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The cornea, subject of this thesis, is a transplantable tissue due to its avascularity and immunologic privilege. It consists of five anatomical layers from anterior to posterior: the epithelium and its basement membrane, Bowman layer, the stroma, Descemet membrane and the endothelium (Figure 2).
Figure 2. The anatomical layers of the cornea.
Both Descemet membrane and the endothelium are the most important layers involved in endothelial keratoplasty, hence, we will focus on the description of these structures.
2.2.1 DESCEMET MEMBRANE
Descemet membrane is a corneal structure anteriorly in contact with the stroma and posteriorly with the endothelium. It conforms the basement membrane of the endothelium and it is composed by collagen (mainly type IV) and laminin. Apparently, endothelial cells lay down this membrane by secreting different components, becoming therefore, thicker with advancing age.1-3
Its total thickness in adulthood is about 10 µm and three thinner layers may be distinguished: A thin nonbanded layer (0.3 µm) adjacent to the stroma, an anterior banded zone (2-4 µm) and a posterior amorphous nonbanded zone (>4 µm).2 In Fuchs endothelial dystrophy, an atypical striated collagen deposition has been described in the posterior layer.4 Furthermore, pathological central protrusions or warts (i.e. guttata) can characteristically be observed in the disease, probably related with the presence of an abnormal fibroblastic endothelial phenotype.
Due to the friable interconnections between Descemet membrane and the posterior stroma, Descemet membrane may be easily stripped off, making it feasible to prepare isolated grafts of Descemet membrane and its endothelium suitable for endothelial transplantation, i.e. Descemet membrane endothelial keratoplasty (DMEK).5,6 When Descemet membrane is peeled from a donor corneo-scleral rim, it rolls up with the endothelium on its outer surface, probably due to the elastic properties of the tissue.5-8
2.2.2 THE ENDOTHELIUM
The human corneal endothelium conforms a single layer of cells covering the posterior cornea with an hexagonal pattern. Since birth, endothelial cell density (ECD) decreases 2.9% per year until adulthood, with a further decrease of 0.6% per year thereafter; resulting in an ECD of about 2000-2500 cells/mm2 at an elderly age.9,10
Apart from aging, other factors can induce an ECD decline such as: traumatic intraocular surgery, inflammatory reactions in the anterior chamber and considerable intraocular pressure increase.9-11
Indicators of endothelial cell loss are the presence of variations in the size or cell area (polymegathism) and in shape or hexagonality (polymorphism) of endothelial cells. Both pleomorphism and polymegathism increase with age, and can also be observed in Fuchs endothelial dystrophy.9-12
a) Endothelial migration and proliferation
Endothelial cells in vivo do not replicate as far as the literature is concerned.
However, several studies suggest that healthy endothelial cells inherently present the ability to replicate, but do not proliferate because they 'remain arrested in the G1-phase instead of finishing the cell cycle'.13 Hence, many efforts have been made in order to find the factors that refrain the endothelium from proliferating: lack of autocrine or paracrine stimulation by positive mitogenic growth factors, age, negative regulation from transforming growth factor (TGF-)beta (that in aqueous humor suppresses the S-phase entry), and cell contact inhibition, in part, via the activity of p27kip1, a known G1-phase inhibitor.13,14
Relatively recent studies have proved that endothelial cells have the capacity to proliferate in vitro, grow in tissue culture,15,16 and regenerate in donor corneas in eye bank organ culture up to 7 days postmortem.17 Furthermore, several methods have been shown to enhance endothelial cell proliferation such as: mechanical wounding,18 ethylenediaminetetraacetate acid (EDTA) with mitogens by braking endothelial cells' tight junctions,19 viral oncogenes transformation,20,21 culture medium usage with positive growth factors such as epidermal growth factor (EGF), nerve growth factor (NGF) or basic fibroblast growth factor (bFGF),22,25 and addition of an animal-derived extracellular matrix (ECM).23,24 However, these processes may still show some limitations like restricted cell output with untransfected cultures, questionable long-term survival of transfected endothelium, and the 'impossibility of subculturing endothelial cells for more than a few passages'.13,25,26
Replication of central endothelial cells versus peripheral cells
Endothelial cell density in a normal cornea increases from the center toward the periphery.27,28 In the presence of damaged endothelium, the defect is repaired by a process similar to wound healing, that is, by proliferation and migration of mesenchymal cells from the periphery to the central cornea that will most likely stop by cell contact inhibition.13 Hence, it has been suggested that peripheral endothelial corneal cells could present a greater capacity to proliferate than the central endothelium. Nevertheless, several studies in vitro have demonstrated that there is no significant difference in the progression of endothelial cells depending on its relative location within the endothelium, and that it may be the presence of a low endothelial cell density (< 2000 cells/mm2) more than its position, what seems to be the indirect stimulus for cell replication in culture medium. However, the different processes involved may still need to be elucidated.
Replication of endothelial cells depending on age-related differences
Another determinating factor in the ability of endothelial cells to replicate under mitogenic stimulation may be age.29 Consequently, it has been shown that cells from older donors (>50 years old), although able to finish the cell cycle, present a longer G1-phase and need a more intense mitogenic stimulation than younger donors (<30 years old).18,29 Furthermore, older donors respond slower and to a less extent to mitogenic stimulation, cell culture and mechanical wounding, in comparison with younger donors.30
In vivo, some modifications related with age may also influence the relative proliferative capacity of the cells. It seems that with age, endothelial cells enter a 'replicative senescence-like state in which they become increasingly refractive to mitogenic stimulation'.30 However, this senescent status is not generally found in vivo because cell replication is actively inhibited.
Even though the etiology for these age-related changes have not been clearly determined, 31,32 it seems that they could be related with cyclin-dependent kinase inhibitors. Consequently, the presence of CKIs, p27KIP1, p16INK4a, and p21CIP1 has been shown in endothelial cells in vivo.33
Cellular telomere shortening has been considered a possible mechanism to estimate the number of cell-cycle divisions undergone by the cells.34 Moreover, endothelial cells in young and old donors seem to present relatively long telomeres, suggesting a low replication rate during their lifetime. Hence, the refractive response to proliferation in older donors may not be explained by replicative senescence due to short telomeres.30
Furthermore, it has been proposed that environmental stresses, including oxidative stress, could be related with the age-related replicative modifications observed in the corneal endothelium.31,35
Viral oncogene transformation of human corneal endothelial cells
The replication of endothelial cells can be achieved by its transformation with the expression of viral oncogenes such as the SV40 large-T antigen and E6/E7 from the human papilloma virus.36,37 These viral oncogenes, have been reported to provide a high proliferative capacity, by 'bypassing the physiologic cell cycle inhibitory mechanisms'.13 Furthermore, these cells have been shown to maintain their physiologic morphology.
Growth factor-induced signal transduction in corneal endothelial cells
There is limited knowledge about growth factor-induced proliferation of corneal endothelial cells; moreover, efficient culture techniques for endothelial cells still require further development.26
It has been proven a positive response of endothelial cells in vitro to several growth factors such as epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2) and serum.13,22
Furthermore, it has been recently demonstrated that 25% ESC-CM (mouse embryonic stem cell conditioned medium) enhances endothelial cell proliferation, by stimulating cell colony formation, decreasing cell apoptosis and necrosis, and inhibiting the expression of the cell-cycle related protein p21.26,38-41 Moreover, many cytokines/growth modulators have been described to be secreted by ESC: interleukins IL-1α, IL-10, and IL-11, colony stimulating factors M-CSF (macrophage-colony stimulating factor) and GM-CSF (granulocyte macrophage-colony stimulating factor), EGF (epidermal growth factor), FGF-basic (fibroblast growth factor-basic), FGF-9 (fibroblast growth factor-9), OSM (oncostatin M), SCF (stem cell factor), VEGF (vascular endothelial cell growth factor), IFN-γ (interferon-γ), insulin, LIF (leukemia inhibitory factor), and TNF-α (tumor necrosis factor-α).26
b) Corneal transparency and hydration: endothelial barrier and pump function
Corneal architecture is a major determinant of corneal transparency. The regular arrangement of stromal collagen fibers and the constant distance between each other maintained by the interfibrillar proteoglycans,42-45 may explain in part this phenomenon. Since both, fibers' diameter and interfibrillar distances are smaller than light's wavelength, a destructive interference of the scattered light and a constructive interference of the incident light is created; allowing a transmission of 90% of the incident light through the cornea.46,47
The dysfunction in any of the corneal components can reduce corneal transparency and cause an important function loss. For example, if corneal edema occurs, stromal swelling induces an increase in the interfibrillar distance, producing light scattering and corneal haze.48
Both corneal epithelium and endothelium, play important roles in maintaining corneal hydration and transparency. Physiologically, they behave as mechanical barriers to fluid diffusion (tears or aqueous humour)42 and create a gradient by cellular active ion transport mechanisms inducing the osmotic movement of water out of the hydrophilic stroma. The ideal physiological corneal water content is approximately 78% (3.8 mg water/mg dry weight) and an increase or decrease from this ideal level can result in corneal opacity.42,49-51
Endothelial barrier and pump function
Several mechanisms in the cornea draw fluid out from the stroma, preventing stromal swelling and loss of transparency.
Endothelial cells are laterally interconnected at the most apical part by tight junctions, that selectively allow the movement of nutrients from the aqueous humour into the stroma.52 Mainly three ion transport systems have been identified or postulated to exist in the corneal endothelium (Figure 3). First, the Na,K-ATPase enzyme, is the better characterized ion transporter of corneal endothelial cells. Localized at the basolateral membrane, it actively pumps sodium ions from the stroma into the aqueous humour 42,53-57 creating an osmotic gradient that induces the passive diffusion of water in the same direction. The adequate functioning of this system is critical in order to maintain a normal corneal hydration. Secondly, at the basolateral membrane of the endothelial cell, a sodium-hydrogen exchanger has been identified, moving sodium into the cell and hydrogen ions outward.58 Third, some evidence has been found on the existence of a bicarbonate-sodium cotransport, moving both ions out of the cell (Figure 3).51
Figure 3. Ion transport systems and anhydrase carbonic (CA) function in the corneal endothelium.
Next to the ion transport systems, the carbonic anhydrase has an important role in the intracellular conversion of carbon dioxide to hydrogen ion and bicarbonate, being both transported out of the cell by the basolateral hydrogen exchanger and the apical bicarbonate transport, respectively. The transport of bicarbonate creates a negative potential in the aqueous humour in respect with the stroma, contributing to the sodium osmotic gradient by generating a movement of sodium ions from the lateral spaces between cells into the aqueous humour. Moreover, the sodium-hydrogen exchanger acidifies the extracellular fluid, increasing the diffusion of carbon dioxide into the cell, and therefore the production of bicarbonate and the concentration of sodium in the aqueous humour. Hence, the active transport of bicarbonate is presumed to be a major contributor to the endothelial pump system. Carbonic anhydrase inhibitors can result in corneal swelling by inhibition of this process.59,60
After endothelial trauma, adjacent undamaged cells migrate and enlarge covering the defect, creating a temporary barrier with minimal pump sites and incompletely formed tight junctions. Furthermore, the pump sites and tight junctions are completely restored and corneal transparency is accomplished, even though endothelial cells may still form irregular hexagons. Finally, endothelial cells are remodeled creating regular polygons.61
However, this condition may be mainly observed in healthy corneas,62 because in patients with diseased endothelium, i.e. Fuchs endothelial dystrophy, the wound healing activity may be somehow altered. If endothelial cell density decreases sufficiently with no proliferation (range 750-250 cells/mm2), the maximal metabolic pump function may not be enough to compensate the 'leak' and the cornea may not clear up, resulting in corneal edema.63