Title: The Role of SPARC in Aqueous Humor Outflow and TGFß2-mediated Ocular Hypertension in a Murine Model
Description: Glaucoma is the leading cause of irreversible blindness worldwide, and is a major cause of blindness in the United States. It affects approximately 5% of Caucasians and 10% of African- Americans over the age of 60 years. Elevated intraocular pressure (IOP) is currently the only modifiable risk factor for glaucoma. Impaired outflow of aqueous humor from the eye is thought to be the cause of pathologically elevated IOP. However, the etiology of outflow impairment is unknown. Anatomically, the aqueous humor drains into the iridocorneal angle of the eye, where the iris inserts at the transition between the cornea and sclera. In humans, approximately 80-90% of the aqueous traverses through the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), Schlemm’s canal, collector channels and empties into episcleral veins. Abnormalities at these sites are thought to cause impaired outflow.

Abnormal accumulation of extracellular matrix (ECM) in the TM or JCT, abnormal endothelial function in Schlemm’s canal, or a combination of these components have been strongly implicated. Our laboratory has focused on the role of Secreted Protein Acidic and Rich in Cysteine (SPARC) in regulating outflow. SPARC is the prototypical matricellular protein that mediates ECM organization and turnover in numerous human tissues. Our lab was first to demonstrate that SPARC is highly expressed in the TM and JCT regions of the eye, and that the SPARC knockout (KO) mouse has a significant decrease in IOP of 15-20%. SPARC may affect the degree of segmental flow, a theory that states that variable aqueous outflow occurs around the circumference of the eye; only certain portions of the TM are thought to display active outflow at any particular moment. The cytokine transforming growth factor-ß2 (TGFß2) has been shown to modulate multiple ECM proteins, including SPARC.

TGFß2 is significantly upregulated by 2 to 3-fold in the aqueous humor of glaucoma patients compared to controls. In addition, when TGFß2 is overexpressed in rodent eyes, increased ECM deposition is observed within the trabecular meshwork leading to IOP elevation. SPARC is one of the most highly upregulated proteins by TGFß2, and is downstream of TGFß2. We hypothesized that wild-type (WT) mice would demonstrate segmental flow, while SPARC KO mice would display a more continuous pattern of outflow around the eye. We also believed that IOP would be inversely correlated with outflow area. We also hypothesized that SPARC is essential to the process of TGFß2-mediated ocular hypertension, and that the lack of SPARC would impair IOP elevation.

We conducted a tracer study utilizing fluorescent microbeads to determine the location of outflow circumferentially around the mouse TM. Microbeads were injected intracamerally into the eyes of WT and KO mice. After a 45-minute incubation period, the mice were euthanized and eyes were processed for confocal, light, and electron microscopy. During the second group of experiments, empty or TGFß2-containing adenovirus was injected intravitreally into WT and SPARC KO mice and IOP was measured for 2 weeks. Immunohistochemistry was completed on all tissues to assess for changes in major ECM proteins.

Percentage effective filtration length (PEFL), or area of the TM labeled by tracer, was significantly increased in SPARC KO mice (70.61% ± 11.36%, p<0.005; N=11) compared to WT mice (54.68% ± 9.95%; N=11). In addition, the pressures between the two sets of eyes were significantly different with mean pressures of 16.3 mm Hg in WT mice and 12.6 mm Hg in KO mice (p<0.005, N=11 pairs). In addition, PEFL and IOP were inversely correlated with R2 = 0.72 (N=10 pairs); in eyes with higher IOP, PEFL was reduced. Electron microscopy demonstrated that high-tracer TM areas had a greater separation between trabecular beams. Collagen fibril diameter was found to be smaller in the KO (28.272 nm) compared to WT (34.961 nm; p<0.0005, N=3 pairs). These data provided structural correlations to the functional data regarding segmental flow.

In the second set of experiments, IOP was found to be significantly elevated in TGFß2- injected WT mice compared to empty vector-injected WT mice during days 4-11 (p<0.05, N=8). However, IOP was not significantly elevated in TGFß2-injected KO mice compared to controls. Immunohistochemistry demonstrated that TGFß2 increased expression of collagen IV, fibronectin, plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and SPARC within the TM of WT mice, but only PAI-1 and CTGF in KO mice (p<0.05, N=3 pairs).

These data support our hypotheses, indicating that SPARC plays an integral role in the modulation of aqueous humor outflow. In addition, it appears as though SPARC is essential to the regulation of TGFß2-mediated ocular hypertension. Aside from providing further evidence of the importance of ECM in IOP regulation, our work presents the novel discovery of segmental flow in the mouse. Given the potential role of SPARC in TGFß2-mediated ocular hypertension, SPARC may not only play an integral role in ECM homeostasis within the trabecular meshwork, but may be a valuable target for pharmacologic therapy in treating primary open-angle glaucoma.