Overview
The human cornea consists of an outer stratified epithelium, and an inner monolayer of epithelial cells referred to as the corneal endothelium. The middle layer, or stroma, constitutes 90% of the thickness of the cornea and is primarily a structural matrix of collagen fibrils embedded with transparent cells (keratocytes). The structural integrity of the stroma is essential for maintaining corneal shape, strength, and transparency. All of these features are attributed to the precise alignment and spacing of the stromal collagen fibrils and associated proteoglycans, which provide a clear, undistorted optical path for vision. If the cornea is damaged by trauma, surgery, or disease, a wound-healing response rapidly begins in order to prevent infection and restore vision. In other tissues it is sufficient for wounds to heal with replacement connective tissue, in which the collagen structural organization appears to be random, resulting in scarring. Since wound healing in the cornea has the additional requirement for transparency in order to maintain clear vision, precise repair of the matrix by the corneal cells must occur while maintaining the organization of the stromal connective tissue.
Stromal keratocytes ( Figure 2.1 ) are quiescent, mesenchymal-derived cells that form a network connected by gap junctions. Keratocytes appear transparent because they have a refractive index similar to that of the surrounding extracellular matrix (ECM). This has been attributed to the presence of high concentrations of soluble proteins (corneal crystallines) in the cytoplasm of the keratocytes. The first step in corneal repair is apoptosis of keratocytes immediately surrounding the site of trauma. Following that, keratocytes bordering the acellular zone are activated and become visible corneal fibroblasts. The fibroblasts proliferate and migrate to the margin of the wound in response to a number of growth factors and cytokines derived from the epithelial cells, the adjacent basement membrane, or tears. In response to transforming growth factor-β (TGF-β) some of the fibroblasts differentiate into nonmotile myofibroblasts containing α-smooth-muscle actin (α-SMA) and large focal adhesions, which promote a strong adherence to the ECM ( Figure 2.2 ). After attachment, alpha-SMA stress fibers (a defining characteristic of the myofibroblast phenotype) are formed ( Figure 2.3 ). These are required for myofibroblasts to exert tension on the matrix and close the wound. The fibroblasts and myofibroblasts secrete new ECM that initially appears opaque, resulting in a visual haze experienced by individuals during the corneal repair process. If the wound heals correctly, the myofibroblasts and fibroblasts gradually disappear, leaving a properly organized, transparent network of collagen fibrils once again embedded with a quiescent network of keratocytes. Conversely, if normal wound healing is compromised, for example if myofibroblasts persist or the source of the trauma remains, corneal fibrosis may develop due to the presence of excessive repair cells and consequently an excessive build-up of ECM in the stroma ( Box 2.1 ).
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After wounding, transparent keratocytes differentiate into migratory fibroblasts
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Fibroblasts migrate into the wound margin
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At the wound margin fibroblasts differentiate into nonmotile, contractile myofibroblasts
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After wound closure, myofibroblasts disappear
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The persistence of myofibroblasts in a wound correlates with fibrotic healing
Clinical manifestations of wound healing
The key sign of corneal fibrosis is the presence of haze in the cornea that impairs an individual’s ability to see clearly. A variety of conditions lead to fibrosis including corneal ulcers that can result from genetic factors such as hereditary keratitis, which is passed on through autosomal dominant inheritance ; a secondary response to an autoimmune disease; infectious keratitis due to fungi, bacteria, or viruses; persistent inflammation; or a change in neurotrophic factor related to a decrease in corneal innervation. If the ulcer extends into the stroma, corneal fibrosis may occur as the tissue attempts to repair the breach. Symptoms of corneal ulcers are red, watery eyes, pain, colored discharge, and light sensitivity. A deficiency in vitamin A increases the chances of developing a corneal ulcer, consistent with increased prevalence of corneal ulcers and fibrosis in developing countries. Corneal ulcers are one of the leading causes of blindness in the world, estimated to account for 1.5–2 million new cases of monocular blindness per year.
If a patient displays signs of corneal haze, a diagnosis of corneal fibrosis is likely. Wounds or ulcers are detected using a slit-lamp microscope in conjunction with a fluorescent dye. If detected early enough, most ulcers can be reversed before irreversible damage occurs. Advances in treating neurotrophic and autoimmune ulcers with topical nerve growth factor drops have recently been successful for previously incurable conditions. Currently, there are no pharmaceutical solutions for fibrosis, but surgical procedures such as phototherapeutic keratectomy have proven effective in treating subepithelial corneal scars. The procedure uses an excimer laser to vaporize corneal scars while minimizing damage to the surrounding tissue ( Figure 2.4 ). If the haze is advanced enough to impair vision severely, a corneal transplant may be required. Although considered a highly successful procedure, about 15% of corneal grafts are rejected due to either a buildup of corneal edema from an immune response or a recurrence of opacification ( Box 2.2 ).
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Key sign of corneal fibrosis is corneal haze
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In many cases corneal ulcers lead to corneal scarring
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Currently, no pharmaceutical intervention is available for fibrosis
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If haze is advanced enough, corneal transplant may be required
Clinical studies show that maintaining an intact basement membrane prevents fibrosis, presumably because it prevents epithelial–stromal cross-talk (see below). For example, debridement of the corneal epithelium without removing the basement membrane leads to apoptosis of the underlying stromal keratocytes. This is followed by proliferation of neighboring keratocytes, but they remain quiescent and do not differentiate into a repair phenotype, thus maintaining corneal clarity. Conversely, when the basement membrane is penetrated or removed, the epithelial cytokines reach the stroma, leading to formation of fibroblasts and myofibroblasts and at least a temporary loss of vision due to stromal haze, such as is observed after photorefractive keratectomy (PRK) to correct refractive errors.
Several techniques have been developed to prevent or minimize haze. Applying an amniotic membrane to the eye after PRK has been shown to limit inflammation, apoptosis, and TGF-β effects, resulting in a decrease of postoperative haze in cases of severe fibrosis. In addition, adding mitomycin C, a reagent that acts to limit cellular proliferation, after PRK for severe nearsightedness has been shown to reduce haze by limiting myofibroblast formation. Conversely, in refractive surgery using laser in situ keratomileusis (LASIK), an epithelial–stromal hinged flap is cut with a microkeratome or laser and then the underlying stroma is ablated with a laser to modify corneal curvature. Because the epithelium and basement membrane are penetrated only at the edges of the flap, the stromal wound-healing response is limited and myofibroblasts have been found only at the flap margin (see below).
The science of fibrosis
The immune response and angiogenesis
The cornea is considered an immune-privileged tissue. Normally, few inflammatory cells are detectable in the stroma. A full-blown immune response, such as observed in the skin, would disrupt corneal transparency. Nevertheless, there are circumstances when immune cells from the surrounding limbic vessels, such as T cells and macrophages, are attracted into the stroma by the cytokines released from epithelial cells and keratocytes. Severe trauma or persistent infection leading to the enhanced immunological reaction appears to coincide with the growth of new blood vessels (neovascularization) into the normally avascular cornea, consistent with the observed secretion of proangiogenic chemical mediators by the invading leukocytes. Extensive neovascularization causes severe corneal opacity, sometimes leading to blindness. In the USA, neovascularization is observed in about 1.4 million patients annually, and blinds about 7 million people worldwide.
Epithelial–stromal interactions
In vascularized tissues platelets secrete many factors that recruit inflammatory cells and fibroblasts to the wound site. However, since the cornea is normally avascular, during wound repair, the source of cytokines such as interleukin-1 and TGF-β is the corneal epithelium and its basement membrane. A penetrating wound to these layers permits diffusion of released cytokines that are quickly sensed by keratocyte receptors. Interleukin-1 is a master regulator that stimulates keratocytes to secrete secondary cytokines such as hepatocyte growth factor, keratinocyte growth factor, and platelet-derived growth factor. A wound that penetrates the basement membrane also permits epithelial TGF-β to diffuse into the stroma, which is considered one of the primary factors in fibrotic healing. This epithelial–stroma communication promotes the proliferation, migration, and differentiation of the underlying stromal cells and initiates a cascade of keratocyte cytokine expression ( Box 2.3 ).
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Interleukin-1 is a master regulator that stimulates keratocytes to secrete secondary cytokines
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Maintaining an intact epithelial basement membrane is the key to preventing epithelial–stromal interactions
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Transforming growth factor-β crossing the basement membrane is a primary factor in fibrotic wound healing
The importance of TGF-β
Decades of research have focused on the role of TGF-β during wound healing. To date, three TGF-β isoforms have been identified. Normally in most ocular tissues TGF-β 2 is the dominantly expressed isoform. Low levels of TGF-β 1 and TGF-β 2 promote fibroblast proliferation and migration but do not promote the differentiation to the myofibroblast phenotype. Cell migration and proliferation to the wound site are critical because fibroblasts secrete matrix molecules that act as “glue” to seal the wound. When fibroblast migration is inhibited, the wound never heals properly. Fibroblasts must produce properly oriented collagen fibers to generate the transparency and strength of a properly healed wound. This process is not currently understood but is critical to regenerative healing.
After wounding, all three isoforms are expressed in the cornea. High levels of TGF-β 1 and TGF-β 2 result in the persistence of the myofibroblast phenotype and overproduction of ECM molecules, including collagen, fibronectin, vitronectin, and their cell surface receptors (integrins). When expression of TGF-β 1 and TGF-β 2 is exaggerated and sustained, an imbalance between: (1) proteases that degrade the matrix (metalloproteases, plasmin); (2) protease inhibitors (tissue inhibitors of metalloproteases (TIMPs) and plasminogen activator inhibitor-1 (PAI-1)); and (3) secretion of ECM components results in improper degradation and buildup of unorganized collagen fibrils. Studies show that administering a therapeutic dose of a pan-TGF-β antibody prevents myofibroblast differentiation and corneal haze after wounding, but other functions of TGF-β, such as cell migration and cell proliferation into the wound margin, were also reduced. Thus, targeting TGF-β signaling pathways instead of TGF-β isoforms may be a more selective approach to fighting corneal fibrosis.
TGF-β 3 appears to have a different function than that of TGF-β 1 and TGF-β 2 . No fibrosis is observed during embryonic wound healing in mice before day 16, which coincides with elevated levels of TGF-β 3 and reduced expression of TGF-β 1 and TGF-β 2 . However, from day 17 until birth (day 21), the formation of a scar is evident. This suggests that increasing TGF-β 3 expression in a wound may be a useful approach to reducing fibrosis. Fibrotic healing probably developed as an important evolutionary adaptation to prevent infection, because a quickly healed scar, even if accompanied by a partial loss of function, yielded better chances of survival than the possible deadly consequences of infection. These ideas are consistent with the observation that dermal wounds treated with TGF-β 3 have reduced scarring. Thus, treating corneal wounds with TGF-β 3 may be a useful therapeutic tool. More research is needed to understand the significance of the tissue-specific and temporally regulated TGF-β isoform expression during wound healing.
Unhealed wounds
Some wounds in the cornea never heal because keratocytes do not repopulate the wound and the stroma remains hypocellular. This occurs after refractive surgery with LASIK. In the hinged flap, the majority of the epithelial–stromal interface is not disrupted. Only in the area where the laser has made the cut, around the edge of the flap, is there the potential for a fibrotic response. Consequently, since after laser ablation of the stroma the keratocytes do not proliferate and repopulate the anterior stromal tissue under the flap, there is no challenge to the transparency, there is little trauma to the corneal nerves, and millions of patients enjoy the restoration of visual acuity. However, the structural integrity of the flap is compromised because new stromal connections are not created and thus the flap never heals completely, resulting in a dramatic decrease in tensile strength. For this reason, eye banks do not accept corneal donors who have had LASIK refractive surgery. In vivo confocal studies have shown a progressive decrease in keratocyte density in the anterior stroma each year after treatment, and after 5 years the keratocytes in the posterior stroma also begin to decrease in number.
Another consequence of hypocellularity in the anterior stroma is an increase in the potential for corneal edema because the unhealed wound creates a space where fluid may accumulate. This is critical because the stroma is normally maintained in a deturgescent state. Fluid is constantly removed by active transport of salt and water out of the stroma by the underlying corneal endothelial cells. Disturbed endothelial cell function and/or sustained high intraocular pressure increase the fluid load in the stroma which rapidly accumulates in the interface between the flap and ablated stromal ECM, leading to edema or interface fluid syndrome and blurry vision ( Figure 2.5 ). Endothelial cell density and function decrease with age, suggesting that post-LASIK, a rise in stromal edema due to LASIK is likely to increase ( Box 2.4 ).
