Skip Navigation LinksHome > April 2010 - Volume 87 - Issue 4 > Dynamic Regulation of Barrier Integrity of the Corneal Endot...
Optometry & Vision Science:
doi: 10.1097/OPX.0b013e3181d39464
Reviews

Dynamic Regulation of Barrier Integrity of the Corneal Endothelium

Srinivas, Sangly P.*

Free Access
Article Outline
Collapse Box

Author Information

*PhD

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.

Collapse Box

Abstract

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.

Figure 1
Figure 1
Image Tools
Back to Top | Article Outline
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.

Figure 2
Figure 2
Image Tools
Figure 3
Figure 3
Image Tools
Back to Top | Article Outline
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).

Figure 4
Figure 4
Image Tools

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).

Figure 5
Figure 5
Image Tools
Back to Top | Article Outline
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.

Figure 6
Figure 6
Image Tools
Back to Top | Article Outline
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.

Back to Top | Article Outline
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.

Back to Top | Article Outline
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.

Figure 7
Figure 7
Image Tools
Figure 8
Figure 8
Image Tools
Back to Top | Article Outline
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.

Figure 9
Figure 9
Image Tools
Back to Top | Article Outline
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).

Figure 10
Figure 10
Image Tools

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.

Figure 11
Figure 11
Image Tools
Back to Top | Article Outline
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.

Figure 12
Figure 12
Image Tools
Figure 13
Figure 13
Image Tools
Figure 14
Figure 14
Image Tools
Back to Top | Article Outline
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.

Figure 15
Figure 15
Image Tools
Back to Top | Article Outline

ACKNOWLEDGMENTS

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

e-mail: srinivas@indiana.edu

Back to Top | Article Outline

REFERENCES

1.Edelhauser HF. The balance between corneal transparency and edema: the Proctor Lecture. Invest Ophthalmol Vis Sci 2006;47:1754–67.

2.Maurice DM. The location of the fluid pump in the cornea. J Physiol 1972;221:43–54.

3.Dikstein S, Maurice DM. The metabolic basis to the fluid pump in the cornea. J Physiol 1972;221:29–41.

4.Riley M. Pump and leak in regulation of fluid transport in rabbit cornea. Curr Eye Res 1985;4:371–6.

5.Fischbarg J, Maurice DM. An update on corneal hydration control. Exp Eye Res 2004;78:537–41.

6.Bonanno JA. Identity and regulation of ion transport mechanisms in the corneal endothelium. Prog Retin Eye Res 2003;22:69–94.

7.Srinivas SP, Guan Y, Bonanno JA. Swelling activated chloride channels in cultured bovine corneal endothelial cells. Exp Eye Res 1999;68:165–77.

8.Srinivas SP, Bonanno JA, Lariviere E, Jans D, Van Driessche W. Measurement of rapid changes in cell volume by forward light scattering. Pflugers Arch 2003;447:97–108.

9.Srinivas SP, Bonanno JA, Hughes BA. Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Biophys J 1998;75:115–23.

10.Srinivas SP, Bonanno JA. Measurement of changes in cell volume based on fluorescence quenching. Am J Physiol 1997;272:C1405–14.

11.Riley MV, Winkler BS, Peters MI, Czajkowski CA. Relationship between fluid transport and in situ inhibition of Na(+)-K+ adenosine triphosphatase in corneal endothelium. Invest Ophthalmol Vis Sci 1994;35:560–7.

12.Bonanno JA, Yi G, Kang XJ, Srinivas SP. Reevaluation of Cl-/HCO3- exchange in cultured bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 1998;39:2713–22.

13.Bonanno JA, Srinivas SP, Brown M. Effect of acetazolamide on intracellular pH and bicarbonate transport in bovine corneal endothelium. Exp Eye Res 1995;60:425–34.

14.Bonanno JA, Srinivas SP. Cyclic AMP activates anion channels in cultured bovine corneal endothelial cells. Exp Eye Res 1997;64:953–62.

15.Srinivas SP, Satpathy M, Guo Y, Anandan V. Histamine-induced phosphorylation of the regulatory light chain of myosin II disrupts the barrier integrity of corneal endothelial cells. Invest Ophthalmol Vis Sci 2006;47:4011–8.

16.Srinivas SP, Satpathy M, Gallagher P, Lariviere E, Van Driessche W. Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells. Exp Eye Res 2004;79:543–51.

17.Shivanna M, Srinivas SP. Microtubule stabilization opposes the (TNF-alpha)-induced loss in the barrier integrity of corneal endothelium. Exp Eye Res 2009;89:950–9.

18.Satpathy M, Gallagher P, Lizotte-Waniewski M, Srinivas SP. Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells. Exp Eye Res 2004;79:477–86.

19.Satpathy M, Gallagher P, Jin Y, Srinivas SP. Extracellular ATP opposes thrombin-induced myosin light chain phosphorylation and loss of barrier integrity in corneal endothelial cells. Exp Eye Res 2005;81:183–92.

20.Riley MV, Winkler BS, Starnes CA, Peters MI, Dang L. Regulation of corneal endothelial barrier function by adenosine, cyclic AMP, and protein kinases. Invest Ophthalmol Vis Sci 1998;39:2076–84.

21.Ma L, Kuang K, Smith RW, Rittenband D, Iserovich P, Diecke FP, Fischbarg J. Modulation of tight junction properties relevant to fluid transport across rabbit corneal endothelium. Exp Eye Res 2007;84:790–8.

22.Jalimarada SS, Shivanna M, Kini V, Mehta D, Srinivas SP. Microtubule disassembly breaks down the barrier integrity of corneal endothelium. Exp Eye Res 2009;89:333–43.

23.Fischbarg J, Diecke FP, Iserovich P, Rubashkin A. The role of the tight junction in paracellular fluid transport across corneal endothelium. electro-osmosis as a driving force. J Membr Biol 2006;210:117–30.

24.Sanchez JM, Li Y, Rubashkin A, Iserovich P, Wen Q, Ruberti JW, Smith RW, Rittenband D, Kuang K, Diecke FP, Fischbarg J. Evidence for a central role for electro-osmosis in fluid transport by corneal endothelium. J Membr Biol 2002;187:37–50.

25.Levin MH, Verkman AS. Aquaporin-dependent water permeation at the mouse ocular surface: in vivo microfluorimetric measurements in cornea and conjunctiva. Invest Ophthalmol Vis Sci 2004;45:4423–32.

26.Kuang K, Yiming M, Wen Q, Li Y, Ma L, Iserovich P, Verkman AS, Fischbarg J. Fluid transport across cultured layers of corneal endothelium from aquaporin-1 null mice. Exp Eye Res 2004;78:791–8.

27.Diamond JM, Bossert WH. Standing-gradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia. J Gen Physiol 1967;50:2061–83.

28.Hill AE. Fluid transport: a guide for the perplexed. J Membr Biol 2008;223:1–11.

29.Hill AE, Shachar-Hill B. A new approach to epithelial isotonic fluid transport: an osmosensor feedback model. J Membr Biol 2006;210:77–90.

30.Murakami M, Murdiastuti K, Hosoi K, Hill AE. AQP and the control of fluid transport in a salivary gland. J Membr Biol 2006;210:91–103.

31.Shachar-Hill B, Hill AE. Paracellular fluid transport by epithelia. Int Rev Cytol 2002;215:319–50.

32.Fischbarg J. Mechanism of fluid transport across corneal endothelium and other epithelial layers: a possible explanation based on cyclic cell volume regulatory changes. Br J Ophthalmol 1997;81:85–9.

33.Srinivas SP, Maertens C, Goon LH, Goon L, Satpathy M, Yue BY, Droogmans G, Nilius B. Cell volume response to hyposmotic shock and elevated cAMP in bovine trabecular meshwork cells. Exp Eye Res 2004;78:15–26.

34.Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability. Physiol Rev 2006;86:279–367.

35.Maurice DM, Srinivas SP. Fluorometric measurement of light absorption by the rabbit cornea. Exp Eye Res 1994;58:409–13.

36.Chiba H, Osanai M, Murata M, Kojima T, Sawada N. Transmembrane proteins of tight junctions. Biochim Biophys Acta 2008;1778:588–600.

37.Takakuwa R, Kokai Y, Kojima T, Akatsuka T, Tobioka H, Sawada N, Mori M. Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B. Exp Cell Res 2000;257:238–44.

38.Mandel LJ, Bacallao R, Zampighi G. Uncoupling of the molecular ‘fence’ and paracellular ‘gate’ functions in epithelial tight junctions. Nature 1993;361:552–5.

39.Pleyer U, Schlickeiser S. The taming of the shrew? The immunology of corneal transplantation. Acta Ophthalmol 2009;87:488–97.

40.Tan DT, Anshu A, Mehta JS. Paradigm shifts in corneal transplantation. Ann Acad Med Singapore 2009;38:332–8.

41.Patel SV, Bachman LA, Hann CR, Bahler CK, Fautsch MP. Human corneal endothelial cell transplantation in a human ex vivo model. Invest Ophthalmol Vis Sci 2009;50:2123–31.

42.Fu H, Larkin DF, George AJ. Immune modulation in corneal transplantation. Transplant Rev (Orlando) 2008;22:105–15.

43.Patel SV, Hodge DO, Bourne WM. Corneal endothelium and postoperative outcomes 15 years after penetrating keratoplasty. Am J Ophthalmol 2005;139:311–9.

44.Niederkorn JY. Immune mechanisms of corneal allograft rejection. Curr Eye Res 2007;32:1005–16.

45.George AJ, Larkin DF. Corneal transplantation: the forgotten graft. Am J Transplant 2004;4:678–85.

46.Bourne WM, McLaren JW. Clinical responses of the corneal endothelium. Exp Eye Res 2004;78:561–72.

47.Bourne WM. Biology of the corneal endothelium in health and disease. Eye (Lond) 2003;17:912–8.

48.Koch S, Nusrat A. Dynamic regulation of epithelial cell fate and barrier function by intercellular junctions. Ann N Y Acad Sci 2009;1165:220–7.

49.Capaldo CT, Nusrat A. Cytokine regulation of tight junctions. Biochim Biophys Acta 2009;1788:864–71.

50.Bruewer M, Samarin S, Nusrat A. Inflammatory bowel disease and the apical junctional complex. Ann N Y Acad Sci 2006;1072:242–52.

51.Utech M, Bruwer M, Nusrat A. Tight junctions and cell-cell interactions. Methods Mol Biol 2006;341:185–95.

52.Ivanov AI, Nusrat A, Parkos CA. The epithelium in inflammatory bowel disease: potential role of endocytosis of junctional proteins in barrier disruption. Novartis Found Symp 2004;263:115–24.

53.Bruewer M, Luegering A, Kucharzik T, Parkos CA, Madara JL, Hopkins AM, Nusrat A. Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. J Immunol 2003;171:6164–72.

54.Nusrat A, Turner JR, Madara JL. Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest Liver Physiol 2000;279:G851–7.

55.Walsh SV, Hopkins AM, Nusrat A. Modulation of tight junction structure and function by cytokines. Adv Drug Deliv Rev 2000;41:303–13.

56.Dudek SM, Garcia JG. Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol 2001;91:1487–500.

57.Garcia JG, Davis HW, Patterson CE. Regulation of endothelial cell gap formation and barrier dysfunction: role of myosin light chain phosphorylation. J Cell Physiol 1995;163:510–22.

58.Turner JR. ‘Putting the squeeze’ on the tight junction: understanding cytoskeletal regulation. Semin Cell Dev Biol 2000;11:301–8.

59.Turner JR. Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application. Am J Pathol 2006;169:1901–9.

60.Madara JL, Barenberg D, Carlson S. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J Cell Biol 1986;102:2125–36.

61.Madara JL, Moore R, Carlson S. Alteration of intestinal tight junction structure and permeability by cytoskeletal contraction. Am J Physiol 1987;253:C854–61.

62.Turner JR, Rill BK, Carlson SL, Carnes D, Kerner R, Mrsny RJ, Madara JL. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation. Am J Physiol 1997;273:C1378–85.

63.Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 2003;83:1325–58.

64.Ding HL, Ryder JW, Stull JT, Kamm KE. Signaling processes for initiating smooth muscle contraction upon neural stimulation. J Biol Chem 2009;284:15541–8.

65.Kamm KE, Stull JT. Dedicated myosin light chain kinases with diverse cellular functions. J Biol Chem 2001;276:4527–30.

66.Ramachandran C, Satpathy M, Mehta D, Srinivas SP. Forskolin induces myosin light chain dephosphorylation in bovine trabecular meshwork cells. Curr Eye Res 2008;33:169–76.

67.D'Hondt C, Ponsaerts R, Srinivas SP, Vereecke J, Himpens B. Thrombin inhibits intercellular calcium wave propagation in corneal endothelial cells by modulation of hemichannels and gap junctions. Invest Ophthalmol Vis Sci 2007;48:120–33.

68.D'Hondt C, Ponsaerts R, Srinivas SP, Vereecke J, Himpens B. Reduced intercellular communication and altered morphology of bovine corneal endothelial cells with prolonged time in cell culture. Curr Eye Res 2009;34:454–65.

69.D'Hondt C, Srinivas SP, Vereecke J, Himpens B. Adenosine opposes thrombin-induced inhibition of intercellular calcium wave in corneal endothelial cells. Invest Ophthalmol Vis Sci 2007;48:1518–27.

70.Guo Y, Ramachandran C, Satpathy M, Srinivas SP. Histamine-induced myosin light chain phosphorylation breaks down the barrier integrity of cultured corneal epithelial cells. Pharm Res 2007;24:1824–33.

71.Guo Y, Satpathy M, Wilson G, Srinivas SP. Benzalkonium chloride induces dephosphorylation of myosin light chain in cultured corneal epithelial cells. Invest Ophthalmol Vis Sci 2007;48:2001–8.

72.Ponsaerts R, D'Hondt C, Bultynck G, Srinivas SP, Vereecke J, Himpens B. The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells. Invest Ophthalmol Vis Sci 2008;49:4816–27.

73.Ramachandran C, Srinivas SP. Actomyosin contraction regulates formation and disassembly of adherens and tight junctions in the corneal endothelium. Invest Ophthalmol Vis Sci 2009 Dec 17, PMID: 20019371. (Epub)

74.Shivanna M, Rajashekhar G, Srinivas S. Barrier dysfunction of the corneal endothelium in response to TNF-α involves activation of p38 MAP kinase. Invest Ophthalmol Vis Sci 2009 Sep 24. PMID: 19797215. (Epub)

75.Shivanna M, Jalimarada SS, Srinivas SP. Effects of lovastatin on actin cytoskeleton in cultured bovine corneal endothelial cells. J Ocul Pharmacol Ther 2009. (In Press)

76.Tiruppathi C, Malik AB, Del Vecchio PJ, Keese CR, Giaever I. Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function. Proc Natl Acad Sci U S A 1992;89:7919–23.

77.Yin F, Watsky MA. LPA and S1P increase corneal epithelial and endothelial cell transcellular resistance. Invest Ophthalmol Vis Sci 2005;46:1927–33.

78.Rayner SA, Larkin DF, George AJ. TNF receptor secretion after ex vivo adenoviral gene transfer to cornea and effect on in vivo graft survival. Invest Ophthalmol Vis Sci 2001;42:1568–73.

79.Rayner SA, King WJ, Comer RM, Isaacs JD, Hale G, George AJ, Larkin DF. Local bioactive tumour necrosis factor (TNF) in corneal allotransplantation. Clin Exp Immunol 2000;122:109–16.

80.Watsky MA, Guan Z, Ragsdale DN. Effect of tumor necrosis factor alpha on rabbit corneal endothelial permeability. Invest Ophthalmol Vis Sci 1996;37:1924–9.

81.Petrache I, Birukova A, Ramirez SI, Garcia JG, Verin AD. The role of the microtubules in tumor necrosis factor-alpha-induced endothelial cell permeability. Am J Respir Cell Mol Biol 2003;28:574–81.

82.Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva I, Garcia JG, Verin AD. Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms. J Cell Physiol 2004;201:55–70.

83.Gomes P, Srinivas SP, Vereecke J, Himpens B. Gap junctional intercellular communication in bovine corneal endothelial cells. Exp Eye Res 2006;83:1225–37.

84.Gomes P, Srinivas SP, Vereecke J, Himpens B. ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 2005;46:104–13.

85.Gomes P, Srinivas SP, Van Driessche W, Vereecke J, Himpens B. ATP release through connexin hemichannels in corneal endothelial cells. Invest Ophthalmol Vis Sci 2005;46:1208–18.

Cited By:

This article has been cited 4 time(s).

Bioessays
The limbal epithelium of the eye A review of limbal stem cell biology, disease and treatment
Osei-Bempong, C; Figueiredo, FC; Lako, M
Bioessays, 35(3): 211-219.
10.1002/bies.201200086
CrossRef
Eye
Corneal endothelium: developmental strategies for regeneration
Zavala, J; Jaime, GRL; Barrientos, CAR; Valdez-Garcia, J
Eye, 27(5): 579-588.
10.1038/eye.2013.15
CrossRef
Plos One
Telomerase Immortalization of Human Corneal Endothelial Cells Yields Functional Hexagonal Monolayers
Schmedt, T; Chen, YM; Nguyen, TT; Li, SM; Bonanno, JA; Jurkunas, UV
Plos One, 7(): -.
ARTN e51427
CrossRef
Investigative Ophthalmology & Visual Science
Serine Protease Inhibitor A3K Protects Rabbit Corneal Endothelium From Barrier Function Disruption Induced by TNF-alpha
Hu, JY; Zhang, ZH; Xie, H; Chen, LL; Zhou, YP; Chen, WS; Liu, ZG
Investigative Ophthalmology & Visual Science, 54(8): 5400-5407.
10.1167/iovs.12-10145
CrossRef
Back to Top | Article Outline
Keywords:

cornea; endothelium; pump-leak hypothesis; tight junctions; actomyosin contraction

© 2010 American Academy of Optometry

Login

Search for Similar Articles
You may search for similar articles that contain these same keywords or you may modify the keyword list to augment your search.