Optometry & Vision Science:
Dynamic Regulation of Barrier Integrity of the Corneal Endothelium
Srinivas, Sangly P.*
School of Optometry, Indiana University, Bloomington, Indiana.
This work was supported by research grants from NIH, Ocumetrics, Inc., Alcon, Inc., and Office of Vice President for Research, Indiana University, Bloomington, Indiana.
Received September 16, 2009; accepted December 8, 2009.
The corneal endothelium maintains stromal deturgescence, which is a prerequisite for corneal transparency. The principal challenge to stromal deturgescence is the swelling pressure associated with the hydrophilic glycosaminoglycans in the stroma. This negative pressure induces fluid leak into the stroma from the anterior chamber, but the rate of leak is restrained by the tight junctions of the endothelium. This role of the endothelium represents its barrier function. In healthy cornea, the fluid leak is counterbalanced by an active fluid pump mechanism associated with the endothelium itself. Although this pump-leak hypothesis was postulated several decades ago, the mechanisms underlying regulation of the balance between the pump and leak functions remain largely unknown. In the last couple of decades, the ion transport systems that support the fluid pump activity have been discovered. In contrast, despite significant evidence for corneal edema secondary to endothelial barrier dysfunction, the molecular aspects underlying its regulation are relatively unknown. Recent findings in our laboratory, however, indicate that barrier integrity (i.e., structural and functional integrity of the tight junctions) of the endothelium is sensitive to remodeling of its peri-junctional actomyosin ring, which is located at the apical junctional complex. This review provides a focused perspective on dynamic regulation of the barrier integrity of endothelium vis-à-vis plasticity of the peri-junctional actomyosin ring and its association with cell signaling downstream of small GTPases of the Rho family. Based on findings to date, it appears that development of specific pharmacological strategies to treat corneal edema in response to inflammatory stress would be possible in the near future.
As a monolayer of cells forming a regular hexagonal mosaic at the posterior surface of the cornea, the corneal endothelium forms a critical epithelial interface between the anterior chamber and the corneal stroma. From a cell physiologic perspective, despite its name, the corneal endothelium is quintessentially a leaky epithelium. It is considered to be of neural crest origin, ∼5 μm thick, and typically exhibits a cell density of 2500 cells/mm2 in adult human eyes.1 In the early 1970s, the physiological activity of the corneal endothelium became well known from a series of experiments by Maurice2 and Dikstein and Maurice,3 using rabbit cornea in vitro. Even to this day, their experimental paradigm is routinely used in studies on the endothelial physiology. Their experiments demonstrated, without a doubt, that the corneal endothelium acts as a metabolic fluid pump. Specifically, it was shown that the endothelium drives fluid from stroma into the anterior chamber and thereby prevents corneal edema, which would have otherwise ensued in response to the inherent fluid leak into the stroma (Fig. 1). This mechanism, referred to as the pump-leak hypothesis,1,4–6 is undisputed to date and remains an accepted physiological basis for stromal hydration control. In the absence of the latter, a strict organization of the collagen fibrils in the stroma, critical for transparency of the cornea, would not be possible. Given this vital role for the endothelium, there has been a sustained interest in the molecular basis of endothelial fluid transport.6–14 In parallel, details about the molecular and pathophysiological aspects of the barrier function have also been accrued, but to a limited extent.1,15–23 The main purpose of this review is to provide a fresh perspective on the regulation of barrier function of the corneal endothelium, and thereby provide impetus for further progress. In the interest of brevity, however, we highlight sensitivity of the barrier integrity to plasticity of a pool of actin cytoskeleton at the apical junctional complex (AJC) after a brief consideration into salient aspects of endothelial fluid transport.
Fluid Transport by the Corneal Endothelium
The fluid pump activity, akin to that in other fluid transporting epithelia, is associated with a host of ion channels/exchangers/pumps positioned strategically at the apical and basolateral membranes. These mechanisms are driven by ionic gradients set up by the sodium pump (i.e., Na+/K+-ATPase), which is in the basolateral membrane of the endothelium.6,11 The role of Na+/K+-ATPase in the endothelium and the importance of the fluid pump function toward stromal hydration control becomes readily apparent by prompt stromal swelling when the monolayer is challenged with the cardiac glycoside ouabain.1,11,21 Similar pharmacological maneuvers seem to suggest that the corneal endothelium brings about a net Na+-HCO3− movement across its apical membrane.6 This elicits fluid secretion into the anterior chamber, presumably through resultant local osmotic gradients. Accordingly, several ion transport mechanisms that are likely to contribute to the vectorial ionic movement have been examined extensively, and such studies have been reviewed.6
Although the molecular identity of the transport mechanisms and their putative role toward net ionic secretion are being established with relative flair,6 the exact nature of the solute-solvent coupling leading to fluid secretion remains uncertain.5,23,24 With the discovery of aquaporins and the specific involvement of AQP1 in endothelial osmotic water permeability,25,26 there is impetus to accept the classical standing gradient model27 as a plausible mechanism underlying the solute-solvent coupling (Fig. 2). In contrast, however, Fischbarg et al.,23 have recently invoked the theory of electro-osmotic coupling to explain fluid transport by the endothelium. Interestingly, in the realm of electro-osmotic coupling, water secretion can also assume the paracellular pathway.5,28–31 Because we strive to further delineate the nature of the solute-solvent coupling, it appears that mechanisms of cell volume regulation in response to acute osmotic shocks are invaluable.32 This is because cell volume changes, which can be measured in realtime,7–10,33 are also brought about by ion transport mechanisms that are implicated in fluid transport (Fig. 3). Hence, measurement of cell volume regulation provides a novel window to observe the regulation of ion transport mechanisms by extracellular stresses and bioactive factors found in the aqueous humor.
Barrier Integrity: Leakiness and Relationship to Stromal Deturgescence
As suggested earlier, the barrier function represents resistance to facile permeability to solutes, and most importantly, to fluid leak through the paracellular route. This resistance is conferred by the tight junctions (TJs) of the endothelium. In general, the TJs are supramolecular assemblies forming the intercellular junctions and are found close to the apical domains of the epithelial/endothelial cells in a monolayer (Fig. 4A). The trans-membrane molecules of the TJs (namely, occludins, claudins, and the junctional adhesion molecule) interact with their homophilic counterparts in the neighboring cells.34 Such interactions, facilitated by the intercellular tethering forces evoked by the cadherin-dependent adherens junctions (AJs), occlude the paracellular space. Accordingly, AJs are proximal to TJs, and together, for the most part, they form the AJC. In the cytoplasm, each of the components of the AJC forms a microdomain comprising a large number of linker proteins.34 The components of such microdomains possess significant ties to several major cell signaling molecules as well as a proximal pool of actin cytoskeleton (Fig. 4A).
In the corneal endothelium, the TJs offer only a very weak resistance to paracellular passage of solutes and water, presumably due to fewer TJ strands. The leakiness of the endothelium is reflected in the very small trans-endothelial resistance (TER) of 15 to 25 Ω.cm21,16,21 and also by a significant permeability to macromolecules of MW > 150 kDa.35 Nonetheless, a breakdown of the barrier integrity by pharmacological or pathological insults is known to readily cause stromal swelling.1 The biophysical basis for the stromal edema lies in the swelling pressure put forth by the hydrophilic glycosaminoglycans of the stroma (50 mm Hg). The corneal epithelium poses a significant barrier for potential water influx from tears, so the main challenge for stromal deturgescence is at the posterior surface of the cornea in the form of the fluid entry across the endothelium from the anterior chamber. The fluid pump activity in the opposite direction, as discussed earlier, balances this leak (Fig. 1). In summary, the barrier function of the endothelium restrains facile leak into the stroma so that a state of deturgescence of the tissue is maintained.
In addition to a direct block of the paracellular route for water influx from the anterior chamber (i.e., the “paracellular gate” function of TJs), the TJs in the endothelium could be envisaged to influence the fluid pump activity by at least two other mechanisms (Fig. 4B). First, stable TJs are essential for preventing dissipation of electrolyte gradients set up by the ion transport mechanisms; they restrain potential futile solute back-flux through the paracellular space. In other words, if the TJs break down and consequently occlusion of the paracellular space is lost, it is unlikely that sustained local osmotic gradients would be developed to promote trans-endothelial fluid movement. The second mode of coupling to fluid transport lies in the so-called “molecular fence” function of TJs (Fig. 4B). Specifically, it is known that intact TJs are indispensable for the maintenance of apical-basal polarity of the integral membrane proteins (including those of the ion transport mechanisms) by limiting their lateral diffusion.36–38 Therefore, a breakdown of the TJs would adversely impact the polarity of the membrane proteins and thereby possibly reduce the rate of fluid pump in the face of increase in paracellular fluid leak. In summary, the barrier integrity of the corneal endothelium not only restrains the rate of fluid leak into stroma secondary to the swelling pressure of the glycosaminoglycans but may also modulate the molecular determinants of the endothelial ion transport and the solute-solvent coupling. Therefore, barrier integrity of the endothelium is a prerequisite for the maintenance of corneal transparency (Fig. 5).
Pathophysiology of the Corneal Endothelium
Despite a continuous age-related loss of endothelial cells (∼0.6% per year), corneal transparency is maintained as long as the cell density is >700 cells/mm2, a minimum density needed to counteract the basal fluid leakage.1 Thus, a cell density of 2500 cells/mm2, which is found in adult human corneas, suggests that a “functional reserve” is associated with the endothelium. Nevertheless, when the corneal endothelium is subjected to inflammation (e.g., uveitis allograft rejection), disease (e.g., Fuchs' dystrophy), or surgical trauma, there is a loss of stromal hydration control (Fig. 6). Among the pathological insults that result in corneal edema, the potential for barrier dysfunction in response to inflammation poses a clinically significant threat in the context of corneal transplantation and anterior uveitis. Given a lack of pharmacological approaches to a variety of corneal disorders including decompensated endothelium (e.g., Fuch's dystrophy and bullous keratopathy), corneal transplantation is being performed in large numbers (∼40,000 annually in the United States), and this may increase further given new approaches such as Descemets Stripping Endothelial Keratoplasty.39–42 Even after transplantation, survival of the endothelium is a major concern. The central endothelial cell loss of the donor cornea over the first postoperative year exceeds 30%.43–47 At the present time, there are no pharmacological strategies to prevent the progressive loss of endothelial function in response to acute or chronic allograft rejection. Therefore, understanding the basic mechanisms of endothelial barrier in healthy conditions and in the presence of pro-inflammatory factors, which are released secondary to immune response during allograft rejection and uveitis, is of prime importance.
Plasticity of the Actin Cytoskeleton and its Influence on the Barrier Integrity
A plethora of mechanisms are implicated in the dynamic regulation of the barrier integrity in response to extracellular stresses, neurohormonal factors, growth factors, bacterial/viral products, cytokines, and nutrients in a variety of epithelial/endothelial monolayers.34,48–55 For example, as a direct effect, altered expression and/or changes in the tyrosine phosphorylation of the components of TJs and AJs are known to break down the barrier integrity.34 However, an indirect mechanism frequently noted in response to inflammatory stress involves plasticity of the actin cytoskeleton.34,52,56–59 In this mechanism, agents that induce actomyosin contraction and/or depolymerization of the cortical actin cytoskeleton at the AJC bring about disruption of the barrier integrity.34,57,58,60–62 The pool of actin cytoskeleton at the AJC, which forms a dense band around the cells, has been referred to as the peri-junctional actomyosin ring (PAMR).56,58,62 The PAMR is structurally and functionally linked to the trans-membrane components of AJs and TJs through a variety of linker proteins such as zonula occludens-1 (ZO-1).56,58,62 Accordingly, many molecules involved in inflammatory stress (e.g., thrombin, histamine, TNF-alpha) break down the barrier integrity through mechanisms that increase contractility of the PAMR. The breakdown occurs with concomitant disruption of the PAMR, redistribution of the components of the AJs and TJs, and formation of intercellular gaps. In agreement with these findings, agents that promote relaxation of the PAMR (e.g., agents known to elevate cAMP, such as adenosine and adrenomedullin) are known to oppose the loss of barrier integrity by agents such as thrombin and histamine and also oppose structural rearrangements alluded to above. These observations have led to the notion that a significant increase in the actomyosin contraction of the PAMR is detrimental to the integrity of AJs and TJs owing to onset of a centripetal force that would oppose the intercellular tethering forces at AJC. In the absence of cell-cell tethering, trans-membrane proteins of the AJs and TJs cannot maintain a stable interaction, which consequently results in the breakdown of the barrier integrity. Interestingly, although direct actomyosin contraction of the PAMR is not implicated, cytokines (e.g., TNF-α) and oxidative stress are known to induce profound redistribution of the components of the TJs and AJs, along with loss of barrier integrity and significant disruption of actin cytoskeleton. In essence, PAMR, which has structural interactions with components of AJs and TJs on one hand and coupling to cell signaling on the other, is able to focus diverse extracellular signals into regulation of the barrier integrity.
In light of the above observations, we began characterizing the actomyosin contraction in corneal endothelium in the context of model agents and drugs to establish potential involvement of actin cytoskeleton (specifically, PAMR) in the regulation of the barrier integrity. We are now investigating how the cytokines and proteinases found in the aqueous humor and cornea during uveitis and allograft rejection disrupt the barrier integrity (Fig. 5). In the following, after a general introduction to cell signaling that couples to actomyosin contraction, we highlight our recent experimental findings on the regulation of the barrier integrity of corneal endothelium.
PAMR and the Actomyosin Contraction in the Corneal Endothelium
As in smooth muscle cells, a variety of endothelial and epithelial cells show increased contractility of their actin cytoskeleton in response to the phosphorylation of myosin light chain (MLC), a regulatory protein associated with the motor protein Myosin II (Fig. 6). MLC phosphorylation is determined by two opposing pathways: MLC kinase (MLCK)-driven phosphorylation and MLC phosphatase (MLCP)-driven dephosphorylation.63–65 MLCK, activated on binding to the Ca2+-calmodulin complex, is dedicated to MLC phosphorylation (Fig. 6A). MLCP is a trimeric complex consisting of PP1CΔ (catalytic subunit), MYPT1/2 (myosin-binding subunit), and M20 (unknown function) (Fig. 6C). Phosphorylation of MYPT1 by Rho kinase, a downstream effector of RhoA, inhibits the phosphatase activity of PP1CΔ. Elevated cAMP inhibits the activation of RhoA by phosphorylation of the small GTPase itself by cAMP-dependent protein kinase (PKA).66 This is thought to increase affinity of the RhoA-GDP to RhoA-GDI (Fig. 6D). In contrast with Rho kinase, PKC inhibits the activity of MLCP by phosphorylation of CPI-17 (PKC-activated 17 kDa inhibitor protein of type 1 phosphatase) and consequent inactivation of PP1CΔ. Thus, activation of Rho kinase and/or PKC results in contraction of the actin cytoskeleton whereas elevated cAMP promotes relaxation.66 These mechanisms surrounding MLC phosphorylation, which is well characterized in vascular endothelial cells, are found in corneal endothelial cells, as shown in our studies,15,16–19,22,66–75 described below.
Effect of Actomyosin Contraction on the Barrier Integrity of the Endothelium
Based on pharmacological agents to modulate MLC phosphorylation, we have examined the influence of actomyosin contraction on the barrier integrity of corneal endothelium. In our first study,18 we demonstrated that the thrombin activates RhoA–Rho kinase axis through Gα12/13-coupled PAR-1 receptors in bovine corneal endothelial cells (Fig. 7). The resulting increase in MLC phosphorylation led to a disruption of the PAMR with a concomitant breakdown of the barrier integrity. In two subsequent studies,16,19 we showed that thrombin-induced MLC phosphorylation and the breakdown in the barrier integrity could be suppressed through cAMP-mediated inhibition of RhoA signaling (Fig. 8). We have also investigated the Ca2+- and PKC-dependent mechanisms involved in MLC phosphorylation upon activation of H1 receptors,15 which are not known to activate RhoA significantly. Our data showed that histamine-induced MLC phosphorylation and the resulting loss of endothelial and epithelial barrier integrity are suppressed by ML-7 and a PKC inhibitor, indicating a role of MLCK and PKC as well as actin cytoskeleton.15,70 Taken together, our published findings to date emphasize a strong role for actomyosin contraction along the locus of the AJC in the regulation of the endothelial barrier integrity. These data have thus provided insights into how many bioactive factors found in the aqueous humor may influence the barrier function of the corneal endothelium and thereby alter the stromal hydration control.
A High Throughput Assay for Barrier Integrity
In our early studies, we assessed the barrier integrity in terms of flux of FITC Dextran (10 kDa) and/or horse radish peroxidase (40 kDa), applied at the apical side of the corneal endothelial cells (Fig. 9A). To increase the throughput of our experiments and enable rapid testing of other bioactive factors in the aqueous humor, we began to examine TER by measurement of electrical impedance (Fig. 9B) as a measure of barrier integrity of confluent monolayers. We used electrical cell-substrate impedance sensing (ECIS, Applied Biophysics, NY), a commercial device designed to assess TER in up to 16-indepedent monolayers at a time. For such measurements, cells are grown to confluence on small, gelatin-coated gold electrodes. A small AC current is applied across the cells. The impedance for the current flow is measured by a lock-in amplifier at ∼0.1 Hz. The resistive component of the impedance for current flow is taken as TER because the plasma membrane resistance is relatively large.76 Fig. 9C shows evolution of the resistive component of the measured impedance with cell spreading on the gold electrodes. The steady state resistance is indicative of the barrier integrity. Fig. 9D shows the dynamics of TER measurements in response to cytochalasin D, which is known to break down barrier integrity in epithelia.60 As an inhibitor of actin polymerization, cytochalasin D breaks apart the PAMR, and consequently the barrier integrity is lost. These results clearly show that TER, as measured using the ECIS system, reflects the status of the barrier integrity. Similar findings have been reported contemporaneously by Yin and Watsky.77 Moreover, the ECIS protocol offers a sensitive and high-throughput approach to assess the barrier integrity of the leaky corneal endothelium.
Reformation of Adherens and Tight Junctions inthe Endothelium
As opposed to the indirect influence of enhanced actomyosin contraction of the PAMR, cell loss during Fuch's dystrophy, iatrogenic injury (e.g., phacoemulsification), and keratoplasty present a direct threat to the barrier property of the corneal endothelium. Given that the corneal endothelium is non-mitotic in human eyes, reassembly of cell-cell junctions after cell spreading becomes crucial for preserving the barrier integrity. To investigate the influence of actin cytoskeleton on the dynamics of disassembly and reassembly of AJs and TJs, we used the so-called Ca2+ switch maneuver. In this protocol, Ca2+ depletion breaks down the AJs and subsequent Ca2+ add-back allows them to re-assemble. Disassembly of the AJs, and as a consequence, TJs, was induced by replacing Ringers (2 mM Ca2+) with Ca2+-free Ringers (with 2 mM EGTA). On the same cells, reassembly of the AJs, and thus resealing of TJs, was induced by exposing to Ca2+-rich Ringers (2 mM). As shown in Fig. 10A, Ca2+ depletion leads to precipitous loss in TER. Repletion of Ca2+ led to a recovery of TER (Fig. 10B).
To probe the role of actomyosin contraction, we used Y-27632 and blebbistatin, the selective inhibitors of Rho kinase and Myosin II ATPase, respectively. Preexposure to Y-27632 and blebbistatin led to significant inhibition in the rate of decline in TER (Fig. 1C, D). In addition, the same agents also reduced the rate of recovery in TER in response to Ca2+ add-back (Fig. 10E, F). Because cadherin-dependent AJs require external Ca2+, the reduction in the rate of decline in TER on Ca2+ removal in the presence of blebbistatin and Y-27632 indicates that disassembly of AJs is dependent on actomyosin contraction. Significant inhibition in the rate of increase in TER after Ca2+ add-back by blebbistatin and Y-27632 suggests that a contractile tone is necessary for assembly of TJs. These findings, summarized in Fig. 11, indicate the need to determine whether AJ assembly, which precedes TJ assembly, is also influenced by actomyosin contractility and inflammatory stress.
Effect of TNF-α on Barrier Integrity of theCorneal Endothelium
As noted above, after corneal transplantation, survival of the endothelium is a major concern because of cytokines produced secondary to allograft rejection. Among the cytokines found at significant levels in the cornea and aqueous humor, TNF-α is thought to play a major role in corneal allograft rejection.39,44,45,78,79 TNF-α is known to increase permeability of epithelial and endothelial monolayers by mechanisms that are independent of apoptosis. Its influence on the corneal endothelium has also been demonstrated in an in vitro model of rabbit eyes.80 This study by Watsky et al., showed that exposure of the corneal endothelium to the cytokine led to increase in the permeability to hydrophilic dye carboxyfluorescein with a concomitant disruption of the actin cytoskeleton. Recent studies on (TNF-α)-induced loss of barrier integrity in vascular endothelium have implicated microtubule disassembly.81,82 One mechanism shown in vascular endothelium involves activation of RhoA through a release of certain RhoA–specific guanine nucleotide exchange factors (GEFs), which are anchored to the microtubules and released in response to its disassembly.82 In a recent study from our laboratory,22 we showed that microtubule disassembly by exposure to nocodazole results in disruption of the actin cytoskeleton, leading to a loss of barrier integrity in corneal endothelium (Fig. 12). On the basis of nocodazole findings and given the importance of TNF-α in allograft rejection, we have begun to investigate the effects of the cytokine on barrier integrity.17,74 Exposure to TNF-α led to disruption of microtubules and dispersion of ZO-1 (Fig. 13A, B). These events occurred in parallel with a sustained reduction in TER, indicating a loss of barrier integrity. All effects of TNF-α were opposed by pretreatment with paclitaxel (microtubule stabilizing agent) (Fig. 13)17 and SB-203580, a p38 MAPK (mitogen-activated protein kinase) inhibitor (Fig. 14).74 The latter inhibited the effect of TNF-α completely.74 Based on these findings, we have suggested that the loss of barrier integrity in response to TNF-α involves activation of p38 MAP kinase and cytoskeletal reorganization, and that these can be inhibited to rescue barrier dysfunction in corneal endothelium.
Summary and Future Perspective
The corneal endothelium is a leaky epithelium, but its barrier integrity is critical to corneal transparency. As in many other epithelia, corneal endothelial cells possess a dense band of actin cytoskeleton at the AJC. This PAMR is structurally and functionally associated with AJs, TJs, and gap junctions. These intercellular junctions are strongly influenced by the status of MLC phosphorylation. Increased MLC phosphorylation, which induces increased contractility of the PAMR, breaks down the barrier integrity, presumably by opposing cell-cell adhesion at the AJs. Elevated cAMP, which opposes MLC phosphorylation by inhibiting activation of RhoA, opposes loss of barrier integrity induced by enhanced actomyosin contraction. Measurement of TER by ECIS is a sensitive approach for high throughput assessment of the barrier integrity of the corneal endothelial monolayers. Similar to the data in this review, there are reports to suggest that connexin-dependent gap junctions and connexin hemichannels83–85 are significantly impacted by actomyosin contraction in corneal endothelium.67–69,72 Given this understanding on the plasticity of the actin cytoskeleton and its kaleidoscope of its effects on intercellular junctions including TJs, AJs, and connexin-dependent gap junctions/hemichannels, the challenge ahead of us is to sort out the bioactive factors whose influence are likely to converge on the variety of signaling pathways that impact the PAMR and consequently modulate the barrier integrity (Fig. 15). We have already embarked on such an investigation of the impact of TNF-α on the corneal endothelium, and it appears that tweaking the tone of the PAMR to modulate the leak could be one approach to overcome corneal edema in response to inflammation.
I thank all my mentors (Drs. J. A. Bonanno, OD, PhD, D. Maurice, PhD, G. Lowther, OD, PhD, R. Mutharasan, PhD, S. Soni, OD, MS), collaborators (Drs. D. Jans, PhD, B. Himpens, MD, PhD, J. Vereecke, PhD, W. Van Driessche, PhD), postdoctoral fellow (Dr. M. Satpathy, PhD), and students (Dr. C. D'hondt, PhD, Dr. Gomes, PhD, Dr. Y. Guo, PhD, S. Jalimarada, R. Ponsaerts, C. Ramachandran, Dr. M. Shivanna, PhD). I also thank Dr. Tony Adams for organizing this review series and the anonymous reviewer for many useful suggestions and revisions.
Sangly P. Srinivas
800 East Atwater Avenue
Indiana University, School of Optometry
Bloomington, Indiana 47405
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