Accepted Manuscript The aqueous humor outflow pathways in glaucoma: A unifying concept of disease mechanisms and causative treatment Barbara M. Braunger, Rudolf Fuchshofer, Ernst R. Tamm PII: DOI: Reference:

S0939-6411(15)00209-X http://dx.doi.org/10.1016/j.ejpb.2015.04.029 EJPB 11920

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Revised Date: Accepted Date:

12 March 2015 24 April 2015 29 April 2015

Please cite this article as: B.M. Braunger, R. Fuchshofer, E.R. Tamm, The aqueous humor outflow pathways in glaucoma: A unifying concept of disease mechanisms and causative treatment, European Journal of Pharmaceutics and Biopharmaceutics (2015), doi: http://dx.doi.org/10.1016/j.ejpb.2015.04.029

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The aqueous humor outflow pathways in glaucoma: A unifying concept of disease mechanisms and causative treatment

Barbara M. Braunger, Rudolf Fuchshofer and Ernst R. Tamm* Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg, Germany

*Corresponding author: E.R. Tamm, Institute of Human Anatomy and Embryology, University of Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany. Tel.: +49-941/943-2839, Fax: +49-941/943-2840, E-mail: [email protected]

Abstract Intraocular pressure (IOP) is the critical risk factor for glaucoma, a neurodegenerative disease and frequent cause of blindness worldwide. As of today, all effective strategies to treat glaucoma aim at lowering IOP. IOP is generated and maintained via the aqueous humor circulation system in the anterior eye. Aqueous humor is secreted by the ciliary processes and exits the eye through the trabecular meshwork (TM) or the uveoscleral outflow pathways. The TM outflow pathways provide resistance to aqueous humor outflow and IOP builds up in response to it. In the normal eye, the resistance is localized in the inner wall region, which comprises the juxtacanalicular connective tissue (JCT) and the inner wall endothelium of Schlemm’s canal (SC). Outflow resistance in the inner wall region is lowered through the contraction of the ciliary muscle or the relaxation of contractile myofibroblasts in the posterior part of the TM and the adjacent scleral spur. Patients with primary open-angle glaucoma (POAG), the most frequent form of glaucoma, typically suffer from an abnormally high outflow resistance of the inner wall region. There is increasing evidence that the increase in TM outflow resistance in POAG is the result of a characteristic change in the biological properties of the resident cells in the JCT, which increasingly acquire the phenotype of contractile myofibroblasts. This scenario strengthens simultaneously both their actin cytoskeleton and their directly associated extracellular matrix fibrils, leads to overall stiffening of the tissue, and is modulated by transforming growth factor-β (TGF-β)/connective tissue growth factor (CTGF) signaling. Essentially comparable changes appear to occur in SC endothelial cells in glaucoma. Causative therapy concepts targeting the aqueous outflow pathways in glaucoma should aim at interfering with this process either by attenuating TM or SC stiffness, and/or by modulating TGF-β/CTGF signaling.

Introduction Glaucoma is the leading cause of irreversible blindness worldwide [1-3] and more than 60 million people throughout the world suffer from this disease [4, 5]. The number of people that are blind from glaucoma has not markedly changed over the years [1, 3], but is expected to rise [5] indicating quite strongly that glaucoma remains an unsolved problem of high significance in ophthalmology. Characteristics of glaucoma are that of a chronic, progressive optic nerve neuropathy which occurs in parallel to typical structural changes at the optic disc where the optic nerve axons exit the eye [6-8]. Primary damage is restricted to the retinal ganglion cells and the optic disc is thought to be the site of initial damage. A substantial number of prospective randomized multi-center studies convincingly documented that the level of intraocular pressure (IOP) is the major modifiable risk factor for the incidence of glaucoma and its progression [9-15]. It is generally assumed that an IOP level, which is too high for the health of the optic nerve initiates events that result in a chronic and progressive deformation of the optic nerve head [16], a scenario that is observed by examining ophthalmologists as excavation or cupping of the optic disc [6, 8, 17]. The deformation of the optic nerve head causes or contributes to the chronic degeneration of optic nerve axons and finally leads to apoptotic death of the retinal ganglion cells. As of today, the molecular pathogenesis that directly causes the damage of retinal ganglion cells and their axons in glaucoma has not been clarified. Impairment of axonal transport, reactive changes in macro- or microglial cells or optic nerve macrophages, or changes in blood flow, which were all observed in patients and various animal models of glaucoma, are currently discussed as causative or contributing events [6, 8, 18-21]. Even though retinal ganglion cell death can be prevented in animal models of glaucoma by treatment with neuroprotective molecules, no evidence is currently available indicating that such a direct neuroprotective therapy would be feasible for human patients [22, 23]. In contrast, there is overwhelming evidence from several prospective randomized multi-center studies showing that the reduction of IOP is neuroprotective in the sense that it delays or even prevents the structural

and functional damage of optic nerve axons in glaucoma [9-15]. Here we will review the mechanisms that are thought to generate IOP in the normal eye and in that with primary openangle glaucoma (POAG), the most common form of glaucoma. Moreover, we will propose a unifying concept to explain the pathological molecular changes that affect IOP in POAG, and discuss novel or established pharmaceutical IOP-lowering options for causative treatment [24].

Intraocular pressure and aqueous humor outflow IOP is generated through the aqueous humor circulation system in the anterior eye segment (Fig. 1). Aqueous humor is secreted into the posterior chamber, a process which involves ultrafiltration from the fenestrated capillaries of the ciliary processes followed by an active secretion from the two epithelial layers of the ciliary body (inner nonpigmented epithelium and outer pigmented epithelium) that function as syncytium coupled through gap junctions (Fig. 1B) [25]. Aqueous humor passes from the posterior chamber through the pupil into the anterior chamber and exits the eye via two pathways: (1) the conventional or trabecular outflow pathway, or (2) the unconventional or uveoscleral outflow pathway (Fig. 1). In the trabecular outflow pathways, aqueous humor passes through the trabecular meshwork, the juxtacanalicular tissue (JCT) and Schlemm’s canal endothelium to reach the lumen of Schlemm’s canal, a circumferential capillary vessel that is connected through radial collector channels with the episcleral veins [26]. In the unconventional or uveoscleral outflow pathways, aqueous humor diffuses through the interstitial spaces of the ciliary body, a route that is made possible by the fact that the anterior surface of the ciliary body is not separated from the anterior chamber of the eye by an epithelial layer [27]. In consequence, aqueous humor passes into the interstitial spaces between the bundles of the ciliary muscle to reach the capillaries of the ciliary body [28]. In addition, the supraciliary and suprachoroidal spaces are used by aqueous humor to reach the lymphatic vessels of the orbit [28]. There is currently considerable controversy

regarding the existence of lymphatic vessels in the eye proper [29]. In case such vessels exist, they may contribute to the uveoscleral outflow of aqueous humor. The percentage of the unconventional outflow was reported to be between 10 to 57% of total aqueous humor outflow, depending on the methods used for its measurement [30]. Under physiological conditions, only the trabecular outflow pathways are relevant for the generation and maintenance of IOP [31]. The trabecular outflow pathways provide resistance against the flow of the aqueous humor and in response to this resistance IOP is generated until IOP is high enough to drive aqueous humor against the resistance to the outside of the eye. Passage of aqueous humor in the trabecular outflow pathways occurs as bulk flow driven by pressure gradient only. Neither diffusion processes nor active transport mechanisms play a role in this passage as neither metabolic drugs nor temperature changes modify the rate of trabecular aqueous humor outflow [31]. If IOP is in a steady state, the outflow of the aqueous humor equals its secretion from the epithelium of the ciliary body. The resistance in the human and non-human primate trabecular outflow system increases with age, but as secretion of aqueous humor decreases in parallel, IOP is normally not affected [32-36]. In eyes with POAG though, outflow resistance in the trabecular outflow pathways is higher than in age-matched normal control eyes [31], while secretion of aqueous humor is commonly not changed in POAG [37, 38]. Contrary to conventional outflow, the rate of unconventional outflow is not dependent on the levels of IOP [31].

The trabecular meshwork The tissues that contribute to the trabecular outflow pathways are embedded in the scleral sulcus, a circular groove located at the inner side of the sclera next to the corneoscleral limbus [26, 39]. The sulcus extends from the peripheral end of Descemet’s membrane to the scleral spur, a circumferential ridge that protrudes from the inner sclera into the eye. Schlemm’s canal, a circular vessel, is located at the outer aspect of the scleral sulcus while the trabecular

meshwork is localized at its inner aspect. The trabecular meshwork comprises of connective tissue strands or beams, termed lamellae, containing a core of collagenous and elastic fibers. The lamellae are entirely covered by flat trabecular meshwork cells that are separated by a basal lamina from the core of the lamellae. Trabecular meshwork cells have a considerable phagocytotic activity, which is thought to serve as self-cleaning mechanism to keep the porous structure of the trabecular meshwork open and intact [40]. Moreover, cells containing pigment granules that most likely originate from the iris are frequent and accumulate in regions of high flow such as opposite collector channels [41]. The trabecular meshwork cells that cover the trabecular lamellae show many biological characteristics that are common to epithelial cells, even though they are frequently referred to as trabecular meshwork endothelial cells, due to their flat morphological shape. In any case, trabecular meshwork cells do not share with vascular endothelial cells their typical biological characteristics and are of different origin as they derive from the neural crest [42]. The lamellae of the trabecular meshwork are partly connected through their connective tissue core as well as through their cellular processes, resulting in a porous, sponge-like structure. As Schlemm’s canal dimensions are shorter in anterior-posterior orientation than the trabecular meshwork (Fig. 1C), one distinguishes a posterior, filtering part from an anterior non-filtering part of the trabecular meshwork. Anteriorly, the trabecular lamellae are attached near the end of Descemet’s membrane (Schwalbe’s line) and extend posteriorly to the stroma of the ciliary body and iris at their junction, and to the scleral spur (Fig. 1C). Those cells of the non-filtering portion of the trabecular meshwork, which reside close to its attachment at Schwalbe’s line, differ in structure from that of the trabecular meshwork proper [43]. There is considerable evidence that this region serves as a niche for cells with adult stem cell-/progenitor cell-like properties that are capable of dividing and repopulating the filtering part of the trabecular meshwork after injury [43]. The trabecular meshwork consists of three regions that differ in structure (Fig. 2A): The uveal trabecular meshwork comprises of 1 to 3 layers of lamellae. The corneoscleral portion of the trabecular meshwork consists of 8-15 lamellae that

are thicker than those of the uveal trabecular meshwork. Between the lamellae of the corneoscleral trabecular meshwork and the endothelium of the Schlemm’s canal lays the juxtacanalicular tissue, the smallest part of the trabecular meshwork with a depth of about only 2-20 µm. The juxtacanalicular tissue is not built up by lamellae, but largely consists of loose connective tissue with 2-5 layers of cells showing no epithelial, but rather a star-like, mesenchymal morphology, comparable to that of fibroblasts. The cells of the juxtacanalicular tissue are embedded in an extracellular matrix, consisting of fibrillar components and an amorphous ground substance of hyaluronan and proteoglycans [44]. A major structural characteristic of the juxtacanalicular tissue is its network of elastic fibers, the cribriform plexus, which spans tangentially to the endothelium of Schlemm’s canal [45, 46]. The elastic fibers of the cribriform plexus consist of an elastin-containing core and of banded sheath material with a periodicity of about 50 nm. Up until now, the exact molecular nature of the sheath material is not fully understood, but collagen type VI and fibronectin appear to be associated with it [45, 47]. The fibers of the cribriform plexus are connected with Schlemm’s canal through connecting fibrils that emerge from the sheath material (Fig. 2B). These cell-matrix connections contain fibronectin and appear to be important for the adhesion of the endothelium that otherwise only rests on an incomplete basal lamina. If those connections are disrupted experimentally, Schlemm’s canal endothelium will detach in response to the force exerted by aqueous humor flow [48].

Schlemm’s canal Schlemm’s canal is a modified capillary blood vessel with a specific hydraulic conductivity (K < 65 x 10-4 cm2) [31] that is the highest in the body and even higher than that in the fenestrated capillaries of the glomeruli in the kidney or the sinusoids in the liver [31]. To allow for the high hydraulic conductivity, the endothelium of Schlemm’s canal transiently forms intra- and

intercellular pores (Fig. 2C), which can acquire unusually large diameters of up to 1 µm [31]. Moreover the endothelial cells of Schlemm’s canal inner wall rest on an incomplete basal lamina and considerable areas of their basal cell membrane are not supported by extracellular matrix, but are in direct contact with the open spaces of the juxtacanalicular tissue and aqueous humor flow. Unlike the transendothelial flow in capillaries, aqueous humor flow passes Schlemm’s canal endothelial cells in a basal-luminal direction, a fact that results in the formation of cellular outpouchings of Schlemm’s canal cells (so-called giant vacuoles) in response to the pressure gradient associated with aqueous humor flow (Fig. 2A, C). As mentioned before, the contacts of Schlemm’s canal endothelium with the connecting fibrils of the cribriform plexus prevent the complete detachment of the endothelium in response to aqueous humor flow. The pores of Schlemm’s canal likely develop from mini-pores with a size of 60 nm, which are covered by a diaphragm built of the molecule plasmalemma-vesicle associated protein (PLVAP) [49, 50]. The molecular processes of pore formation are presumably very important for aqueous humor outflow, but are ill understood. PLVAP is likely involved, as the formation of vascular pores is largely impaired in PLVAP-deficient mice [49, 51].

Trabecular outflow resistance The pores in the uveal and corneoscleral trabecular meshwork are too spacious to generate an outflow resistance for aqueous humor. Therefore, it is broadly accepted that the outflow resistance for aqueous humor in the trabecular outflow pathways is localized in the inner wall region composed of the juxtacanalicular tissue and the inner wall endothelium of Schlemm’s canal [31]. Three potential mechanisms are discussed to be involved in the generation of trabecular outflow resistance:

1.) Outflow resistance is localized in the extracellular components of the juxtacanalicular tissue and is largely dependent on the quality and quantity of the extracellular matrix in that particular region. 2.) Outflow resistance is localized in the inner endothelial layer of Schlemm’s canal. 3.) Outflow resistance is generated through a synergistic functional interplay between the extracellular outflow pathways of the juxtacanalicular tissue and the pores of Schlemm’s canal endothelium. In support of a critical role for the juxtacanalicular tissue extracellular matrix in generating outflow resistance are experiments, which show that the perfusion of anterior segment organ cultures with metalloproteinases, enzymes that degrade extracellular matrix compounds, lowers outflow resistance [52]. Arguments against this hypothesis are based on experiments using morphometry in conjunction with transmission electron microscopy [53, 54]. According to those studies, the areas in the juxtacanalicular outflow pathways occupied by electron-dense extracellular matrix are much too small to generate the physiological outflow resistance. On the other hand, the procedures that are required to process tissue for conventional transmission electron microscopy are well known to cause a substantial loss of non-fibrillar extracellular matrix components such as hyaluronan and proteoglycans that are hardly detectable with this method. In fact, studies using quick-freeze deep-etch electron microscopy reported on considerable higher amounts of extracellular matrix in the juxtacanalicular tissue as seen with conventional electron microscopy [55]. Arguments against a critical role of Schlemm’s canal endothelium in generating outflow resistance come from studies in which the endothelial pores were visualized by scanning electron microscopy (SEM) and quantitatively evaluated to assess their role in outflow resistance [56]. According to those data only 10% of the outflow resistance would be generated

in Schlemm’s canal endothelium. Still, more recent observations show that the number of pores in Schlemm’s canal endothelium is influenced by the duration of chemical fixation [57, 58], a fact that clearly indicates that the number of pores in vivo might actually be significantly smaller than seen with SEM. The possibility of a synergistic functional interplay between juxtacanalicular tissue and Schlemm’s canal endothelium is based on the hypothesis that the extracellular matrix of the juxtacanalicular tissue generates a funneling effect by guiding the aqueous humor right to the pores in Schlemm’s canal endothelium (funneling hypothesis) [59]. Funneling should result in a restriction of aqueous humor flow and thus generate outflow resistance. The outflow resistance would be considerably reduced if either the number of pores in the Schlemm’s canal endothelium or the flow pathways in the juxtacanalicular tissue would increase and disrupt the funneling effect. Currently, the funneling hypothesis appears to be in agreement with the available experimental data.

The uveoscleral outflow and intraocular pressure Even though uveoscleral outflow has no relevant physiologic impact on the generation of IOP, it can be modified efficiently using pharmacological drugs to significantly reduce IOP. Prostaglandin analogues stimulate the activity of matrix metalloproteinases [60], an effect that causes a modification of the ciliary muscle extracellular matrix resulting in wide interstitial spaces between the muscle bundles and, consequently, in a widening of the uveoscleral outflow pathways [61-63]. There is some evidence from studies using perfused human anterior eye segment organ cultures indicating that prostaglandins analogues may reduce also outflow resistance in the conventional trabecular outflow pathways [64], probably by a similar mechanism.

Contractile mechanisms in the trabecular outflow pathways Two contractile systems influence the geometry of the trabecular outflow pathways and thereby the outflow resistance in the inner wall region. One system comprises the ciliary muscle, which is attached to the scleral spur via elastic and collagenous fibers that form distinct tendon-like structures (Fig. 3) [65, 66]. Since the elastic fibers of the cribriform plexus are also anchored to the scleral spur [65, 67], contraction of the ciliary muscle results in a changed geometry of the trabecular angle outflow pathways and, consequently, to a reduction of outflow resistance in the inner wall region. This effect is even more pronounced as some of the ciliary muscle tendons pass the scleral spur and reach into the lamellae of the trabecular meshwork (Fig. 3) [66]. The ciliary muscle is among the most densely cholinergic innervated tissues of the whole organism [27] and its anatomical connection to the scleral spur and the trabecular meshwork is the basis of the pharmacologically-induced reduction of IOP through cholinergic drugs [68]. Experimental disinsertion of ciliary muscle’s anterior insertion to the scleral spur completely abolishes the effects of cholinergic drugs on outflow resistance and IOP in primates [69]. The second contractile system is comprised of cells in the trabecular meshwork and the juxtacanalicular tissue, which show properties of contractile myofibroblasts [70]. The cells are particularly numerous in the posterior part of the tissues, the region next to the scleral spur (scleral spur cells) [65] (Fig. 4). The cells in this region contain high amounts of α-smooth muscle actin, which is the characteristic actin isoform of smooth muscle cells (Fig. 4). In contrast to the cells of the ciliary muscle, myofibroblasts in the trabecular meshwork outflow pathways contain no desmin, the intermediate filament typically found in smooth muscle cells (Fig. 4). Ultrastructural studies show that scleral spur cells have all the structural characteristics of myofibroblasts like a cytoplasm that contains numerous myofilaments or cellular processes that form tendon-like contacts with the neighboring extracellular matrix fibers fibers (Fig 5). One

major structural difference between the myofibroblasts in the trabecular outflow pathways and the cells of the neighboring ciliary muscle is the fact that the myofibroblasts are orientated in a circumferential direction. This orientation is perpendicular to that of the longitudinally orientated bundles of the ciliary muscle (Fig. 4, 6). Numerous neuronal endings with structural characteristics of mechanosensors are located in the scleral spur and thus right between both contractile systems. The mechanosensitive neuronal endings are most likely part of a proprioceptive system controlling the tone of both contractile systems [71]. The contractile myofibroblasts are parasympathetically innervated by nervous endings containing neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) [72]. Myofibroblasts are especially numerous in the trabecular outflow pathways of bovine eyes [73], and experimental data show that treatment with muscarinic and α-adrenergic agonists, or with endothelin result in the contraction of the cells, while treatment with β-adrenergic agonists and nitric oxide lead to their relaxation [74]. In summary, an elevated tone of contractile cells in the trabecular outflow pathways results in an elevated outflow resistance in the trabecular meshwork while their relaxation leads to a reduction of the outflow resistance [74, 75]. Apparently, both of the contractile systems, ciliary muscle and myofibroblasts of the trabecular outflow system, appear to act as functional antagonists when it comes to the modulation of the outflow resistance. This phenomenon can easily be explained through the observation that the contractile cells in both systems are differently oriented and at an angle of 90° to each other allowing modifying the geometry of the trabecular outflow pathways in an antagonistic manner (Fig. 6). Recent studies of genetically modified mice show that the enzyme endothelial nitric oxide (eNOS) which leads to the release of nitric oxide (NO), plays a critical autoregulative role for the tone of the cells in the trabecular meshwork outflow pathways. Stretching of the trabecular meshwork e.g. through an increased IOP results in a release of nitric oxide due to the

activity of eNOS, as part of a mechano-regulatory system. Subsequently, outflow resistance and IOP are decreased most likely due to the nitric oxide-mediated relaxation of the cells of the trabecular outflow system [76].

Trabecular outflow pathways in primary open angle glaucoma In patients with POAG, the trabecular outflow pathways show a very characteristic structural change in the juxtacanalicular tissue [77]. There is a distinct increase in the quality and quantity of the fibrillar extracellular matrix, which mostly affects the sheath material of the elastic fibers of the cribriform plexus, a finding originally described as the formation of “sheath-derived plaques” [67, 78]. The severity of optic nerve damage in POAG correlates with the increase in the amount of sheath-derived plaque material in the juxtacanalicular tissue [79]. Still, the amount of sheathderived plaque material does not correlate with the level of IOP in eyes of patients with POAG [79], strongly indicating that the increase in extracellular matrix is not the cause, but a symptom of the changes in trabecular outflow resistance in POAG. Most likely, the increase in extracellular matrix in the juxtacanalicular tissue is directly related to the elevated concentrations of transforming growth factor (TGF)-β2, which are observed in the aqueous humor of most patients with POAG [80]. Cultured trabecular meshwork cells are capable of producing the isoforms TGF-β1 and TGF-β2, and increase their extracellular matrix synthesis upon treatment with TGF-βs [80]. In addition, treatment with TGF-βs increases the contractile properties of the TM actin cytoskeleton [80]. The effects of TGF-β are embedded in a very complex homeostatic signaling system of molecules that stimulate or inhibit its activity in the trabecular outflow pathways [81]. The effects on extracellular matrix synthesis and the actin cytoskeleton are mediated through TGF-β’s downstream mediator connective tissue growth factor (CTGF) [82, 83]. CTGF

is critically required for the effects of TGF-β on the extracellular matrix synthesis and for increasing the contractile properties of mesenchymal cells including that of the trabecular meshwork [83, 84]. In a mesenchymal cell, the force generated by the actin cytoskeleton is transmitted to fibrillar components of the extracellular matrix via integrin-based cell-matrix contacts (Fig. 7). High activity of TGF-β signaling appears to cause, via the downstream mediator CTGF, a switch to a myofibroblast-like phenotype including the augmentation of the actin cytoskeleton and its directly associated fibrillar extracellular matrix (Fig. 7). There is recent evidence from studies in genetically engineered animals, which supports the assumption that the TGF-β/CTGF-mediated switch is a critical causative factor for the increase in outflow resistance and IOP in eyes with POAG. Transgenic mice with ectopic overexpression of CTGF in their eyes develop an increase in IOP that correlates with the loss of optic nerve axons [83]. Since the chamber angle of CTGF-overexpressing mice is wide open, CTGF-overexpressing mice constitute an animal model with essential characteristics of POAG. The trabecular meshwork outflow pathways of the mice accumulate fibrillar extracellular matrix components and show signs of an increase in actin-based contractility. Treatment of the mice with a Rho-kinase inhibitor that interferes with actin contractility reduces within hours their high IOP to levels seen in normal animals. Overall, the effects of CTGF on IOP appear to be mediated by a modification of the trabecular meshwork actin cytoskeleton. The observation leads to the assumption that the high activity of TGF-β signaling in POAG causes, via the downstream mediator CTGF, a similar change in the phenotype of trabecular outflow cells in human patients as seen in CTGF-overexpressing mice with glaucoma. The cells may switch to a myofibroblast-like phenotype, a scenario, which strengthens simultaneously both their actin cytoskeleton and their directly associated extracellular matrix fibrils (Fig. 7). Through the increased tone and accumulation of extracellular matrix material the trabecular meshwork is likely to get stiffer and to lose its capability to respond to endogenous signals that lead to their

relaxation and consequently to the decrease in outflow resistance under physiologic conditions. Findings that strengthen this hypothesis are based on atomic force microscopy. Using this technique, Last and colleagues could showed that the trabecular meshwork of patients with POAG is stiffer than that of healthy persons [85]. Additional support comes from a recent study in rats with a vector-based expression of a constitutively active RhoAGTPase (RhoAV14) in the trabecular outflow pathways [86]. The rats exhibited a significantly elevated IOP associated with an increase in extracellular matrix and F-actin in the trabecular meshwork outflow pathways. Most of the changes were ameliorated by topical application of Rho kinase inhibitor [86]. Quite intriguingly, the switch to a stiffer cellular phenotype in POAG appears not to be restricted to the cells in the juxtacanalicular tissue, but rather to occur simultaneously in Schlemm’s canal endothelial cells, the other cell type in the trabecular meshwork outflow pathways that is very likely involved in the generation of outflow resistance. Cells from the canal of Schlemm of glaucomatous eyes were recently shown to have an increased cytoskeletal stiffness that leads to reduced pore formation in these cells, likely contributing to an increase in outflow resistance [87]. The stiffness positively correlates with elevated altered expression of several key genes, particularly CTGF [87].

Conclusions New insights into the molecular pathobiology of the trabecular meshwork outflow pathways strongly indicate that treatment of the cellular stiffness in this region is likely to address the cause for the POAG-associated increase in trabecular outflow resistance. Currently, Rhoassociated kinase inhibitors appear to be a promising option [88]. Alternative strategies could target components of the TGF-β/CTGF signaling pathway.

Acknowledgements: Part of the research reviewed in this article was supported by the DFG (FOR 1075).

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Fig. 1. The tissues of the aqueous humor circulation system in the anterior eye. A. Aqueous humor is secreted from the ciliary processes (CP, white arrow) into the posterior chamber (PC). It passes around the iris (Ir) into the anterior chamber (AC) to leave the eye via the conventional

or trabecular outflow pathways (black arrow), or the unconventional or uveoscleral outflow pathways (red arrow) along the supraciliary space between sclera (Sc) and ciliary muscle (CM) or through the ciliary muscle bundles. B. Higher magnification of the ciliary processes showing the structures in involved in aqueous humor secretion. Ca. Capillary; NPE. Nonpigmented ciliary epithelium; PE. Pigmented ciliary epithelium. White arrow indicates secretion of aqueous humor into the posterior chamber. From [25]. C. Higher magnification of trabecular (black arrow) and uveoscleral (red arrow) outflow pathways. TM: Trabecular meshwork; SC: Schlemm’s canal. From [26].

Fig. 2. A. Light micrograph of a meridional section through the trabecular meshwork. AC: Anterior chamber; JCT: Juxtacanalicular tissue; CTM: Corneoscleral trabecular meshwork; UTM: Uveal trabecular meshwork. Arrows point to giant vacuoles in the inner wall endothelium of SC. B and C. Transmission electron microscopy of the SC inner wall region. B. Connecting fibers (CF) in the juxtacanalicular tissue, which emerge from the cribriform elastic plexus, connect the plexus with the inner wall endothelium of Schlemm’s canal (SC). The connection with the inner wall endothelium is made via the banded sheath material of the fibers or via fine fibrils that emerge from it (arrows). C. A giant vacuole in the inner wall of SC forms an intracellular pore (arrow). From [26] .

Fig. 3. Light (A, B) and electron microscopy (C) of anterior ciliary muscle tendons. A, B. The muscle bundles of the radial portion of the ciliary muscle (RCM, A) and the inner bundles of its longitudinal portion (LCM, B) form tendons ( arrows ) in region of their anterior insertion that are continuous with the extracellular matrix of the trabecular meshwork beams. CCM, circular portion of the ciliary muscle. A. The tendons of the radial portion continue to the uveal trabecular meshwork (UTM). B. The tendons of the inner muscle bundles of the longitudinal portion pass the scleral spur (SS) at its inner aspect to continue to the corneoscleral trabecular

meshwork (CTM). C. The banded material of the ciliary muscle tendons ( solid arrows ) comes in direct contact with the cell membrane of the muscle cell (MC) which forms dense bands at the cytoplasmic site ( open arrows ). In region of contact with the tendon, the muscle cell forms deep furrows which are filled with banded material. From [39]. Fig. 4. Meridional (A, B) and tangential sections (C, D) through ciliary muscle (CM), scleral spur (SS), and trabecular meshwork (TM) stained with antibodies against a-smooth muscle actin (A, C) or desmin (B, D). A. Ciliary muscle cells and vascular smooth muscle cells stain positively with antibodies against a-smooth muscle actin. Arrows indicate the scleral spur, where all cells stain show intense immunoreactivity for a-smooth muscle actin. B. Immunostaining with antibodies against desmin. Ciliary muscle cells stain brightly positive, whereas the scleral spur is not labeled for desmin. C. Tangential section of scleral spur, trabecular meshwork and ciliary muscle. Positively stained cells oriented in a circular direction are seen throughout the entire spur tissue. While ciliary muscle cells also stain positive, no staining is seen in the trabecular meshwork. D. Tangential section of scleral spur and ciliary muscle after staining for desmin. The plane of the section is the same as in (C). Ciliary muscle cells stain brightly positive, whereas the cells of the scleral spur remain unstained. From [65].

Fig. 5. Electron microscopy of scleral spur cells. A. Scleral spur cells (SSC) are in close contact with banded sheath material (asterisks) of elastic fibers. The cytoplasm of the cells is filled with abundant 6–7 nm thin (actin) filaments which run parallel to the long axis of the cells (solid arrows). The cell membrane shows numerous membrane bound caveolae (open arrows). B. Scleral spur cells may form long processes to contact the elastic fibers (asterisk) in the scleral spur. In regions of contact, dense bands (arrows) are formed at the cell membrane of the scleral spur cell. C. Scleral spur cells are connected by gap junctions (arrow). From [65].

Fig. 6. Schematic drawing (orientation analogue to Fig. 4C) depicting the different orientation of the contractile systems that influence the geometry of the trabecular outflow pathways. The anterior longitudinal or meridional ciliary muscle bundles, which attach to scleral spur and trabecular meshwork are oriented perpendicularly to the circumferential or equatorially oriented contractile myofibroblasts of posterior trabecular meshwork (TM) and scleral spur. Arrows indicate movements in a circumferential direction (myofibroblasts in TM and scleral spur) or in a meridional direction (ciliary muscle bundles) following contraction. From [89]. Fig. 7. Schematic drawing of the actin cytoskeleton and the fibrillar extracellular matrix of cells in the juxtacanalicular tissue. The force generated by the actin cytoskeleton (red) is transmitted to the fibrillar extracellular matrix (green) by integrin based cell-matrix contacts (yellow). The high activity of TGF-β signaling in POAG causes, via the downstream mediator CTGF, a switch to a myofibroblast-like phenotype including an augmentation of the actin cytoskeleton and its directly associated extracellular fibrillar matrix. Overall, the changes cause an increase in TM rigidity and aqueous humor outflow resistance. From [89]

Graphical abstract