Ординатура / Офтальмология / Английские материалы / Dry Eye and Ocular Surface Disorders_Pflugfelder, Beuerman, Elliot Stern_2004

Figure 3 Autoradiograms showing the response of corneal and limbal epithelia to treatment with phorbol ester [16]. Low tritiated thymidine incorporation is evident is the control panels a and b. A single exposure of phorbal myristate markedly increases tritiated thymidine incorporation in panels c and d, whereas 2 days of phorbal myristate traetment results in decreased incorporation. (a) Corneal epithelium, petrolatum (control) treatment.

(b) Limbal epithelium, petrolatum (control) treatment. (c) Corneal epithelium, 1 phorbal myristate treatment. (d) Limbal epithelium, 1 phorbal myristate treatment. (e) Corneal epithelium, 2 days of phorbal myristate treatment. (f) Limbal epithelium, 2 days of phorbal myristate treatment.

microenvironment of these cells, as indicated by their eventual senescence. Therefore, the true proliferative reserve of stem cells relative to the life span of the organism is impossible to determine at present.

Clinical evidence supports the limbal region as the site of corneal stem cells [36,42–45]. Destruction of the limbal epithelium by physical or chemical insult induces a stem cell-deficient state. Clinical features of limbal stem cell deficiency include abnormal wound healing with persistent or recurrent epithelial

defects, conjunctivalization (conjunctival epithelial ingrowth), vascularization, loss of corneal clarity, and chronic inflammation. Additionally, the limbus is the most common site of ocular surface neoplasias. They likely arise from altered growth behavior of undifferentiated progenitor cells, suggesting that a corneal intraepithelial neoplasm is essentially a stem cell tumor.

IV. THE LIMBUS: A STEM CELL MICROENVIRONMENT

Since the corneal epithelium must provide a transparent medium for vision, it is devoid of pigmentation, and has a smooth stromal-epithelial junctional structure. Accordingly, corneal epithelial cells are vulnerable to shearing injury because of their poor adhesion to the underlying stroma, as evident in patients with recurrent corneal erosions following relatively minor corneal injuries.

The anatomical structure of the limbus is significantly different from the adjacent cornea because it need not be transparent. It is well suited to harbor and protect corneal stem cells. Stem cells in the body are usually located in deeper tissue layers, presumably for protection. The limbal epithelium is 8–10 cell layers thick, compared with 5 layers in the corneal epithelium. The limbus tends to be heavily pigmented, especially in pigmented races; this may protect basal cells from carcinogenic effects of ultraviolet radiation [28,46]. In addition, the palisades of Vogt have an undulating epithelial–stromal junction, which provides greater adhesion properties, thereby rendering the limbal epithelium resistant to shearing forces. These folds also greatly increase the surface area of the basal cells. The stromal component of the limbus is well innervated, and is supplied by a rich vascular network, allowing regulation of limbal stem cell growth and proliferation through various cytokineand neural-mediated pathways. An appropriate stromal microenvironment (stem cell niche) is probably important for correctly regulated stem cell activity.

V.TRANSIENT AMPLIFYING CELLS

Transient amplifying cells play an important role in wound healing. When slowcycling limbal stem cells are activated by a demand for tissue regeneration, such as wounding, they give rise to daughter transient amplifying cells that migrate centrally or superficially to replenish the population of corneal epithelial cells [5]. Transient amplifying cells have shorter cell cycle times, resulting in rapid cell division, and have a limited proliferative capacity. They probably undergo a predetermined number of cell divisions before differentiating into postmitotic cells, which in turn terminally differentiate and replenish the diminished epithelial cell population.

Z vector represents the overall direction of corneal epithelial cell movement due to a combination of X and Y forces, from the basal peripheral region, to the superficial central aspects of the cornea. Maintenance of the corneal epithelium involves a balance of these processes, defined by the equation: X + Y = Z. Constant renewal results in complete turnover of corneal epithelial cells every 7–10 days. Following corneal injury, these regenerative mechanisms must be accelerated to replace lost corneal epithelial cells. Other investigators have also demonstrated epithelial cell migration from the peripheral cornea and limbus [33,48–50].

Corneal epithelial defects, regardless of the nature of injury, result in a fairly consistent pattern of re-epithelization [36,51]. Three to six convex leading fronts of migrating epithelial sheets develop around the circumference of the defect and progress toward the center. The advancing fronts of epithelium eventually meet and merge imperceptibly to repopulate the entire surface [51]. Notably, healing rates for larger (8-mm-diameter) corneal epithelial defects are more rapid (mean rate 0.91 mm2/h) than for smaller (4-mm-diameter) defects (mean rate 0.37 mm2/h), consistent with a greater proliferative response of cells in the peripheral cornea and limbus than in the central cornea [52]. Regarding the mechanism of centripetal migration, Lavker et al. suggested that they are drawn inward by preferential desquamation of central corneal epithelial cells rather than by forcing their way toward the center [46].

VII. CONJUNCTIVAL STEM CELLS

Corneal and conjunctival epithelia are now believed to arise from different stem cell populations [53]. Consistent with this, transdifferentiation of conjunctival epithelial cells in a corneal stromal environment appears incomplete [10,54]. Conjunctival epithelium transplanted to the cornea of limbal stem cell-deficient patients retained many characteristics of conjunctival tissue, such as its glycogen content and goblet cells [55,56].

Patterns of cytokeratin expression under identical cell culture conditions provided direct evidence for separate lineages of conjunctival and corneal cells [53]. Conjunctival epithelial cells expressed K4 and K13 cytokeratins, whereas corneal epithelial cells expressed K3 and K12 [36,53]. Additionally, when conjunctival and corneal epithelial cell suspensions were injected subcutaneously into the flanks of athymic mice, cysts resulting from injection of limbal and corneal epithelial cells retained features of normal corneal epithelium, a stratified squamous epithelium without goblet cells, whereas cysts derived from conjunctival epithelial cells displayed normal conjunctival morphology, a stratified epithelium interspersed with numerous goblet cells [37,53].

The mixed population of keratinocytes and goblet cells observed in these cysts was ultimately derived from a single cultured conjunctival epithelial cell.

Therefore, both cell types must have descended from a bipotent progenitor cell. Conjunctival keratinocytes differentiate into goblet cells at fairly specific times during the life span of transient amplifying cells, suggesting that the decision to differentiate into a goblet cell depends on an intrinsic cell doubling clock [35]. The preponderance of evidence indicates that conjunctival epithelial stem cells are bipotent, and divergence of some keratinocytes into a goblet cell differentiation pathway probably occurs late in the process.

VIII. LOCATION OF CONJUNCTIVAL STEM CELLS

The nonkeratinized conjunctival epithelium extends from the corneal limbus to the lid margin, where it gradually transitions to keratinized, stratified squamous epithelium. The conjunctival epithelium provides a mechanical and immunological barrier to injury and infection, and its numerous mucin-secreting goblet cells contribute to the production and stability of the tear film.

The forniceal conjunctiva of rabbits and mice appears to be enriched in conjunctival stem cells. Using the pulse labeling approach described earlier, a significantly greater percentage of slow-cycling cells (probable stem cells) were found in the murine forniceal epithelium than in the bulbar and palpebral conjunctiva [55]. Following stimulation with a tumor-promoting compound, forniceal basal cells displayed a significantly greater and more sustained proliferative response than cells from other regions (Fig. 5). Cells from the forniceal conjunctiva of rabbits showed greater colony-forming efficiency and greater numbers of serial subcultures than cells from the bulbar and palpebral regions, which generated small colonies and could not be subcultured more than once [36]. Thus, a cell population with the properties of stem cells is evident in the forniceal epithelium of rabbits and mice (Fig. 6).

Although the conjunctival fornix appears to contain the greatest proportion of stem cells, pockets of conjunctival stem cells may also exist throughout the conjunctival epithelium. This could explain observations by other investigators who analyzed the in-vitro proliferative capacities of the different conjunctival regions, and concluded that stem cells may be uniformly distributed over the bulbar and forniceal conjunctiva [35]. The high density of goblet cells in the fornix may be due to the concentration of bipotent conjunctival stem cells there [55,56]. More scattered conjunctival stem cells in the bulbar and palpebral conjunctiva may give rise to the dispersed pockets of goblet cells seen within these regions.

Another study suggested that the mucocutaneous junction at the lid margin might also be enriched in conjunctival stem cells, and serve as a source of epithelial cell replacement for the palpebral and forniceal conjunctiva [57]. However, the pulse labeling method employed in these experiments essentially labeled transient amplifying cells rather than conjunctival stem cells.

Figure 5 Autoradiograms showing the response of bulbar, forniceal, and palpebral conjunctival epithelia to treatment with phorbol ester [16]. A single exposure to phorbal myristate markedly increases tritiated thymidine incorporation (d, e, and f), most notably in the fornical epithelium. After 2 days of treatment, incorporation into bulbar and palpebral epithelia has decreased (g and j), whereas the fornical epithelium shows a greater proliferative capacity, indicated by continued incorporation (h). (a) Bulbar epithelium, petrolatum (control) treatment. (b) Fornical epithelium, petrolatum (control) treatment. (c) Palpebral epithelium, petrolatum (control) treatment. (d) Bulbar epithelium, 1 phorbal myristate treatment. (e) Fornical epithelium, 1 phorbal myristate treatment. (f) Palpebral epithelium, 1 phorbal myristate treatment. (g) Bulbar epithelium, 2 days of phorbal myristate treatment. (h) Fornical epithelium, 2 days of phorbal myristate treatment. (i) Palpebral epithelium, 2 days of phorbal myristate treatment.

Clustering of stem cells into localized regions is a recurring finding among stratified epithelial tissues, for example, corneal stem cells are located in the limbal region, interfollicular epidermal stem cells are located in the bottom of deep rete ridges, and epidermal stem cells are concentrated in the bulge area of the hair follicle. Consistent with this, the fornix is located well within the upper and lower recesses created by the closely apposed eyelid and globe, farther from the external environment than the other conjunctival regions, and is able to protect conjunctival stem cells from extrinsic insult and injury. The stroma of the fornix comprises a network of collagen and elastic fibers, protecting the epithelial cells from shearing and mechanical forces. The fornix is also the most richly

Figure 6 Schematic diagram showing the relative densities of label-retaining cells in the palpebral, forniceal, and bulbar conjunctiva in the mouse model. The highest concentration is noted in the forniceal conjunctiva (F), which is believed to be the site enriched in conjunctival stem cells. It also represents the site with the highest density of goblet cells. E, epidermis; T, transitional zone between palpebral conjunctiva and epidermis (muco–cutaneous junction); P, palpebral conjunctiva; F, fornix conjunctiva; B, bulbar conjunctiva; L, limbus; C, cornea.

vascularized and innervated region of the conjunctiva, allowing prompt response to cytokine or neural stimuli. Thus, the fornix provides a suitable microenvironment for maintaining conjunctival stem cells.

IX. EPITHELIAL–STROMAL INTERACTIONS AND THE STEM CELL MICROENVIRONMENT

Both intrinsic factors (inherent to the cell), and extrinsic factors (environmental factors surrounding the cell) are thought to be involved in the regulation of stem

cells [58,59]. Schofield proposed that stem cells existed in a microenvironment that helped maintain their undifferentiated state [59]. In-vitro cultures of limbal and corneal epithelial cells have demonstrated effects of growth factors and extracellular calcium on the growth and proliferation of these cells [38–41,59–61]. Under similar culture conditions, limbal epithelial cells proliferated faster and were more resistant to tumor-promoting compounds than central corneal epithelial cells [62].

Limbal basal cells express higher levels of epidermal growth factor receptor (EGFR) compared with more mature and differentiated cells, such as basal cells of the central cornea [63]. Greater concentrations of EGF receptors might allow these cells to be rapidly stimulated by growth factors to undergo cell division during development and following wounding.

Limbal basal cells also express intermediate filaments, cytokeratin 19, vimentin, α6 β4-integrin, metallothionein, transferrin receptor, and a protein bound by monoclonal antibody AE1 [64–66]. Intermediate filaments are involved in maintenance of cell cytoarchitecture, and may play a role in anchorage of these cells to underlying tissues. This expression profile is unique to limbal basal cells, and differs from that of surrounding basal cells.

Other proteins present in higher concentrations in limbal basal cells than in central corneal basal cells include metabolic enzymes, such as Na-K-ATPase, cytochrome oxidase, and carbonic anhydrase [3,61,67]. Differences in the concentrations of these proteins may reflect inherent differences in the physiological and metabolic characteristics of these cells.

Long-term survival and serial propagation of epidermal cells is possible when they are co-cultured with 3T3 fibroblast feeder layers [68,69]. This system has been used successfully for cultivation of epithelial cells, including ocular surface epithelial cells. Growth properties of epidermal and ocular surface stem cells appear to be preserved in the 3T3 feeder system [30,34,68,70]. The identitiy of growth-promoting factors or antiapoptotic factors arising from the mesenchymal–epithelial interaction remains to be determined.

The basement membrane of the limbus differs from that of the central cornea. The basement membrane of the central corneal epithelium contains a protein identified by monoclonal antibody AE27, also present in low amounts in the limbal area [71]. Conversely, collagen type IV is abundant in the basement membrane of the limbus, but absent from the cornea. The basement membranes of human corneal and conjunctival epithelium can be divided into at least three domains: the conjunctival basement membrane (type IV collagen-positive, AE27weak), the limbal basement membrane (type IV collagen-positive, AE27-strong), and corneal basement membrane (type IV collagen-negative, AE27-strong). Basement membrane heterogeneity may play a functional role in regulating keratin expression and other aspects of corneal epithelial differentiation. These features, together with the anchoring fibrils of the limbus, might enhance the adhesion of the basal cells to the underlying stroma.

Stromal–epithelial interactions are believed to be extremely important in supporting normal corneal function. Intercellular communications between the corneal stromal and epithelial cells that are critical during early development, homeostasis, and wound healing are mediated by a variety of cytokines [72–74]. Various growth factors, such as transforming growth factor-β (TGF-β), plateletderived growth factor B (PDGF-B), and interleukin-1 (IL-1) are synthesized by epithelial cells, while receptors for these factors are found among stromal fibroblasts [72]. The best characterized stromal to epithelial interaction in the cornea is mediated by hepatocyte growth factor (HGF), expressed by corneal fibroblasts, and by keratinocyte growth factor (KGF), expressed mostly by limbal fibroblasts [73–75]. Because KGF plays an important role in wound healing, the uniquely high expression of KGF may be important for regulation of proliferation, motility, or differentiation during epithelial stem cell division in wound healing. These findings suggest regulation of limbal stem cells by their microenvironment, including epithelial–stromal exchange of growth factors and cytokines.

Identification of stem cells is critical to our understanding of the normal homeostatic mechanisms that regulate proliferation and maintenance of tissues in the body. Stem cells remain poorly characterized because molecular markers that can conclusively distinguish stem cells from transient amplifying cells have yet not been identified. Strategies and markers used for identification of epithelial stem cells can also be applied to limbal stem cells.

X.STRATEGIES FOR IDENTIFICATION OF EPITHELIAL STEM CELLS

Keratinocytes with high levels of α6-integrin and low to undetectable expression of the transferrin receptor (CD71) represented a small, quiescent subpopulation of cells with a high nuclear to cytoplasmic ratio and a high proliferative capacity. Approximately 70% of these cells retained tritiated thymidine after a prolonged chase. All of these are properties of stem cells. Conversely, the majority of actively cycling basal cells, most likely transient amplifying cells, contained high levels of both α6-integrin and CD71 receptor. Cells with low α6-integrin expression had limited proliferative capacity, and expressed differentiation markers, indicating they were postmitotic [76].

Stem cells can also be identified by their proliferative capacities in vitro [29,30]. Three types of keratinocytes with different capacities for proliferation have been identified from the human epidermis, holoclones, meroclones, and paraclones. The holoclone has the highest proliferative capacity, is able to undergo 120–160 divisions with less than 5% terminally differentiated colonies, and is considered an epidermal stem cell. The paraclone is able to undergo 15 cell divisions with 95% of colonies containing terminally differentiated cells, and

represents a transient amplifying cell. The meroclone is an intermediate cell type which represents a reservoir of transient amplifying cells [29,30]. Clonal analysis showed that nuclear protein p63 was abundantly expressed by epidermal and limbal holocolones, but was undetectable in paraclones, suggesting that p63 might be a marker for keratinocyte stem cells [77]. Transient amplifying cells displayed greatly reduced p63 expression immediately after their withdrawal from the stem cell compartment (meroclones), even though they still possessed appreciable proliferative capacity.

Alpha-enolase, another possible candidate corneal epithelial stem cell marker, was localized to K3-negative limbal basal cells [14,78,79]. The number of K3-negative, enolase-positive limbal basal cells increased following corneal epithelial injury. However, α-enolase is not a specific marker of stem cells because it is also present in basal cells of other stratified epithelia [80].

Cell–cell communication plays an important role in cellular development and differentiation. Gap junction channels, formed by a family of related amphipathic polypeptides called connexins, allow direct passive diffusion of low- molecular-weight solutes between neighboring cells. Two connexins, connexins 43 and 50, are abundantly expressed in corneal epithelial cells but are absent from the limbus [81]. Consistent with this, dye transfer studies indicate no gap junction mediated cell–cell communication in the limbus.

XI. CAUSES OF LIMBAL STEM CELL DEFICIENCY

Limbal stem cell deficiency can be caused by a variety of hereditary or acquired disorders. Inherited disorders include aniridia keratitis and keratitis associated with multiple endocrine deficiency, in which limbal stem cells may be congenitally absent or dysfunctional. Acquired disorders associated with deficient or destroyed stem cells are the majority of cases seen clinically, including StevensJohnson syndrome, chemical injuries, ocular cicatricial pemphigoid, contact lensinduced keratopathy, multiple surgeries or cryotherapies to the limbal region, neurotrophic keratopathy, and peripheral ulcerative keratitis.

XII. CLINICAL PRESENTATION OF LIMBAL STEM CELL DEFICIENCY

Limbal stem cell deficiency results in abnormal healing and epithelization of the cornea. It is characterized by persistent or recurrent epithelial defects, ulceration, corneal vascularization, stromal inflammation and scarring, and conjunctivalization (conjunctival epithelial ingrowth), with resultant loss of the clear demarcation between corneal and conjunctival epithelium at the limbal region [5,44,45,82].