Current Understanding of Conventional Outflow Dysfunction in Glaucoma

Abstract

Purpose of review

Regulation of intraocular pressure by the conventional (trabecular) outflow pathway is complicated, involving a myriad of mechanical and chemical signals. In most, intraocular pressure is maintained within a tight range over a lifetime. Unfortunately in some, dysfunction results in ocular hypertension and open-angle glaucoma. In the context of established knowledge, this review summarizes recent investigations of conventional outflow function, with the goal of identifying areas for future inquiry and therapeutic targeting.

Recent findings

Mechanical stimulation of conventional outflow cells due to intraocular pressure fluctuations impacts contractility, gene expression, pore formation enzyme activity and signaling. Numerous local signaling mediators in the conventional pathway such as bioactive lipids, cytokines, nitric oxide and nucleotides participate in the regulation of outflow. Interestingly outflow through the conventional pathway is not uniform, but segmental, with passageways constantly changing due to focal protease activity of trabecular cells clearing extracellular matrix materials. The relationship between extracellular matrix expression and trabecular meshwork contractility appears to coordinately impact outflow resistance and is the target of a new class of drugs, the rho kinase inhibitors.

Summary

The conventional outflow pathway is a dynamic, pressure-sensitive tissue that is vulnerable to pathology on many fronts, each representing a therapeutic opportunity.

Introduction

The principal risk factor and only currently modifiable ocular parameter for millions of patients having primary open-angle glaucoma (POAG) is ocular hypertension (elevated intraocular pressure, IOP). The pathology responsible for ocular hypertension is located in the pressure-dependent, conventional (trabecular) outflow pathway. Over the past 60 years, several key observations implicate trabecular meshwork (TM) dysfunction in POAG. First, untreated POAG patients with ocular hypertension have elevated conventional outflow resistance compared to age-matched controls and surgical removal of TM from POAG eyes, eliminates the extra resistance [1, 2]. Second, activation of TM cells with directed laser energy temporarily reduces outflow resistance and brings down IOP in POAG eyes [3, 4]. Third, water drinking test of POAG patients reveals retarded IOP recovery [5]. Fourth, circadian IOP fluctuations are more dramatic in those with glaucoma, indicating hindered capacitance of TM [6]. Fifth, clinical features of corticosteroid-induced glaucoma mirror those of POAG, particularly effects on outflow facility [7, 8]. Lastly, mutations in a prominent TM gene product, myocilin, result in increased outflow resistance, ocular hypertension and POAG [9, 10].

While it is clear that the pathology responsible for ocular hypertension is located in the conventional outflow pathway, the molecular and cellular mechanisms that underpin the manifestation of disease are largely unknown. Sadly, glaucoma patients do not currently have a daily medication that targets conventional outflow (dys)function. The purpose of the present article is to review the recent knowledge concerning the regulatory mechanisms that underlie IOP control in the conventional outflow pathway, identifying areas for further study and candidates for therapeutic targeting.

Conventional Outflow Regulation

Regulation of conventional outflow resistance is dynamic and likely involves multiple redundant signaling systems and processes. There is much higher incidence of ocular hypertension than hypotony in the general population suggesting that ocular hypertension is better tolerated [11] and further suggesting that the regulatory machinery in the pressure-dependent conventional pathway is biased toward preventing hypotony. Such a design makes sense considering that under conditions of elevated IOP, the visual pathway and thus high acuity vision is maintained. In addition, it is difficult to medically lower IOP in most patients below a critical level (~12–13 mmHg), suggesting that “compensatory systems” are activated as IOP drops [12]. Thus, glaucoma surgeries such as trabeculectomy or the placement of tubes bypass these normal regulatory pathways and are a primary cause of hypotony [13, 14]. Since ocular hypertension is tightly linked to disease progression in POAG [15], current research has been directed at better understanding the mechanical and biochemical elements responsible for maintaining homeostasis in the conventional outflow pathway in health and those that go awry in disease with the goal of future therapeutic intervention.

Location of Outflow Resistance and Funneling

IOP is a byproduct of outflow resistance generation deep in the conventional outflow tract; in the juxtacanalicular tissue (JCT) region, where TM and Schlemm’s canal (SC) inner wall cells interact [2, 16–18]. Importantly, the “extra” resistance found in glaucomatous eyes is also located within the JCT region [2]. However it is not known if the source of resistance is due to TM cells and their extracellular matrix (ECM) or the inner wall of SC. By itself, neither structure appears to generate enough resistance to explain IOP. Recent evidence suggest that the TM and SC cells work together to synergistically regulate resistance likely via a hydrodynamic effect known as “funneling” [19]. Funneling arises because the inner wall is relatively impermeable except at discrete pore sites, and aqueous humor flowing through the JCT must thereby converge or “funnel” to pass through the pores of the inner wall. The convergence of flow caused by funneling reduces the area of the JCT that is actively involved in filtration, thereby increasing its effective hydraulic resistance. Thus, the immediate juxtaposition between the two tissues (facilitated by TM cell tethering to the inner wall), and hence their hydrodynamic coupling, gives rise to the funneling effect such that resistance decreases when they are separated.

Three separate studies, looking at two pharmacological agents (H7 and Y27632) and one experimental phenomenon called “washout” emphasize the importance of separation of TM and SC cells in the JCT. During washout [20], or perfusion with H7 or Y27632 [21, 22] outflow facility increases and alters the distribution of a flow tracers in the conventional tract of living monkeys or bovine eyes, respectively. While tracer is deposited in distinct foci along the inner wall in control eyes, consistent with the distribution of pores, the tracer pattern dramatically changes in treated eyes becoming more uniform over the surface of the inner wall. Moreover, the distance between the cribiform plates and the inner wall in the JCT expands in treated eyes, a phenomenon that is reversible. All three of these observations are in accordance with the loss of TM tethering and consequent disruption of funneling. The funneling model also predicts that changes in pore density of the inner wall will impact funneling and outflow facility. Consistent with the model, perfusion of certain compounds increased both facility and pore density; however, other compounds failed to reveal a relationship. Despite this, two other observations are also consistent with the model: First, if pores are selectively occluded with a flow tracer such as cationic ferritin, outflow facility dramatically decreases [23]. Second, glaucomatous eyes in two studies had significantly lower pore density, suggesting that those with ocular hypertension have a defect in pore formation/closure and thus higher resistance according to the funneling hypothesis [24, 25]. Importantly, recent in vitro studies of SC cells show that pore formation is pressure-dependent and impaired in glaucomatous cells [26].

Local Mediators of Outflow Facility

Due to its unique architecture, directional flow of aqueous humor and close proximity of cells to one another, the conventional outflow pathway utilizes autocrine and paracrine mediators to regulate outflow resistance. Local mediators from several categories including lipid-derived (lysophospholipids, prostaglandins (PG), cannabinoids), cytokines (TGFβ, BMP, Wnt, IL-1, TNFα), nucleotides (ATP/adenosine) and gases (nitric oxide) all impact conventional outflow resistance to varying degrees and in either direction (i.e.: increase or decrease, see table 1).

Table 1.

Summary of Effects of Autocrine/Paracrine Mediators of Conventional Outflow Function (original)

Local Mediator	Receptor(s)	Impact on C	Species	References
PM/PG F2α	PG FP (sv)	↑ 26%–67%	human	Brubaker et al., 2001; Christiansen et al., 2004; Wan et al., 2007, Toris et al., 2007, Fautsch et al., 2008
PG E2	EP4	↑ 35%–69%	monkey, human	Woodward et al., 2009; Millard et al., 2011
2-AG/AEA	CB1/CB2	↑ 0%–80%	porcine, feline, monkey	Zhong et al., 2005; Njie et al., 2006; Njie et al., 2008; Colasanti, 1990; Chien et al., 2003
LPA	LPAR	↓ 37%	porcine	Mettu et al., 2004
S1P	S1PR2	↓ 31%–41%	porcine, human	Mettu et al., 2004; Stamer et al., 2009; Sumida and Stamer, 2011
TGFβ	RI/RII	↓ 27%–45%	mouse, rat, porcine, monkey, human	Sheppard et al., 2010; Bhattacharya et al., 2008; Bachmann et al., 2006; Fleenor et al., 2006; Gottanka et al., 2004
IL-1α/β	IL1-R	↑ 25%–100%	rat, porcine, human	Birke et al., 2011; Bradley et al., 1998; Kee and Seo, 1997; Kelly et al., 2007
TNFα	TNFR	↑ 20%	porcine	Kelley et al., 2007
Gremlin	BMP RI/RII	↓ 36%	human	Wordinger et al., 2007
Wnt/sFRP	Fzd	↓ 55%	human	Wang et al., 2008
Nitric oxide	sGC	↑ 10%–115%	porcine, human	Dismuke et al., 2008; Ellis et al., 2008; Stamer et al., 2011; Shneemann et al., 2002
ATP/adenosine	A1/KATP	↑ 28%–71%	bovine, human	Tian et al., 1997; Crosson et al., 2005; Chowdhury et al., 2011

Abbreviations: C: outflow facility; PM: Prostamide; PG: Prostaglandin; sv: splice variant; 2-AG: 2-arachindonyl glycerol; AEA: anandamide; LPA: lysophosphatidic acid; S1P: sphingosine-1-phosphate; TGF: transforming growth factor; IL: interleukin; TNF: tumor necrosis factor; sFRP: soluble frizzled-related protein; CB: cannabinoid; R: receptor; BMP: bone morphogenetic protein; Fzd: frizzled receptor; sGC: soluble guanylate cyclase

Over the past decade the involvement of lipid mediation of conventional outflow function has become apparent [27]. For example, prostaglandin E2 and prostaglandin/prostamide F_2a, acting at EP4 or FP (and/or FP splice variant) receptors, respectively increase outflow facility [28–33]. Early clinical observations suggesting that PG drugs increase outflow facility was confirmed recently in controlled experimental systems that isolate the conventional outflow pathway. Interestingly, there appears to be two different mechanisms of action of these compounds because effects on outflow are either within minutes or can take days. Acute affects of prostaglandins likely affect the contractility status (relaxation) and/or cell-cell junction assembly/disassembly of cells in the conventional pathway, while long-term effects likely involve alterations in extracellular matrix turnover [28, 34].

Like the PGs, endocannabinoids acting on either CB1 or CB2 receptors increase outflow facility [35–38]. Perfusion of porcine anterior segments with endogenous cannabinoid agonists such as 2-arachidonoylglycerol or anandamide (or synthetic cannabinoids) results in an immediate and significant increase in outflow facility; effects that were blocked in part by CB1 and/or CB2 selective antagonists. In addition to cannabinoid receptors, endogenous ligands and their metabolizing enzymes are also present in the conventional outflow pathway [35, 36, 39].

While the types of lipid mediators described above increase outflow facility, lysophospholipids such as lysophosphatidic acid and sphingosine-1-phosphate (S1P) decrease outflow facility in porcine and human eyes [40, 41]. For example, perfusion of human eyes in organ culture with S1P results in an immediate decrease in outflow facility. The effects of S1P appear to be mediated by both TM and SC cells, involving increased contraction and cell-cell junction assembly, respectively [42]. Use of selective antagonists to S1P receptor subtypes showed that S1P effects on outflow facility are mediated by the S1P2 receptor subtype [43].

To add further complexity to local mediation of conventional outflow, many secreted cytokines impact conventional outflow function. Several cytokines, particularly those that mediate local inflammatory processes have been known to alter conventional outflow homeostasis for some time. For example, TGFβ levels are elevated in the aqueous humor of glaucoma patients and treatment of eyes with TGFβ gradually and substantially decreases outflow facility over the course of days [44] [45]. It appears that TGFβ affects ECM homeostasis in the JCT over time [46]. At least two key types of molecules, BMP4/7 and IL-1, have been shown to antagonize TGFβ signaling and contribute to outflow tissue homeostasis [47, 48]. The lipophilic secreted class of cytokines, Wnts, is expressed and signals in the conventional tract. Hence, an antagonist to Wnt signaling, sFRP increases outflow facility in perfused human anterior segments, while overexpression of sFRP in mice increases IOP [49].

Small signaling molecules such as nucleotides and nitric oxide (NO) modify conventional outflow. For instance, ATP release from TM cells through several different molecular conduits can be induced by stretching of the TM due to IOP fluctuations [50, 51]. Once outside, ATP interacts with the KATP potassium channel to increase outflow facility and/or is rapidly converted to adenosine by ectoenzymes and is free to interact with one of several adenosine receptors [52]. Importantly, adenosine activation of the A1 receptor in the conventional pathway rapidly increases outflow facility in cultured bovine eyes through effects involving MMP activity [53]. Importantly for patients, a synthetic agonist (INO-8875) that specifically activates A1 receptors is currently in clinical trials as a glaucoma therapeutic (clinicaltrials.gov ID# NCT01123785). In addition, the labile gas, nitric oxide, increases outflow facility [54–58]. Using NO-donating compounds or mice overexpressing endothelial nitric oxide synthase, liberation/generation of NO in the conventional tract results in approximately two-fold increase in outflow facility. Possible mechanisms underlying the NO effects are decreased volume, contractility and/or cell-cell junction assembly in conventional outflow cells.

Mechanoregulation of Outflow Facility

Most people do not develop ocular hypertension and glaucoma, even at advanced ages, suggesting the existence of effective IOP homeostatic mechanisms [59, 60]. Since conventional outflow tissues are constantly subjected to a pressure gradient, it makes sense that biomechanics play a role in outflow regulation. This was demonstrated experimentally several years ago [61, 62] and studies of the mechanistic details in recent years have clarified the process to some extent [59, 60]. Elevated IOP is sensed by cells within the JCT region as mechanical stretch or distortion [63]. TM cells, perhaps with assistance from SC cells, respond by adjusting the outflow resistance, which ultimately corrects the IOP. Changes in cell signaling, gene expression, ECM turnover, and cytoskeletal organization are all associated with, and appear to be involved in, this stretch-induced adjustment of the outflow resistance [60, 64–75]. For example, elevation of IOP in perfused eyes increases the net activity of MMPs within the JCT region, including ADAMTS-4 [76]. In terms of gene expression and signaling, TM cells exposed to mechanical stress increase expression of the micro RNA, miR-24, which regulates a panel of genes including those that process cytokines like TGFβ that regulate outflow facility [77]. Lastly, mechanical stimuli from daily oscillations in IOP, such as ocular pulse, help set contractile tone in the TM [71]. Taken together, mechanical stimulation of conventional outflow cells triggers the coordination of many regulatory processes that contribute to homeostasis.

Trabecular Tone, Cytoskeletal Organization and Outflow Facility

The trabecular meshwork is under constant tension, contracting to counter (i) pull from the ciliary muscle that extends elastic tendons through the TM, anchoring onto the inner wall of Schlemm’s canal and (ii) a constant pressure gradient. The role of trabecular tone or contractility in regulating outflow facility has been known for some time, but the molecular sophistication that underlies its homeostatic role is only recently becoming understood [42, 71, 78–82]. Early on, compounds such as the cytochalasins, which inhibit actin polymerization, were shown to dramatically affect outflow facility [83]; and other direct-acting cytoskeletal agents have similar effects [84]. The therapeutic importance of relaxing the TM is evidenced by clinical trials that are currently underway for several related agents, including Rho kinase inhibitors, that have primary effects on trabecular cytoskeleton/tone that increase outflow facility (figure 1) [85–87]. In contrast, off target effects from agents such as corticosteroids which increase cellular rigidity and promote formation of cytoskeletal structures known as CLANS (cross-linked actin networks) decrease outflow facility [88–92]. The precise mechanism(s) by which cytoskeletal manipulations of TM or SC cells affect outflow resistance remains unclear, but they could be acting directly through cell shape and effect on active flow pathways or through the cellular control of ECM organization via integrins and other ECM interactions [81, 93–95]. Two recent studies highlight the intimate connection between TM cell contractility and ECM-integrin expression [81, 93].

Figure 1. Dose-related intraocular pressure-lowering effects of a rho kinase inhibitor, AR-12286, in ocular hypertensive patients enrolled in phase 2A trial.

Shown are mean change in diurnal intraocular pressure measurements from baseline for each treatment group. bid: twice daily, q.d.: daily

Source: reproduced with permission from Robert D. Williams, Gary D. Novack, Thomas van Haarlem, Casey Kopczynski and AR-12286 Phase 2A Study Group; Ocular Hypotensive Effect of the Rho Kinase Inhibitor AR-12286 in Patients With Glaucoma and Ocular Hypertension; Am J Ophthalmol 2011: 152: 838

ECM-Mediated Resistance and Segmental Outflow

While the relationship between TM contractility and ECM expression/organization is a recent finding, the contribution of glycosaminoglycans (GAGs) and their proteoglycan core proteins to outflow resistance generation has been hypothesized since the 1950s [59, 96]. Hence several ECM components likely participate in the generation of resistance, including versican, a large proteoglycan with numerous GAG side chains, and hyaluronan, which forms long GAG chains and fibrils, and appear to provide much of the outflow resistance [59, 73, 97]. Interestingly, shape changes of TM and SC cells in the JCT is hypothesized to modify the relative orientation of GAGs and proteoglycans within flow channels of the conventional tract, acting to fine tune the resistance to outflow; again emphasizing the relationship between cellular contraction and associated ECM.

In addition to the abundance and position of ECM in flow pathways, the number and location of such flow pathways in the conventional tract also contribute to the regulation of outflow facility. It has long been known that outflow channels are not uniform around the circumference of the eye [97–100]. Rather, outflow is segmental, exhibiting regions of relatively high and low conductance. This variability is reflected by non-uniform pigment, tracer or stiffness patterns in the conventional outflow tissues [97, 101, 102] (figure 2). Some of this segmentation has an anatomical component in that it corresponds closely to the positions of collector channels and some of it does not, but reflects some larger organizational pattern [99]. Interestingly, the distribution of versican is segmental and levels correlate inversely with relative outflow rates [97]. Recent studies suggest that flow pathways are dynamic, constantly changing over time. Moreover, the dynamics may be mediated by local ECM degradation by podosome/invadopodia like structure (PILS) activity of TM cells (figure 3) [103]. This high degree of segmental distribution of flow demands adjustments of theoretical estimates of outflow resistance and further suggests that Schlemm’s canal may not exhibit free radial flow around the eye. It also has obvious implications for surgical stent placement or other trabecular surgeries.

Figure 2. Segmental flow patterns through the human trabecular meshwork.

Enucleated human donor eyes were perfused with a bolus of green fluorescent tracer microspheres (200 nm) and perfusion fixed. The trabecular meshwork was imaged en face after removing the iris and ciliary body. The non-uniform pattern of fluorescent tracer decoration suggests segmental flow within the trabecular meshwork. Arrows indicate areas of high flow and arrowheads show areas of low/no flow through the trabecular meshwork. Sc: sclera; C: cornea

Source: image provided by Darryl Overby, Ph.D.

Figure 3. Effects of mechanical stretch on podosome/invadosome-like structures (PILS) component distribution.

TM cells were exposed to ~10% radial stretch for 24 hours (stretch) or were not stretched (control). Component localization was imaged by confocal microscopy examining microtubules (green), MMP-14 (red) and actin microfilaments (blue). Yellow (arrowheads) indicate colocalization of microtubule clusters and MMP-14 at sites of PILS

Source: image provided by Ted Acott, Ph.D

Oxidative Damage and Outflow Dysfunction

TM cells are terminally differentiated and long-lived. In addition to accumulated stress due to normal metabolism over a lifetime, TM cells are subjected to oxidative stress and cellular debris that are continuously introduced via aqueous humor flow. Unfortunately, all four proteolytic enzyme systems responsible for processing cellular material have been shown to be compromised or strained in TM cells with age, in POAG, upon exposure to chronic oxidative stress [104] or upon genetic mutations that produce excess amounts of misfolded protein [105]. The accumulation of nondegradable and oxidized materials in the conventional tract over time has extreme potential to impair normal cellular function including outflow regulation observed in POAG. Accordingly, many hallmarks of oxidative damage in the conventional pathway have been reported in people with POAG; such as decreased antioxidant potential plus increased expression of oxidative stress markers, oxidative DNA damage, peroxidized lipids and mitochondrial reactive oxygen species (ROS) [104]. For example, a recent study of 79 trabeculectomy specimens from patients with POAG showed a 5-fold increase in mitochondrial DNA deletions, a 4-fold decrease in number of mitochondria and 16 fold fewer cells than age-matched controls [106]. Another study showed that in aging and/or chronic exposure to oxidative stress damage may be due to altered iron homeostasis resulting in abnormal production of intralysosomal ROS, possibly leading to lysosomal membrane permeabilization and release of cathepsin-D into the cytosol with consequent TM cell death [107]. Efforts to limit oxidative damage in the conventional pathway are underway. Recently, a mitochondrial targeted antioxidant was shown to attenuate ROS generation in TM cells exposed to oxidative stress in vitro [108]. Moreover, treatment of TM cells with resveratrol, an antioxidant found in red wine and berries, limits appearance of markers of oxidative damage [109]. Taken together, these studies suggest compromised outflow function in POAG may result from diminished capacitance to quench oxidative byproducts, a weakened ability to process cellular material or excessive oxidative load over time.

Conclusion

The pressure-dependent, conventional outflow tract is the diseased tissue responsible for ocular hypertension. Regulation of outflow is dynamic and complicated, involving many signaling molecules and cellular processes that for most people maintain IOP within a narrow range over a lifetime. Understanding the homeostatic mechanisms that regulate conventional outflow will reveal (i) susceptible pathways responsible for ocular hypertension plus (ii) therapeutic targets that have the potential to medically lower IOP below current therapeutic “floor” limits. Most importantly, intervention holds promise to restore function to this vital pressure-responsive and pressure-dampening tissue of the eye.

Key Points.

• Molecular/cellular defect(s) in the conventional outflow pathway are responsible for ocular hypertension in glaucoma.

• Regulation of conventional outflow facility and thus IOP is dependent upon complex orchestration of many local signaling molecules including bioactive lipids, cytokines, nucleotides and gases.

• The contractile tone of the trabecular meshwork is a target for rho kinase inhibitors, a new class of anti-glaucoma medications that increase conventional outflow.

• Only a fraction of total flow pathways through the TM are utilized at any one time; these preferential pathways appear to be transient, dynamic and a result of podosome activity.

• As the final destination for aqueous humor circulation, the trabecular meshwork is continually challenged with oxidative, mechanical and phagocytic stressors that likely contribute to dysfunction over time.