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

suggesting the possibility that both anions – Cl and HCO3 – participate directly in the pump mechanism. In fact, the basolateral membrane expresses the Naþ: Kþ:2Cl cotransporter and the cytoplasmic [Cl ] is 35 mM, which is above electrochemical equilibrium (Figure 3). Thus the potential for apical anion channel mediated Cl flux is in place. However, the highly specific inhibitor of the Naþ:Kþ:2Cl cotransporter – bumetanide

– has no effect on corneal thickness in vitro. In addition, studies examining transendothelial Cl fluxes have been equivocal. This indicates that a basal Cl flux across the endothelium is unlikely to have a role in the pump mechanism. The presence of Cl may have two other roles that could support HCO3 fluxes. First, because NBCe1 is electrogenic (1 Naþ : 2HCO3 ), membrane potential hyperpolarization (e.g., from 50 to 65 mV) will stop NBCe1 dependent HCO3 influx. Cl efflux through CFTR or other unidentified anion channels could dissipate the hyperpolarizing effects of NBCe1 activity. Second, the anion exchanger AE2 is expressed on the basolateral membrane as shown in the rabbit corneal endothelium. Thus, the transmembrane Cl gradient can help regulate intracellular [HCO3 ]. Further studies are needed to elucidate the role of Cl or Cl transporters in the corneal endothelial pump mechanism. Figure 3 illustrates a possible model for bicarbonate transport.

Aquaporin-1 (AQP1)

Aquaporin-1 (AQP1) water channels are highly expressed on both apical and basolateral membranes of corneal endothelium. No other AQP channel is expressed in the corneal endothelium. AQP1 confers a very high osmotic permeability and allows for rapid cell-volume regulation in response to anisosmotic solutions. In AQP1-knockout mice, corneal endothelial cell osmotic permeability is reduced, as expected. Corneal de-swelling rates are also significantly reduced. This indicates that a significant amount of water flux across the endothelium – at least under non-steady-state conditions – is transcellular. However, steady-state corneal thickness of these mice is slightly thinner than wild-type mice. This result may indicate that the pathway for water fluxes driven by the pump and the leak (GAGs-dependent stromal swelling pressure) is the same, which would result in no net effect. Thus AQP1 (and AQP5 in the corneal epithelium) may be present to increase the rate of water fluxes in response to osmotic changes that can occur, for example, following eye closure or exposure to hypotonicity that occurs during swimming.

Pump Mechanism

The conventional view of epithelial cell secretion and absorption of water is that these cells create local osmotic

differences in the lateral spaces between the cells and/or on the apical surfaces of cells within an unstirred layer. These osmotic gradients are the driving forces for water movement across cellular membranes. This standing gradient osmotic theory, first developed by Diamond and Bossert, has come under fire as a general mechanism for fluid transport. For example, in many epithelial cells – including the corneal endothelium – there is no evidence that these gradients exist. This has led to the consideration of other mechanisms, such as electro-osmosis, which has been championed by Jorge Fischbarg for the corneal endothelium. In this theory, cells generate a transepithelial potential (0.5 mVapical-side negative in corneal endothelium) that draws counter-ions – for example, Naþ – through a paracellular pathway that is ion specific. This produces electro-osmotic coupling across the TJ. Another mechanism for fluid transport across epithelial cells that has been recently developed is the cotransporter model. In this model, water is directly transported or coupled with the movement of the associated ions and metabolites within the cotransporter protein. Since the endothelium expresses several cotransporters – for example, 1Naþ:2HCO3 and Naþ:Kþ:2Cl cotransporters – and at least two monocarboxylic acid (lactic acid) cotransporters – MCT1 and MCT2 – there is potential for downhill fluid transport by this mechanism.

Barrier Integrity

Barrier integrity – which implies resistance to diffusion of solutes or fluid leak through the paracellular pathway – is dependent on the TJs. TJs are supramolecular assemblies localized at the apical domain of epithelial/endothelial monolayers. The transmembrane molecules (occludins, claudins, and junctional adhesion molecule ( JAM)) – which are all expressed in corneal endothelium associated with the TJs – these transmembrane molecules of one cell interact with their homotypic counterparts in the neighboring cells. This interaction – facilitated by intercellular tethering forces through Ca2þ-dependent adherence junctions (AJs) – brings about occlusion of the paracellular space. The cytoplasmic domains of the transmembrane molecules of the TJs are structurally linked to a thick band of cortical actin cytoskeleton (called peri-junctional actomyosin ring (PAMR)) via adapter molecules such as zona occludens-1 (ZO-1). For AJs, the association with PAMR is mediated through catenins. The adapter molecules of both AJs and TJs also form a scaffold for a number of signaling molecules which are now implicated in the control of the stability of AJs and TJs and hence in the regulation of barrier integrity. Recent studies have demonstrated that increased contractility of the PAMR induces a breakdown of the barrier integrity through a reduction of the intercellular tethering forces at the TJs and AJs and also possibly through redistribution

of the AJ and TJ molecules at the apical junctional complex. An increase in contractility of the actin cytoskeleton is induced by an increase in the phosphorylation of the regulatory light chain of myosin II (also called myosin light chain or MLC; 20 kDa). Recent studies have shed light on the importance of actomyosin contraction in the regulation of corneal endothelial barrier integrity.

MLC Phosphorylation and Actomyosin

Contraction

Phosphorylation of myosin light chain (MLC) – which is bound to the motor protein myosin II – induces actomyosin interaction resulting in increased contractility of the actin cytoskeleton (Figure 4). The extent of MLC phosphorylation is regulated by two opposing pathways: myosin light-chain kinase (MLCK)-driven phosphorylation, and myosin light-chain phosphatase (MLCP)-driven dephosphorylation. MLCK is activated after binding to the Ca2þ-calmodulin complex and its activity is dedicated to MLC phosphorylation. MLCK activity is also modulated by other protein kinases by direct phosphorylation, especially the large-size isoform of MLCK called vascular

endothelial cell MLCK (EC-MLCK; 220 kDa). MLCP is a hetero-trimeric complex consisting of PP1Cd (the catalytic subunit), the myosin-binding subunit (MYPT1; 130 kDa), and a small subunit of unknown function (M20). Phosphorylation of MYPT1 by Rho kinase (at Thr-696 and Thr-850) – a downstream effector of the small GTPase RhoA – inhibits the phosphatase activity of PP1Cd. Protein kinase C (PKC) isoforms also inhibit the activity of MLCP through phosphorylation of CPI-17 (PKC-activated 17-kDa inhibitor protein of type 1 phosphatase) and consequent inactivation of PP1Cd. Thus, activation of Rho kinase and/or PKC results in contraction of the actin cytoskeleton and a breakdown in barrier integrity.

Effect of MLC Phosphorylation on Corneal Endothelium Barrier Integrity

The mechanisms associated with MLC phosphorylation – which are well characterized in smooth muscle cells – are also present in corneal endothelial cells as demonstrated in several recent studies. Thus, it is now known that corneal endothelial cells – similar to vascular endothelial

cells – possess both EC-MLCK and a smaller smooth muscle isoform of MLCK (SM-MLCK; 120 kDa). In addition, myosin II isoforms (myosin IIA and IIB) are also expressed in the endothelium. To show the importance of MLC phosphorylation and its effects on the corneal endothelial barrier integrity, several G-protein-coupled receptor (GPCR) agonists have been employed. Thus, thrombin – which activates the RhoA–Rho-kinase axis through Ga12/13-coupled PAR-1 receptors in corneal endothelial cells – led to an increase in MLC phosphorylation. This was found to disrupt PAMR with a concomitant breakdown of the barrier integrity. Pretreatment with ML-7 – a selective inhibitor of MLCK – could not significantly block the thrombin-induced MLC phosphorylation. However, the Rho-kinase inhibitor Y-27632 was effective in blocking the thrombin effect. These findings indicate the dominance of the RhoA-mediated Ca2þ- independent pathway downstream of PAR-1 activation. The importance of the Ca2þ- and PKC-dependent mechanisms in MLC phosphorylation, however, have also been demonstrated independently through activation of histamine H1 receptors, which are coupled to Gaq/11 G-protein and are not known to activate RhoA. Thus, histamine-induced MLC phosphorylation and the resultant loss of endothelial barrier integrity could be suppressed by ML-7 and chelerythrine (a nonspecific PKC inhibitor). Taken together, these data have emphasized a strong role for actin cytoskeleton in the regulation of the endothelial barrier integrity and also suggest that bioactive factors and proinflammatory mediators found in the aqueous humor may influence the barrier function through their respective cell surface receptors.

Transplantation Failure and Tumor

Necrosis Factor

Tumor necrosis factor (TNF-a) is a 17-KDa proinflammatory cytokine implicated in corneal endothelial failure during graft rejection (Figure 2). TNF-a mRNA has been detected in corneal allografts undergoing rejection, and TNF-a protein levels are significantly elevated in the aqueous humor and the serum of hosts that reject corneal allografts. A study with rabbit corneas has demonstrated that TNF-a breaks down the barrier integrity of the endothelial cells concomitant with disruption of actin cytoskeleton. The cytokine is well known to breakdown barrier integrity in vascular endothelial cells. Some of the important molecular mechanisms involved include activation of RhoA, MLC phosphorylation, significant loss of PAMR, microtubule disassembly secondary to activation of p38 MAP kinase, mobilization of oxidative stress, and formation of stress fibers. In recent studies with bovine corneal endothelium, it is becoming evident that TNF-a also induces disassembly of microtubules concomitant

with a gradual loss of barrier integrity and disappearance of PAMR. These effects could be blocked by pretreatment with paclitaxel, SB-203580 (a selective inhibitor of p38 MAP kinase), and inhibitors of matrix metalloproteinases. These results suggest that a number of mechanisms are involved in the breakdown of the barrier integrity in response to TNF-a which converges directly and indirectly on actin and microtubule cytoskeleton.

Regulation of Transport Activity

and Barrier Integrity

Adenosine, Soluble Adenylate Cyclase, and cAMP

The corneal endothelial fluid pump is thought to proceed at one rate and when this rate matches the leak rate driven by the stromal GAGs, corneal hydration and thickness reaches a steady state. There are other forces that can influence the leak rate including evaporative loss across the epithelial surface and contact lens-induced hypoxic stimulation of epithelial lactate production, which can produce substantial additions to stromal osmotic pressure. There is no evidence that the pump speeds up when the cornea is edematous or slows down if the cornea thins.

Shortly after it was clear that the endothelium was actively transporting water, it was discovered that the rate of fluid transport could be increased by the addition of adenosine. Later, it was shown that adenosine increases intracellular [cAMP] in endothelial cells through activation of adenosine A2b receptors. Other approaches that increase cAMP within the cells – for example, stimulating adenylate cyclase (AC) directly or inhibiting phosphodiesterase – also increased corneal endothelial fluid-transport rates. More recently, the expression of a new type of AC, called soluble AC (sAC), was shown in corneal endothelium. Unlike the transmembrane-linked adenylate cyclases, sAC is distributed throughout the cytoplasm and it is activated by HCO3 and Ca2þ. Because of the robust 1Naþ:2HCO3 cotransporter in endothelial cells, the sAC is active and raises the basal [cAMP] by 50%, suggesting that it may have a small role in maintaining basal fluid transport rates. Raising cAMP activates protein kinase A (PKA) and this demonstrably phosphorylates the apical CFTR channel, increases apical Cl and HCO3 permeability, and increases transendothelial HCO3 flux. Together, this could contribute to the increased fluid transport observed by increasing cAMP. Corneal endothelial cells can produce adenosine from ATP at the apical surface. When stressed, corneal endothelial cells release more ATP – which, when converted to adenosine, will enhance fluid transport and could help counter the negative effects of the stress.

Role of cAMP–PKA axis in the Regulation of Barrier Integrity

An in vitro study with rabbit cornea first showed that adenosine could also promote corneal deturgescence through enhanced barrier integrity. Consistent with this finding, several recent studies have shown that adenosine induces MLC dephosphorylation through mobilization of cAMP–PKA axis via A2b receptors as noted above. More importantly, consistent with MLC dephosphorylation, exposure to adenosine led to an increase in the barrier integrity as measured by the trans-endothelial electrical resistance. Similar findings were noted with extracellular ATP – which was found to undergo extracellular hydrolysis resulting in formation of adenosine and subsequent activation of A2b receptors. In addition to these findings, forskolin (direct activator of adenylate cyclase), adenosine, and ATP have been found to overcome thrombinand histamine-induced MLC phosphorylation as well as loss of barrier integrity. At the molecular level, the locus of action of elevated cAMP is also becoming evident. PKA is known to induce MLC dephosphorylation through modulation of the RhoA–Rho-kinase axis. One potential mechanism involves direct phosphorylation of RhoA by PKA and consequent increase in the affinity of the small guanosine triphosphatase (GTPase) to its guanosine diphosphate (GDP) dissociation inhibitor (GDI). An alternative mechanism involves direct phosphorylation of MYPT1 by PKA. The latter is known to prevent Rho kinase from phosphorylating MYPT1 leading to inactivation of MLCP. cAMP may also influence cell–cell adhesion through activation of a small GTPase, namely Rap1. This involves activation of Epac – a guanine nucleotideexchange factor (GEF) for Rap1. Activated Rap1 promotes formation of AJs, presumably through enhanced cadherin ligation.

Summary and Perspective

The corneal endothelium is responsible for maintaining corneal hydration and transparency. Active transport processes – through mechanisms that are not fully elucidated – provide a Pump that exactly counterbalances the stromal glycosaminoglycan induced Leak. The Pump

is regulated through cAMP-dependent signaling that acts on components of ion transport and the barrier integrity of the endothelial monolayer. Further understanding of the Pump and Leak mechanisms are needed to provide medical therapies that could maintain stromal deturgescence in diseased or traumatized corneal endothelium.

See also: Corneal Endothelium: Overview; The Corneal Stroma; Regulation of Corneal Endothelial Cell Proliferation.

Further Reading

Bonanno, J. A. (2003). Identity and regulation of ion transport mechanisms in the corneal endothelium. Progress in Retinal and Eye Research 22(1): 69–94.

Dikstein, S. and Maurice, D. M. (1972). The metabolic basis to the fluid pump in the cornea. Journal of Physiology 221(1): 29–41.

Doughty, M. J. and Maurice, D. M. (1988). Bicarbonate sensitivity of rabbit corneal endothelium fluid pump in vitro. Investigative Ophthalmology and Visual Science 29(2): 216–223.

Fischbarg, J., Diecke, F. P., Iserovich, P., and Rubashkin, A. (2006). The role of the tight junction in paracellular fluid transport across corneal endothelium. Electro-osmosis as a driving force. Journal of Membrane Biology 210(2): 117–130.

Fischbarg, J. and Lim, J. (1974). Role of cations, anions, and carbonic anhydrase in fluid transport across rabbit corneal endothelium.

Journal of Physiology 241: 647–675.

Hodson, S. and Miller, F. (1976). The bicarbonate ion pump in the endothelium which regulates the hydration of rabbit cornea. Journal of Physiology 263: 563–577.

Li, J., Sun, X. C., and Bonanno, J. A. (2005). Role of NBC1 in apical and basolateral HCO3 permeabilities and transendothelial HCO3 fluxes in bovine corneal endothelium. American Journal of Physiology. Cell Physiology 288(3): C739–C746.

Maurice, D. (1972). The location of the fluid pump in the cornea. Journal of Physiology 221: 43–54.

Riley, M., Winkler, B., Starnes, C. A., and Peters, M. I. (1996). Adenosine promotes gulation of corneal hydration through cyclic adenosine monophosphate. Investigative Ophthalmology and Visual Science

37: 1–10.

Satpathy, M., Gallagher, P., Lizotte-Waniewski, M., and Srinivas, S. P. (2004). Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells.

Experimental Eye Research 79: 477–486.

Srinivas, S. P., Satpathy, M., Gallagher, P., Larivie`re, E., and Van Driessche, W. (2004). Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells. Experimental Eye Research 79: 543–551.

Srinivas, S. P., Satpathy, M., Guo, Y., and Anandan, V. (2006). Histamine-induced phosphorylation of the regulatory light chain of myosin II disrupts the barrier integrity of corneal endothelial cells.

Investigative Ophthalmology Visual Science 47: 4011–4018.

Glossary

Cell cycle – Series of events that occur in order for a cell to divide. This cycle is divided into phases where the G0 (gap 0) is resting or quiescent phase; G1 (gap 1) is the synthesis of enzymes necessary for DNA replication; S phase is when DNA synthesis occurs; G2 (gap 2) phase involves the production of microtubules; M phase is the division of the cell into daughter cells.

Corneal dystrophies – Conditions in which the cornea is altered without the presence of any inflammation, infection, or other eye disease.

Cyclins and cyclin-dependent kinases (CDKs) –

Cyclins act as the regulatory subunits while CDKs act as the catalytic subunits of an activated heterodimer. Neither cyclins nor CDKs are active in the absence of one another. CDKs are constitutively expressed in cells, whereas cyclins are synthesized at specific stages of the cell cycle, in response to various molecular signals.

Descemet’s membrane – A specialized form of extracellular matrix separating corneal endothelial cells from corneal stroma.

E2F – A group of genes that encodes a family of transcription factors.

Epidermal growth factor (EGF) – Compound that promotes cell growth and differentiation.

Mitogen – A chemical substance that induces cell division.

Retinoblastoma gene – A gene whose protein is dysfunctional in many types of cancer.

Tight junction – A junction between two cells composed of the junctional membrane. This acts as a selective barrier to small molecules and as a total barrier to large molecules.

Transforming growth factor (TGF) – Used as a polypeptide growth factors, TGF is produced in many cell types and is involved in cellular development.

Background

Mammalian corneal endothelial cells (CECs) are derived from neural crest cells of mesenchyme and form a monolayer in the inner portion of the cornea (Figure 1). The corneal

endothelium is attached to the Descemet’s membrane (DM) and is in direct contact with the aqueous humor. Its main function is to maintain corneal transparency by regulating corneal hydration. CECs contain numerous Na+–K+-adenosine triphosphatases (ATPases) that pump fluid out of the stroma to counteract the corneal tendency to swell. In addition, tight junctions between the CECs provide a barrier that prevents the influx of fluid from aqueous humor into the stroma. CEC number gradually declines with age (Figure 2). Since CECs do not proliferate in vivo and have a limited ability to regenerate, the loss of endothelial cells is permanent. Certain corneal pathologies, such as dystrophies, infections, and trauma, can lead to an accelerated loss of CECs and a compromise in the endothelial cell layer integrity. As a result, the cornea is unable to maintain its water balance and corneal edema ensues, clinically resulting loss of clarity and a decline in visual acuity.

Cell Cycle Progression, G1/S Transition and its Cell Cycle Regulators

Mammalian cell cycles can be divided into four phases, including G1 phase (gap phase 1), S phase (DNA synthesis), G2 phase (gap phase 2), and M phase (mitosis). The entire process of cell cycle progression is controlled by a variety of regulatory proteins (Figure 3). Among them, cyclindependent kinases (CDKs) are the engine cores that promote cell cycle progression. CDKs generally remain at a constant level throughout the cell cycle, while their binding partners, cyclins, and post-translational modifiers, kinases and phosphatases, undergo periodic fluctuations throughout the cell cycle. During this process, negative regulatory protein cyclin-dependent kinase inhibitors (CDKIs) also play an important role in cell cycle progression.

During the G1/S transition (Figure 3), CDKs 2, 4, 6 and their regulators control the cell transition to the S-phase. Association of CDK4 or CDK6 with D-type cyclins (Cyc D) is critical for G1 phase progression, whereas the association of CDK2 and cyclin E (Cyc E) fosters the initiation of the S phase. Both complexes, Cyc D–CDK4/6 and Cyc E–CDK2, are involved in phosphorylation of retinoblastoma gene (Rb) and a subsequent G1/S transition. During this process, two main families of CDKIs play an important role in regulating G1/S transition: inhibitor of CDK (INK) and CDK-interacting protein/cyclin-dependent kinase inhibitory protein (CIP/KIP). The INK protein, p16, is a

a

b

c

Figure 1 Ultrastructure of a human cornea: (a) epithelium,

(b) stroma, and (c) endothelium.

(a)

(b)

Figure 2 Physiological loss of human corneas endothelial cells:

(a) in a newborn (cell density (CD) = 4347 cells mm–2) and (b) in a 60-year-old normal eye (CD = 2392 cells mm–2).

competitive inhibitor of CDK4/6-Cyc D complex, while the CIP/KIP proteins, p21 and p27, serve as competitive inhibitors of CDK2–Cyc E complex. The G1/S phase transition features two consecutive steps: (1) Early G1 progression involves mitogen-dependent accumulation of cyclin D, sequestration of p21, and cyclin D-dependent phosphorylation of retinoblastoma gene (Rb) (2) Late G1 progression depends on phosphorylation and degradation of p27 and additional phosphorylation of Rb by Cyc E/CDK2. Full phosphorylation of Rb leads to its dissociation from E2F and ultimately activation of E2F, which is a main transcription factor involved in the G1/S transition (Figure 3).

G1 Phase Cell Cycle Arrest in CECs in vivo

Human corneal endothelial cells (HCECs) do not divide in vivo sufficiently to replenish lost cells due to aging, trauma, or disease. HCECs exhibit no positive staining with Ki67, a marker of actively cycling cells. However, HCECs exhibit in situ staining with the key cycle cell regulators, such as cyclins D, E, and A. Such staining pattern which is similar to the one seen in limbal epithelium known to contain slow-cycling stem cells, points to the fact that CECs are arrested in the G1 phase rather than having permanently exited from the cell cycle. Possible mechanisms accounting for such G1 phase cell cycle arrest are contact inhibition, interaction with the extracellular matrix (ECM), and presence of transforming growth factor (TGF)-b2 in the aqueous humor (Figure 4).

Cell–Cell Contact Inhibition

Contact-dependent inhibition of cell proliferation is a well described phenomenon. In corneal endothelium, majority of studies on cell–cell contact inhibition have been performed in neonatal rats, since rats have an immature corneal endothelium at birth, unlike the other species. In neonatal rats, the number of CECs staining positively for bromo-deoxyuridine (BrdU), an S-phase marker, gradually decreased between postnatal days 1 and 13. After postnatal day 13, positive BrdU staining was no longer detectable. Stable cell–cell and cell–substrate contacts gradually formed, and monolayer maturation was complete between postnatal days 14 and 21. These studies showed a correlation between decreased proliferation and increased monolayer formation, underlying the importance of cell–cell contact inhibition in the maturation of endothelial monolayer.

Consistent with these findings, treatment of corneal endothelial monolayer with ethylenediaminetetraacetate (EDTA), a calcium chelator which releases cells from

Cell−cell contact-dependent inhibition

Cell adhesion molecules in CEC = cadherin, connexin 43, ZO-1

¥Releasing cell−cell contact by EDTA

¥Knocking down connexin 43

?

Inhibition of

CDK2

cell–cell contact, has been shown to promote cell proliferation. CEC proliferation can be regulated by calcium levels due to the presence of several cell adhesion proteins that are calcium sensitive and maintain cells in the state of contact inhibition. The main cell adhesion molecules involved in CEC physiology are cadherins, zonula occludens, and connexins (Cx). Exposure of CECs to calciumfree medium resulted in loosening of apical junctions, loss of barrier function, and corneal edema. This effect was reversible by exposing the endothelial cells to calcium. The attempts to break these intercellular interactions have been successful in promoting endothelial cell proliferation, and providing promise to regenerating CEC in

traumatic and degenerative situations. These findings indicate that cell–cell contact inhibition may play an important role in the growth arrest status of CECs.

It was demonstrated that knocking down one of the major connexins, Cx43 resulted in a significant increase in the number of actively proliferating CECs. Using the rat model, corneal endothelial scrape injuries were simultaneously applied with Cx43 antisense oligodeoxynucleotide, small interfering RNA, or adenovirus (CMV– Cx43–mRFP1) into the anterior chamber. Changes in Cx43 expression were analyzed by immunolabeling (ZO-1, alpha-smooth muscle actin (SMA), Cx43). While, the endothelial–mesenchymal transition/transformation

after injury was inhibited, Cx43 knock-down induced proliferation of the corneal endothelium.

DNA-binding transcription factors, including the members of the BTB/POZ-zinc finger protein family, might be involved in the signaling pathway of cell–cell contactinduced growth inhibition as well. Among the family members, the promyelocytic leukemia zinc finger protein (PLZF) gene specifically inhibits the transcription of genes involved in G1/S transition, such as cyclin A2 and c-myc. The expression of PLZF is closely related with cell–cell contact. Its expression is high in confluent cells, and treatment with EDTA to disrupt cell–cell contact decreases PLZF messenger RNA (mRNA) levels. Similarly, overexpression of PLZF has been found to inhibit cell proliferation. N-Cadherin is thought to be a potential upstream signaling molecule that regulates PLZF mRNA levels.

Studies showed that p27, a CDKI-type protein, is also involved in the mediation of cell cycle arrest induced by cell–cell contact. Increased expression of p27 in the developing corneal endothelium is well correlated with the cessation of the proliferation in wild-type mice. On the other hand, p27 knock-out mice show prolonged postnatal period of CEC proliferation when compared with their wild-type counterparts. These data indicate that p27 plays an important role in contact-inhibition-mediated growth arrested in CEC.

The Presence of Antiproliferative TGF-b2 in the

Aqueous Humor

In many cell types, TGF-b2 inhibits proliferation by inducing G1-phase arrest. CECs are in direct contact with aqueous humor containing TGF-b2. Both exogenous TGF-b2 and active TGF-b2 in rat aqueous humor inhibit S-phase entry in rat CECs. The effect of TGF-b2 may be mediated by CDK4 and p27. In CECs treated with TGF-b2, CDK4 synthesis is inhibited and p27 is mobilized from the cyclin D-CDK 4 complex into the cyclin E-CDK 2 complex to inhibit CDK 2 activity. Furthermore, TGF-b2 prevents phosphorylation of p27 and maintains p27 in an active form.

The Interaction Between CECs and Extracellular

Matrix

ECM provides an important microenvironment for cell adhesion, migration, growth, differentiation, and signal transduction. DM is a specialized form of ECM separating CECs from corneal stroma. One difference between infant and adult human corneal DM is that collagen VIII, the major component of DM, shifted from the endothelial face of DM in infant to the stromal side of the DM in the adult. It is not fully understood whether compositional differences of collagen VIII are responsible for the differences in CECs

growth potential from young and adult humans. In a mouse model, the complete lack of type VIII collagen leads to dysgenesis (abnormal development) of the anterior ocular segment and enlarged CECs with reduced cell density. Hence, collagen VIII may serve as an important component of matrix that stimulates CEC growth during embryonic development. It is possible that adult DM has structural differences that foster endothelial growth arrest, associated with adult corneas. Proteomic analysis revealed that there is an age-related increase in TGFb-induced protein content and proteolytic processing of the human corneal endothelium and DM complex.

What Do We Know From Primary and

Subcultured CECs

Human and other mammalian CECs can be isolated and subcultured in vitro, indicating that endothelium still retains some replicative capacity. Such studies may provide better understanding of the regulatory mechanisms involved in CECs proliferation. Age-related decrease in sensitivity to mitogen or growth factors has been observed in cultured HCECs. It is still controversial whether the corneal periphery has more replicative capacity compared to central region. E2F is a key transcription factor involved in the G1/S transition of CECs, and overexpression of one of its isoforms E2F2 has been shown to promote proliferation. Various upstream signals including protein kinase C (PKC) and fibroblast growth factor (FGF)-2 might also play an important role in the proliferation of cultured CECs.

Age-Related Decrease in Sensitivity to Mitogen or Growth Factors in Cultured HCECs

Donor age negatively affects the proliferative capacity of cultured HCEC. In cultured HCEC a decrease in proliferative capacity is accompanied by changes in morphology and cell density. CECs cultured from older donors show an increase in cell size and a decrease in overall cell density. Different expression levels of cell cycle regulators were studied in cultured HCEC from younger and older donors. Increased expression of CDKIs such as p21 and p16 were seen in HCEC from older donors when compared with those from younger donors. On the other hand, transfection of p27 siRNA was sufficient to promote proliferation in confluent cultures of HCECs from younger (<30 years old), but not from older donors (>60 years old). This suggests that inhibition of proliferation in older donors is regulated by other mechanisms in addition to p27. It is known that an age-dependent increase in expression of negative cell cycle regulators p21 and p16 might be among those mechanisms.

Protein tyrosine phosphatase (PTP) plays a negative role in regulating epidermal growth factor (EGF) signaling pathway. PTP1B is a widely expressed nonreceptor PTP originally identified in placentas. It downregulates the EGF signaling pathway by dephosphorylating epidermal growth factor receptor (EGFR) and inhibition of PTB1B promotes S phase entry. Increased PTP1B activity was detected in HCECs from older donors and reduced proliferative activity in response to EGF in those cells is partly due to increased PTP1B activity.

Comparison of Proliferative Capacity of HCECs from Central and Peripheral Regions

It is still controversial whether the peripheral endothelium has more potential for cell division than the central endothelium. p53 is a negative cell cycle regulator; it inhibits cell division primarily through a p21 pathway. It was found that p53 and its family member TAp63 are highly expressed in central rather than in peripheral endothelium, supporting that there is a greater potential for cell proliferation in the peripheral. However, studies by other group have shown that HCECs cultured from both the central and peripheral areas are capable of cell division in response to serum and their proliferation rates are same.

Overexpression of E2F2 Promotes Proliferation

of CEC

E2F family proteins are key transcription factors for genes involved in the cell cycle progression. Three isoforms of this family (E2F1, E2F2, and E2F3) play an important role in G1/S transition. Overexpression of E2F2 promotes cell proliferation in rabbit CECs by increasing the proliferation marker ki67 and cylin B (G2 phase cell cycle regulator).

PKC Signaling Pathways in Regulating

Proliferation of CECs

PKC comprises a family of serine/threonine protein kinases, which play an important role in regulating proliferation in many cell types. Several PKC isoforms, including PKC-alpha, -betaII, -delta, -epsilon, -iota, -eta, -gamma, and –theta, were detected in CECs. PKC activity, in particular PKC-alpha and -epsilon activity, is important in promoting CEC proliferation. Inhibition of PKC activity prohibits G1/S-phase progression and reduces cyclin E protein levels in cultured rat CECs.

FGF-2 Signaling Pathway

FGF-2 is a component of DM. As a member of the FGF family, it is a multifunctional regulator of cell development,

differentiation, regeneration, senescence, proliferation, and migration. The biological actions of FGF-2 are mediated through transmembrane cell surface receptors that possess tyrosine kinase activity. There are four isoforms of FGF-2. Only the 24-kDa nuclear FGF-2 isoform induced by corneal endothelium modulation factor (CEMF) may be involved in cell proliferation. Phospholipase C gamma (PLC-gamma) or phosphoinositide 3-kinases (PI3 kinase) serve as a downstream signaling pathway in FGF-2- mediated proliferation of rabbit CEC proliferation. Both PLC-gamma and PI3 kinase may utilize Cdk4 and p27 while exerting the mitogenic signal.

Summary

Understanding of the regulatory mechanisms involved in CEC proliferation is important in the context of regenerative medicine. Since corneal endothelium does not divide in vivo, manipulation of the factors involved in this cell cycle can be used to expand endothelium for regenerative purposes. Such developments would bring a great promise to the development of the treatment strategies for both exogenous and endogenous corneal endotheliopathies, hopefully bypassing the need for allogeneic corneal transplantation.

See also: Corneal Endothelium: Overview; Regulation of Corneal Endothelial Function.

Further Reading

Bednarz, J., Teifel, M., Friedl, P., and Engelmann, K. (2000). Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells. Acta Ohthalmologica Scandinavica 78: 130–136.

Coqueret, O. (2002). Linking cyclins to transcriptional control. Gene 299: 35–55.

Enomoto, K., Mimura, T., Harris, D. L., and Joyce, N. C. (2006). Age differences in cyclin-dependent kinase inhibitor expression and Rb hyperphosphorylation in human corneal endothelial cells.

Investigative Ophthalmology and Visual Science 47: 4330–4340. Harris, D. L. and Joyce, N. C. (2007). Protein tyrosine phosphatase,

PTP1B, expression and activity in rat corneal endothelial cells.

Molecular Vision 13: 785–796.

He, Y., Weng, J., Li, Q., Knauf, H. P., and Wilson, S. E. (1997). Fuchs’ corneal endothelial cells transduced with the human papilloma virus E6/E7 oncogenes. Experimental Eye Research 65: 135–142.

Hopfer, U., Fukai, N., Hopfer, H., et al. (2005). Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye. FASEB Journal 19: 1232–1244.

Joko, T., Nanba, D., Shiba, F., et al. (2007). Effects of promyelocytic leukemia zinc finger protein on the proliferation of cultured human corneal endothelial cells. Molecular Vision 13: 649–658.

Joyce, N. C. (2003). Proliferative capacity of the corneal endothelium.

Progress in Retinal and Eye Research 22: 359–389.

Joyce, N. C., Harris, D. L., and Zieske, J. D. (1998). Mitotic inhibition of corneal endothelium in neonatal rats. Investigative Ophthalmology and Visual Science 39: 2572–2583.

Jurkunas, U. V., Bitar, M. S., and Rawe, I. M. (2009). Co-localization of increased transforming growth factor beta induced protein (TGFBIp) and clusterin expression in guttae of Fuchs endothelial corneal dystrophy patients. Investigative Ophthalmology and Visual Science

50(3): 1129–1136.

Kabosova, A., Azar, D. T., Bannikov, G. A., et al. (2006). p27kip1 siRNA induces proliferation in corneal endothelial cells from young but not older donors. Investigative Ophthalmology and Visual Science 47:

4803–4809.

Konomi, K., Zhu, C., Harris, D., and Joyce, N. C. (2005). Comparison of the proliferative capacity of human corneal endothelial cells from the central and peripheral areas. Investigative Ophthalmology and Visual Science 46: 4086–4091.

McAlister, J. C., Joyce, N. C., Harris, D. L., Ali, R. R., and Larkin, D. F. (2005). Induction of replication in human corneal endothelial cells by E2F2 transcription factor cDNA transfer. Investigative Ophthalmology and Visual Science 46: 3597–3603.

Nakano, Y., Oyamada, M., Dai, P., et al. (2008). Connexin43 knockdown accelerates wound healing but inhibits mesenchymal transition after corneal endothelial injury in vivo. Investigative Ophthalmology and Visual Science 49: 93–104.

Paull, A. C. and Whikehart, D. R. (2005). Expression of the p53 family of proteins in central and peripheral human corneal endothelial cells.

Molecular Vision 11: 328–334.

Sasaki, T., Sorokin, L. M., Steiner-Champliaud, M. F., et al. (2007). Compositional differences between infant and adult human corneal basement membranes. Investigative Ophthalmology and Visual Science 48: 4989–4999.

Yoshida, K., Kase, S., Nakayama, K., et al. (2004). Involvement of p27KIP1 in the proliferation of the developing corneal endothelium.

Investigative Ophthalmology and Visual Science 45: 2163–2167. Zhu, C. and Joyce, N. C. (2004). Proliferative response of corneal

endothelial cells from young and older donors. Investigative Ophthalmology and Visual Science 45: 1743–1751.