Matricellular proteins in the trabecular meshwork: review and update

Abstract

Abstract Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide, and intraocular pressure (IOP) is an important modifiable risk factor. IOP is a function of aqueous humor production and aqueous humor outflow, and it is thought that prolonged IOP elevation leads to optic nerve damage over time. Within the trabecular meshwork (TM), the eye's primary drainage system for aqueous humor, matricellular proteins generally allow cells to modulate their attachments with and alter the characteristics of their surrounding extracellular matrix (ECM). It is now well established that ECM turnover in the TM affects outflow facility, and matricellular proteins are emerging as significant players in IOP regulation. The formalized study of matricellular proteins in TM has gained increased attention. Secreted protein acidic and rich in cysteine (SPARC), myocilin, connective tissue growth factor (CTGF), and thrombospondin-1 and -2 (TSP-1 and -2) have been localized to the TM, and a growing body of evidence suggests that these matricellular proteins play an important role in IOP regulation and possibly the pathophysiology of POAG. As evidence continues to emerge, these proteins are now seen as potential therapeutic targets. Further study is warranted to assess their utility in treating glaucoma in humans.

FIG. 1.

Schematic diagram showing key ocular structures involved in aqueous humor inflow and outflow (adapted, with permission, from Tomarev 2001, Nature Med. 7, 294–295; Copyright © 2001 Nature Publishing Group). (A) Secreted by the ciliary body (CB), aqueous humor passes from the posterior chamber (PC) to the anterior chamber (AC) via the pupil. Aqueous humor outflow through the conventional outflow pathway involves the trabecular meshwork (TM) and juxtacanalicular (JCT) region, Schlemm's canal (SC), and the episcleral venous system. (B) An enlargement of the TM, JCT, and SC from (A).

FIG. 2.

(A, B) Representative pair of matched WT and SPARC-null (KO) mouse eyes (adapted, with permission, from Swaminathan et al., Copyright © 2013 Association for Research in Vision and Ophthalmology). Fluorescence microscopy was complete en face through the corneal side. Tracer bead distribution in the TM of wild-type (WT) eyes was heterogeneous, with large sections having little staining. Greater fluorescence in a more homogeneous pattern was seen in SPARC KO eyes, reflecting more uniform outflow. Gray portions of these images are from the projection of the brightfield lamp on the tissue and medium. Central portions of these images were removed due to iris autofluorescence. (C, D) Representative confocal microscopy of frontal sections of WT and KO high-tracer sections, respectively. Sclera, TM, SC, CB, collector channel (CC), and episcleral vein (EV) are labeled. Tracer is strongly present throughout the TM. Tracer can also be noted within SC. (E) En face image of a high-tracer section demonstrating microbeads within the TM and along the walls of EVs. (F) Percentage effective filtration length (PEFL) values in WT and KO eyes with SEM error bars. KO PEFL was significantly higher (asterisk) than WT PEFL (n=11 pairs, P<0.005).

FIG. 3.

Representative immunolabeling of ECM proteins after infection with adenovirus overexpressing human SPARC versus vector only control (adapted, with permission, from Oh et al., Copyright © 2013 Association for Research in Vision and Ophthalmology). Collagen I (A), collagen IV (B), and fibronectin (D) were increased, but collagen VI (C) and laminin (E) were not changed. Collagen I was increased throughout the TM with some increase in the JCT region. Collagen IV was detected more prominently in the JCT region and under the outer wall of SC. Fibronectin was increased throughout the TM, but prominently within the JCT region. The secondary staining (F) was shown as a negative control. Scale bar: 30 μm; C, control, S+, SPARC+; magnification=× 100.

FIG. 4.

Analysis of SPARC levels following TGF-β2 treatment (2 ng/mL) in cultured TM cells, n=6 (adapted, with permission, from Kang et al., Copyright © 2013 Association for Research in Vision and Ophthalmology). SPARC mRNA was analyzed by qPCR and normalized with β-actin. SPARC protein levels in conditioned media were analyzed by immunoblotting and calculation of integrated band intensity. Representative immunoblot shown.

FIG. 5.

(A) Substratum stiffness impacts SPARC mRNA and cellular response to treatment with the actin cytoskeletal disruptor Latrunculin-B (Lat-B) in human TM cells (adapted, with permission, from Thomasy et al., Copyright © 2012 Association for Research in Vision and Ophthalmology). Cells grown on stiff hydrogels and treated with DMSO had significantly different SPARC mRNA expression in comparison with DMSO-treated cells on tissue culture polystyrene (TCP; >1 GPa) or more compliant hydrogels. Cells grown on hydrogels and treated with Lat-B had significantly less SPARC mRNA expression versus treatment with DMSO. In contrast, cells grown on TCP and treated with Lat-B had significantly greater SPARC mRNA expression versus treatment with DMSO. Data are mean±SEM (*P<0.05 for hydrogel versus TCP; ^a,b,cP<0.05 between the different hydrogels; ^†P<0.05 for DMSO versus Lat-B). (B) Substratum stiffness modulates expression of SPARC protein and the impact of Lat-B treatment on SPARC protein expression. TM cells on 75 kPa hydrogels (mimicking the stiffness of glaucomatous TM) have markedly greater SPARC expression than on the much stiffer (>1 GPa) substrates. Treatment with 2 μM Lat-B decreases SPARC protein expression on the 75 kPa hydrogels. Exposure time for each image was 3.0 s. Scale bar, 50 μm.