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

from a keratocyte to an activated keratocyte and then possibly to a myofibroblast depends on the TGF-b ligand/receptor occupancy rate and the duration of this occupancy rate. The myofibroblast phenotype may also require synergistic PDGF co-stimulation. Behaviorally, activated keratocytes promote provisional fibronectin ECM synthesis and deposition, while myofibroblasts promote mature collagenous fibrotic ECM synthesis and deposition. It has been postulated that at least partial ECM regeneration may still be possible in vivo since an adult keratocyte stem cell subpopulation has been discovered near the limbus. These progenitor cells express the ocular development gene Pax6, which is an embryonic marker not expressed by resident stromal keratocytes. Overall, it has been suggested that variations in these two repair phenotypes (activated keratocyte and myofibroblast), especially the degree and duration of myofibroblast differentiation, ultimately determine the final clinical outcome of the wound-healing process, resulting in either primitive, incomplete wound repair, acceptable tissue fibrosis, or excessive fibrotic scarring.

Three Phases of Corneal Wound Healing

Classically, corneal wound healing is typically divided into three overlapping phases (Figure 5): (1) inflammatory phase – cytokine release and amplified expression, keratocyte apoptosis, and inflammation; (2) active wound-healing

phase – epithelial cell migration, proliferation, and regeneration, keratocyte migration and proliferation, keratocyte metabolic activation and eventual quiescence, myofibroblast development, ECM synthesis and deposition (immature primitive scar formation with or without transition to early mature fibrotic scar formation); and (3) remodeling phase – decrease in cellular density, persistence or disappearance of myofibroblasts, persistence or disappearance of primitive scar, and collagen fibril re-organization/remodel- ing (mature fibrotic scar formation).

Inflammatory phase (lasts up to 2–4 weeks after injury)

As described earlier, after injury, damaged resident cells in the cornea release or adjacent surviving cells secrete a variety of cytokines and GFs into the wound. As the local concentration of these cytokines and GFs increases and these compounds are converted to their active form, one of the first morphologically observable changes in the corneal stroma following injury is a 50–75 mm zone of keratocyte apoptosis surrounding the actual direct injury site. This is followed by a subsequent transient influx of mixed acute and chronic inflammatory cells. Keratocyte apoptosis usually peaks at approximately 4 h after injury, but may still occur minimally up until approximately 1 week after the initial insult. After the initial peak of keratocyte apoptosis, an increased proportion of cells die

TGF-β ligand/receptor signaling/# of myofibroblasts

Pathological fibrotic scar repair response

through necrosis. Within 8 h of injury, neutrophils are attracted to the wound site by chemokines and these inflammatory cells function by killing microbes and even host resident cells with their secretion of free radicals. Neutrophils serve no direct role in amplifying or suppressing the fibrotic repair response through its secreted cytokines or GFs, but may indirectly amplify it by damaging more resident tissue cells. Within 24 h of injury, monocytes are attracted to the wound site where they differentiate into macrophages, which serve to degrade and remove dead or damaged cells and ECM. Keratocytes and epithelial-derived proteases and collagenases help macrophages in this degradation and removal process. Macrophages also directly amplify the fibrotic repair response through secretion of pro-fibrotic TGF-b 1 and some PDGF, which can potentially result in a paracrine positive feedback loop, if intense enough. Finally, within 3 days of injury, lymphocytes enter the wound, where they secrete antifibrotic cytokines and GFs. Overall, this direct injury-mediated innate inflammatory response typically adds more pro-fibrotic cytokines and GFs to the original direct resident tissue injury-mediated cytokine and GF cascade. The innate inflammatory response amplifies the cascade of events already taking place, commonly resulting in additional keratocyte proliferation and migration, further keratocyte differentiation to one of the various fibrotic repair phenotypes including the myofibroblastic stage. Thus, inflammation has both positive and negative consequences of preventing infection and amplifying the fibrotic repair response, respectively.

Active wound-healing phase (lasts up to 4–6 months after injury)

Proliferation and migration of residual surviving keratocytes begins 12–24 h after injury to reconstitute the cellularity of the injured area and typically continues for only several days after injury. The migrating keratoctyes are spindle-shaped in appearance and highly motile. Subsequently, migratory keratocytes differentiate into a metabolically activated repair phenotype called an activated keratocyte. Within 1–2 weeks after injury, myofibroblasts first begin to appear in the stromal wound under the epithelium and then develop in the deeper stroma down to a depth of approximately 50–75 mm. Myofibroblasts are a fibrotic repair phenotype characterized by significant deposition of a disorganized collagenous ECM, significant hypercellularity, and extensive wound contraction. An important physical characteristic of myofibroblasts in the cornea is their reduced transparency relative to other cell types in the corneal stroma, which may be due to a loss of soluble cytoplasmic corneal crystallins in combination with assembly of insoluble actin stress fiber bundles.

Remodeling phase (occurs from 4–6 months to 3–4 years after injury)

During the remodeling phase, corneal cellularity in the repair tissue decreases closer to normal because of the disappearance or persistence of residual keratocytes or myofibroblasts. The stromal cells also revert back to a more normal nonfibrotic reparative phenotype as actin stress fibril bundles disappear and TGF-b receptor site density gets closer to baseline. The ECM reorganizes closer to normal as collagen fibrils become larger in diameter, more regular, more orderly in arrangement, richer in collagens type I molecules, less rich in type III molecules, and more cross-linked. In addition, any residual hyaluronic acid in the provisional ECM is replaced by more mature, sulfated PGs. The remodeling phase is the dominant process guiding the early mature scar to complete the maturation process. As such, this phase may be considered more or less a functional improvement phase. After remodeling, corneal scars gain increased tensile strength, have less haze and less wound contracture, and are more cosmetically pleasing with a reduction in size and improvement in structure.. During the remodeling phase, the collagen fibril turnover rate is still higher than normal, most likely due to ongoing MMP activity. Unfortunately, if only an immature provisional fibronectin ECM is present after completion of the active wound-healing phase, then the remodeling phase is incapable of making the transition from the immature provisional matrix to the mature collagenous ECM and a provisional fibronectin matrix remains permanently in the corneal stroma.

Termination of the fibrotic repair response (about 3–4 years after injury)

In humans, the long-term result of corneal wounding is the production of a hypercellular fibrotic scar (i.e., chronically hazy fibrotic scar) or a normocellular-to-hypercellular tissue fibrosis (i.e., transparent fibrotic tissue) in wound regions where epithelial– and endothelial–stromal interactions took place and a hypocellular primitive matrix (i.e., transparent immature provisional fibronectin ECM) in wound regions where keratocyte injury pathways took place in the absence of other supplemental healing responses (Figure 4). These three long-term histological tissue repair types have functional differences as the hypercellular fibrotic scar is strong (tensile strength approximately 40% of normal), but clinically hazy because of persistent myofibroblasts present in the repair tissue longterm. In contrast, the hypocellular primitive matrix is transparent, but it is very weak in tensile strength since it is composed of very little collagen fibrils (tensile strength approximately 2–3% of normal). Normocellular-to-hyper- cellular tissue fibrosis more or less is a hybrid of the two just described as it is macroscopically transparent and somewhat strong (tensile strength approximately 30% of normal). An additional variable to consider in this scheme

is the fact that more precisely realigned wounds, such as sutured and unsutured wounds with minimal gaping and no epithelial cell plugging, heal better than poorly aligned wounds, such as wounds with wide wound gaping; epithelial plugging; or incarceration of Bowman’s layer, Descemet’s membrane, or uvea (Figure 6).

Modulation of Scarring

Currently Available Scar-Reducing Therapies

Corneal fibrotic scars remain difficult to cure. Thus, the best approach is prevention. At present, there is no pharmacologic agent or surgical procedure that is universally effective in ameliorating fibrotic scarring. The best strategy currently entails a polytherapeutic empirical approach involving short-term (approximately 1–3 months in duration) topical corticosteroids, a single intraoperative local application of mitomycin C (MMC), and possibly several alternative or emerging options (Table 2). The

Table 2 Mechanism of action of scar-reducing agents

easiest way to understand the place that a drug is located in the range of possible treatment options is to consider the TGF-b signaling pathway or the wound-healing phase that the compounds act on.

Corticosteroids reduce scar formation primarily by suppressing the TGF-b-mediated inflammatory cell–stromal interactions and, secondarily, by possibly depressing steps in the active wound-healing phase, such as inhibition of fibroblast proliferation and diminished collagen synthesis. Regarding the fibroblasts, a previous study demonstrated that corticosteroids have a direct antiproliferative effect on ocular fibroblasts by altering the intracellular activity and expression of multiple genes that participate in fibrotic scar formation, including inhibition of TGF-b1, TGF-b2, and collagen expression. Thus, it should not be surprising that corticosteroids affect stromal wound healing to a greater degree than epithelial wound healing. Unfortunately, in randomized, placebo controlled, prospective clinical trials, corticosteroids produced no clinically significant longterm effects on either haze or refractive regression due to fibrotic scarring compared to placebo, but were definitely associated with a few unwanted side effects (high intraocular pressure and posterior subcapsular cataract formation). Thus, the role of steroids in the prevention of fibrotic scarring remains limited. Moreover, in view of their side-effect profile, steroids clearly would be unacceptable for longterm use in scar prevention and should be strictly used short-term (about 1–3 months from injury). Overall, corticosteroids will likely be used most effectively in future scar-reducing protocols as part of a polytherapeutic strategy since inflammation is just one of the four TGF-b signaling pathways involved in the avascular fibrotic repair response. When used, however, the timing of corticosteroid administration is crucial as the best results occur if used within hours of injury before neutrophils and profibrotic macrophages enter the wound. Once the injury-induced inflammatory response is initiated in the wound, it is somewhat self-amplifying, making it quite difficult to immediately suppress the response with corticosteroids.

The mechanism by which MMC prevents or reduces fibrotic scar formation has not been fully elucidated. It appears to work by inhibiting cell proliferation due to its potent alkylating chemotherapeutic effect by crosslinking DNA after metabolic activation by way of reduction. This effect is downstream from the initiating pro-fibrotic events in the four TGF-b-mediated signaling pathways of the cornea. Thus, MMC may affect all the four pathways, at least acutely while it remains in the tissue at therapeutic concentrations. MMC is potent because it affects cells in all phases of the cell cycle. In high concentration, MMC also is known to promote direct keratoctye and myofibroblast apoptosis in the inflammatory phase via free radical injury, which may be another mechanism by which it works. MMC combined with low-dose corticosteroids is the

conventional treatment approach used today for corneal fibrotic scar prevention in high-risk cases. This combination is also used in the treatment of corneal fibrotic scars after additional maneuvers are performed to remove as much of the scar tissue as possible by surgical debridement (e.g., phototherapeutic keratectomy (PTK) before direct MMC local application).

An alternative or supplementary scar-reducing treatment is that of using fresh or cryopreserved amniotic membrane (AM) allografts to cover treated areas of exposed injured corneal stroma to prevent fibrotic scarring. Human fetal AM is composed of three layers: a cuboidal to columnar epithelial monolayer, a 200–300-nm thick BM, and an avascular stroma. This membrane suppresses the inflammatory and active wound-healing phases of the fibrotic repair response. Intact AM–BM suppresses epithelial–stromal interactions since the stroma has intrinsic antifibrotic, anti-inflammatory, and antiangiogenic properties, presumably due to cytokine and GF binding to sulfated PGs in the AM stroma. AM also aids in re-epithelization due to its epithelial surface and its BM, which contains cell adhesion molecules, such as fibronectin. The primary disadvantages of AM are its expensive cost and increased surgical time. Thus, AM is used more often for severe corneal and/or limbal stem cell disease rather than repair of simple corneal wounds.

Newer advanced surface ablation techniques, such as laser-assisted subepithelial keratectomy (LASEK) or epikeratome laser-assisted in situ keratomileusis (EpiLASIK), modify the PRK surgical technique using natural means to suppress direct epithelial–stromal interactions. For example, in LASEK surgery, the replaced epithelial flap retains as much intact epithelial BM as possible, which serves to bind and store direct injury-induced epithelialderived TGF-b. Thus, LASEK ingeniously takes advantage of natural antifibrotic cytokine and GF–ECM interactions to reduce the degree of fibrosis. EpiLASIK goes one step further in that the epithelial flap retains much healthier, living corneal epithelium as well as an intact epithelial BM. Thus, EpiLASIK additionally blunts profibrotic tear film–stromal interactions by natural means since a healthy, viable superficial squamous epithelial layer containing zonula occludens tight junctions serves as a physical barrier to direct stromal exposure to cytokines and GFs factors in the tear film.

Emerging Scar-Reducing Therapies

Extensive research has been performed to elucidate the fibrotic repair response, particularly in the skin, with the long-term goal of iatrogenically manipulating this response toward obtaining a clinical advantage. Recent molecular therapeutic investigations have concentrated on inhibiting myofibroblast differentiation by targeting TGF-b. A promising strategy currently being evaluated

in phase-I and-II clinical trials in the skin entails manipulating the ratio of the three TGF-b isoforms in the wound in favor of TGF-b3. Three independent, randomized, placebo controlled, prospective clinical trials have shown that exogenous TGF-b3 supplementary treatment resulted in clinically significant scar prevention or reduction with primary and multiple secondary endpoints in the skin. Inhibition of TGF-b1 and b2 binding to their associated receptors with topical anti-TGF-b antibodies has also been shown to reduce myofibroblastic differentiation, ECM deposition, and cell haze induced by PRK in animal models. By subtly altering the ratio of cytokines or GFs present in the wound during corneal wound healing, it is ultimately hoped that one day postnatal wounds could be induced to heal like embryonic wounds through tissue regeneration. An important caveat to these molecular therapies is that the timing of the application of these agents is critical, usually with the best results occurring if used immediately after injury or, at the very least, within 48 h of injury.

There have been a few published studies, limited to animal models, using gene therapy for scar reduction. To date, fibroblasts have been used as the primary targets. Further investigation into the role of gene therapy for scar reduction is still needed since the practical use of such an approach is not presently feasible.

Other possible emerging fibrotic scar-reducing therapies include systemically administered minocycline antibiotics, which significantly reduce the severity of hypertrophic scarring in a rabbit ear scar model. The mechanism by which minocycline reduces scar formation in this model remains unanswered, but the most plausible mechanism involves inhibition of MMPs resulting in the inhibition of fibroblast migration. Anti-intracellular Smad signaling pathway agents are another promising possibility.

Conclusion

Significant advances have been made in understanding the mechanisms controlling the adult fibrotic wound-repair response in comparison to the embryonic scar-free regenerative response. Discovery of four TGF-b signaling pathways and the central role of myofibroblasts in corneal wound healing have been crucial to this end. Currently, the most effective corneal scar-reducing therapies involve a rather crude polytherapeutic empirical strategy of management involving short-term topical steroids and single intraoperative MMC application. Hopefully, in the near future, the recent advances in understanding the molecular and cell biology of fibrotic wound repair can translate into the development and clinical use of more promising agents, such as molecular, gene, or stem cell therapies. The capability to successfully manipulate the fibrotic repair process in the cornea in vivo offers many tantalizing

prospects from preventing blindness from corneal scarring to obtaining 20/8 perfect vision with optimal corneal wound healing after keratorefractive surgery to perfect tissue regeneration due to converting the adult fibrotic repair response back to an embryonic scar-free regenerative response to the possibility of restoring vision through tissue engineering of a human healthy cornea for replacement purposes.

Acknowledgments

This work was supported in part by NIH Grants P30 EY06360 (Departmental Core Grant), T32EY07092 (DGD), R01EY00933 (HFE), and Research to Prevent Blindness, Inc., New York, New York.

See also: Corneal Endothelium: Overview; Corneal Epithelium: Wound Healing Junctions, Attachment to Stroma Receptors, Matrix Metalloproteinases, Intracellular Communications; Cornea Overview; The Corneal Stroma; Refractive Surgery and Inlays; Regulation of Corneal Endothelial Cell Proliferation; Regulation of Corneal Endothelial Function; The Surgical Treatment for Corneal Epithelial Stem Cell Deficiency, Corneal Epithelial Defect, and Peripheral Corneal Ulcer.

Further Reading

Dahlmann, A. H., Mireskandari, K., Cambrey, A. D., et al. (2005). Current and future prospects for the prevention of ocular fibrosis.

Ophthalmology Clinics of North America 539–559.

Ferguson, M. W. J. and O’Kane, S. (2004). Scar-free healing: From embryonic mechanisms to adult therapeutic intervention.

Philosophical Transactions of the Royal Society B: Biological Sciences 359: 839–850.

Fini, M. E., Stramer, B. M., Fini, M. E., and Stramer, B. M. (2005). How the cornea heals: Cornea-specific repair mechanisms affecting surgical outcomes. Cornea 24(8 supplement): S2–S11.

Gabison, E. E., Huet, E., Baudouin, C., and Mensashi, S. (2009). Direct epithelial–stromal interaction in corneal wound healing: Role of EMMPRIN/CD147 in MMPs induction and beyond. Progress in Retinal Eye Research 28(1): 19–33.

Klenkler, B., Sheardown, H., Jones, L., et al. (2007). Growth factors in the tear film: Role in tissue maintenance, wound healing, and ocular pathology. Ocular Surface 5(3): 228–239.

Klenkler, B., Sheardown, H., Klenkler, B., and Sheardown, H. (2004). Growth factors in the anterior segment: Role in tissue maintenance, wound healing and ocular pathology. Experimental Eye Research 79(5): 677–688.

LaGier, A. J., Yoo, S. H., Alfonso, E. C., et al. (2007). Inhibition of human corneal epithelial production of fibrotic mediator TGF-beta2 by basement membrane-like extracellular matrix. Investigative Ophthalmology and Visual Science 48(3): 1061–1071.

Netto, M. V., Mohan, R. R., Ambrosio, R., Jr, et al. (2005). Wound healing in the cornea: A review of refractive surgery complications and new prospects for therapy. Cornea 24(5): 509–522.

Obata, H., Tsuru, T., Obata, H., and Tsuru, T. (2007). Corneal wound healing from the perspective of keratoplasty specimens with special

reference to the function of the Bowman layer and Descemet membrane. Cornea 26(9 supplement 1): S82–S89.

Peled, Z., Liu, W., Levinson, H., et al. (2000). Cellular strain upregulated profibrotic growth factors and collagen gene expression. Surgical Forum 51: 591–593.

Saika, S., Yamanaka, O., Sumioka, T., et al. (2008). Fibrotic disorders in the eye: Targets of gene therapy. Progress in Retinal and Eye Research 27: 177–196.

Stramer, B. M., Mori, R., and Martin, P. (2007). The inflammation–fibrosis link? A Jekyll and Hyde role for blood cells

during wound repair. Journal of Investigative Dermatology 127: 1009–1017.

Stramer, B. M., Zieske, J. D., Jung, J. C., et al. (2003). Molecular mechanisms controlling the fibrotic repair phenotype in cornea: Implications for surgical outcomes. Investigative Ophthalmology and Visual Science 44(10): 4237–4246.

Wilson, S. E., Liu, J. J., and Mohan, R. R. (1999). Stromal–epithelial interactions in the cornea. Progress in Retinal and Eye Research

18(3): 293–309.

Glossary

Autosomal dominant – The property of inheritance of a disease or trait from a single parent.

Chamber angle – This is the natural angle formed at the junction of the posterior limbus (at the trabecular meshwork), the ciliary body, and the iris.

Glycosaminoglycan – A general term for any sugar polymer of alternating sugars and aminosugars (aka a GAG).

Homeobox protein – A protein that is concerned with the embryological development of a multicellular organism. It is synthesized by a homeobox gene. Keratocyte – A cell type found in the corneal stroma that makes and maintains collagens and glycosaminoglycans (aka a stromal cell). Knock-out mouse – A genetically engineered mouse in which one or more specific genes have been made nonfunctional.

Schlemm’s canal – A vessel behind the trabecular meshwork through which the aqueous fluid of the eye can drain into the venous system.

Stem cell – A primitive cell that has not differentiated into a functional cell of a multicellular organism. Trabecular beam – A portion of the trabecular meshwork of the posterior limbus that consists of a rod of largely collagen material. When assembled in its typical complex pattern, trabecular beams offer resistance to the outflow of the aqueous fluid.

Transient amplifying cell – A cell in the process of differentiation from a stem cell as a functioning cell.

Anatomy

General Description

The corneal endothelium consists of a monolayer of polygonal cells, primarily hexagonal in shape, such that each has dimensions of 18–20 mm (width) by 5 mm (thickness). These cells produce a predominately collagenous product known as Descemet’s membrane that is sandwiched between the anterior basal side of the endothelial cells and the posterior layer of the stroma. Descemet’s membrane is a basement membrane that may act as a cushion for endothelial cell trauma and, in general, such basement membranes have been assigned roles associated

with cell adhesion, migration, differentiation, and signal transduction. However, the exact roles of this membrane are presently unknown. Endothelial cells continually synthesize Descemet’s membrane throughout life. On the posterior apical side, endothelial cells make direct contact with the anterior aqueous fluid in the anterior chamber. This pH neutral fluid (pH 7.4) is virtually absent in serum proteins and lipids, but contains nourishing glucose, antioxidants, and a variety of cytokines. At the boundary of the endothelium, endothelial cells are joined to a limbus whose transitional area is composed of cells that may contain precursor stem cells for the endothelium. The circular diameter of the corneal endothelium is approximately 11.7 mm in the adult. As with the remainder of the cornea, there are normally no blood vessels in this tissue. Although the anterior cornea does possess nerve endings, none are found in the endothelium (see Figures 1–3).

Cell-to-Cell Junctions

Corneal endothelial cells number about 3000 mm–2 in healthy, young adults and slowly decrease in number with age. The cells have a well-defined nucleus and their cytoplasm is packed with mitochondria necessitated by a high-energy requirement for the cells to act as a fluid pump. The cells also have a well-developed Golgi apparatus associated with the production of extracellular proteins needed for the assembly of Descemet’s membrane. The cell-to-cell junctions are somewhat tortuous and interdigitated, a form that helps to keep the cells together. The cellular membranes at these junctions are held together with well-described joining proteins, of which one assembled complex has been called a zonula occludens (tight junction). However, there is still a controversy about whether or not this is a true zonula occludens. The reason for this is that the channel joining adjacent endothelial cells must allow both sodium and water to flow into the anterior chamber. This junctional form has been described to be more focal or point-like than that of the true riveted tight junctions of a zonula occludens. Gap junctions have also been shown to exist between the cells for the purpose of transporting small molecules between each cell. Additionally, adhesion junctions to strengthen cell-to-cell fastening can be found there. The membranes facing the anterior chamber have a number of microvilli, typical of many endothelial cell types. However, no roles for the microvilli in these cells have been described (see

Figures 3 and 4).

Lens 11.7 0.50

0.70

Endothelium

Figure 1 Overview of the anterior segment of the eye showing the cornea with the endothelium labeled in red. Numbers

within the cornea are the cross-sectional dimensions in millimeters. The vertical line with the number represents the approximate diameter of an adult cornea in millimeters. Modified from Hogan, M. J., Alvarado, J. A., and Weddell, J. E. (1971). Histology of the Human Eye, p. 61. Philadelphia, PA: Saunders.

a b

c

d e

Figure 2 Cross-section of the human cornea. a, epithelium; b, Bowman’s membrane; c, stroma; d, Descemet’s membrane; e, endothelium. Adapted from Hogan, M. J., Alvarado, J. A., and Weddell, J. E. (1971). Histology of the Human Eye, p. 65. Philadelphia, PA: Saunders.

Embryology

Initial Development

It has been known for a long time that the corneal endothelium originates from neural crest, stem cells. These

a

b

Figure 3 Three-dimensional, representational sketch of the corneal endothelium (c) with attached Descemet’s membrane

(b) and posterior stroma (a). Endothelial cell microvilli are shown at (d). Intercellular channels are indicated at (e) while quasi-tight junctions occur at (f). Adapted from Hogan, M. J., Alvarado, J. A., and Weddell, J. E. (1971). Histology of the Human Eye, p. 101.

Philadelphia, PA: Saunders.

A

A

B

C

C

D E

Figure 4 Cross-section of human corneal endothelial cells. The large population of mitochondria are evident at (A) while an intercellular channel may be seen at (B). Two Golgi apparatus are pointed out at (C). The intercellular channel, labeled at (B) is shown to empty out at the aqueous chamber at (D). At (E) can be seen a microvillus. Modified from Hogan, M. J., Alvarado, J. A., and Weddell, J. E. (1971). Histology of the Human Eye, p. 103. Philadelphia, PA: Saunders.

cells migrate from folds of the neural ectoderm at weeks 4–5 of embryonic development. This is illustrated in Figures 5–7. Since the developing embryo is relatively small and neural crest cells are localized along each section of the neural folds, from which they detach, migration is not very distant for each group of cells. In eye development, optic sulci (the primordial eyes) appear as shallow pits along the neural plate at week 4. These sulci begin to protrude outward as hollow optic vesicles from the proencephalon or forebrain of the neural plate. At just over 30 days, a wave of mesenchymal cells (in this case, neural crest cells) migrates over the optic cup into the space between the anterior surface of the lens and the surface ectoderm (corneal epithelium). These cells will become

Neural folds

Neural crest

cells

Surface ectoderm

Neural

tube

(a)

Migrating neural crest cells

(b)

Figure 5 The developing neural folds and neural tube. As the neural folds move away from the neural tube (a and b), neural crest cells break away from the neural fold tissue (a) and begin their migration (b) to specific developing tissues. Modified from Forrester, J. V., Dick, A. D., McMenamin, P. G., and Roberts, F. (2008). The Eye. Basic Science in Practice, 3rd edn., p. 111. Edinburgh: Saunders.

Rhombencephalon

Mesencephalon

Migrating mesencephalic neural crest cells

Figure 6 Migration pathways of neural crest cells. Neural crest cells are shown in green. The neural crest cells migrating to the eye originate primarily from the proencephalon (developing forebrain and brainstem) and the mesencephalon (developing midbrain). Migration is shown by dark blue arrows. Adapted from Forrester, J. V., Dick, A. D., McMenamin, P. G., and

Roberts, F. (2008). The Eye. Basic Science in Practice, 3rd edn., p. 119. Edinburgh: Saunders.

the corneal endothelium. A second wave of mesenchymal cells (also neural crest cells) migrates around day 49 and places itself between the endothelium and epithelium. These cells are destined to become the keratocytes of the corneal stroma. Based on mouse model studies, it appears that those neural crest cells that will define the corneal endothelium at first remain in contact with the anterior lens and flatten out into a monolayer of cells. Then, the lens detaches from the endothelial monolayer to provide space for an anterior chamber where the aqueous fluid may enter (Figures 8 and 9). During this

process, a third wave of neural crest cells migrates to the angle between the posterior cornea (endothelium) and the anterior edge of the optic cup (Figure 10). These cells eventually develop into the ciliary body and the iris.

During this development, the tissues anterior to the chamber angle between the anterior eye cup and the endothelium becomes occupied by a mass of mesenchymal (neural crest) cells that remain, at first, undifferentiated. These cells develop into flat endothelial-like cells that bring about the trabecular beams (trabecular meshwork) and, separately, Schlemm’s canal cells. Some of the stellate cells, between the trabecular beams and the endothelial lining of Schlemm’s canal, appear to remain undifferentiated. Evidence to support this is seen in a number of stem cell markers in this area which change with wounding (Figure 11). This is an important observation as it suggests the retention of stem cells in a niche for the replacement of cells in the posterior limbus and for the corneal endothelium. The point is made again that evidence strongly suggests that nearly all the mesenchymal cells that invade these areas of the anterior segment are neural crest in origin.

Role of Transcription Factors

Molecular biological mechanisms that control the overall development of the anterior segment remain incompletely described. This is also true for the corneal endothelium. What is known can explain some developmental effects. It is understood, for example, that inductive signals from the lens are partly responsible for endothelial cell differentiation. Defects in three lens genes that produce the homeobox proteins – MAF, FOXE3, and PITX3 – result in the inability of the lens to separate from the cornea (Figure 12).) Homeobox proteins assemble in a specific binding area of DNA to signal a specific RNA synthesis. They are required to bind to DNA in a set order for synthesis to begin. The three genes mentioned are concerned with producing genetic transcription factors that cause the synthesis of proteins necessary for lens–cornea separation. Other genetic abnormalities may also occur in the mesenchymal (stem) cells themselves that cause corneal development. In the endothelium itself, mutations in the genes PITX2, FOXC1, and PAX6, for example, are known to prevent satisfactory, functional corneal development. Normally, these genes produce the transcription factors for protein synthesis related to such development. PAX6, in particular, is required for making signaling molecules that cause transport of neural crest cells into the eye as well as the phenotypical development of the corneal endothelium itself. In addition, the amounts and origins of PAX6 proteins appear to be critical for the sequence signaling of corneal development. One signaling molecule that may be produced as a result of DNA binding of such transcription factors is transforming

End of week 4

Neural crest cells streaming over optic cup and stalk

Lens placode

Retinal disk

Cavity of optic vesicle

Optic stalk

Start of week 7

Day 44 (13–17 mm)

Tunica vasculosa lentis

Hyaloid artery

Developing sclera

Developing choroid

Lids forming

Mesenchyme between lens and surface ectoderm – future corneal endothelium and stroma

growth factor, beta 2 (TGF-b2). In TGF-b2 knock-out mice, that cannot produce this protein due to a lack of PAX6, the corneal endothelium is completely absent. It is also known that the overexpression of TGF-b1 (a related molecule using similar receptors) results in an absent endothelium. So, the case is made that these controlling proteins must be present in specific amounts and at the right time sequence to allow proper corneal endothelial cell development to occur.

Biochemistry and Metabolism

Glucose and Energy Metabolism

The corneal endothelium is a cell type that is required to have large outputs of energy to maintain the process of

deturgescence (discussed in the section entitled ‘Proteins synthesized for external transport’). In this regard, corneal endothelial cells maintain a high amount of adenosine triphosphate (ATP)-producing mitochondria as well as a defined Golgi apparatus for the complete synthesis, retention, and export of proteins. In studies of comparative carbohydrate metabolism, it has been shown that the amount of aerobic glycolysis (glucose breakdown to produce ATP) is 3 times higher than it is in the cells of the corneal epithelium and stromal keratocytes. Compared to the cells of the lens, corneal endothelial cells use aerobic glycolysis at better than 6 times the amount used by the lens. On a per cell basis, it is even estimated that corneal endothelial cells produce more ATP energy than individual cells of the ciliary body by 40%. Only cells of the retina exceed the cells of the corneal endothelium in