altering the synthesis and turnover of specific ECM molecules and by inducing alternative splice variants of ECM molecules such as fibronectin (Li et al., 2000) and versican (Zhao and Russell, 2005). In addition to eVects on the ECM, TGFb inhibits TM cell proliferation (Wordinger et al., 1998), increases the expression of smooth muscle a actin (Tamm et al., 1996), and increases TM cell phagocytosis (Cao et al., 2003). The TGFb induced alteration of the TM ECM, cytoskeleton, cell proliferation, and rate of phagocytosis all could account for the decreased TM cellularity seen in glaucoma (Alvarado et al., 1984). TGFb receptor inhibitors block the TGFb2 induction of PAI 1 and fibronectin in TM cells (Fleenor et al., 2006), but it is still not known whether this signaling in the TM is via the canonical Smad pathway or a non Smad pathway(s).

In addition to this circumstantial evidence, there is even more compelling evidence for the involvement of TGFb in glaucomatous damage to the outflow pathway. Perfusion culture of human and porcine anterior segments with TGFb2 increased outflow resistance and elevated IOP (Gottanka et al., 2004; Bachmann et al., 2006; Fleenor et al., 2006). The TGFb2 increased outflow resistance was accompanied by the induction of fibronectin and PAI 1 (Bachmann et al., 2006; Fleenor et al., 2006) and the accumulation of fine fibrillar material within the JCT (Gottanka et al., 2004). Very recent studies have shown that transduction of rat and mouse eyes by intraocular injection with an adenoviral expression vector encoding a bioactivated form of TGFb2 significantly elevated IOP over the course of weeks (Clark et al., 2006). It is hoped that this will be a valuable animal model that mimics many features of human glaucoma.

2. CTGF

Connective Tissue Growth Factor (CTGF) is one of the genes induced by TGFb treatment of TM cells (Shepard et al., 2003; Fuchshofer et al., 2007). CTGF is also present in the aqueous humor (van Setten et al., 2002), and aqueous humor levels of CTGF are elevated in patients with pseudoexfoliation glaucoma (Ho et al., 2005). Although CTGF plays important roles in development, it is generally expressed in adult tissues only during pathological states of fibrogenesis. CTGF can regulate ECM metabolism, and its eVects are often synergistic to TGFb. In fact, some of the TGFb eVects on the TM may be mediated by CTGF. Over expression of CTGF in the anterior segment of rodent eyes elevates IOP (Shepard, personal communication), further supporting a role for CTGF in the regulation of aqueous humor outflow.

3. BMP

Bone morphogenic proteins (BMPs) were originally identified as osteogenic growth factors, but they are expressed in a variety of tissues where they regulate embryogenesis and other cell functions (Wordinger and Clark, 2007). Four BMPs, all three BMP receptors, and several BMP antagonists are expressed in adult TM cells and tissues (Wordinger et al., 2002). Although Bmp4 null mice are not viable, mice with a heterozygous Bmp4 deficiency develop elevated IOP (Chang et al., 2001). Several recent studies support an important functional role for BMPs in the TM. BMPs can block the eVects of TGFb on ECM metabolism in cultured TM cells. Fuchshofer and colleagues showed that BMP7 inhibited TGFb2 induction of CTGF, fibronectin, TSP 1, collagens, and PAI 1 (Fuchshofer et al., 2007). Wordinger et al. showed that BMP4 was also able to block TGFb2 induction of fibronectin and PAI 1 (Wordinger et al., 2007). Further evidence for the involvement of BMP signaling in the TM is the finding of increased expression of the BMP antagonist Gremlin in glaucomatous TM cells (Wordinger et al., 2007). The addition of Gremlin to the medium of perfusion cultured human anterior segments significantly increased IOP, demonstrating that perturbation of BMP signaling aVects the outflow pathway.

4. Wnt

Another new signaling pathway that regulates IOP has been recently discovered. The Wnt signaling pathway plays important roles in embryogenesis and morphogenesis, including development of the eye. Wnt is a secreted extracellular protein that binds to frizzled membrane receptors to signal via three diVerent pathways. The canonical pathway involves b catenin and Tcf transcription factors to regulate gene expression. Adult TM cells and tissues express all the components required for Wnt signaling (Wnts, FZDs, coreceptor LRP5, b catenin, Tcf, and several Wnt antagonists) (Clark et al., 2007; Wang et al., 2008). Increased expression of the Wnt signaling antagonist secreted frizzled related protein 1 (sFRP1) was found in studies comparing gene expression between normal and glaucomatous TM (Clark et al., 2007; Wang et al., 2008). To determine whether the TM has a functional Wnt signaling pathway and whether Wnt regulates IOP, sFRP1 was added to the perfusion medium of ex vivo perfusion cultured human anterior segments. Perfusion of human anterior segments with sFRP1 decreased the aqueous outflow facility and also decreased b catenin protein levels in the TM. Suppression of Wnt signaling would cause decreased b catenin levels due to enhanced b catenin phosphorylation by glycogen synthase kinase 3b (GSK3b) and subsequent degradation of b catenin by the proteosome. Increased expression of sFRP1 by transduction of mouse eyes with an Ad5. sFRP1 expression vector caused elevated IOP, and the degree of IOP

Altered TM functions

(e.g., ECM)

IOP

FIGURE 2 Potential interactions between TGFb, CTGF, Wnt, and BMP pathways in the regulation of intraocular pressure.

elevation correlated with aqueous humor levels of sFRP1. Topical ocular administration of a GSK3b inhibitor reversed this sFRP 1 mediated ocular hypertension (Wang et al., 2008). These results clearly demonstrate that the TM contains a functional Wnt signaling pathway that regulates IOP, which appears to be altered in glaucoma.

Growth factors in most cases do not work independently, and there often is a complex interplay between the growth factor signaling pathways. This appears to be the case for TGFb, CTGF, BMP, and Wnt signaling, at least during development. During chondorogenesis and osteogenesis, BMP2 can induce b catenin mediated signaling via Wnt ligands (Chen et al., 2007), and CTGF is an important target of Wnt and BMP signaling in the diVerentiation of mesenchymal stem cells (Luo et al., 2004). Wnt and BMP pathways also appear to interact in cancer cell diVerentiation and tumor suppression (Nishanian et al., 2004). Interactions between the TGFb and BMP pathways already have been shown in the adult TM (Fuchshofer et al., 2007; Wordinger et al., 2007), and studies are underway to determine whether there are similar interactions involving the Wnt and BMP/TGFb signaling pathways in the TM (Fig. 2).

F. Cytokines and Other New Pathways

1. IL 1

Interleukin 1 (IL 1) is one of the cytokines induced in the anterior segment by laser trabeculoplasty (Bradley et al., 2000; Alvarado et al., 2005), and IL 1 plays a key role in regulating matrix metalloproteinase expression in the TM via several diVerent signaling pathways, including AP 1 (Fleenor et al., 2003), JNK (Hosseini et al., 2006), and p38MAPK (Kelley et al., 2007a).

Commensurate with this MMP activation, IL 1 also increases trabecular outflow ex vivo in perfusion cultured anterior segments (Bradley et al., 1998) and in vivo when injected into rat eyes (Kee and Seo, 1997). IL 1 also induces ELAM 1 (E Selectin), and ELAM 1 mRNA and protein expression is increased in glaucomatous TM cells and tissues (Wang et al., 2001). Liton and colleagues independently confirmed that ELAM 1 gene expression was elevated in glaucomatous TM tissues (Liton et al., 2006). Interestingly, a constitutively active IL 1 signaling pathway is present in glaucomatous TM cells, which is no longer regulated by IL 1 autocrine signaling (Zhang et al., 2006). IL 1/NF kB signaling also protects cultured TM cells from oxidation induced apoptosis (Wang et al., 2001).

2. CD44

Levels of the glycosaminoglycan hyaluronan are lower in glaucomatous TM tissues (Knepper et al., 1996a), which has led to additional investigation of the hyaluronan receptor CD44. Immunohistochemical analysis of normal and glaucomatous eyes showed decreased levels of membrane associated CD44H in the glaucomatous TM (Knepper et al., 1998). This was accompanied by increased aqueous humor levels of soluble CD44 (sCD44) in POAG patients compared with non glaucomatous controls (Knepper et al., 2005; Nolan et al., 2007). Aqueous humor sCD44 concentrations in POAG patients were significantly correlated with the degree of visual field loss in POAG patients (Nolan et al., 2007). There are multiple isoforms of sCD44 in aqueous humor due to varying degrees of phosphorylation, and there are greater levels of hypophosphorylated sCD44 in glaucomatous aqueous humor (Knepper et al., 2005). sCD44 is toxic to cultured TM cells and retinal ganglion cells but not several other cell types (Choi et al., 2005), with hypophosphorylated sCD44 being more toxic than standard sCD44 (Knepper et al., 2005). Recently, Shepard and colleagues used viral vectors to over express CD44 in the anterior segments of mouse eyes, causing a significant increase in IOP that correlated with aqueous humor levels of

SCD 44 (Shepardet al,. 2008). This intrigui ng new pathw ay war rants add tional study to determine whether sCD44 is directly involved in the genera-

tion of glaucomatous ocular hypertension and if so, to discover the molecular mechanisms involved.

3. Cochlin

Proteomics analysis of normal versus glaucomatous TM tissues led to the discovery of increased cochlin levels in the glaucoma samples. Cochlin is an ECM protein highly expressed in the inner ear, but is also expressed in the eye. PAGE/MS analysis of TM proteins extracted from normal donor eyes and from trabeculectomy specimens from glaucoma patients showed

4.SAA

deposition, but there was no evidence of amyloid deposition in the outflow pathway of either human glaucoma eyes or the SAA induced ocular hypertensive mouse eyes (Wang, unpublished observation). In vivo or in vitro treatment of TM cells with SAA altered TM gene expression (Wang et al., 2008), which may be responsible for the glaucomatous changes to the TM.

IV. FUTURE THERAPEUTIC OPPORTUNITIES

Almost all current glaucoma therapy lowers IOP either by suppressing aqueous humor formation or by increasing uveoscleral or trabecular outflow. However, none of these therapies address the underlying cause of the increased outflow resistance that occurs in glaucomatous eyes. Our inability to alter glaucomatous disease progression in the glaucomatous outflow pathway may be one important reason that glaucoma patients become ‘‘resistant’’ to their medical therapies over time because outflow damage continues to progress.

C3 transferase in organ cultured monkey eyes (Liu et al., 2005), and caldesmon in cultured human and monkey anterior segments (Gabelt et al., 2006), have increased aqueous outflow, providing proof of principle for this potential new therapeutic approach. However, a number of basic questions remain