that the GSTM1-positive genotype and GSTT1-null genotype or the combination of both may be associated with an increased risk of development of POAG in the Turkish population.

Free radicals probably play a fundamental role in the decrease of HTM cells: indeed, oxidative DNA damage is significantly increased in the TM of glaucoma patients than in controls and a statistically significant correlation has been found among HTM DNA oxidative damage, visual field damage, and IOP (Izzotti et al., 2003; Sacca`et al., 2005). These data confirm weakened antioxidant defenses and the elevation of oxidative stress in glaucoma patients.

Glaucomatous cascade

Nitric oxide and endothelins

HTM is a complex organ that is stably engaged in the AH and it is able to respond to vasoactive

substances, including vasoconstrictors, such as endothelins (ET), and vasodilators, such as nitric oxide (NO). A balance between vasoconstrictors and vasodilators is necessary for the maintenance of the physiological structure and function of endothelia (Wiederholt et al., 2000). Whenever this balance is disrupted, as in glaucoma, the outcome is endothelial dysfunction and injury, triggering the glaucomatous pathogenic cascade (Gibbons, 1997).

NO is produced by NO synthase (NOS) enzyme. This enzyme can have three different forms: neuronal NOS (NOS1), macrophage — or inducible — NOS (NOS2), and endothelial NOS (NOS3). NOS2 seems to be the most important isoenzyme involved in glaucoma pathogenesis (Liu and Neufeld, 2000). Indeed, NOS2 is expressed only in the ONH of patients with glaucoma (Neufeld et al., 1997). The induction of NOS2 expression generates high levels of NO, which have been associated with neural tissue toxicity (Dawson et al., 1993, 1994).

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Vitamin C protects NO by scavenging superoxide and peroxynitrite within endothelial cells, which enhances NO exit from the cells, and spares NOS or tetrahydrobiopterin from oxidative modification (May, 2000).

NO production increases after elevation of the pressure gradient over the TM (Schneemann et al., 2003). In an experimental animal model of glaucoma, increased IOP appears to be a major causative factor for the overproduction of NO through inducible NOS (NOS2) activation, resulting in RGC death and optic nerve damage (Shareef et al., 1999). The presence of free radicals may induce NO to generate toxic products interacting with oxygen, iron, and/or copper: this can aggravate the metabolic conditions of the TM and alter its mobility (Tamm and Lutjen Drecoll, 1998; Wiederholt, 1998; Haefliger et al., 1999). NO has multiple effects: it is a neural messenger having a fundamental role in the nervous system, and it may modulate the sodium pump that regulates the neuronal energy metabolism of the brain (Royes et al., 2005). Through this mechanism, NO, glutamate, and certain other intercellular messengers bring about a marked and prolonged alteration in Na+, K+-ATPase activity and, so, causing an alteration of cellular energy usage, form a focal point for the action of several cellular messengers that have been implicated in neuronal viability in certain degenerative diseases and under conditions of stress (Ellis and Nathanson, 1998).

Moreover, the increase of the NO concentration, reacting with anion superoxide to form ONOO (peroxynitrite) or other substances derived from oxidative stress, can lead to neurotoxicity (Lipton, 1999).

Also, ET can participate in the regulation of IOP, and despite TM mobility, ET levels in the glaucomatous AH are more elevated than in controls (Wiederholt, 1998; Haefliger et al., 1999; Orgul et al., 1999). From this point of view, ET-1 may induce TM contraction, and this contraction increases outflow resistance, whereas TM relaxation increases outflow facility (Wiederholt et al., 2000). In any case, ET-1 has a variety of pathophysiological ocular functions, depending on receptor subtype and tissue involved: ET induces an increase in intracellular calcium

(Marsden and Brenner, 1991) and vasoconstriction (Marsault et al., 1993). Therefore, ET-1 would be the main effector of the glaucoma ischemia (Orgul et al., 1999). Surely, optic nerve ischemia could contribute to visual field loss and neuropathy; for this reason, ET-1 has a primary role in the vascular theory of glaucoma (Flammer, 1994). ET may be involved in vasoconstriction and/or vasospasms from abnormal autoregulation of the retinal microcirculation (Cioffi et al., 1995; Haefliger et al., 1999). Besides, chronic administration of low doses of ET into the perineural region in primates produces damage to the ONH that is characteristic of that seen in glaucoma and independent of IOP changes (Cioffi and Sullivan, 1999). Indeed, ET-1 induces ECM remodeling in the ONH, influencing the regulation of matrix metalloproteinases (MMPs) (He et al., 2007).

Furthermore, the effect of ET-1 is related to a reduction of Na+, K+-ATPase activity, underlining its vasoconstrictive properties, and it may contribute to the decrease of AH formation (Prasanna et al., 2001; Petzold et al., 2003) (Fig. 2).

In any case, free radicals play an important role in the development of ischemia/reperfusion (I/R) injury. There is not only a dysregulation of the vascular response to increased levels of ET, but there are also direct effects of ET on target tissues, depending on the expression and distribution of their ET receptor (Yorio et al., 2002). NO can modulate the expression, sensitivity, and signal termination of ET receptors (Redmond et al., 1996).

ET-1 and NO have been considered important mediators of apoptotic RGCs death in glaucoma, where lamina cribrosa appears to be the primary site of injury of the ONH (Haefliger et al., 1999; Quigley, 2005).

In animal models of glaucoma, RGCs die via apoptosis (Kerrigan et al., 1997); apoptosis is a genetically predetermined program of cell death, which can be activated by many different factors (Levin, 1999; Kikuchi et al., 2000; Tatton et al., 2001). There are different stimuli for apoptotic RGCs death: hypoxia, neurotrophin withdrawal, and glutamate-mediated toxicity, and in this context, oxidative stress seems to play an important role (Meldrum and Garthwaite, 1990;

Lambert et al., 2004; Moreno et al., 2004; Tezel and Yang, 2004). Therefore, mechanical and vascular factors might work synergistically, leading to the same end result (Prasanna et al., 2005). IOP increase may induce factors such as mechanical stretching on the TM; mechanical strain on glial cells supporting ganglion cell axons; the scleral mechanical properties that should have such a large influence on ONH biomechanics, where the lamina cribrosa appears to be the primary site of injury (Burgoyne et al., 2005; Quigley, 2005; Sigal et al., 2005).

Lamina cribrosa is a specific connective region through which RGC axons exit the eye (Birch et al., 1997). In normal conditions, lamina cribrosa provides metabolic and mechanical support to nerve fibers against a pressure gradient, during course of glaucoma, and a remarkable overthrow of its architecture is observable: deformation, collapse, and ECM reorganization are manifest (Miller and Quigley, 1988). Elevated IOP induces both axonal transport and cytoskeleton changes in

the ONH. Changes to the cytoskeleton may contribute to the axonal transport abnormalities that occur in elevated IOP (Balaratnasingam et al., 2007). Furthermore, this axonal transport blockage results in RGCs death and optic nerve degeneration (Quigley et al., 1983).

Extracellular matrix

The expression of a variety of TM genes is significantly affected by mechanical stretching and age-related variations. These are involved in apoptosis, cellular proliferation, and in other major aspects of cellular metabolism (Vittal et al., 2005). A particularly interesting data is provided by the involvement of the ECM: indeed, several ECM proteins may contribute to homeostatic modifications of AH outflow resistance, being upor downregulated (Vittal et al., 2005). ECM remodeling occurring in TM during POAG is similar to the remodeling of other tissues, in particular the same pathological process occurs in

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atherosclerosis: trabecular cells are endothelial-like cells similar to the fibrogenic cells in these disorders (Veach, 2004). In any case, lower concentrations of oxidized low-density lipids have stimulated ECM remodeling (Bachem et al., 1999).

A recent study indicates that increased fibronectin synthesis could result in concomitant increase in IOP (Fleenor et al., 2006). Increased laminin and collagen type IV synthesis by TM cells exposed to ascorbic acid was also demonstrated (Zhou et al., 1998). A tight correlation between reduced permeability and increased expression of the ECM components has been observed, suggesting a possible link between excess matrix deposition and potential blockage in aqueous outflow (Tane et al., 2007). In vitro researches on human eyes have found a decrease in glycosaminoglycans (GAGs) synthesis, particularly hyaluronic acid, in glaucomatous eyes compared with normal eyes, and an increase in chondroitin sulfate content (Knepper et al., 1996b; Navajas et al., 2005).

Transforming growth factors (TGFs) are a family of cytokines that control a large variety of cellular processes like inflammation, wound healing, and ECM accumulation (Sporn and Roberts, 1992; Saika, 2004; Kottler et al., 2005). TGF regulates production of a wide variety of ECM genes, including elastin, collagens, fibrillin, laminin, and fibulin. Three structurally similar isoforms have been identified: TGF-b1, TGF-b2, and TGF- b3 (Massague, 1990). TGF-b2 levels are elevated in glaucomatous human AH (Tripathi et al., 1994) and alter ECM metabolism (Wordinger et al., 2007).

Bone morphogenetic proteins (BMP) are groups of growth factors known for their ability to induce the formation of bone and cartilage, which are expressed in the human TM and ONH (Wordinger et al., 2002). Recently, it has been discovered that BMP-7, which is expressed in the adult TM, modulates and antagonizes the effects of TGF-b2 signaling on tissues of the outflow pathways in vivo and leads to increased ECM deposition and elevated IOP (Kane et al., 2005; Fuchshofer et al., 2007). In any case, elevated levels of TGF-b2 in the AH may have the dual effect of both a direct increase of ECM components production in TM (e.g., fibronectin) and an enhanced production of gene products inhibiting ECM degradation

(Fleenor et al., 2006). Furthermore, TGF-b2 increases the expression of plasminogen activator inhibitor-1 (PAI-1). However, elevated PAI-1 levels have been shown to be linked to both decreased adhesion and increased detachment of a variety of cell types (Czakey and Loskutoff, 2004). This phenomenon may act with oxidative stress in cellular decay in TM.

TGF in the AH is also responsible for anterior chamber-associated immune deviation, a mechanism that protects the eye from inflammation and immune-related tissue damage (Wilbanks et al., 1992). Indeed, TGF-b2 is one of the most important immunosuppressive cytokines in the anterior chamber of the eye and has a fibrogenic effect in trabecular cells (Alexander et al., 1998). A direct correlation between oxidative stress and TGF-b2 expression has been demonstrated (Poli and Parola, 1997).

Moreover, TGF rules the expression levels of hyaluronan synthases (Usui et al., 2000). Among the GAGs of TM, hyaluronan is the most abundant, and it has been suggested to be an important modulator of aqueous outflow resistance and TM cell survival (Acott et al., 1985; Knepper et al., 1996a; Lerner et al., 1997). It represents a significant factor in outflow resistance in POAG, particularly during elevated IOP (Knepper et al., 2005).

ECM production in the TM may be mediated by vitamin C (Epstein et al., 1990; Sawaguchi et al., 1992). Ascorbic acid is reported to stimulate increased hyaluronic acid synthesis in glaucomatous trabecular cells compared with normal human trabecular cells (Schachtschabel and Binninger, 1993). Also, ascorbate reduces the viscosity of hyaluronic acid, thus increasing outflow through the trabeculum (McCarty, 1998). Researchers seem to be in disagreement about the trend of glaucoma patients having an ascorbate deficiency (Asregadoo, 1979; Lane, 1980; Beit-Yannai et al., 2007). However, there is compelling research on its effectiveness in treating glaucoma: high doses of vitamin C, first in animals and then in humans, showed that ascorbate decreases IOP (Virno et al., 1966). Other authors have confirmed this capacity of vitamin C, used both orally and topically (Linner, 1996). It is possible that ascorbic acid