The dysfunction of the trabecular meshwork during glaucoma course.

REVIEW ARTICLE

Journal of

Cellular Physiology

The Dysfunction of the Trabecular Meshwork During Glaucoma Course SERGIO CLAUDIO SACCÀ,1* ALESSANDRA PULLIERO,2

AND

ALBERTO IZZOTTI2,3

1

Department of Head/Neck Pathologies, St Martino Hospital, Ophthalmology Unit, Genoa, Italy

2

Department of Health Sciences, Section of Hygiene and Preventive Medicine, University of Genoa, Genoa, Italy

3

Mutagenesis Unit, IST National Institute for Cancer Research, IRCCS Hospital-University San Martino Company, Genoa, Italy

Primary open angle glaucoma is a multi-tissue disease that targets, in an ascending order, the trabecular meshwork, the optic nerve head, the lateral geniculate nuclei, and the visual cortex. Oxidative stress and vascular damage play major roles in triggering apoptotic cell loss in these tissues. Molecular alterations occurring in the ocular anterior chamber during the early course of glaucoma trigger this cell loss. These molecular events are mainly of endogenous origin and related to the long-term accumulation of oxidative damages arising from mitochondrial failure and endothelial dysfunction. This situation results in decreased antioxidant defences in aqueous humour and apoptosis activation in trabecular meshwork cells as triggered by severe mitochondrial damage altering tissue function and integrity. The presence of neural proteins in glaucomatous aqueous humour indicate that a molecular interconnection exists between the anterior and the posterior chamber tissues. Trabecular meshwork and lamina cribrosa share a common neuro-ectodermal embryological, which contribute to explain the interconnection between anterior and the posterior chamber during glaucoma pathogenesis. During glaucoma, proteins deriving from the damage occurring in endothelial trabecular meshwork cells are released into aqueous humour. Accordingly, aqueous humour composition is characterised in glaucomatous patients by the presence of proteins deriving from apoptosis activation, mitochondrial damage, loss of intercellular connections, antioxidant decrease. Many questions remain unanswered, but molecular events illuminate TM damage and indicate that trabecular cell protection plays a role in the treatment and prevention of glaucoma. J. Cell. Physiol. 230: 510–525, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

Glaucoma is composed of several diseases that are characterized by an optic neuropathy in which retinal ganglion cell degeneration leads to a characteristic cupping of the optic nerve head. This neuropathy is associated with typical visual field defects (Shields et al., 1996). The glaucomas have been traditionally grouped into open angle, angle closure (this name refers to the configuration of the irido-corneal angle), and congenital types. Each type is subdivided into primary or secondary subtypes that denote when no cause for glaucoma can be identified or when underlying ocular or systemic conditions cause glaucomas, respectively (Shields et al., 1996). To date, the literature indicates that the majority (60–70%) of primary glaucomas are open angle; furthermore, primary open-angle glaucoma (POAG) is a chronic optic neuropathy that is a significant cause of blindness throughout the world (Leske, 2007). Although the precise molecular mechanisms that lead to glaucoma are far from understood, we know that POAG has three target tissues, that is the fluid-filled space inside the eye between the iris and the cornea's innermost surface, the endothelium. The first is the trabecular meshwork (TM) in the anterior chamber (AC) of the eye (Saccà and Izzotti, 2008); the second is the optic nerve head, specifically, the retinal ganglion cells (RGCs) that comprise the head in the posterior segment of the eye that is a narrow space behind the peripheral part of the iris, and in front of the suspensory ligament of the lens and the ciliary processes, a small space directly posterior to the iris but anterior to the lens, (Howell et al., 2007) (Fig. 1); the third is the visual cortex given that the central nervous system of adult primates are affected by the lateral geniculate nucleus (LGN) (Gupta et al., 2006). Thus, glaucoma is an ascending disease that begins in the anterior segment with the increase of intraocular pressure (IOP) (Weinreb and Khaw, 2004). Furthermore, it spreads to the posterior segment and finally along the neuronal chain before arriving at the visual cortex. The biomolecular

process that leads to cell death in various areas is always the same: apoptosis; however, the mechanisms that trigger it can differ. For example, the death of a retinal ganglion cell might be due to oxidative stress (Izzotti et al., 2006; Wang and Michaelis, 2010) high levels of nitric oxide (Brown, 2010), glutamate excitotoxicity (Brandt et al., 2011), mitochondrial damage (Izzotti et al., 2010b), defective axonal transport (Band et al., 2009), or glial cell pathology (Johnson and Morrison, 2009). Nevertheless, at the end, we will check the same morphological (neuronal death) and functional (visual field alteration) events. In fact, the visual field defects due to RGC degeneration are directly proportional to TM oxidative damage (Izzotti et al., 2003) and therefore linked to cellularity (Saccà et al., 2005). TM is a crucial tissue with regard to glaucoma pathogenesis because its malfunction increases IOP, which occurs during most types of open-angle glaucoma (Saccà et al., 2007). Although the precise mechanism is not well understood, increases in IOP create a mechanical stress that is transmitted to the back of the eye and damages the RGCs of

Conflict of interest: none. *Correspondence to: Sergio Claudio Saccà, Department of Head/ Neck Pathologies, St Martino Hospital, Ophthalmology Unit, Viale Benedetto XV, 16132 Genoa, Italy. E-mail: [email protected] Manuscript Received: 20 February 2014 Manuscript Accepted: 5 September 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 12 September 2014. DOI: 10.1002/jcp.24826

© 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C . , A W I L E Y C O M P A N Y

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Fig. 1. Scheme of eye's anatomy. Colored left panel reports details of the irido-corneal angle, which is the target region of glaucoma pathogenic processes. The anterior chamber (AC) is the fluid-filled space inside the eye between the iris and the cornea's innermost surface. This also includes the trabecular meshwork which is placed between these two tissues.

the optic nerve and their axons. IOP might be partially controlled by the genes expressed in the eye, specifically in the ocular sites involved in aqueous production and outflow: the ciliary body, trabecular meshwork, or both (Dan et al., 2005). In addition, the genetic risk variant for POAG is located in the genes that are expressed in the TM and RGCs (Thorleifsson et al., 2010). This recent discovery indicates that the pathogenesis of molecular damage is common in both areas even when the insult is expressed in different tissues. In 2005, LütjenDrecoll (2005) stated that “common factors are involved in the pathogenesis of both the TM and the optic nerve changes”. A few years later, Steely et al. (2000) showed that the proteins in TM- and LC-cultured cells and tissue were exceptionally similar; in fact, these ocular tissues (which are intimately linked to the pathogenesis of primary open-angle glaucoma) display remarkable similarities with regard to protein expression. These structures are different but linked together as a “unicum” in the eye by the pathogenetic events that determine the beginning of glaucomatous cascades. In the current paper, we examine the molecular alterations involved in the anterior segment during glaucoma. Two common types of cellular damage exist in the pathogenesis of glaucoma with regard to both the TM and optic nerve: oxidative stress (Tezel, 2006) and vascular damage (Flammer, 1994). Oxidative/Nitrosative Stress

Free radicals are chemical species with a single, unpaired electron; these radicals are highly reactive because they seek to pair with free electrons. This pairing produces another free JOURNAL OF CELLULAR PHYSIOLOGY

radical. The newly produced free radical is unstable and can trigger a chain reaction that causes molecular damage. Oxidative stress consists of the molecular damage that occurs within a cell or organ (rather than in tissue) in response to exposure from oxidizing agents. If reactive oxygen species (ROS) generation overwhelms antioxidant defense, then free radicals are able to interact with endogenous macromolecules and alter cellular function (Fig. 2). The majority of the ROS effects on cells occur through their actions via signalling pathways rather than the nonspecific damage of intracellular macromolecules (Maher and Schubert, 2000). Although many biological effects have been ascribed to various free radicals, superoxide radicals might also be important mediators of cellular effects. Indeed, nitric oxide (NO) radical plays a role in controlling ocular vascular tone and blood flow in humans (Mann et al., 1995). NO is a potent signalling molecule in blood vessels, where the continuous formation from endothelial cells acts on the underlying smooth muscle cells to maintain vasodilatation and blood flow. NO is synthesized from L-arginine via a family of nitric oxide synthase (NOS) isozymes: neuronal (n)NOS, endothelial (e)NOS, and inducible-NOS (i)NOS. The first two isozymes are constitutive while iNOS is Ca2 þ -independent (Bredt and Snyder, 1994). iNOS expression is regulated by redox-responsive transcription factors NF-kB (Fig. 3) and mitogen-activated protein kinases (MAPKs) (MacMicking et al., 1997). NF-kB is activated by ROS thus inducing the expression of proinflammatory markers including ELAM-1, interleukin (IL)-1a, IL-6, and IL-8 (Li et al., 2007). NO production is modulated by Glutamate (Kosenko et al., 2003). Furthermore, the mediators released by the ciliary epithelium influence eNOS activity in cells regulating inflow and

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Fig. 2. Impaired balance between antioxidant mechanisms and reactive oxygen species leads to cell death via mitochondrial damage, apoptosis, and loss of tissue integrity as documented with regard to glaucomatous TM using an electron scanning microscope.

Fig. 3. NF-kB is involved in cellular responses to stimuli such as stress, ultraviolet irradiation, free radicals, cytokines, oxidized LDL, and bacterial or viral antigens. This protein is responsible for cytokine production and cell survival. In trabecular meshwork, NF-kappaB can be activated via increased intraocular pressure, increased age, vascular diseases, and oxidative stress.

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TRABECULAR MESHWORK DURING GLAUCOMA outflow pathways (Coca-Prados and Ghosh, 2008). “Oxidative/ nitrosative stress”, the “pervasive condition of increased amounts of reactive oxygen/nitrogen species, is now recognized” as a “prominent feature of many acute and chronic diseases” as well as “the normal aging process (Dalle-Donne et al., 2006).” Although ROS might originate from various cell sources, the highest amounts are produced in mitochondria under certain physiological and pathophysiological conditions (Freeman and Crapo, 1982; Rappaport et al., 1998). Mitochondria are membrane-enclosed organelles (1- to 10-mm diameter) that generate most of the cell's supply of ATP; they also play a role in cell signalling and differentiation, Ca2þ buffering, apoptosis, and cell death. Moreover, mitochondria control the cell cycle and cell growth (Kadenbach et al., 2009). Many of these processes are triggered or mediated by Ca2þ, ROS, or both. Mitochondrial ROS are generated by oxidative phosphorylation, a process that occurs in the inner mitochondrial membrane and involves the oxidation of nicotinamide adenine dinucleotide (NADH) to produce energy via the transfer of unpaired electrons from semiubiquinone or semi-reduced flavins of complexes I, II, and III to O2; these forms are converted to H2O2 via the Mn-dependent superoxide dismutase (SOD) in the mitochondrial matrix (Kadenbach et al., 2009). In the AC, the oxidant effect of H2O2 on the adhesion of TM cells to extracellular matrix proteins rearranges cytoskeletal structures, which might lead to HTM disruption (Zhou et al., 1999). Under physiological conditions, this effect is reversed with glutathione (GSH), catalase, and ascorbic acid (Saccà, 2007). When the H2O2 concentration is low, the GSH redox system protects ocular tissues, whereas catalase likely protects ocular tissues from the damage induced by higher H2O2 concentrations (Costarides et al., 1991). Patients with glaucoma exhibit low levels of circulating glutathione (Gherghel et al., 2005), and this condition increases oxidative stress. Furthermore, the basal level of the oxidative nucleotide modifications in the AC is higher in the cornea than the iris or TM. The TM is the most sensitive tissue to oxidative damage because this damage dramatically increases in the TM but not the cornea or iris after exposure to hydrogen peroxide. Thus, even when the cornea and iris are directly exposed to light, they are not harmed because they possess antioxidant defense mechanisms that are not activated in the TM. In fact, the TM is the most sensitive AC tissue to oxidative damage (Izzotti et al., 2009). Nerve cells are also sensitive to oxidative damage, and the oxidative protein modifications that occur during glaucomatous neurodegeneration increase neuronal susceptibility to damage and lead to glial dysfunction (Tezel, 2006). The interaction between nitric oxide and oxygen radicals produces peroxynitrite (ONOO) (Beckman et al., 1990), which is a potent oxidant that causes the following problems: the initiation of lipid peroxidation; DNA breakage and base modification; tyrosine amino acid nitration and nitrosylation; alterations in cell signalling; and the induction of cell necrosis and apoptosis in cases of severe damage (Virag et al., 2003). An increase in reactive oxygen species production from mitochondria by CO is probably associated with the glaucoma. CO is a potent inhibitor of cytochrome c oxidase and is the terminal electron acceptor in the respiratory chain of mitochondria. By binding avidly to cytochrome c oxidase, CO would be predicted to cause cellular toxicity. Recent literature shows that safe and tolerable levels of CO increases ATP production or prevents stress-induced damage and enhances overall mitochondrial biogenesis (Lancel et al., 2009). The results obtained so far, point out to mitochondria as a direct target for which the signal of CO could be transduced into a functional (and possibly beneficial) effect. An interesting review on CO and glaucoma has been recently published on this topic (Bucolo and Drago, 2011), focuses on CO as a fine modulator of intraocular pressure and on its potential implications in JOURNAL OF CELLULAR PHYSIOLOGY

glaucoma. Indeed, exogenous CO can re-equilibrate the environment in disease states, but it does not control the homeostasis in physiological environment per se. Alternatively, another simpler explanation could be that, from a high baseline IOP, a small increase in trabecular outflow facility or a decrease in aqueous humor production or episcleral venous pressure causes a greater decrease in IOP due to the exponential ocular pressure–volume relationship (Kiel and van Heuven, 1995; Zamora and Kiel, 2010). The episcleral venous pressure is the pressure that must be overcome for aqueous humour to leave the eye via the trabecular outflow pathway. Consequently, it is considered a key determinant in IOP. In addition, ROS are highly reactive molecules that can extensively damage proteins, lipids, and (especially) DNA molecules (Beckman and Ames 1998; Valko et al., 2007). Moreover, ROS are required to trigger apoptosis (Dröge, 2002). Interestingly, the peroxisomal b-oxidation of fatty acids is a second source of oxygen radicals, and this process generates H2O2 as a by-product. Peroxisomes are responsible for degrading fatty acids and other molecules (Singh et al., 1996). The exogenous sources of ROS include UV light, visible light, ionizing radiation, chemotherapeutics, and environmental toxins. Its endogenous sources include the activity of peroxisomes, lipooxygenases, NADH oxidase, cytochrome P450, and mitochondrial respiration (Dröge, 2002). At moderate concentrations, NO, superoxide anion, and ROS play important roles as regulatory mediators in signalling processes. If the level of pro-oxidants exceeds that of antioxidants in cells, then cellular components are oxidized and cellular function is lost (Valko et al., 2007). The most frequent ocular diseases are ROS-mediated (Saccà et al., 2009), most likely because UV-B exposure generates ROS through multiple sources (Saccà et al., 2013) and NO through increased NOS activity. ROS engender parallel signalling pathways that mediate the responses of specific genes to UV-B radiation (A-H-Mackerness et al., 2001). Mitochondrial integrity declines as a function of age; in fact, mitochondria appear to be morphologically altered, and they produce more oxidants and less ATP (Shigenaga et al., 1994). Age-dependent increases in the level of damaged DNA have been commonly assessed using biomarkers such as the formation of 8-Hydroxy-20 -deoxyguanosine (8-OHdG). Mitochondria and Trabecular Meshwork

The amount of OH8dG increases progressively with normal aging in both nDNA and mtDNA; however, the rate of this increase with age was much greater for mtDNA (Mecocci et al., 1993; Ames et al., 1993; Yakes and van Houten, 1997). The reasons for these differences likely include the proximity of mitochondrial DNA to the source of oxidants and the lack of any protective histone covering. The “postulated and observed increased sensitivity of mitochondrial DNA to oxidative damage has led to the concept of the ‘vicious cycle’ in which the initial ROS-induced impairment of mitochondria leads to an increase in oxidant production that leads to additional mitochondrial damage (Balaban et al., 2005).” Thus, both the presence of mitochondria and the changes to their quantitative and qualitative parameters constitutes an important regulation mode of the oxygen-peroxide state and the signalling pathways that depend on it. In fact, peroxynitrite can affect mitochondrial respiration by causing cellular energy failure, contractile dysfunction, and cell death (Ghafourifar et al., 1999; Estevez et al., 1999). As in several neurodegenerative diseases, the normal antioxidant defence mechanisms decline in the aging eye. This decline increases the vulnerability of the eye to the deleterious effects of oxidative damage (Finkel and Holbrook, 2000). Furthermore, the activity of mitochondrial respiratory chain

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complex I decreases in the TM cells of patients with POAG (He et al., 2008b). An error in mtDNA replication might cause a significant deletion to the mitochondrion genome; a shorter genome will replicate more quickly and lead to the formation of malfunctioning or completely inactive mitochondria. Given this state of things, the core responds by upregulating the mitochondria formation; for the reasons mentioned above, this process leads to an accumulation of mitochondria with short, inactive genomes, thereby leading to energy deficits and atrophy (Morris, 1990). The level of mtDNA damage detected in glaucomatous TM is remarkably high (Izzotti et al., 2010b), which supports the theory that mitochondrial dysfunction impairs ATP production, limits the oxidative capacity of the TM endothelium, and leads to an intracellular calcium overload (He et al., 2008a). Apoptosis can occur when radical-scavenging cellular antioxidants cannot handle the amount of ROS produced in the mitochondria (Lennon et al., 1991; Suzuki et al., 1999). Izzotti et al. (2011) demonstrated that the amount of mitochondrial deletion does not significantly differ from control TM samples with regard to all glaucoma types (except for POAG and pseudoexfoliative glaucoma; PEXG). Similar findings were obtained with regard to oxidative damage to nuclear DNA, which significantly increased only in patients with POAG or those with PEXG but not those with other glaucoma types. Specifically, the number of mitochondria in the TM decreased in all patients, regardless of glaucoma type; this finding is related to the cell loss observed during the course of glaucoma. Thus, the mechanism that causes glaucoma is not identical across type even when the target tissues are the same. In addition, the oxidative stress and the AH-related defenses play important roles with regard to glaucoma pathogenesis in the absence of evident mitochondrial damage. For example, the antioxidant enzymes SOD, catalase, glutathione peroxidase, and glutathione reductase display reduced activities in the eyes of patients with glaucoma (Saccà et al., 2007; Ferreira et al., 2004). Oxidative DNA damage might be important with regard to degenerative, mutagenic, carcinogenic, and aging processes. Several studies have found increased levels of 8-OHdG in the mtDNA of elderly patients' brains (Agarwal and Sohal 1994; Izzotti et al., 1999). The oxidative DNA damage and 8-OH-dG levels measured in the TMs of patients with glaucoma were significantly higher than those in controls (Izzotti et al., 2003). Furthermore, human TM primarily expresses the eNOS isoform with a much smaller amount of nNOS in the outflow pathway (Nathanson and McKee, 1995), and physiological eNOS regulates the aqueous outflow in the eye by maintaining vascular endothelial cell function. AC endothelial cells play a major rule in glaucoma pathogenesis; in fact, numerous studies have demonstrated that declines in human (H)TM cellularity are linearly related to age (Alvarado et al., 1981, 1984a). Moreover, patients with glaucoma have lower HTM cellularity than controls (Alvarado et al., 1981, 1984a). Interestingly, an NOS-like deficit in the TM and Schlemm's canal manifests in the outflow pathways of patients with glaucoma, and subordinate NADPH-reactivity loss might affect outflow resistance more directly through the altered tone of contractile cells (Nathanson and McKee, 1995). In addition, peroxynitrite can affect mitochondrial respiration, thereby causing cellular energy failure, contractile dysfunction, and cell death. Interestingly, ROS modulate cell signalling via mitochondrially generated ROS; in fact, ROS (typically H2O2)-induced signalling (Rhee, 1999) inhibit tyrosine phosphatase (Salmeen et al., 2003), which leads to the following outcomes: cell proliferation; translocation and activation of the serine/ threonine kinases (e.g., protein kinase C) (Novalija et al., 2003); the activation of the MAPK family of protein kinases that mediate mitogen and stress-activated signals; and the activation JOURNAL OF CELLULAR PHYSIOLOGY

of NF-kB, a transcription factor that regulates the gene expression involving immune and inflammation responses (Thannickal and Fanburg, 2000). Figure 4 summarizes the molecular alterations related to ultrastructural alterations that were detected in the TMs of patients with glaucoma at DNA and gene expression levels. Is the Anterior Chamber a Vessel?

From a structural point of view, the AC is a specialized vessel whose walls are formed by corneal and trabecular endothelial cells and whose “blood“ is the aqueous humour (AH). Furthermore, the discovery of atherosclerotic plaque markers in the aqueous humours of patients with glaucoma confirms this interpretation (Saccà et al., 2012). The iris loses its endothelial lining at birth (Vrabec, 1952). This important concept helps us to understand how the malfunction of its endothelium can lead to outflow deterioration and ultimately to increases in IOP. Although the corneal endothelia are clearly visible when exposed to direct light, the TM endothelium (E) is hidden between them in the iris corneal corner. The three-dimensional architecture of the HTM considerably increases the filtration surface between the TME and AH; in fact, the TM consists of arrays of collagen beams covered by endothelium-like cells, with extracellular material/matrix (ECM) occupying the spaces between the beams (Chen and Kadlubar, 2003). “The outermost juxtacanalicular or cribriform region has no collagenous beams; rather, it has several cell layers immersed in loose ECM. The adjacent Schlemm's canal is a continuous endothelium-lined channel that drains aqueous humour into the general venous circulation (Tian et al., 2000).” These cells display an endothelial cell-like morphology and are avid phagocytes (Zhou et al., 1995) that possess contractile and migratory apparatuses (Calthorpe and Grierson, 1990; Stumpff and Wiederholt, 2000), have the capacity to produce ECM elements (Yue, 1996) and can transduce signals upon attachment to the ECM via protein kinase C (PKC) (Zhou et al., 2000) The cellular mechanisms that underlie the changes in AH outflow via the TM are not well understood. The mechanisms that regulate the cellular mechanisms of outflow and that might be involved in the pore formation of the inner wall of Schlemm's canal (Johnson et al., 1992; Ethier et al., 1999) include ciliary muscle and TM cell contractions (Tian et al., 2000; Wiederholt et al., 2000); TM cells changes in shape and size (Epstein et al., 1999; Freddo et al., 1984; Al-Aswad et al., 1999), alterations in TM intracellular volume and permeability (O'Donnell et al., 1995); volume-regulatory ion flux pathways that remodel the extracellular matrix of the JCT (Putney et al., 1999; Maepea and Bill, 1992; Lutjen-Drecoll and Rohen, 1996); and the progressive accumulation of damaged and cross-linked proteins within aging tissues with malfunctioning proteolytic cellular systems (Liton et al., 2009). Finally, oxidative damage might induce mitochondrial damage and trigger apoptosis and cell loss (Izzotti et al., 2010b). Although the molecular mechanisms responsible for mitochondrionmediated disease processes are not well understood, this last mechanism occurs only among patients with POAG or PEXG, where the oxidative damage due to mitochondrial failure plays a pathogenic role in the functional decay of the TM (Izzotti et al., 2011). Conversely, in other glaucoma types, TM cell loss is independent of mitochondrial damage and loss. Nevertheless, cell death might cause a free radical attack (Yan et al., 1991; Padgaonkar et al., 1994), and the loss or altered functionality of HTM cells might be the result of an increase in oxidative stress (De La Paz and Epstein, 1996). The Endothelial Barrier

The endothelium plays an important role in AC homeostasis. In particular, the endothelium of the TM plays a fundamental role


Fig. 4. Molecular alterations targeting the trabecular meshwork in patients with primary open angle glaucoma (POAG) (upper panels) with regard to the histopathological alterations detected using scanning electron microscopy (2,000) under physiological conditions (left-low panel) and in POAG (right-low panel).

in the AH transit from the AC to Schlemm's canal (SC). In fact, Alvarado et al. (2005a,b) showed that the AH leaves the AC through two barriers: the TME and the endothelium that lines the lumen of Schlemm's canal. The same type of cells forms both barriers, but these cells have different functions. The fluid flow resistance across the SCE is greater than that across the TME (Alvarado et al., 2004); however, the TME releases cytokines into the media, and these factors increase the permeability of the SCE barrier upon binding to the SCE (Alvarado et al., 2005a). Next, these factors flow downstream with the aqueous humour to influence the barrier function of the SCE to regulate the exit of the aqueous humour (Alvarado et al., 2004). When the IOP is greater than the venous pressure, the increased tension most likely tightens the trabecular beams and cords, thereby triggering the stretch receptors to activate the TME to release vasoactive factors that will then increase flow across the SCE (Alvarado et al., 2005b). Recent literature reviews suggesting that epithelial sodium channel proteins function as sensors of pressure-induced vascular stretch and laminar flow support the hypothesis regarding the presence of the stretch receptors on the TM (Drummond, 2009) . In fact, the transport of AH across the epithelial structures of the eye regulates IOP, and this regulation is associated with ion flux. The specific upregulation of epithelial sodium channel proteins JOURNAL OF CELLULAR PHYSIOLOGY

might serve as a protecting mechanism against IOP increases (Dyka et al., 2005). Therefore, the endothelial cells of the TM and Schlemm's canal constitute a system that governs the outflow because their interactions are complex and proceed bidirectionally, involve the TME-SCE and SCE-TME relationship, and mutual exchanges involving TME-TME and SCE-SCE associations (Alvarado et al., 2005b). Of course, many factors might influence these biomolecular results. First, light (Saccà et al., 2013) can cause cell dysfunction through the action of reactive oxygen species on DNA. This effect might contribute to cellular aging, age-related pathologies, and tumorigenesis (Godley et al., 2005). In fact, light has a direct oxidizing effect that is exerted both locally and at a systemic level in exposed organisms (Wei et al., 1998; Balansky et al., 2003). The apoptotic death of photoreceptor cells via the lightinduced stress present in the eyes of patients with retinal degenerative disorders is well documented (Osborne et al., 2006; Kanan et al., 2007). UV irradiation induces typical, caspase-dependent conjunctival cell death (Buron et al., 2006). Furthermore, light induces the formation of oxidative radicals in the AC that can indirectly target the TM by altering the oxidant/antioxidant balance in the AH, thereby contributing to the pathogenesis of glaucoma (Saccà et al., 2013; Wood et al., 2007). In fact, on one hand, this mechanism decreases the total

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antioxidant potential of the AC (Ferreira et al., 2004); on the other hand, it damages the TM with regard to 8-oxo-dG formation and DNA fragmentation (Izzotti et al., 2003), thereby contributing to endothelial dysfunction. Endothelial Dysfunction

Vascular dysfunction is a common symptom of many diseases and closely related to oxidative stress in many conditions (Griendling and FitzGerald, 2003a,b). Hypertension, hyperlipidaemia, atherosclerosis, and diabetes mellitus are associated with higher levels of reactive oxygen and nitrogen species within the vasculature, and these species induce oxidative damage in vascular tissue (Minuz et al., 2006). Chronic endothelial dysfunction diseases are characterized by a decrease in NO biosynthesis, bioavailability, or both (Napoli and Ignarro, 2001); an excess of superoxide (Daiber et al., 2009); and an excess of endothelin production (Ruschitzka et al., 2001). TNF-a regulates NOS expression and activity, thereby directly affecting NO production. TNF-a might increase iNOS expression by activating NF-kB (Fig. 3), and this increased TNF-a expression induces the production of ROS. TNF-a activates the transcription of NF-kB, which regulates the expression of the genes involved in inflammation, oxidative stress, and endothelial dysfunction (Rimbach et al., 2000; Kumar et al., 2004; Dela et al., 2007). In addition, the glaucoma syndrome is characterized by these biohumoural changes. In fact, a balance between vasoconstrictors and vasodilators is necessary to maintain the physiological structure and function of endothelia (Wiederholt et al., 2000), which are implicated in modulating the permeability of the endothelial barrier as well as the release of ET and NO. This balance is impaired and can cause major consequences among patients with glaucoma. Cyclic Guanosine monophosphate (GMP) and NO2 concentrations were lower within the aqueous humours of patients with POAG compared with those in patients with normal eyes; moreover, cyclic GMP and NO2 plasma levels were decreased among patients with glaucoma (Doganay et al., 2002). NO donors decrease IOP by increasing their aqueous outflow facility in the TM, Schlemm's canal, or both via cellular mechanisms that regulate outflow facility, including changes in cell volume and cellular contractility. In fact, NO (which is closely linked with guanylate cyclase) promotes the conversion of GTP in cGMP. This promotion activates protein kinase G (pkg), which in turn determines the phosphorylation and the activation of channel ionic BKCa (Dismuke et al., 2008). These potassium (Kþ) ion channels are activated by calcium (Caþþ), located at the cell membrane level, and activated by electric potential membrane changes or intracellular Caþþ concentration increases. Their activation determines the exit of Kþ from cells and the consequent cell volume reduction with regard to osmotic pressure cytoplasmic reduction (Dismuke and Ellis, 2009). The volume reduction in TM cells allows for intertrabecular spaces enlargement and a greater exposure of surface cells, thereby enhancing AH outflow facility. Furthermore, the NO-dependent solubleguanylate cyclase/ cyclic guanosine monophosphate system plays an important role in regulating aqueous humour dynamics via its production in the ciliary processes (Ellis et al., 2001; Shahidullah and Delamere, 2006), with subsequent decreases in IOP (Kotikoski et al., 2002). In addition, endothelin helps to regulate IOP. Despite TM mobility, ET-1 might induce TM contraction and increase outflow resistance, whereas TM relaxation increases outflow facility (Wiederholt et al., 2000). The Relationship Endothelin/NO

In fact, ET-1 is the most powerful substance because it constricts endothelial production and acts on the specific JOURNAL OF CELLULAR PHYSIOLOGY

receptors ETA and ETB. ETA receptors are only present on smooth muscle cells and cause vasoconstriction and cell growth, whereas ETB receptors are present on both smooth muscle cells (which leads to vasoconstriction) and another type of cell (which determine expansion and stimulate the production of NO), thereby acting as a negative feedback loop by inhibiting the continued production of ET-1. When the negative feedback mechanism of NO reduced bioavailability is compromised, the vasoconstriction effect is increased (Haynes and Webb, 1998). Vasoconstriction decreases blood flow to the endothelia in the anterior region of the eye, followed by pathological changes in the retina and the optic nerve head; these effects likely contribute to the degeneration of retinal ganglion cells. Nevertheless, endothelin affects TM contractility, which modulates trabecular outflow (Rosenthal and Fromm, 2011). High ET-1 levels were reported in the aqueous humours of patients with glaucoma (Noske et al., 1997; Tezel et al., 1997), and abnormal vascular responses to endothelin or receptor antagonists have been shown in patients with normal tension glaucoma (Buckley et al., 2002). Although direct evidence of local ocular endothelial dysfunction is difficult to obtain, Henry et al. (1999) found evidence of generalized endothelial dysfunction in a group of patients with NTG. This finding might be due to attenuated ETA-receptor-mediated tone, increased ETB-receptormediated contraction, or the impaired ETB-receptor-mediated release of endothelial NO (Henry et al., 2006). Not only might dysregulation occur with regard to the vascular response to increased levels of endothelin, but also endothelin might have direct effects on target tissues depending on the expression and distribution of their receptor (Yorio et al., 2002). NO can modulate the expression, sensitivity, and signal termination of endothelin receptors (Redmond et al., 1996). Nevertheless, POAG is associated with peripheral vascular endothelial dysfunction (Su et al., 2008); clearly, this dysfunction includes the TM endothelium mitochondrial dysfunction among patients with POAG (Izzotti et al., 2010b) and (usually) the higher susceptibility to ROS compared with the other tissues that constitute the AC (Izzotti et al., 2009). Moreover, patients with POAG have a significant impaired endothelial function, which Siasos et al. (2011) linked with the increased inflammatory status that causes endothelial dysfunction. Furthermore, ET-1 has been linked to various other glaucoma-related effects on the optic nerve and RGC, including astrogliosis, ECM remodelling, and NO-induced damage (Good and Kahook, 2010). Additional evidence of endothelial dysfunction arises from data showing increased AH levels of the endothelial leukocyte adhesion molecule-1 (ELAM-1), which is the earliest marker of atherosclerotic plaque in the vasculature. This marker is activated in the HTM cells collected from patients with glaucoma (Wang et al., 2001) and represents a sustained stress response that results in the expression of proinflammatory markers (Luna et al., 2009). Moreover, it is most likely an endothelial index of damage. In fact, the increased production of NO due to the iNOS present in the TMs of patients with POAG might contribute to TM cell death (Fernandez-Durango et al., 2008). The activation of nuclear factor (NF)-kB is an essential requirement for the expression of the iNOS gene. This factor mediates inflammatory responses and is activated by a wide variety of noxious stimuli (e.g., UV light, ionising radiation, free radicals, and many cytokines) (Baldwin, 1996; Beverley and Buchwald, 1997). Oxidative stress releases prostaglandins and NO from the ocular vascular endothelium, both of which participate in the regulation of ocular vascular smooth muscle tone. Figure 4 summarizes the pathogenic cascade that targets the TMs of patients with POAG. The molecular alterations that result in endothelial dysfunction, mitochondrial damage, and cell loss drive these pathogenic events.


Trabecular Meshwork Cytoskeleton

Although the mechanisms that control IOP are unknown, the cytoskeletons of the cells within outflow tissues are known to play a key role in influencing aqueous outflow resistance (Tian et al., 2000). In fact, Schlemm's canal endothelial cells are subjected to a basal-to-apical pressure gradient due to transendothelial flow that depends on F-actin distribution (Ethier et al., 2004). The TM possesses smooth muscle-like properties and is actively involved in the regulation of AH outflow and IOP. Furthermore, the TM and ciliary muscles are functional antagonists; ciliary muscle contraction leads to TM distension with a subsequent reduction in outflow, whereas TM contraction leads to the opposite effect (Wiederholt et al., 2000). The intracellular Ca2þ signal primarily regulates smooth muscle contraction (Hirano et al., 2003). “The cytosolic Ca2þ level activates the myosin light chain kinase that phosphorylates the 20-kDa regulatory myosin light chain and activates myosin ATPase.” Major cytoskeletal cell components (including the protein filaments F-actin, microtubules, and neurofilaments) are liable for maintaining cell shape, positioning cellular organelles, and transporting intracellular proteins (Goode et al., 2000). Glaucoma damages retinal the nerve fibre layer (RNFL), which usually precedes detectable visual field loss. Therefore, the direct assessment of the RNFL is a sensitive predictor of disease progression. RNFL alteration occurs in the cytoskeleton during the course of glaucoma; irregular F-actin staining, microtubules, and neurofilaments were found within bundles (Huang et al., 2011). In addition, decreases in RNFL birefringence might indicate a loss of microtubules (Huang and Knighton, 2005). Changes to the F-actin cytoskeleton are clearly associated with microvascular leakage (Yu et al., 2005). The formation of gaps between endothelial cells (e.g., those that occur during inflammation) is based on an actin–nonmuscle myosin contraction process at the cell margins (Van Hinsbergh, 1997). These changes also indicate that TM damage is reflected both in cell motility and endothelial function. From a molecular point of view, the RhoA/ROK pathway is constitutively active in numerous organs under physiological conditions. Moreover, Rho/Rho kinase is involved in the regulation of force, the basal tone of smooth muscle, and the response to contractile agonists (Somlyo and Somlyo, 2003). The Rho-Rho-kinase pathway modulates the phosphorylation level of the myosin light chain of myosin II, primarily through the inhibition of myosin phosphatase, which contributes to agonist-induced Ca2þ sensitization during smooth muscle contraction (Fukata et al., 2001). Rho kinase has a dual role in regulating the endothelial barrier function with opposite effects. Specifically, it shows intrinsic activity at cell margins, which is essential for proper barrier integrity and induced activity at stress fibres that mediate cell contractions, thereby resulting in barrier disruption (van Nieuw Amerongen et al., 2007). “Two Rho family GTPases, Rho and Rac, have emerged as key antagonistic regulators” with regard to “endothelial barrier function: Rho increases actomyosin contractility which facilitates the breakdown of intercellular junctions, whereas Rac stabilizes endothelial junctions and counteracts the effects of Rho (Wojciak-Stothard and Ridley, 2002).” The activation of Rho GTPase in endothelial cells disrupts the permeability barrier due to increased contraction (Wojciak-Stothard and Ridley, 2002). Lysophosphatidic acid (LPA) is a RhoA activator, and LPA-induced barrier dysfunction is accompanied by a reorganization of the F-actin cytoskeleton and the formation of focal attachment sites without changes to the intracellular calcium concentration (van Nieuw Amerongen et al., 2000). The cytoskeletal protein composition of the TM cells is similar to that of the ciliary muscle and vascular endothelial cells. Rho kinase predominantly regulates the activity of myosin II, a crucial regulator of cellular contraction and a major JOURNAL OF CELLULAR PHYSIOLOGY

component of the cytoskeletal protein fractions in TM and CM cells (Inoue et al., 2010). Furthermore, the rho-kinase pathway likely mediates TM cell responses to cyclic mechanical stress (Ramos et al., 2009). In fact, changes in TM cytoskeletal organization affect the response of the monolayers to cyclic biomechanical stress; moreover, Rho-kinase and its downstream effectors are involved in the pulse-mediated response of TM cells (Ramos et al., 2009). Interestingly, myocilin affects TM cell adhesion through the Rho GT-phase and cAMP/protein kinase A signalling pathways (Shen et al., 2008). Specifically, myocilin is a gene that is linked to the most common form of glaucoma (Resch and Fautsch, 2009). Its normal, physiologic function is currently unknown; however, when it is moderately overexpressed, myocilin induces a loss of actin stress fibres and focal adhesions (Wentz-Hunter et al., 2004), inhibits the adhesion of human TM cells to ECM proteins, and compromises the cohesiveness of the TM cell matrix. The Rho/Rho-kinase pathway is an important regulator of vascular smooth muscle cell contraction as well as migration (Wirth, 2010), proliferation (Kamiyama et al., 2003), and differentiation (Owens et al., 2004). The upregulation of the RhoA/ROCK signalling cascade has been observed in cardiovascular disorders such as atherosclerosis, pulmonary hypertension, and stroke. Furthermore, RhoA/ROCK affects NO-signalling and vice versa. The regulation of eNOS mRNA stability is likely the mechanism through which ROCK influences NO production (Laufs and Liao, 1998; Shiga et al., 2005). These suppositions are supported by the sustained activation of the Rho GTPase signalling in the aqueous humour outflow pathway, which “increases resistance to aqueous humour outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells (Zhang et al., 2008).” In particular, RhoA-induced changes in actomyosin contractile activity as well as decreased fibronectin levels, alpha-smooth muscle actin levels, ECM synthesis/ assembly, and Rho kinase activation, together with the fibronectin-induced alpha-SMA expression in TM cells, “reveal a potential molecular interplay between actomyosin cytoskeletal tension and ECM synthesis/assembly (Pattabiraman and Rao, 2010).” Furthermore, ET-1 and TGF-b are Rho GTPase agonists, and their AH levels are elevated in patients with glaucoma (Thorleifsson et al., 2010; Noske et al., 1997). However, the actual functions of Rho-kinase remain largely unknown, despite its ubiquitous expression in the body. For instance, Notch3 plays an important role in the control of resistance arteries and mechanotransduction by modulating the RhoA/Rho kinase pathway, which is involved in pressureinduced (myogenic) tone (Belin et al., 2008). The Notch pathway is a highly conserved cell-signalling system that regulates many aspects of embryonic development (Louvi and Artavanis-Tsakonas, 2006). “Notch activation generally prevents differentiation and maintains progenitor or stem cell proliferation, and it is a classical mediator of lateral inhibition during cell fate determination (Yoon and Gaiano, 2005; Bol os et al., 2007; Rowan et al., 2008).” However, Notch signalling has diverse and almost unlimited cellular outcomes because “it can regulate cell cycle progression, nervous system survival, fate determination, and morphogenesis across different organs and cellular contexts (Artavanis-Tsakonas et al., 1999; Thomas, 2005; Rowan et al., 2008).” Increased cell death is also correlated with reduced Notch signalling (Rulifson and Blair, 1995; Jehn et al., 1999) Interestingly, “RhoA/Rho kinase regulation via Notch signalling in endothelial cells triggers a senescence phenotype associated with endothelial barrier dysfunction (Venkatesh et al., 2011).” The relationships among anterior chamber structures and the alterations that occur in patients with POAG are summarized in Figure 5.

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Fig. 5. DNA lesions directly through photochemical reaction or indirectly through redox reaction-generating ROS, in turn produce DNA lesions. This leads to changes in the transcriptome and the proteome that in turn are used as signals for the activation of other biological processes.

The Aqueous Humour

The AH is continuously formed from plasma by the epithelial cells of the ciliary processes. It is secreted into the posterior chamber, passes from the posterior chamber through the pupil into the anterior chamber, and is drained at the anterior chamber angle. Furthermore, a small loss of liquid occurs through the limbal sclera (Bill, 1971). The selective uptake of certain substances in the iris also influences AH drainage (Raviola and Butler, 1985) . These fluid and solute transitions are not easily quantified. AH is a crystal-clear fluid with a specific gravity between 1.0034 and 1.0036. The index of refraction of this fluid is 1.3336, which is less than that of the lens, which is 1.39 (Kaufman, 1994). The volume of the anterior chamber and the AH contained within is approximately 0.25 cc. The volume of the posterior chamber and theAH contained within is 0.06 cc. The flow of AH is estimated to be approximately 2.5 ml/min during the day in the normal eye, and the tonography value of “C” (i.e., the coefficient of aqueous outflow facility) is 0.3 l mx min1 mmHg1 (Bill and Maepea, 1999). The calculation of C involves certain basic assumptions that are potential sources of inaccuracy (Blondeau, 1985). IOP in ocular-hypertensive patients is caused by a reduction of TM outflow facility and uveoscleral outflow while the aqueous flow remains normal (Toris et al., 2002). The production and elimination rates of this fluid are responsible for intraocular pressure. Interestingly, reductions in the production of AH and its drainage through the uveoscleral outflow pathway have been observed in the healthy aging eye (Toris et al., 1999). JOURNAL OF CELLULAR PHYSIOLOGY

Furthermore, apraclonidine studies among patients with ocular hypertension have shown that IOP is associated with an increase in fluorophotometric outflow facility (Toris et al., 1995). The AH production rate is approximately 2–2.5 cc/min. Approximately 1% of the anterior chamber and 3% of the posterior chamber volume of AH is replaced each minute (Stamper, 1992). AH provides nutrients for the avascular lens and cornea, and it removes the waste products from these structures. AH contains a protein concentration of approximately 1/100th that of serum (Pavao et al., 1989), which enters the AC directly via diffusion through the root of the iris. The aqueous fluid has 0.1–0.2% of the concentration of plasma proteins but a higher concentration of amino acids than plasma. Ascorbate (Ringvold et al., 2000) and lactate concentrations (Tokuda et al., 2007) as well as bicarbonate are also high in the AH (Gerometta et al., 2005). Glutathione and vitamin C defend against free radicals in- and outside of cells (Cardoso et al., 1998). In addition, vitamin E prevents the endogenous mitochondrial production of ROS (Southam et al., 1991). Furthermore, glutathione maintains vitamins C and E in their reduced (active) forms within the cell, and vitamin C helps to protect membrane lipids from peroxidation by recycling vitamin E (May, 1999). Lipid peroxidation occurs when the levels of mitochondrial glutathione and vitamin E are reduced to 20% of their baseline (Augustin et al., 1997). A high level of ascorbic acid in AH is necessary to maintain a filter-like function against UV radiation in both the central corneal epithelium and the AH (Giblin et al., 1994; Ringvold et al., 2000; Izzotti et al. 2009). This effect reacts with O2 to form H2O2, thereby


maintaining the oxidative balance in the AH, whereas vitamin E deficiency increases H2O2 levels (Chow et al., 1999). Furthermore, vitamin E induces a protective effect via a nonoxidant mechanism by inhibiting protein kinase C activity in smooth muscle cells in vivo (Ozer and Azzi, 2000; Ozer et al., 2003). The most interesting feature of a glaucomatous AH is its protein fraction. In fact, certain stimuli, including elevated IOP, exert physical forces on HTM cells that causing mechanical stress. This stress can have a profound effect on their gene expression profile and most likely radically and dramatically changes the AH proteome during the glaucoma (Fig. 6). Currently, several molecules have been identified that are likely involved in the pathogenesis of POAG. Many of these molecules are involved in the control mechanism of the ECM turnover in the TM outflow pathways. Others should provide the key needed to clarify the molecular mechanisms that govern AH outflow through the TM and the pathogenetic mechanisms of POAG (Tamm Ernst, 2008). Proteome in Glaucomatous AH

Specific glaucomatous AH proteins are vascular type proteins because they are markers of atherosclerotic plaque (Saccà et al., 2012). The best known is endothelium leukocyte adhesion molecule 1 (ELAM 1), which was commonly found in the TM of patients with glaucoma (Wang et al., 2001). Our results revealed it in the AH (Saccà et al., 2012). Among others, some are worthy of mention. Apolipoprotein B (ApoB) is the major apolipoprotein of chylomicrons and low-density

lipoproteins. It is present in the AHs of patients with glaucoma because the production of cytokines such as TNF-alpha induces chronic inflammation. This finding implies that the transcription factor NF-kB is also involved in inflammatory, anti-apoptotic, and immune responses (Li and Lin, 2008). TNF-alpha leads to the production of ApoB (Goswami et al., 2010). Apolipoprotein E, which shows antioxidant activity (Miyata and Smith, 1996), is also highly expressed in the AHs of patients with glaucoma and most likely expressed by the TM endothelium to act against the oxidative stress that damages cells. The vasodilator-stimulated phosphoprotein (VASP) is another protein that characterizes the glaucomatous AH. The VASP is associated with the formation of filamentous actin, which plays a major role in cell adhesion and motility. The VASP is also involved in the intracellular signal route that regulates the interaction between integrin and the extracellular matrix (Scott et al., 2006). It plays a major role in the formation of adherens junctions and the remodeling of the actin cytoskeleton. These events are involved in the maintenance of endothelial function (Aziz et al., 2010). Therefore, their presence indicates that the endothelial barrier of the TM has been altered. The muscles of the eye play a vital role in TM activity. Myogenic factors 3 and 4 are found in glaucomatous AHs. Myogenic factor 3 is the principal regulator of skeletal myogenesis and acts as a proapoptotic factor. In fact, it is involved in secondary apoptosis during muscle regeneration (Aziz et al., 2010; Hirai et al., 2010). On the other hand, myogenic factor 4 (i.e., myogenin) belongs to a family of myogenic proteins that regulate the expression of specific

Fig. 6. Pathogenic cascade targeting trabecular meshwork (TM) in patients with POAG reflects on proteome profile in ocular anterior chamber. These events are the result of an interplay between genetic and environmental factors resulting in DNA damage, alterations in gene expression, and finally proteome changes.


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TABLE I. Proteins undergoing significant alteration in AH of POAG patients as compared to controls, adapted From Table 4.1, Saccà et al. (2014)

Protein name

Trend in POAG vs. control

Ontology

ATPase, Naþ/Kþ transporting, beta 3 polypeptide

"""

Cell homeostasis

Glutamate-ammonia ligase (glutamine synthase)

"""

Gluatamate detoxification glutamine production neural tissue hoemeostasis

A kinase anchor protein 1, mitochondrial

"""

Mitochondria integrity

Calcium-binding mitochondrial carrier protein Aralar1

"""

Mitochondrial transporter

Mitochondrial heat shock 60 kD protein 1 Translocase of inner mitochondrial membrane 23 Cytochrome c

""" """

Mitochondrial protein repair Mitochondrial transporter

"""

Cell respiration Apoptosis

Function Non-catalytic component of the active enzyme, which catalyzes the hydrolysis of ATP coupled with the exchange of Naþ and Kþ ions across the plasma membrane. Membrane protein Expressed in retina and neural tissues. Glutamate catabolism and Glutamine production. Catalyzes glutamate transformation into glutamine by ATPdependent NH3 addiction. Glutamine is a main source of energy and is involved in cell proliferation, inhibition of apoptosis. Anchors the cytoplasmic face of the mitochondrial outer membrane. Located in mitochondrial outer membrane. Calcium-dependent mitochondrial aspartate and glutamate carrier. Located in mitochondria inner membrane. Molecular chaperone. Mitochondrial import inner membrane translocase.

Mitochondrial electron-transport chain. Plays a role in apoptosis. Suppression of the anti-apoptotic members or activation of the pro-apoptotic members of the Bcl-2 family leads to altered mitochondrial membrane permeability resulting in release of cytochrome c into the cytosol. Binding of cytochrome c to Apaf-1 triggers the activation of caspase-9, which then accelerates apoptosis by activating other caspases. Located in mitochondrial matrix. Mitochondrial proteins involved in electron transport chain, trans-membrane transport, protein repair, mitochondrial integrity maintenance. BCL2-associated X protein (BAX) """ Apoptosis Positive regulation of apoptosis. Located in mitochondria. BCL2-interacting killer (apoptosis-inducing) """ Apoptosis Activation of apoptosis through the intrinsic pathway. (BIK) Located in mitochondria. Caspase 8 """ Apoptosis Apoptosis-related cysteine protease. Most upstream protease of the activation cascade of caspases responsible for the TNFRSF6/FAS mediated and TNFRSF1A induced cell death. Cleaves and activates CASP3, CASP4, CASP6, CASP7, CASP9, and CASP10. Caspase 9 """ Apoptosis Involved in the activation cascade of caspases responsible for apoptosis execution. TNF receptor-associated factor 2 """ Apoptosis Links members of the tumor necrosis factor receptor family to different signalling pathways. Mediates activation of NF-kappa-B and JNK. Involved in apoptosis. Fas (TNFRSF6)-associated via death domain """ Apoptosis Activation of apoptosis resulting from inflammation. Activation of apoptosis through the extrinsic pathway in response to inflammation and/or oxidative stress. Apoptosis proteins are directly involved in apoptosis induction mainly through the intrinsic, i.e., mitochondrial-dependent, pathway. Cadherin 3 """ Tissue integrity Idem Cadherin 5 """ Tissue integrity Calcium dependent cell adhesion proteins connecting cells. Control cohesion and organization of the intercellular junctions. Calnexin """ Protein repair Calcium-binding protein playing a major role in the quality control apparatus of the Endoplasmic reticulum by the retention of incorrectly folded proteins. Located in melanosomes. Catenin alpha """ Tissue integrity Cadherin associated protein. The association of catenins to cadherins produces a complex which is linked to the actin filament network, and which seems to be of primary importance for cadherins cell-adhesion properties. Junction plakoglobin """ Tissue integrity. Cell adhesion Junctional plaque protein. Presence of plakoglobin in both the desmosomes and in the intermediate junctions suggests that it plays a central role in the structure and function of submembranous plaques. Connecting cells proteins are component of intercellular junction and contribute to tissue homeostasis and maintenance of cell–cell adhesion. Ankyrin 2, neuronal """ Neuron survival Adapter protein in the postsynaptic density (PSD) of excitatory synapses that interconnects receptors of the postsynaptic membrane including NMDA-type and metabotropic glutamate receptors, and the actinbased cytoskeleton. Chondroitin sulfate proteoglycan """ Neuron survival Growth and differentiation factor involved in 5 (neuroglycan C) neuritogenesis and neuron survival. Expressed in neural tissue. Optineurin """ Neuron survival Apoptosis Plays a neuroprotective role in the eye and optic nerve.



TABLE 1. (Continued)

Protein name

Trend in POAG vs. control

Ontology

Function Probably part of the TNF-alpha signaling pathway that can shift the equilibrium toward induction of cell death. Molecular chaperone.

""" Protein repair in neurons Neural precursor cell expressed, developmentally down-regulated 4 (NEDD 4) Neuronal proteins are typically located in neurons and are involved in various neuronal functions including neuron survival, neuroglycanes production, NMDA-receptor activation, glutamate detoxification, and protein repair. Nitric oxide synthase 2 """ Oxidative stress Produces nitric oxide. Superoxide dismutase (SOD1/2) # Antioxidant defence Destroys radicals which are normally produced within the cells and which are toxic to biological systems. Microsomal glutathione S-transferase 1 # Antioxidant defence Conjugation of reduced glutathione to a wide number of exogenous and endogenous electrophiles. Dynein, cytoplasmic, light polypeptide 1 """ Tissue integrity. Oxidative stress Play a role in maintaining the spatial distribution of cytoskeletal structures. Binds and inhibits the catalytic activity of neuronal nitric oxide synthase. The reduced expression of the antioxidant enzymes SOD and GST could aggravate the unbalance between both oxygen-and nitrogen-derived free radicals production and detoxification.

genes in muscles. This factor is known for its role in embryonic myogenesis as well as with regard to muscle growth and regeneration in adults (Meadows et al., 2011). The PKC signalling pathway mediates the stimulation of the myotrophin activity concerning muscle protein synthesis (Aziz et al., 2010). PKC levels are significantly increased in the AHs of patients with glaucoma. PKC influences runoff, cell flow, contraction, and the morphological changes of the TM and the sclerocorneal cells. Myotrophin activates NF-kB (Adhikary et al., 2005) due to oxidative stress in the TM. From a molecular point of view, the AC behaves as a vessel. This effect is supported by the other proteins found in glaucomatous AHs (i. e., phospholipase C beta 1 and gamma 1) (Saccà et al., 2012). In turn, these intracellular proteins generate two products: inositol 1,4,5-trisphosphate (a universal calcium mobilizing second messenger) and diacylglycerol (a PKC activator that governs muscle contraction) (Rebecchi and Pentyala, 2000). These products have been discovered in glaucomatous AH (Izzotti et al., 2010a). Calcium mobilization plays a key role in muscle contraction. When the ciliary muscle contracts, the insertions of its ligament widen the intercellular spaces in the TM, and the permeability of the tissue co-increases with the exposed surface of the endothelial cells in the AH (Rohen et al., 1981). In addition, the TM is a contractile tissue with similar properties to smooth muscle, and its contractility is linked to Rho kinase A, which can be regulated by PKC isoforms (Llobet et al., 2003; Zhang et al., 2008). The activation of these proteins at the start of the regulatory mechanisms involves cytoskeletal reorganization and intercellular adhesion. Moreover, LA PKC has also been implicated in the transcription regulation of the matrix metalloproteinases that maintain the normal amount of AH outflow (Bradley et al., 1998). Phospholipase C beta and gamma mobilize calcium internally through the activation of PKC. This process involves multiple PKCs that control (or modulate) cell division, transformation, differentiation, shape, motility, and apoptosis (Rebecchi and Pentyala, 2000). Classes of Proteins

The protein profile of the AHs of patients with glaucoma differs completely from that of normal participants even when things do not change quantitatively. The most common profiles are grouped into six classes (Table I). In addition to vascular proteins, mitochondrial proteins, those that induce apoptosis, and those involved in the maintenance of intercellular adhesion JOURNAL OF CELLULAR PHYSIOLOGY

are known. Other proteins play an important role in the oxidative stress prior to the pathogenesis of glaucoma, and two classes of interesting neuronal proteins such as optineurin and the growth and differentiation factors involved in neurogenesis and neuron survival as well as the protein kinase group are key regulators of cell function that constitute one of the largest and most functionally different gene families (Izzotti et al., 2010a). The presence of these proteins in the aqueous humours of patients with glaucoma allows us to understand how the molecular events that occur in the AC signal glaucoma pathogenesis. We do not know what is the prime mover; however, we know that cellularity is important with regard to optimal TM function, and cellularity decreases due to an alteration to its mitochondria. We do not know how or which genetic factors influence this TM mitochondrial alteration, but we know that this effect leads to an alteration of the TM endothelium. On the one hand, the proteins that induce apoptosis include cytochrome C as well as those that connect cells, including catenins, junctional plaque protein, dynein, and cadherins. These proteins lead to the profound alteration that occurs in the barrier between the AC and Schlemm's canal. The outflow decrease mechanism is most likely due to the loss of mechanical integrity of the TM caused by the reduction in cell– cell and cell–matrix adhesion. Catenins are WNT proteins, and the WNT signalling pathway controls the activity of genes needed at specific times during development. Furthermore, this pathway regulates the interactions between cells during the formation of organs and tissues. Interestingly, myocilin might modulate WNT signalling, and this modulation might provide a useful tool in the reorganization of the cytoskeleton of TM cells and the regulation of IOP (Wang et al., 2008). The protein kinase group are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. Modulating the PKC pathway might be relevant in patients with glaucoma. For instance, PKC inhibitors relax the TM as well as affect matrix metalloproteinases and PGF2 alpha. In fact, the increased outflow of PKC inhibitors might play an important role in the modulation of aqueous outflow facility by regulating myosin light chain phosphorylation and, as a consequence, the morphological and cytoskeletal characteristics of TM and SC cells (Khurana et al., 2003). PKC has been implicated in the activation of iROS production by NAD(P)H oxidase in endothelial and smooth muscle cells; PKC might contribute to the induction of iROS production (Inoguchi et al., 2003). PKC gamma is a unique isoform of PKC that is

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Fig. 7. Histopathological and ultrastrctural trabecular meshwork alterations that occur in patients with POAG. Pathogenic events are driven by molecular alterations that result in endothelial dysfunction, mitochondrial damage, and cell loss.

found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. H2O2 activates protein kinase Cg through the C1 domain via an oxidative mechanism, and this results in the inhibition of gap junctions (Lin and Takemoto, 2005) . This effect confirms the key role of endothelial dysfunction in the course of glaucoma. In addition, PKC is involved in apoptosis activation and signal transduction (Liu et al., 2004; Kim et al., 2008). The alteration of the endothelium leads to an alteration of the barrier and the motility of the TM, thereby resulting in a TM malfunction with an important change to the glaucomatous AH proteome (Fig. 7) (Saccà and Izzotti, 2013). It is likely that these proteins signal the continuation of molecular events in the posterior segment, which allows proteins to communicate and exchange among the AC, optic nerve head, and retina. In fact, much of the protein in the AH of the anterior chamber diffuses through the root of the iris (Johnson et al., 1993). From there, they reach the supraciliary space and move as far posterior as the suprachoroidea of the peripapillary retina (Smith et al., 1986). This movement demonstrates how different molecules (in particular, proteins) spread freely in the anterior and posterior segments. The presence of antioxidant proteins is significantly lower in patients with POAG than controls. This finding confirms that antioxidants defences in the AH of patients with glaucoma are loss-making, and the failure of these antioxidant defenses is likely to result in damage to the TM due to the remarkable susceptibility of this tissue to oxidative injury (Izzotti et al., 2009). In addition, “the reduced expression of the” AC “antioxidant enzymes SOD and GST” can “aggravate the unbalance between both oxygen- and nitrogen-derived free radical production and detoxification (Bagnis et al., 2012).” A particularly intriguing report showed that dorzolamide and timolol demonstrated antioxidant effects on TM biopsy JOURNAL OF CELLULAR PHYSIOLOGY

specimens and human TM cells exposed to hydrogen peroxide (Saccà et al., 2011). Furthermore, their antioxidant effects involved mitochondria and were likely to be exerted during the early phases of glaucoma when the mitochondrial damage in the TM tissue occurs at low levels. Conclusions

Although many scientists continue to believe that the TM is only a filter-like tissue, the recent literature suggests that the TM is an organ whose function varies depending on the circumstances. When needed, it opens to expose more endothelial cells to the AH of the anterior chamber and closes for exactly the opposite reasons. If the pores exist, then they most likely have a limited role, and only TM (Sit et al., 1997) impairment results in altered outflows. Thus, the TM causes IOP increases and their associated problems. A better understanding of the function of TM tissue, especially due to elevated pressure, will greatly contribute to the potential management and hopefully cure of glaucoma, a disease with enormous life quality and financial consequences. Many questions remain unanswered, but molecular events illuminate TM damage and indicate that trabecular cell protection plays a role in the treatment and prevention of glaucoma. Literature Cited A-H-Mackerness S, John CF, Jordan B, Thomas B. 2001. Early signaling components in ultraviolet-B responses: Distinct roles for different reactive oxygen species and nitric oxide. FEBS Lett 489:237–242. Adhikary G, Gupta S, Sil P, Saad Y, Sen S. 2005. Characterization and functional significance of myotrophin: a gene with multiple transcripts. Gene 353:31–40. Agarwal S, Sohal RS. 1994. Aging and protein oxidative damage. Mech Ageing Dev 75:11–19.


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The trabecular meshwork in normal eyes and in exfoliation glaucoma.

[Ablation of the trabecular meshwork].

Dexamethasone Stiffens Trabecular Meshwork, Trabecular Meshwork Cells, and Matrix.

The Function of Matricellular Proteins in the Lamina Cribrosa and Trabecular Meshwork in Glaucoma.

Targeting the ER-autophagy system in the trabecular meshwork to treat glaucoma.

Mechanotransduction channels of the trabecular meshwork.

Role of microRNAs in the trabecular meshwork.

HDAC Inhibitor-Mediated Epigenetic Regulation of Glaucoma-Associated TGFβ2 in the Trabecular Meshwork.

Histological Findings in the Trabecular Meshwork of a Patient with Atopic Glaucoma.

Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma.

Lysyl oxidases in the trabecular meshwork.

The role of integrins in the trabecular meshwork.

Biphasic model of the trabecular meshwork in the eye.

Microfilaments in the cells of the human trabecular meshwork.

Role of nuclear factor (erythroid-derived 2)-like 2 in the age-resistant properties of the glaucoma trabecular meshwork.

Cross-talk between ciliary epithelium and trabecular meshwork cells in-vitro: a new insight into glaucoma.

Potential regulatory molecules in the human trabecular meshwork of patients with glaucoma: immunohistochemical profile of a number of inflammatory cytokines.

The Trabecular Meshwork: A Basic Review of Form and Function.

Stem Cells in the Trabecular Meshwork for Regulating Intraocular Pressure.

Eyes on new product development: trabecular meshwork.

Fluctuations in intraocular pressure increase the trabecular meshwork extracellular matrix.

TGF-β induces phosphorylation of phosphatase and tensin homolog: implications for fibrosis of the trabecular meshwork tissue in glaucoma.

Pro-fibrotic pathway activation in trabecular meshwork and lamina cribrosa is the main driving force of glaucoma.

Matricellular proteins in the trabecular meshwork: review and update.