Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

kinase C may play an important role in modulation of aqueous outflow facility.

C. ROCK Inhibitors

Recently, in addition to the broad spectrum kinase inhibitors, ROCK inhibitors, such as Y-27632 and H-1152, have been found to be efficacious in lowering IOP. ROCKs are kinases that can be activated by a cell signaling molecule Rho. Once activated, ROCKs phosphorylate and affect functions of various other proteins, including the myosin light chain phosphatase, LIM kinases, ezrin-radixin-moesin proteins, adducin, sodium hydrogen exchanger NHE1, and intermediate-filament proteins, etc. These molecular actions of ROCKs play a significant role in cell contractility, cell shape, and focal adhesion. Consequently, ROCK inhibitors are expected to affect these cellular functions. Consistent with this expectation, ROCK inhibitors are found to induce changes in cell shape of cultured human TM and Schlemm’s canal cells, and decrease actin stress fibers and myosin light chain phosphorylation in these cells, which is associated with widening of the extracellular spaces in the TM, especially of the JCT (Rao et al., 2005a; Rosenthal et al., 2005). All these actions should modulate the outflow facility of aqueous humor.

Indeed, the ROCK inhibitor Y-27632 was observed to increase outflow facility of aqueous humor in enucleated porcine eyes in a dose-dependent manner. At 100 μM, Y-27632 increases outflow facility by more than 60% from baseline lasting more than 5 hours (Rao et al., 2001). In the rabbit, topical administration of this compound produces a reduction in IOP lasting for 6 hours. The maximal decrease reaches 12 mmHg (Honjo et al., 2001b; Waki et al., 2001). Intracameral perfusion of Y-27632 also produces a significant ocular hypotension in the monkey (Tian and Kaufman, 2005). Another ROCK inhibitor H-1152 also increases outflow facility in perfused porcine eyes (Rao et al., 2005a).

Although Y-27632 and H-1152 are promoted as ROCK inhibitors, they also inhibit other protein kinases. Therefore, their IOPlowering effects alone cannot prove the involvement of ROCK in the regulation of aqueous outflow. Recently, specific inhibition of ROCK activity in human and porcine TM cells with adenoviral vector expressing the dominant negative Rho-binding domain of ROCK results in a reduced myosin light chain phosphorylation, fewer actin stress fibers, decreased focal adhesion, cell rounding, and cell detachment. Furthermore, organ cultured human eye anterior segments transfected with the same adenoviral vector demonstrate a significant increase in outflow facility (Rao et al., 2005b). These results provide critical molecular evidence supporting the involvement of ROCK in the regulation of aqueous humor outflow facility.

The discovery of the ocular hypotensive effects of protein kinase inhibitors, especially the ROCK inhibitors, opens up a very important new direction in the continuous quest of glaucoma therapy. These compounds are topically active and have great IOP-lowering efficacy, which are highly desirable. Nonetheless, since kinases are involved in many cellular functions in most cells of most tissues, vigilance is needed for the potential local and systemic side effects of prolonged use of these compounds. For example, topical ocular administration of ROCK inhibitors results in significant conjunctival hyperemia, which may be due to vasodilation of this vessel bed.

XIII. STATINS

In ex vivo pig eyes, perfusion of lovastatin for 4 days was demonstrated to increase aqueous outflow by 110% (Song et al., 2005). Lovastatin is one of the cholesterollowering statins. They are inhibitors of the 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase). Inhibition of HMGCoA reductase decreases the synthesis of farnesyl pyrophosphate and geranylgeranyl

pyrophosphate. These isoprenoids are required for Rho activation. Therefore, statins can suppress the activity of Rho, and inhibit ROCK indirectly. Hence, the outflow effect of lovastatin may be related to its effect on ROCK. In porcine TM cells, lovastatin and mevastatin decrease myosin light chain phosphorylation, actin depolymerization, cytoskeletal reorganization, cell rounding, and focal adhesions (Song et al., 2005). Some of these changes are very similar to those caused by ROCK inhibitors.

Interestingly, long-term (24 months or longer) oral use of statins was reported to be associated with a lower risk of open angle glaucoma (McGwin et al., 2004). However, in the same study, a lower incidence of open angle glaucoma was also observed among those who used non-statin cholesterollowering agents. At the present time, it is not clear if statins provide an additional reduced risk in glaucoma.

XIV. SEROTONERGIC AGONISTS

Serotonergic receptor agonists and antagonists have long been shown to affect IOP. Unfortunately, because of the multitude of serotonergic receptor subtypes, the lack of specificity of many of the agents tested, and the probable different responses in different animal species, pharmacological actions of this class of compounds on IOP and aqueous hydrodynamics are still controversial. For example, while intracameral injection of serotonin lowers IOP in the rabbit, topical administration was reported to increase rabbit IOP in some studies, and decrease IOP in others. Similarly, topical application of 5-HT1 agonist 5-carboxamidotryptamine (5-CT) raises rabbit IOP (Meyer Bothling et al., 1993), but topical application of 5-HT1A agonists, such as 8-hydroxy-dipropylaminote- tralin (8-OH-DPAT) or flesinoxan, lowers rabbit IOP (Chidlow et al., 2001, 1999; Chu et al., 1999a). Yet all of the above compounds and other selective 5-HT1A agonists do not affect monkey IOP (Gabelt et al., 2001; May

et al., 2003b). Effects of serotonergic antagonists are equally confounding. Topical ocular instillation of the 5-HT2 receptor antagonist ketanserin lowers IOP in the rabbit, cat, and monkey by suppressing aqueous production (Chang et al., 1985). Oral or topical administration of ketanserin also lowers IOP in normal volunteers and glaucoma patients (Costagliola et al., 1990; Tekat et al., 2001), but with an increase in aqueous outflow facility. This compound is a potent antagonist of the α1-adrenergic receptor as well. Its IOP-lowering effect is likely mediated via blockade of the α1-adrenergic instead of 5-HT2 receptor because selective 5-HT2 antagonists, such as cinanserin and SB-242084, do not affect monkey IOP (May et al., 2003b).

Recently, a 5-HT2 agonist R–1-(4-iodo-2, 5- dimethoxyphenyl)-2-aminopropane (R-DOI) was shown to lower IOP. In laser-induced ocular hypertensive monkeys, topical ocular administration of this agent causes a dose-dependent ocular hypotension, with a maximal reduction of more than 30% at 3 and 6 hours after treatment (May et al., 2003a). In normotensive monkeys, R-DOI also decreases IOP significantly, concomitant with a 240% increase in uveoscleral outflow (Gabelt et al., 2005). A series of selective 5-HT2 agonists were synthesized, and these compounds, such as S-( )-1-(2-aminopropyl)- 8,9-dihydropyrano[3,2-e]indole, the 1R,2R isomer of 1-(4-bromo-2,5-dimethoxyphenyl)- 2-aminopropan-1-ol, and 1-((S)-2-amino- propyl)-1H-indazol-6-ol, have high binding affinities for the 5-HT2 receptors, although they are unable to differentiate among the

5-HT2a, 5-HT2b, and 5-HT2c subtypes. They either do not interact or interact with sub-

stantially lower affinities with other 5-HT receptor subtypes (Glennon et al., 2004; May et al., 2003a, 2006). When evaluated in the lasered monkey eyes, these agents lower IOP by 20–40% at 3 and 6 hours after topical dosing (Glennon et al., 2004; May et al., 2003a, 2006). These results, together with the discovery of 5-HT2 receptors in the human ciliary body (Chidlow et al., 2004) and cultured human TM cells (Sharif et al., 2006), indicate

that 5-HT2 agonists may be a new and exciting class of glaucoma therapeutic agents.

XV. ACTIVATORS OF EXTRACELLULAR MATRIX HYDROLYSIS

As discussed earlier in this chapter, an excessive accumulation of ECM material in the TM of glaucomatous eyes likely contributes to decreased aqueous outflow. Therefore, therapeutic manipulations that eliminate the excessive extracellular matrix should theoretically improve outflow facility and consequently lower IOP. Recently, MMPs have been proposed as important enzymes regulating the turnover of ECM in the TM. Matrix metalloproteinases are a family of zinc-containing neutral proteinases involved in the regulated degradation of ECM. There are more than 20 members in this gene family. They share many common structural and functional features, but differ in substrate specificity.

A. Matrix Metalloproteinases

Activation of these enzymes should reduce the excessive accumulation of ECM molecules, such as proteoglycans, collagens, fibronectins, and laminin, in the glaucomatous eye and in turn decrease hydrodynamic resistance of the outflow pathway. In fact, perfusion with purified MMPs, containing equal concentrations of MMP-2 (gelatinase A), MMP-3 (stromelysin-1), and MMP-9 (gelatinase B), in anterior segments of the human eye increases outflow facility by more than 50%, lasting for at least 5 days (Bradley et al., 1998). Similarly, interleukin-1α (IL-1α), a cytokine known to increase the expression of MMPs in the TM, also produces a long-lasting augmentation of outflow facility when perfused in the anterior segment (Bradley et al., 1998). Consistent with these findings, inhibitors of MMPs, such as the tissue inhibitors of metalloproteinases (TIMP), minocycline, or L-tryptophan hydroxamate,

suppresses aqueous outflow (Bradley et al., 1998). These data, taken together, strongly suggest that MMPs control ECM turnover in the TM and play a significant role in the regulation of aqueous humor outflow facility. In fact, TM expression of MMP-3 and MMP-9 is enhanced after clinical laser treatment for glaucoma and this enhancement may be responsible for mediating the ocular hypotensive effect of trabeculoplasty (Parshley et al., 1996, 1995).

B. Inducers of Matrix

Metalloproteinases

Unfortunately, MMPs, being proteins of large molecular mass, are not practical as medical treatment. However, as indicated above, FP agonists can upregulate the expression of MMP in cultured ciliary muscle cells and in monkey uveoscleral outflow pathway. It is thus expected that future studies may discover other small molecules that stimulate the production or activation of these enzymes, which are more suitable as clinically useful therapeutic agents. Recently, it was found that small molecules, such as tert-butylhydroquinone, can upregulate MMP-3 expression in the TM cells and increase aqueous outflow facility in glaucoma and non-glaucoma donor eyes (Pang et al., 2003). The human perfusion organ culture results demonstrated that compounds of this pharmacological class improve trabecular outflow. These data suggest that inducers of MMP expression, especially small molecules that readily cross the cornea, may become interesting and novel pharmacological agents for the management of ocular hypertension and glaucoma.

C. Activator of Glycosaminoglycan

Degradation

In addition to MMPs and MMP inducers, there are other means to stimulate the degradation of ECM in the TM, for example compounds that catalyze the hydrolysis of glycosaminoglycans (GAGs). GAGs,

which include hyaluronate, chondroitin sulfate, dermatan sulfate, keratin sulfate, and heparin sulfate, are a subset of molecules that constitute ECM found in the TM. Theoretical calculations suggest that they may play an important role in the regulation of trabecular outflow. Intracameral injection of chondroitin sulfate raises IOP in the rabbit and cat. GAG-degrading enzymes, such as hyaluronidase and chondroitinase, consistently increase outflow facility and decrease IOP when perfused into bovine, rabbit, guinea pig, dog, and monkey eyes in both in vivo and ex vivo studies (Pedler, 1956; Sawaguchi et al., 1993). In the cynomolgus monkey, intracameral injection of chondroitinases reduces IOP for 5 to 14 days (Sawaguchi et al., 1992). Unfortunately, similar to MMPs, these GAG-degrading enzymes are not practical for clinical use.

GAGs can be degraded by non-enzymatic methods. To wit, ascorbate, in the presence of metal ions, catalyzes GAG depolymerization. Since the aqueous humor already contains a high concentration (approximately 1.1mM) of ascorbate, supplement of a trace amount of metal ion should be sufficient to increase GAG degradation and therefore affect IOP. Indeed, AL-3037A (sodium ferri ethylenediaminetetraacetate), a small molecule with a chelated ferric ion, accelerates the ascorbate-mediated hydrolysis of GAGs (Pang et al., 2001). Perfusion of bovine eyes with this compound enhances outflow facility by 15–20%. In normal rabbits, AL-3037A lowers IOP by 20–35% after oral, intravenous, or ocular topical administration. In dexamethasone-induced ocular hypertensive rabbits, topical application of this molecule causes a reduction of IOP by approximately 20%. In ex vivo perfusion culture studies, AL-3037A lowers IOP by 15% in non-glaucomatous donor eyes, and more than 50% in tissues derived from glaucoma patients (Pang et al., 2001). These results indicate that small molecule compounds can induce the hydrolysis of GAGs and lower IOP. Compounds of this kind may represent

a new and practical method to treat glaucoma.

XVI. COMPOUNDS THAT

INCREASE CYCLIC GMP

A. Cyclic GMP Analogs

Cell permeable analogs of cyclic GMP have long been demonstrated to lower IOP in many animal species. Ocular administration of cyclic GMP derivatives lowers IOP in the rabbit and monkey (Becker, 1990; Kee et al., 1994). The IOP-lowering effect of 8-Br-cyclic GMP lasts for 3 to 10 hours in the rabbit. No reduction in response is apparent with repeated drug administration.

Cyclic GMP appears to affect both aqueous production and outflow. For example, it reduces aqueous humor secretion in anesthetized monkeys and isolated bovine ciliary epithelium. Intra-arterial perfusion of cyclic GMP analogs suppresses aqueous production. Additionally, cyclic GMP also increases aqueous outflow in the monkey and rabbit. The cellular mechanism of action of cyclic GMP likely involves the activation of cyclic GMP-dependent protein kinases, which, by phosphorylation, leads to functional changes of various proteins. In the bovine ciliary processes, an increase in cyclic GMP correlates with an inhibition of Na,K-ATPase, which can explain its inhibitory effect on aqueous production (Ellis et al., 2001). In the TM and ciliary muscle, cyclic GMP stimulates the maxi-K-channel and relaxes these tissues (Stumpff et al., 1997; Wiederholt et al., 1994).

In addition to the direct application of cyclic GMP analogs, intracellular cyclic GMP levels can be increased by the activation of guanylyl cyclases. There are two major types of guanylyl cyclases: cytosolic guanylyl cyclases and cell membrane-bound guanylyl cyclases. Nitric oxide (NO) and compounds that release NO by hydrolysis (NO donors) are activators of the soluble guanylyl cyclases. Natriuretic peptides are activators of

the membrane-bound guanylyl cyclases. Both NO donors and natriuretic peptides are effective IOP-lowering compounds.

B. Nitric Oxide Donors

NO donors, such as nitroglycerin, have been used clinically for a long time as vasodilators. In the rabbit, topical application of nitroglycerin rapidly lowers IOP in a dose-dependent manner. Its peak effect was observed at 1 to 2 hours after treatment. Similarly, isosorbide dinitrate, sodium nitrite, hydralazine, minoxidil, sodium nitroprusside, and sydnone analogs all produce ocular hypotension in the rabbit without affecting systemic blood pressure (Nathanson, 1992). Tonographic studies showed that the NO donors increase outflow facility of aqueous humor. Nonetheless, in the perfused bovine eye, sodium azide, which likely affects cell functions other than just NO, lowers IOP via a reduction in aqueous humor production (Millar et al., 2001).

C. Natriuretic Peptides

Three natriuretic peptides have been studied for their effects on IOP: atrial natriuretic peptide (ANP), brain-derived natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). In rabbits, intracameral injections of ANP, BNP, and CNP lower IOP by 5–6 mmHg lasting several hours (Fernandez Durango et al., 1999). A similar reduction in IOP was also observed when the peptides were injected into the vitreous of the rabbit eye.

Similar to cyclic GMP and NO donors, the mechanism of action of natriuretic peptides may involve both aqueous production and outflow. For instance, ANP decreases aqueous production after intravitreal injection in the rabbit (Korenfeld and Becker, 1989). This is corroborated by studies on ex vivo bovine eyes, where intra-arterial injection of ANP decreases aqueous humor formation (Millar et al., 1997). However, intravitreal injections of BNP and CNP in

rabbits were reported to increase trabecular outflow (Takashima et al., 1998, 1996).

The major drawback of the natriuretic peptides as potential glaucoma medication is that they are peptides. Cornea penetration and degradation by peptidases can be prohibitive hurdles for their clinical usefulness. A logical direction in this research topic is then to search for non-peptide agonists for the natriuretic peptide receptors, or to develop means to increase intrinsic levels of the peptides in the eye.

D. Compounds that Increase Natriuretic Peptides

Natriuretic peptides are degraded partly by a neutral endopeptidase NEP 24.11. Thus, inhibition of this enzyme increases tissue concentration of natriuretic peptides. In normal volunteers, oral administration of candoxatril, a prodrug that is metabolized to an NEP 24.11 inhibitor, increases plasma ANP level and significantly lowers IOP by 2–3 mmHg (11–16%) in both eyes. The IOP-lowering efficacy of candoxatril correlates with the drug-induced increase in ANP level (Wolfensberger et al., 1994).

A garlic-derived compound S-allyl- mercaptocysteine (SAMC) has also been shown to suppress the degradation of natriuretic peptides. In the rabbit, topical ocular application of 100 μg SAMC produces a fivefold increase in ANP level in the aqueous humor within 30 minutes. The treatment also lowers IOPin a dose-dependent fashion. At the highest dose tested, 200 μg reduces the pressure by 4–6 mmHg, lasting 4 hours (Chu et al., 1999a).

Recently, several additional compounds were shown to increase aqueous concentrations of natriuretic peptides and reduce IOP. For example, kappa opioid receptor agonists, such as dynorphin, bremazocine and spiradoline, increase ANP, BNP, and CNP levels in the rabbit aqueous humor. They also lower IOP by more than 30% (Potter et al., 2004; Russell and Potter, 2002; Russell et al., 2000). Naphazoline, an adrenergic

α2 and imidazoline I1 receptor agonist, increases aqueous ANP level and lowers rabbit IOP (Ogidigben et al., 2002). A dopamine D2/D3 receptor agonist, PD128907 induces ocular hypotension and elevates BNP levels in the rabbit eye (Chu et al., 2004).

XVII. CANNABINOIDS

Smoking marijuana was first reported to reduce IOP in some people about 30 years ago. Subsequently, numerous reports confirmed the ocular hypotensive effect of marijuana, as well as its active component

9-tetrahydrocannabinol (THC) and other cannabinoid derivatives.

The pharmacological actions of cannabinoids are mediated by activation of cannabinoid receptors, which include two major subtypes: CB1 and CB2 receptors. The CB2 receptor is mainly involved in functions of the immune system. It probably does not play any significant role in the regulation of IOP, as evidenced by the lack of IOP effect of CB2 agonist JWH-133 (Laine et al., 2003). In contrast, CB1 receptor is found in the human iris, retina, ciliary muscle, non-pigmented ciliary epithelial, TM, and Schlemm’s canal cells (Lograno and Romano, 2004; Porcella et al., 2000; Stamer et al., 2001). Activation of the CB1 receptor can inhibit adenylyl cyclase, stimulate mitogen-activated protein kinase, and change the conductance of calcium and potassium channels (Romano and Lograno, 2006; Stumpff et al., 2005). These cellular effects may be responsible for the effects of cannabinoids on aqueous production and outflow.

Even though THC has been shown to lower IOP after topical, oral, or intravenous administration, its psychotropic side effects prevent it from becoming a useful medication for glaucoma. A non-psychotropic cannabinoid HU-211 also decreases IOP when administered topically onto normotensive rabbit eyes (Naveh et al., 2000). A single dose results in a 5mmHg (24%) reduction

of IOP, first evidenced at 90 minutes after drug application and lasting for 6 hours. Interestingly, the treatment also causes a 12% decrease in IOP in the contralateral eye.

Topical administration of a synthetic CB1 agonist WIN55212-2 lowers rabbit IOP in a doseand time-dependent fashion without affecting the IOP of the contralateral eye (Song and Slowey, 2000). The ocular hypotensive effect is significantly reduced by topically administered SR141716A, a selective antagonist for the CB1 cannabinoid receptor. In normotensive and hypertensive monkey eyes, topical administration of WIN55212-2 lowers IOP by inhibition of aqueous production (Chien et al., 2003). The same treatment also lowers IOP in glaucoma patients by 30% (Porcella et al., 2001). In addition, CP-55,940, another CB1 agonist, also lowers IOP in the rabbit, which can be blocked by SR141716A (Pate et al., 1998).

In recent years, molecules that activate the cannabinoid receptors were found in animals. These endocannabinoids are mainly derivatives of arachidonic acid, such as arachidonylethanolamine (AEA), 2-ara- chidonoyl glycerol, and 2-arachidonyl glyceryl ether. They also lower IOP. For example, topical application of AEA decreases IOP in normotensive rabbits, but causes ocular hypertension at higher doses (Pate et al., 1995). The IOP-lowering effect of AEA is apparently mediated by the undegraded compound (Laine et al., 2002), whereas the hypertension may be a result of the degraded product – arachidonic acid. It is thus expected degradation-resistant derivatives of AEA should lower IOP without ocular hypertension. Indeed, when alpha-isopropyl substituted AEA were tested for their IOP effect, they did not elicit any hypertensive action while lowering IOP (Pate et al., 1997).

The recent development of cannabinoid agonists that possess reduced psychotropic effects has generated renewed hope in this drug class. If consistent efficacy and minimal untoward effects can be demonstrated, they may become another useful armament in the fight against glaucoma.

XVIII. IDENTIFICATION OF NEW THERAPEUTIC TARGETS BASED ON UNDERSTANDING DISEASE PATHOGENESIS

Current glaucoma IOP lowering therapy can prevent pressure-induced damage to the optic nerve head, optic nerve, and retina. However, these therapies do not address the underlying glaucomatous disease process in the aqueous outflow pathway that is responsible for the elevated IOP. Molecular genetic, genomic, and proteomic studies are providing new insights into the mechanisms responsible for glaucomatous damage to TM, the tissue most responsible for increased outflow resistance and elevated IOP. Nonetheless, it is important to realize that many of these findings are associative (for example, altered protein or mRNA expression in glaucomatous vs normal tissues). Validation of these genes or proteins as new therapeutic targets requires that their altered expression leads to the disease phenotype of elevated IOP. Also, these new candidate genes/proteins may not themselves be ideal targets for drug intervention. Using these discoveries to dissect the pathogenic pathways involved in damaging the TM may lead to the identification of more appropriate therapeutic targets.

A family history of glaucoma is an important risk factor for developing glaucoma, implicating the importance of genetics in glaucoma. A number of glaucoma loci and several glaucoma genes have been identified (Wiggs, 2007). Myocilin (MYOC) was the first glaucoma gene identified (Stone et al., 1997), and MYOC is one of the most abundantly expressed genes in TM tissues. Although defects on MYOC account for only 3–5% of primary open-angle glaucoma (Fingert et al., 1999), it still is the most prevalent molecularly-identified cause of glaucoma, and understanding the mechanism by which mutant myocilin causes glaucoma will lead to new insights on glaucoma pathogenesis. Myocilin is a glycoprotein secreted from the TM (Clark et al., 2001; Nguyen et al., 1998),

although its natural function is not known. Glaucomatous mutations in myocilin lead to altered protein solubility (Zhou and Vollrath, 1999) and prevent myocilin secretion from TM cells (Jacobson et al., 2001). Overexpressing (Gould et al., 2004) or knocking out (Kim et al., 2001) myocilin expression in mice does not cause elevated IOP or obvious damage to the optic nerve or retina, suggesting that MYOC glaucoma is due to a gain-of-function phenotype rather than a loss of function. MYOC glaucoma appears to be due to an entirely new disease mechanism involving mutation-induced exposure of a cryptic localization signal that mislocalizes myocilin inside TM cells, thereby damaging the cells (Shepard et al., 2007).

TM cells, like many cells in our body, can sense and react to their environment. Growth factors are important signaling molecules that regulate cell proliferation and metabolism, and the TM makes and reacts to a number of growth factors (Wordinger et al., 1998). The growth factor TGFβ2 is elevated in the aqueous humor of primary open angle glaucoma patients (LütjenDrecoll, 2005). TGFβ2 increases the synthesis and cross-linking of ECM molecules in the TM, and decreases ECM breakdown (Fleenor et al., 2006; Gottanka et al., 2004). Adding TGFβ2 to the medium of perfusion cultured anterior segments (an ex vivo outflow model) results in elevated IOP (Fleenor et al., 2006; Gottanka et al., 2004), and overexpression of TGFβ2 in mouse eyes using a viral delivery vector significantly elevates IOP (Clark et al., 2006). These results suggest that TGFβ2 or the TGFβ2 signaling pathway may be a useful new therapeutic target for glaucoma. However, this will not be as easy as it sounds because of the complexities of the TGFβ signaling pathways (both Smad and non-Smad signaling) and the interaction of other growth factors such as connective tissue growth factor (CTGF), which potentiates TGFβ (Leask and Abraham, 2004), and bone morphogenic proteins (BMPs), which antagonize TGFβ2 (Fuchshofer et al., 2007; Wordinger et al., 2007).

Recent studies have expanded our understanding of growth factor signaling in the TM. Adult TM cells and TM tissues express BMPs, BMP receptors, and BMP antagonists (Wordinger et al., 2002). Altered expression of BMP4 in mice can lead to elevated IOP and optic nerve defects (Chang et al., 2001). Comparison of normal TM and glaucomatous TM cell gene expression profiles showed increased expression of Gremlin, a BMP antagonist.Adding Gremlin to the medium of perfusion cultured human anterior segments resulted in elevated IOP in this ex vivo system, and IOP returned to baseline upon removal of Gremlin (Wordinger et al., 2007). It would appear that one of the roles for BMPs in the TM is to counter the effects of TGFβ2 because BMP4 and BMP7 can block TGFβ2 stimulation of TM extracellular matrix deposition (Fuchshofer et al., 2007; Wordinger et al., 2007). Increased expression of Gremlin would mitigate this positive effect of BMPs.

A second unexpected growth factor signaling pathway was also discovered by differential gene expression profiling of the TM. Expression of the WNT signaling pathway antagonist sFRP1 is higher in glaucomatous TM cells compared to normal TM cells. Like the BMP pathway, WNT signaling involves a class of secreted growth factors (WNTs), membrane receptors (frizzled), and antagonists (sFRP), all of which are expressed in adult TM cells and tissues (Clark et al., 2007). The intracellular protein β-catenin is a key signaling intermediate in the canonical WNT signaling pathway. Addition of recombinant sFRP1 to the medium of perfusion cultured human anterior segments elevated IOP and altered β-catenin levels in the TM of these eyes. In addition, overexpression of sFRP1 in mouse eyes using a viral delivery vector significantly elevated IOP (Clark et al., 2007). These studies show that the TM has a functional WNT signaling pathway that regulates IOP. There are direct interactions between the TGFβ, CTGF, BMP, and WNT signaling pathways in other tissues, and it will be important to determine

whether these pathways also interact in the TM to regulate IOP.

Serum amyloid A (SAA) is another gene whose expression is elevated in glaucomatous TM cells and tissues (Wang et al., 2006). Increased expression of SAA2 mRNA was found when comparing gene expression profiles between normal and glaucomatous TM tissues and cells. SAA2 protein levels were also elevated in glaucomatous TM tissues and in the aqueous humor of glaucoma patients. In addition, increased SAA2 elevated IOP ex vivo in perfusion cultured human anterior segments and in vivo in mouse eyes transduced with an SAA2 expression vector. SAA2 is an acute phase response protein, which appears in the blood as the result of infection or trauma. Long-term expression of SAA2 can cause amyloid deposits and amyloidosis. However, a different mechanism appears to be responsible for SAA2-induced ocular hypertension. SAA2 is produced locally by the TM, and there are no apparent amyloid deposits in the outflow pathway in glaucoma eyes. SAA2 regulates TM cell gene expression, and this appears to be responsible for the increased IOP.

Proteomics studies have identified several proteins that are associated with the glaucomatous TM. Increased expression of ELAM1 (also known as Selectin E) was reported in the TM of glaucomatous eyes and in cultured glaucomatous TM cells, and was suggested to be a marker for glaucoma (Wang et al., 2001). An independent gene expression study of normal vs glaucomatous TM tissue also showed increased Selectin E expression (Liton et al., 2006). Global profiling of protein expression in TM tissues from normal donor eyes and trabeculectomy specimens from glaucomatous eyes found increased levels of cochlin, a protein initially discovered in the inner ear (Bhattacharya et al., 2005). Although these proteins appear to be associated with glaucomatous TM tissues, their potential role in generation of disease remains to be determined.

XIX. CONCLUSIONS

Pharmacological treatment of glaucoma has been available for more than a century. During this period, much progress has been accomplished and many novel compounds introduced (Table 3.1). Despite these achievements, there still are genuine needs for new drug development to continuously improve the efficacy in controlling ocular hypertension and in minimizing untoward effects. More importantly, in addition to or perhaps instead of the current symptomatic relief by lowering IOP, the eventual future breakthrough in glaucoma therapy should address the pathogenesis of the disease and correct the underlying abnormality(ies) of related tissues. The advances in relevant in vitro, ex vivo, and in vivo study models have assisted these objectives considerably (Table 3.2). These models provide a wide range of research tools to understand the etiology and pathology of the disease, as well as to evaluate pharmacological effects of potential new drugs. Based on studies using these models, recent development in the identification of proteomic and genomic changes in glaucomatous tissues offers unique opportunities in the discovery of a new generation of therapeutic agents for this devastating disease.

XX. REFERENCES

AGIS-Investigators (2000). The advanced glaucoma intervention study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration. Am. J. Ophthalmol. 130, 429–440.

Babizhayev, M.A., Brodskaya, M.W. (1989). Fibronectin detection in drainage outflow system of human eyes in ageing and progression of open-angle glaucoma.

Mech. Ageing Dev. 47, 145–157.

Bahler, C.K., Hann, C.R., Fautsch, M.P., Johnson, D.H. (2004). Pharmacologic disruption of Schlemm’s canal cells and outflow facility in anterior segments of human eyes. Invest. Ophthalmol. Vis. Sci. 45, 2246–2254.

Becker, B. (1990). Topical 8-bromo-cyclic GMP lowers intraocular pressure in rabbits. Invest. Ophthalmol. Vis. Sci. 31, 1647–1649.

Bhattacharya, S.K., Rockwood, E.J., Smith, S.D., Bonilha, V.L., Crabb, J.S., Kuchtey, R.W., Robertson, N.G., Peachey, N.S., Morton, C.C., Crabb, J.W. (2005). Proteomics reveal Cochlin deposits associated with glaucomatous trabecular meshwork. J. Biol. Chem. 280, 6080–6084.

Bhattacherjee, P., Paterson, C.A., Percicot, C. (2001). Studies on receptor binding and signal transduction pathways of unoprostone isopropyl. J. Ocul. Pharmacol. Ther. 17, 433–441.

Bradley, J.M., Vranka, J., Colvis, C.M., Conger, D.M., Alexander, J.P., Fisk, A.S., Samples, J.R., and Acott, T.S. (1998). Effect of matrix metalloproteinases activity on outflow in perfused human organ culture.

Invest. Ophthalmol. Vis. Sci. 39, 2649–2658.

Cai, S., Liu, X., Glasser, A., Volberg, T., Filla, M., Geiger, B., Polansky, J.R., Kaufman, P.L. (2000). Effect of latrunculin-A on morphology and

actin-associated adhesions of cultured human trabecular meshwork cells. Mol. Vis. 6, 132–143.

Chang, B., Smith, R.S., Peters, M., Savinova, O.V., Hawes, N.L., Zabaleta,A., Nusinowitz, S., Martin, J.E., Davisson, M.L., Cepko, C.L., Hogan, B.L., and John, S.W. (2001). Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure. BMC Genet. 2, 18.

Chang, F.W., Burke, J.A., Potter, D.E. (1985). Mechanism of the ocular hypotensive action of ketanserin.

J. Ocul. Pharmacol. 1, 137–147.

Chidlow, G., Cupido, A., Melena, J., Osborne, N.N. (2001). Flesinoxan, a 5-HT1A receptor agonist/ alpha 1-adrenoceptor antagonist, lowers intraocular pressure in NZW rabbits. Curr. Eye Res. 23, 144–153.

Chidlow, G., Hiscott, P.S., Osborne, N.N. (2004). Expression of serotonin receptor mRNAs in human ciliary body: a polymerase chain reaction study.

Graefe’s Arch. Clin. Exp. Ophthalmol. 242, 259–264. Chidlow, G., Nash, M.S., De Santis, L.M., Osborne, N.N.

(1999). The 5-HT(1A) receptor agonist 8-OH-DPAT lowers intraocular pressure in normotensive NZW rabbits. Exp. Eye Res. 69, 587–593.

Chien, F.Y., Wang, R.F., Mittag, T.W., Podos, S.M. (2003). Effect of WIN 55212-2, a cannabinoid receptor agonist, on aqueous humor dynamics in monkeys. Arch. Ophthalmol. 121, 87–90.

Chu, E., Socci, R., Chu, T.C. (2004). PD128,907 induces ocular hypotension in rabbits: involvement of D2/ D3 dopamine receptors and brain natriuretic peptide. J. Ocul. Pharmacol. Ther. 20, 15–23.

Chu, T.C., Han, P., Han, G., Potter, D.E. (1999a). Intraocular pressure lowering by S-allylmercaptocysteine in rabbits. J. Ocul. Pharmacol. Ther. 15, 9–17.

Chu, T.C., Ogidigben, M.J., Potter, D.E. (1999b). 8OH-DPAT-induced ocular hypotension: sites and mechanisms of action. Exp. Eye Res. 69, 227–238.

Clark, A.F., McNatt, L.G., Hellberg, P.E., Millar, J.C., Rubin, J.S., Pang, I.-H., Wang, W.-H. (2007). A functional Wnt pathway in the trabecular meshwork that regulates intraocular pressure. ARVO Abstract, 1142.

Clark, A.F., Millar, J.C., Pang, I.-H., Jacobson, N., Shepard, A. (2006). Adenoviral gene transfer of active human transforming growth factor-β2 induces elevated intraocular pressure in rats. ARVO Abstract, 4771.

Clark, A.F., Pang, I.-H. (2002). Advances in glaucoma therapeutics. Exp. Opin. Emerging Drugs 7, 141–163.

Clark, A.F., Steely, H.T., Dickerson, J.E. Jr, English Wright, S., Stropki, K., McCartney, M.D., Jacobson, N., Shepard, A.R., Clark, J.I., Matsushima, H., Peskind, E.R., Leverenz, J.B., Wilkinson, C.W., Swiderski, R.E., Fingert, J.H., Sheffield, V.C., Stone, E.M. (2001). Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues. Invest. Ophthalmol. Vis. Sci. 42, 1769–1780.

CNTGS-Group (1998). Comparison of glaucomatous progression between untreated patients with normal-tension glaucoma and patients with therapeutically reduced intraocular pressures. Am. J. Ophthalmol. 126, 487–497.

Costagliola, C., Fasano, M.L., Iuliano, G., Ferrara, L.A. (1990). Effects of ketanserin on intraocular pressure.

Cardiovasc. Drugs Ther. 4, Suppl. 1, 97–99.

Ellis, D.Z., Nathanson, J.A., Rabe, J., Sweadner, K.J. (2001). Carbachol and nitric oxide inhibition of Na,K-ATPase activity in bovine ciliary processes.

Invest. Ophthalmol. Vis. Sci. 42, 2625–2631.

Ethier, C.R., Read, A.T., Chan, D.W. (2006). Effects of latrunculin-B on outflow facility and trabecular meshwork structure in human eyes. Invest. Ophthalmol. Vis. Sci. 47, 1991–1998.

Fan, H., Rao, S.K., Zhou, Y.S., Lam, D.S. (2005). Effect of latrunculin B on intraocular pressure in the monkey eye. Arch. Ophthalmol. 123, 1456–1457.

Fernandez Durango, R., Moya, F.J., Ripodas, A., deJuan, J.A., Fernandez Cruz, A., Bernal, R. (1999). Type B and type C natriuretic peptide receptors modulate intraocular pressure in the rabbit eye. Eur. J. Pharmacol. 364, 107–113.

Fingert, J.H., Heon, E., Liebmann, J.M., Yamamoto, T., Craig, J.E., Rait, J., Kawase, K., Hoh, S.T., Buys, Y.M., Dickinson, J., Hockey, R.R., Williams Lyn, D., Trope, G., Kitazawa, Y., Ritch, R., Mackey, D.A., Alward, W.L., Sheffield, V.C., Stone, E.M. (1999). Analysis of myocilin mutations in 1703 glaucoma patients from five different populations. Hum. Mol. Genet. 8, 899–905.

Fleenor, D.L., Shepard,A.R., Hellberg, P.E., Jacobson, N., Pang, I.H., and Clark, A.F. (2006). TGF beta 2- induced changes in human trabecular meshwork: implications for intraocular pressure. Invest. Ophthalmol. Vis. Sci. 47, 226–234.

Fuchshofer, R., Yu, A.H., Welge Lussen, U., Tamm, E.R. (2007). Bone morphogenetic protein-7 is an antagonist of transforming growth factor-beta2 in human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 48, 715–726.

Gabelt, B.T., Kaufman, P.L. (2005). Changes in aqueous humor dynamics with age and glaucoma. Prog. Retin. Eye Res. 24, 612–637.

Gabelt, B.T., Millar, C.J., Kiland, J.A., Peterson, J.A., Seeman, J.L., Kaufman, P.L. (2001). Effects of serotonergic compounds on aqueous humor dynamics in monkeys. Curr. Eye Res. 23, 120–127.

Gabelt, B.T., Okka, M., Dean, T.R., Kaufman, P.L. (2005). Aqueous humor dynamics in monkeys after topical R-DOI. Invest. Ophthalmol. Vis. Sci. 46, 4691–4696.

Glennon, R.A., Bondarev, M.L., Khorana, N., Young, R., May, J.A., Hellberg, M.R., McLaughlin, M.A., Sharif, N.A. (2004). Beta-oxygenated analogues of the 5-HT2A serotonin receptor agonist 1-(4-bromo-2, 5-dimethoxyphenyl)-2-aminopropane. J. Med. Chem. 47, 6034–6041.