corneal thickness was measured by optical coherence tomography in young OPN(-/-) and wild-type mice. RESULTS. OPN levels were significantly reduced in donor POAG AH compared with normal AH (0.54 ± 0.18 ng/μg [n = 8] vs. 0.77 ± 0.23 ng/μg [n = 9]; P = 0.039). A similar trend was observed in surgical AH (1.05 ± 0.31 ng/μg [n = 20] vs. 1.43 ± 0.88 ng/μg [n = 20]; P = 0.083). OPN was present in the trabecular meshwork, corneal epithelium and endothelium, iris, ciliary body, retina, vitreous humor, and optic nerve. LFA increased OPN gene expression, but minimal change in OPN protein expression was observed. No difference in intraocular pressure (17.5 ± 2.0 mm Hg [n = 56] vs. 17.3 ± 1.9 mm Hg [n = 68]) but thinner central corneal thickness (91.7 ± 3.6 μm [n = 50] vs. 99.2 ± 5.5 μm [n = 70]) was noted between OPN(-/-) and wild-type mice. CONCLUSIONS. OPN is widely distributed in the human eye and was found in lower concentrations in POAG AH. Reduction of OPN in young mice does not affect IOP.

Figure 1.

OPN concentration in donor and surgical AH. OPN concentrations are reduced in (A) donor and (B) surgical AH derived from POAG when compared with normal or elective cataract controls.

Figure 2.

OPN expression in normal and POAG AH. (A) Western blot shows six OPN protein molecular weights. The 70-kDa OPN protein (band 4) was absent in 3 of the 6 POAG samples. (B) Densitometric analysis of OPN bands 1 to 5 show decreased OPN in POAG compared with normal. Only band 6 shows an increase of OPN in POAG. Total OPN protein was lower in POAG than in normal AH. Human serum albumin (HSA) was used for internal AH control and to normalize each band intensity. *P < 0.02.

Figure 3.

OPN expression in different ocular tissues. (A–D, I–L) Immunofluorescence and subsequent confocal microscopy show strong expression and localization of OPN in eye tissues within both anterior and posterior chambers. (E–H, M–P) Secondary antibody alone controls are shown for comparison. (A, E) Trabecular meshwork (TM). (B, F) Ciliary body (CB). (C, G) Retina. (D, H) Iris. (I, M) Corneal epithelium (C. Epi). (J, N) Corneal endothelium (C. Endo). (K, O) Anterior optic nerve (Ant ON). (L, P) Posterior optic nerve (Post ON). RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; SC, Schlemm's canal. Image exposure for an eye tissue was the same as the corresponding negative control. Pictures are representative of OPN staining performed in three sections obtained from two donor eyes. Scale bar, 10 μm.

Figure 4.

Western blot analysis of OPN in ocular tissues. Considerable variation in OPN expression profile was seen across different ocular samples. OPN was consistently found in retina, optic nerve, and trabecular meshwork (TM). OPN was also identified in iris and vitreous humor in at least 1 of the 3 samples.

Figure 5.

Deglycosylation of OPN. (A) After deglycosylation (+), OPN protein fragments appeared as lower molecular weights compared with nondeglycosylated controls (−). (B) No change in OPN migration was observed when optic nerve (ON) or normal trabecular meshwork (TM) tissue was treated with (+) or without (−) glycosidases.

Figure 6.

Effect of LFA on OPN gene expression in primary NTM cells. (A) OPN mRNA induction increased within 24 hours after LFA treatment in 10% FBS-containing media. (B) No changes in OPN mRNA level were identified when NTM cells remained in serum-free conditions. (C) Intracellular and (D) secreted OPN protein levels did not change within 48 hours after LFA treatment in 10% FBS-containing media. A slight increase (20%) in secreted OPN was found at 72 hours but was not significant.

Figure 7.

Iridocorneal angles of wild-type and OPN^−/− mice. Iridocorneal angles of (A) 8-week-old wild-type and (B) age-matched OPN^−/− mice appeared microscopically indistinguishable. Schlemm's canal, trabecular beams, cellularity, uveoscleral outflow pathway, and ciliary body location all appeared normal. CP, ciliary processes. Asterisk: anterior chamber; arrow: Schlemm's canal. Scale bar, 50 μm.