Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

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Figure 9 A developing eye of a human fetus past 6 weeks.

1. the outer cornea (epithelium). 2. coalescing stem cells forming the endothelium and keratocytes. 3. the developing lens. 4. the inner layer of the retina. Modified from Forrester, J. V., Dick, A. D., McMenamin, P. G., and Roberts, F. (2008). The Eye. Basic Science in Practice, 3rd edn., p. 115. Edinburgh: Saunders.

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PM

OCM

Figure 10 Development of tissues at the angle of the posterior limbus at 12 weeks. The figure shows the pupillary membrane (PM) beginning to form the optic cup margin (OCM) and indentation caused by vascular mesenchyme (double arrows). At

(1)are the corneal endothelium (1st wave of neural crest cells);

(2)the forming keratocytes (2nd wave of neural crest cells) and collagen; and (3) possible 3rd wave of invading neural crest cells to form the trabecular meshwork. Modified from Forrester, J. V., Dick, A. D., McMenamin, P. G., and Roberts, F. (2008). The Eye. Basic Science in Practice, 3rd edn., p. 131. Edinburgh: Saunders.

metabolic energy produced. Although direct evidence is scarce, it seems that corneal endothelial cells are not insulin dependent and, therefore, are not starved of glucose in diabetes. On the contrary, it is perplexing that glycation of sodium, potassium-ATPase (Na, K-ATPase) does not cause an inhibition of the enzyme in the diabetic.

Cell Division and Replenishment

The lack of any ready, apparent replication of endothelial cells has been an ongoing problem when cell replacement

Trabecular meshwork

Nestin

Alkaline phosphatase

Telomerase

Figure 11 Stem cell and transient amplifying cell staining patterns at the peripheral corneal endothelium, transition zone, and trabecular meshwork of the posterior limbus in adult humans. Unwounded tissue is shown at (a) while wounded tissue is shown at (b). Tissues were stained with the following stem cell marker proteins: nestin, alkaline phosphatase, Wnt1, and Oct3/4. Tissues were stained with the following marker for both stem cells and transient amplifying cells: telomerase. Wounded tissues were stained with the following cell differentiation markers: Pax-6 and Sox2. Adapted from McGowan S. L., Edelhauser, H. F., Pfister, R. R., and Whikehart, D. R. (2007). Stem cell markers in the human posterior limbus and corneal endothelium of unwounded and wounded corneas. Molecular Vision 13: 1984–2000. http://www.molvis.org/molvis/v13/a224/.

might be required. In general, the population of these cells decline with age, but usually there remain sufficient numbers of cells at an advanced age to maintain corneal clarity. This has not seemed to be the case with wounding, trauma, and other dramatic forms of endothelial cell loss. Studies of the cell cycle in endothelial cell have indicated that the cells tend to remain in the G1 phase (perhaps even the Go temporary exit of cell division). This could be due to such factors as simple cell-to-cell contact inhibition or the presence of the TGF-b2 protein found in the aqueous humor. Departure from the G1 stage toward replication can be artificially induced with the E2F2 transcription factor since the E2F2 protein is known to bring cells into the S-phase of the cell cycle. Cells that have been transformed either with the SV40 large T-antigen or with the E6/E7 human papilloma virus can also act to initiate replication. The SV-40 and E6/E7 proteins inactivate the activity of retinoblastoma (Rb) and p53 cell cycle suppressor proteins by interacting with

Figure 12 Cross-section of Foxe3 / (deficient) mouse eye showing small lens (A) and persistent attachment of lens to cornea (B). Adapted with permission from Medina-Martinez, O. and Jamrich M. (2007). Foxe view of lens development and disease. Development 134: 1455–1463.

them and allowing the E2F2 transcription factor to initiate the cell cycle. These are not the usual cell processes, however, and the lack of normal cell division remains unresolved. There is evidence, however, that endothelial cells are replaced in vivo. Studies have pointed to the existence of stem cells in the posterior limbus just beyond the boundary of the corneal endothelium (Figure 11). Initially, some investigators noted a higher than usual density of corneal endothelial cells that exist at the endothelial periphery. This suggested that replacement cells or germinating cells were present in this area. Later, stem cells were identified in the posterior limbus. This has been shown by labeling with stem cell markers such as Nestin and Oct3/4 in the limbus and the subsequent appearance of repair or developmental proteins such as PAX6 and Wnt1 in the limbus and the peripheral endothelium following wounding. The appearance of the transient amplifying cell marker telomerase in these areas strongly points to the possibility that stem cells, resident in the posterior limbus, give rise to new corneal endothelial cells. In fact, BrdU studies have confirmed the generation of new cells from the posterior limbus into the peripheral endothelium after wounding (Figure 13). This is analogous to what occurs in the corneal epithelium, but at a significantly lower rate of reproduction.

Cytokines and Immune Privilege

The existence of cytokines, generated by the lens and other cells adjacent to the anterior chamber in the aqueous fluid, suggests that the endothelium may be influenced by its neighboring tissues. Cytokines are a broad mixture of polypeptides or proteins that are able to communicate a signal to a cell to initiate some change or response from the cell. In fact, the distinction between a

Figure 13 BrdU fluorescence shown in the peripheral corneal endothelium and trabecular meshwork 48 h after a mechanical wound to the endothelium. Adapted from Whikehart, D. R., Parikh, C. H., Vaughn, A. V., Mishler, K., and Edelhauser,

H. F. (2005). Evidence suggesting the existence of stem cells for the human corneal endothelium. Molecular Vision 11: 816–824. http://www.molvis.org/molvis/v11/a97/.

cytokine and a local hormone is considered moot by many. Cytokines were originally associated only with immune functions. An example of a cytokine that has both immune and nonimmune effects is the TGF-b, pointed out earlier. In the eye, TGF-b is known to modulate cell migration, proliferation, death, development, tissue repair, and many pathological processes as well. Generally, however, this cytokine is responsible for extracellular matrix production and the suppression of cell proliferation. There are three isoforms of TGF-b. Of the three, TGF-b2 is the predominant cytokine that is found in the aqueous fluid bathing the endothelium. The presence of TGF-b2 is essential to the cornea since its absence results in a failure of the endothelium to develop (see Figure 14).

Immune privilege in the eye is a process in which the eye protects itself from undesirable immune characteristics, such as inflammation, as a device to preserve vision. For the anterior segment, that means the ability of light to pass through its tissues unimpeded. Immune privilege is the summation of many complex molecular and cellular mechanisms whose operation, as a whole, remains incompletely understood. It is also the process by which corneal transplants are more likely to be free of immune reactions than transplants in other parts of the body. In an immuneprivileged site, such as the corneal endothelium, active processes are set in motion to suppress immune reactions when an antigen enters the site. The aqueous humor is responsible for this action since it contains the necessary suppression cytokines that are released by cells bordering the anterior chamber. TGF-b2, in addition to its roles mentioned previously, inhibits T-cell activation and differentiation that are necessary to initiate inflammation.

Other examples of cytokines that contribute to immune privilege are: a-melanocyte stimulating hormone, vasoactive stimulating peptide, calcitonin gene-related peptide, and somatostatin. A more recent finding has been the discovery of another cytokine: cluster of differentiation 95 ligand (CD95L) at immune-privileged sites. CD95L acts as a death signal that triggers apoptosis (programmed cell death) in CD95 sensitive T cells. The membrane protein form of CD95L, expressed on corneal endothelial cell membranes, has been shown to be important for the preservation of orthoptically placed corneal grafts and it does this by interacting with these T cells.

Some contradictions in the understanding of immune suppression also exist. For example, it is not understood how the soluble versus membrane forms of CD95 function and why the CD95L form is important for graft acceptance. In addition, many of the cells that surround the anterior chamber have receptors for tumor necrosis factor-a. This cytokine, unfortunately, plays an important role in intraocular inflammation (endotoxin-induced uveitis) when it occurs.

Proteins Synthesized for External Transport

In human corneas, there are known anatomical subdivisions found in Descemet’s membrane: an anterior, banded zone and a nonbanded, amorphous zone (Figure 15). The banded zone is formed before birth while the amorphous zone is continuously synthesized during life. Four principal proteins have been found to constitute Descemet’s membrane in both zones: collagens type IV and VIII as well as laminin and fibronectin. All are considered to be synthesized by corneal endothelial cells throughout the lifetime of an individual. However, recent suggestions

BR

NBR

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Figure 15 The banded (BR) and nonbanded (NBR) regions of Descemet’s membrane. The BR is formed before birth while the NBR is made continuously throughout life. E, the

endothelium. 7000. Adapted from Forrester, J. V., Dick, A. D., McMenamin, P. G., and Roberts, F. (2008). The Eye. Basic Science in Practice, 3rd edn., p. 21. Edinburgh: Saunders.

contend that some evidence points to the posterior keratocytes as sources for some of this synthetic work. Collagen type IV was originally considered to be the principal collagen of Descemet’s membrane. It is described as the main structural protein of all basement membranes. The a-1–a-6 chains are found in Descemet’s membrane facing the stromal and endothelial sides of the membrane in both

infants and adults. Type VIII collagen is a short nonfibrillar protein that may determine cell phenotype. In the infant, it is found on the endothelial face of the membrane, whereas it occurs on the stromal face of the membrane in the adult. Laminin is a noncollagenous glycoprotein that binds to other proteins to form sheets. Its association with type IV collagen in basement membranes is well known. Laminin occurs on the stromal and endothelial faces of infants, but it is not found on the stromal face of adult Descemet’s membrane. Fibronectin is a high-molecular-weight protein that can bind to membrane receptor proteins as well as collagen. It is involved in cell adhesion. Fibronectin only occurs on the stromal face of Descemet’s membrane in both infants and adults. Mucin-1 (MUC-1), a cell surface protein that has largely been considered to be present on the surface of the corneal epithelium, has also been found to be synthesized by the endothelium and transported to its apical surface. Its function there would be as an interfacing protein to the aqueous fluid.

The Role of the Corneal Endothelium in

Deturgescence

Introduction

The average human cornea in the adult is maintained at a thickness of 500 mm in the center to about 700 mm at the periphery (Figure 1). Keeping these parameters stable is important for corneal clarity. Deturgescence is the physiological process of maintaining such a clear cornea. The process is active and continuous. Two forces are at work during deturgescence. In one process, the cornea swells and tends to become cloudy. Due to the large volume of negatively charged glycosaminoglycans (GAGs) bound to the proteoglycans in the cornea, there is a constant influx of cations into the stroma from fluids outside of this tissue. These cations are accompanied by water as an osmotic compensation and bring about corneal swelling. The stroma is composed of layers of collagenous lamellae with each collagen strand separated from its neighbor by an aquous space. Disruption of the geometric regularity in the aqueous spaces that contain the GAGs brings about a loss in the mutual interference of light that is refracted through the cornea. This results in the beginning of a loss of clarity which can continue through progressive degrees of opacity (cloudiness). If unchecked, this process would result in functional blindness. In the second process of deturgescence, excess water is actively transferred out of the stroma by the corneal endothelium into the aqueous fluid of the anterior chamber. The corneal epithelium takes on a rather passive role in this process by acting more as a barrier to water flow. Early experiments demonstrated the existence of this active process to be predominately in the endothelium. This was accomplished

by the selective removal of corneal outer layers to determine which side (epithelium or endothelium) was involved in pumping out water. A metabolic component was shown to exist when the deturgescent process was compared in experimental corneas at cold versus physiological temperatures (Figure 16).

The Biochemistry of Active Deturgescence

Active deturgescence is considered to occur as the result of the catalytic activity of two enzymes: sodium, potassiumstimulated adenosine triphosphatase (Na, K-ATPase) and bicarbonate-stimultated adenosine triphosphatase (–HCO3- ATPase ). Na, K-ATPase resides in the basolateral membranes of corneal endothelial cells in sufficient quantities to ensure significant pumping activity.

As an enzyme, Na, K-ATPase exists with a minimal structure of four polypeptide chains of which two (the a- chains) are catalytic and two (the b-chains) are structural stabilizers in the cell membrane. In carrying out pumping activity, the enzyme is energized by the hydrolysis of ATP to adenosine diphosphate (ADP) in which energy, contained in the released inorganic phosphate group, is transferred to an a-subunit. This energy provides for the kinetic transfer of two potassium ions into an endothelial cell while three sodium ions are virtually, simultaneously moved to the cell exterior. The result is the net movement of one cation outside the cell (Figure 17). The osmotic consequence of this is the simultaneous flow of water into the anterior chamber either directly or through the channels that lie between endothelial cells. Water therefore flows osmotically, due to ion transfer, through endothelial cells by means of proteins known as aquaporins.

Figure 16 A temperature-reversal experiment in which a cornea is previously allowed to swell in the cold and then demonstrated to decrease in thickness when placed at room temperature for 2 h. From Dr. Henry Edelhauser.

Figure 18 Aquaporin-1 consists of four polypeptide chains (shown here in red and blue). The channel for water that they form is shown as a green arrow. Modified from Wikipedia/Aquaporin.

experimentally, a decrease in bicarbonate, and the use of carbonic anhydrase inhibitors clearly shows a loss of deturgescence. Carbonic anhydrase converts water and carbon dioxide into bicarbonate and a proton. At this point, whether chloride or bicarbonate is of prime importance is unclear.

The Physiological Control of Active

Deturgescence

It is evident that moving a net amount of cations from the corneal endothelium (via Na, K-ATPase) to the anterior chamber will result in transferring water from the endothelium to the aqueous to maintain an osmotic balance. This activity serves to remove excess water from the stroma (via aquaporin transport through the corneal endothelium) while water leaks back into the stroma simultaneously. Normally, the concentration of Na+ ions is higher in the anterior chamber than in the stroma, so the activity of Na, K-ATPase is required to push Na+ ions against the gradient of the anterior chamber. The two processes (pump and leak) operate in a fashion analogous to water leaking into a basement while it is being removed by a sump pump at the same time. Since Na, K-ATPase requires a substantial supply of ATP as an energy source from mitochondria, it can be speculated that the membrane potential and pH of that organelle requires that the potential and pH stability be well maintained. To what degree the operation of a –HCO3-ATPase in the mitochondria contributes to these phenomena is unknown.

There remain other physiological observations that challenge further investigation of the pump-leak hypothesis of deturgescence. For example, do the aquaporins (AQP-1 in the endothelium and AQP-5 in the epithelium) play more than a passive role in deturgescence? What function might TRPV4 (an osmolar-sensing protein in the epithelium) play in regulating stromal volume?

Genetic Diseases of the Corneal

Endothelium

Fuchs’ Endothelial Dystrophy

This disease is considered to be the result of an autosomal dominant disorder. It is a true disease of the corneal endothelium rather than one that originates from another area of the cornea (Figure 19). It was first described in

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(b)

Figure 19 Endothelial cell layers as viewed from the posterior side of the cornea: (a) is a normal endothelium showing a regular pattern of polygonal cells and (b) is an example of moderately advanced Fuch’s dystrophy. It shows a marked decrease in cell numbers, abnormal nuclei, and cell displacement by guttatae from Descemet’s membrane. Adapted from Kratchmer, J. H., et al. (eds.) (2005). The Cornea, 2nd edn., vol. 1, p. 942. Philadelphia, PA: Elsevier.

1910. There are two forms of the disease: early onset (mutations associated with chromosomes 13 and 18) and late onset (perhaps found on chromosome 1 or at other locations). The early onset form is associated with a mutation of the COL8A2 gene which synthesizes collagen VIII, a major collagen of Descemet’s membrane. The exact identity of the gene defect in the late onset form has been proposed to be SLC4A11, a gene that causes the synthesis of a transport protein in endothelial cells. Both forms of the disease manifest themselves initially in the formation of guttae (gutattae) or buttons on the endothelium. Later development of the condition occurs with the formation of edema or swelling in the posterior stroma. The edema spreads anteriorly and leads to the development of corneal cloudiness. The stroma may take on the appearance of ground glass as the disease progresses. This disease progresses slowly and is more common in females than in males. Effective treatment is a penetrating keratoplasty (corneal transplant).

Related Posterior Membrane Dystrophies

Two dystrophies that have some similarities to Fuch’s dystrophy are posterior polymorphous dystrophy (PPMD) and congenital hereditary endothelial dystrophy (CHED). PPMD has been associated with four gene mutations on chromosomes 10 and 20 that affect the synthesis of collagens IV and VIII. In PPMD, there are differences in the effects on Descemet’s membrane and the corneal endothelium versus the effects that occur in Fuchs’ dystrophy. The nonbanded zone of Descemet’s membrane becomes absent or minimal while the endothelial cells can take on the appearance of epithelial cells. PPMD patients are often asymptomatic, but those receiving penetrating keratoplasties sometimes develop increased intraocular pressure associated with iris attachments (synechiae). By contrast in CHED, the genetic abnormalities have been associated with one or more gene mutations only on chromosome 20. There is a decided loss of endothelial cells rather than a thinning of cells as occurs with Fuchs’ dystrophy. The outcome of penetrating keratoplasty for CHED patients is mixed.

See also: Regulation of Corneal Endothelial Cell Proliferation; Regulation of Corneal Endothelial Function.

Further Reading

Cveki, A. and Tamm, E. R. (2004). Anterior eye development and ocular mesenchyme: New insights form mouse models and human diseases. BioEssays 26: 374–386.

Forrester, J. V., Dick, A. D., McMenamin, P. G., and Roberts, F. (2008).

The Eye. Basic Sciences in Practice, 3rd edn. Edinburgh: Saunders/ Elsevier.

Gerencher, G. A. and Zhang, J. (2003). Chloride ATPase pumps in nature: Do they exist? Biological Reviews 78: 197–218.

Hogan, M. J., Alvarado, J. A., and Weddell, J. E. (1971). Histology of the Human Eye. Philadelphia, PA: Saunders.

Krachmer, J. H., Mannis, M. J., and Holland, E. J. (eds.) (2005). Cornea, 2nd edn., vol. 1, Philadelphia, PA: Mosby/Elsevier.

Kratchmer, J. H., Mannis, M. J., and Holland, E. J. (eds.) (2005). The Cornea, 2nd edn., vol. 1, p. 942. Philadelphia, PA: Elsevier.

McCarey, B. E., Edelhauser, H. F., and Lynn, M. J. (2008). Review of corneal endothelial specular microscopy for FDA trials of refractive procedures, surgical devices, and new intraocular drugs and solutions. Cornea 27: 1–16.

McGowan, S. L., Edelhauser, H. F., Pfister, R. R., and Whikehart, D. R. (2007). Stem cell markers in the human posterior limbus and corneal endothelium of unwounded and wounded corneas. Molecular Vision 13: 1984–2000.

Medina-Martinez, O. and Jamrich, M. (2007). Foxe view of lens development and disease. Development 134: 1455–1463.

Mimura, T. and Joyce, N. C. (2006). Replication competence and senescence in central and peripheral human corneal endothelium.

Investigative Ophthalmology and Visual Science 47: 1387–1396. Streilein, J. W. and Stein-Streilein, J. (2000). Does innate immune

privilege exist? Journal of Leukocyte Biology 67: 479–486. Verkman, A. S. (2005). Aquaporins in endothelia. Kidney International

69: 1120–1123.

Vithana, E. N., Morgan, P. E., Ramprasad, V., et al. (2008). SLC4A11 mutations in Fuchs endothelial corneal dystrophy. Human Molecular Genetics 17: 656–666.

Whikehart, D. R. (2003). Biochemistry of the Eye, 2nd edn. Philadelphia, PA: Butterworth-Heinemann/Elsevier.

Whikehart, D. R., Parikh, C. H., Vaughn, A. V., Mishler, K., and Edelhauser, H. F. (2005). Evidence suggesting the existence of stem cells for the human corneal endothelium. Molecular Vision 11: 816–824.

Wilson, S. E., Weng, J., Blair, S., He, Y. G., and Lloyd, S. (1995). Expression of E6/E7 or SV40 large T antigen-coding oncogenes in human corneal endothelial cells indicates regulated highproliferative capacity. Investigative Ophthalmology and Visual Science 36: 32–40.

Zhu, C., Rawe, I., and Joyce, N. C. (2008). Differential protein expression in human corneal endothelial cells cultured from young and older donors. Molecular Vision 14: 1805–1814.

Relevant Websites

http://www.eyesite.org – Diagrams and a video on corneal endothelial transplantation, The Eyesite.

http://dev.biologists.org – The Company of Biologists Ltd: Development. (This on-line paper reports about the deleterious effects of either TGFa or EGF on corneal endothelial development in a transgenic mouse.).

Glossary

Aquaporins – The water channels that facilitate water movement across the plasma membrane. Deturgescence – The removal of water from the cornea stroma to counteract edema.

Guttata – Excresances of Descemet’s membrane (basement membrane of the corneal endothelium) produced by abnormal endothelial cells.

Peri-junctional actomyosin ring (PAMR) –

A Dense band of actin cytoskeleton found proximal to the apical junction complex.

Pump-leak mechanism – The maintenance of corneal deturgescence in which endothelial active fluid transport (the pump) exactly counters the passive leak directed into the stroma.

Tight junctions – The intercellular junctions at the apical domain of endothelial cells which occlude the paracellular space.

Transendothelial electrical resistance (TER) –

Dependent on the integrity of the tight junctions.

Corneal Endothelial Function

Stromal Swelling Pressure and Maintenance

of Transparency

The transparency of the cornea stroma is dependent on the tissue hydration. The stroma is composed of 200 or more lamellae, 2-mm thick with widths varying from 10 to 250 mm that span from limbus to limbus and overlap each other at varying orientations. The total thickness of the human stroma varies from 450 to 550 mm. Each lamella is composed of parallel strands of collagen (type I) and associated glycosaminoglycans (GAGs). Between the layers of overlapping lamellae are the keratocytes – a very flat, stellate-shaped cell that is responsible for producing collagen and GAGs and maintaining the stromal structure. The GAGs act as spacers between the collagen fibers. At normal stromal hydration, this space is 30 nm and is very uniform. Because the refractive index of collagen and the GAGs ground substance are significantly different, a random orientation of collagen fibers and varying fiber diameter (which is characteristic of the sclera) would produce an opaque tissue. However, because there is almost no variation in fiber diameter and the spacing between fibers is uniform, light scattered offaxis through the stroma is <10%. GAGs, however, are very

hydrophilic and exert a swelling pressure leading to imbibition of water across the relatively leaky endothelium at the posterior surface. Thus, if a bare piece of stroma is placed in saline, it will swell to many times the normal thickness. This stromal edema produces large variations in collagen-fiber spacing and consequentially increased light scatter, corneal haze, and in vivo produces diminished visual acuity. Loss of endothelial cells from surgical trauma or disease, for example, Fuchs’ endothelial dystrophy, results in corneal edema. These observations together with in vitro physiological studies indicate that the maintenance of stromal hydration is primarily dependent on the corneal endothelium.

The pioneering work of David Maurice first showed that the corneal endothelium actively pumps water from stroma to anterior chamber. This pump exactly counterbalances the leak into the stroma, which is driven by the GAG-dependent swelling pressure. Therefore, the stromal hydration is maintained relatively constant and stromal transparency is preserved. This is often called the Pump–Leak hypothesis for maintenance of corneal hydration and is illustrated in

Figure 1.

Endothelial Barrier Function

The tight junctions (TJs) of the corneal endothelium, although leaky (trans-endothelial resistance (TER)25 O cm 2), restrain fluid leak into the stroma. This constitutes the barrier function of the endothelium, and complements fluid-pump function in the regulation of stromal hydration. Despite the leakiness of the endothelium, breakdown of its TJs in the absence of an increase in fluid-pump activity results in corneal edema. In addition to this direct effect, edema could be enhanced further by the fact that TJs also influence the fluid pump function through two indirect mechanisms. First, intact TJs prevent dissipation of local osmotic gradients across the endothelium set up by ion-transport mechanisms by restraining solute back-flux through the paracellular space (i.e., gate function of TJs). Secondly, intact TJs are indispensable for the maintenance of apical-basal polarity of the ion-transport proteins. This is achieved by limiting their lateral diffusion of the membrane proteins (i.e., fence function of TJs). When the polarity of the transport mechanisms is compromised, a vectorial ionic movement and hence fluid transport cannot occur. Thus, the TJs of the endothelium not only restrain fluid leak into the stroma, but also form a principal determinant of the endothelial fluid-pump activity (Figure 2).

Nutrition/Waste Removal

One requirement for corneal transparency is the absence of blood and blood vessels. As such, corneal nutrition must come from the tears, the limbus, and/or the anterior chamber. The stratified squamous epithelium is a very tight barrier, and there are no transporters for nutrients such as glucose. However, the epithelium is permeable to hydrophobic nutrients such as oxygen and wastes, for example, carbon dioxide (CO2). Because of the long diffusion distances from the limbus, its contribution to overall corneal nutrition is minimal. Therefore, most of the nutrition and removal of wastes – for example, lactic acid – occurs across

Tears

Figure 1 Pump–Leak hypothesis for maintenance of corneal hydration. Stromal glycosaminoglycans (GAGs) are negatively charged hydrophilic molecules that exert a swelling pressure that draws water across the limiting layers (epithelium and endothelium). This is the tissue leak. The endothelial pump must exactly counterbalance the leak so that stromal hydration and transparency are maintained.

the endothelium. The corneal endothelium expresses glucose transporters to facilitate uptake into the stroma to nourish keratocytes and the corneal epithelium. Similarly, the endothelium expresses lactic acid transporters to remove this end product of anaerobic glycolysis. Approximately 85% of the glucose consumed by the cornea goes through anaerobic glycolysis because of the relative paucity of mitochondria in the epithelial cells and keratocytes. The relative lack of mitochondria is presumably another strategy to minimize light scatter and enhance transparency.

Corneal Endothelial Transport

Active Transport

Following discovery that the endothelium was responsible for maintaining stromal hydration, it was shown that the endothelial pump was dependent on active transport. In contrast to corneal epithelial cells and keratocytes, endothelial cells have a very high mitochondrial density. Poisoning the mitochondria reduces the pump activity indicating that it is dependent on the availability of adenosine triphosphate (ATP). Furthermore, exposure of endothelium to the cardiac glycoside ouabain – which blocks the membrane Naþ,Kþ-ATPase – also inhibits the pump activity. The Naþ,Kþ-ATPase provides the ion gradients across cell membranes that drive secondary transporters, for example, Naþ:Kþ:2Cl cotransport, Naþ/Hþ exchange, and Naþ : 2HCO3 cotransport, and anion channels that may participate in ion-coupled fluid secretion. A classic fluid-transport mechanism might include basolateral cotransporters and apical anion channels providing a pathway for vectorial ion fluxes that could be osmotically coupled to water transport.

Normal endothelium

SP = 50 mmHg

Leak

Pump

Phaco emulsification

SP < 50 mmHg

Corneal graft rejection

Inflammatory mediators (e.g., TNF-α)

???

Endothelial dysfunction

Bicarbonate/Carbonic Anhydrase

Early studies have shown that the endothelial pump is significantly inhibited in the absence of bicarbonate suggesting that bicarbonate transporters and anion channels may be components of the endothelial pump. A role for bicarbonate was strengthened when it was found that carbonic anhydrase inhibitors (CAIs) – applied directly to the endothelium – also slowed the pump. With the advent of topical carbonic anhydrase inhibitors to lower intraocular pressure (IOP), there was some concern that this could cause corneal edema. However, numerous clinical studies have shown that in humans with normal endothelial cell counts, topical CAIs do not cause corneal edema. Only when cell counts are low and/or in the presence of significant endothelial guttata have topical CAIs been shown to cause corneal edema. This suggests that whatever role bicarbonate/CA activity has in pump activity, there must be a large functional reserve. Interestingly, endothelial pump activity can be maintained in the absence of bicarbonate, but only when bicarbonate is substituted with a high concentration of another buffer. These observations suggest that part of the role of bicarbonate/CA in the endothelial pump may be its buffering capacity, possibly for lactic acid.

Anion Transporters and Channels

Several anion transporters and channels that could participate in a bicarbonate secretory pump mechanism are expressed in corneal endothelium. At the basolateral (stromal side) membrane, the sodium bicarbonate cotransporter (NBCe1) is highly expressed (Figure 3). This protein

transports one Naþ with two HCO3 ions into the cells from the stroma. Application of the anion transport inhibitor 4,40-diisothiocyanatostilbene-2,20-disulphonic acid (DIDS) or genetic knockdown of NBCe1 expression using siRNA significantly reduces bicarbonate uptake and transendothelial bicarbonate flux. DIDS also results in corneal swelling in vitro, suggesting that this anion-transport process has an important role in the endothelial pump activity. NBCe1 is responsible for the large Naþ-dependent (Cl independent) bicarbonate permeability of the basolateral membrane. The bicarbonate permeability of the apical membrane of the corneal endothelium is about one-third of basolateral membrane and it is independent of Naþ or Cl , suggesting that the smaller apical permeability is conferred by a channel. To date, only two apical anion channels have been described in the corneal endothelium – the cystic fibrosis transmembrane regulator (CFTR) and a calciumactivated chloride channel (CaCC). These channels are permeable to both Cl and HCO3 in about a 5:1 ratio. Physiological experiments with rabbit corneas have shown, however, that CFTR-channel inhibitors have no effect on the endothelial pump rate. Furthermore, siRNA knockdown of CFTR in cultured cells – while inhibiting cyclic adenosine monophosphate (cAMP)-activated anion flux, did not change the basal bicarbonate flux. This is consistent with clinical studies that indicate normal corneal thickness and function in cystic fibrosis patients. Similarly, siRNA knockdown of CaCC channels had no effect on basal bicarbonate permeability, but reduced calcium-activated flux. To date, the nature of the apical HCO3 transport remains unknown.

Recent studies have also shown that removal of Cl will also produce significant corneal swelling in vitro,