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

Glossary

Corticosteroid – A class of steroid hormones that are produced in the adrenal cortex and are involved in a wide range of physiologic systems, such as the stress response, the immune response, regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. In wound healing, its primary role is in suppressing the immune response and reducing the release of inflammatory cell mediators.

Epithelial Laser in situ Keratomileusis (epi-LASIK) – An anterior surface ablative refractive surgical procedure that uses a unique epikeratome (a microkeratome with a blunt, oscillating blade) to mechanically separate the epithelium from the Bowman’s layer and stroma, while suction is applied, to make a hinged epithelial flap, similar to a traditional LASIK flap. Unlike LASIK, no sharp blades or intrastromal cuts are made. It is most similar to LASEK, but no alcohol is required.

Extracellular matrix (ECM) – Any material produced by cells and secreted into the surrounding medium, but it usually is applied to the noncellular portion of living tissues. It typically is classified as basement membrane or interstitial ECM.

Fibrosis – Formation of excessive fibrotic scar tissue in an organ or specific tissue of the body. Hypertrophic scar – An abnormal, excessively large fibrotic scar due to a pathologic reparative woundhealing response, where the scar is raised above the surrounding adjacent normal tissue, but it does not grow beyond the boundaries of the original wound. It often regresses in size and improves in appearance over a few years after.

Keloid – An abnormal, excessively large fibrotic scar due to a pathologic reparative fibrotic wound-repair response, where the scar extends beyond the boundaries of the original wound in addition to being raised above the surrounding adjacent normal tissue. It is a more severe degree of pathologic scarring than a hypertrophic scar because it can carry on growing indefinitely into a large, benign growth that can present a significant cosmetic issue to the individual.

Laser-assisted subepithelial keratomileusis (LASEK) – An anterior surface ablative refractive surgical procedure that reshapes the cornea to correct refractive errors of the eye and is basically a modified form of photorefractive keratectomy (PRK).

With LASEK, instead of removing or excising the epithelium completely as in PRK, the surgeon cuts a round semi-circular area of epithelium with a finebladed instrument called a trephine. A dilute alcohol solution is then applied to loosen the cut epithelium inside the semi-circle where it attaches to Bowman’s layer and then the surgeon gently lifts and folds back the hinged loosened epithelial flap, exposing the underlying Bowman’s layer and corneal stroma for the excimer laser treatment.

Mitomycin C (MMC) – Mitomycins are a family of aziridine-containing natural products isolated from the soil fungus, Streptomyces caespitosus. One of these compounds, MMC, finds use as a chemotherapeutic agent or as a scar-reducing agent by virtue of its potent DNA cross-linking. Myofibroblast – An epithelial-, endothelial-, or mesenchymal-derived cell (e.g., keratocyte) that acquires morphological and biochemical features of smooth muscle cells, including the expression of alpha-smooth muscle actin (a-SMA). It can contract by using the actin smooth muscle filament complex, which can help speed up wound repair by contracting the edges of the wound and enhancing ECM deposition compared to that of simply an activated fibroblast. It has been suggested that in several fibrotic diseases (e.g., liver cirrhosis, kidney fibrosis, retroperitoneal fibrosis, corneal fibrotic scarring) that persistence of myofibroblasts and chronic wound contracture leads to a long-term fibrotic scar.

Platelet-derived growth factor (PDGF) – A family of growth factors (GFs) involved in stimulating fibrotic wound repair and angiogenesis. Three PDGF isomers are found in humans: PDGF-AA, PDGF-AB, and PDGF-BB. In wound healing, its primary cellular source is platelets, which degranulate and release PDGF-AB. This initiates a fibrotic repair response by chemotactic and proliferative effects on fibroblasts and inflammatory cells.

Regeneration – Regrowth of a damaged organ or tissue so that the original structure and function are restored back to normal.

Transforming growth factor-beta (TGF-b) –

A family of cytokine proteins that are expressed by almost all mammalian cells in three isoforms (TGF-b1, TGF-b2, and TGF-b3), which have different functions in wound healing. All three TGF-b isoforms play major roles in wound healing including chemotaxis

(signaling migration of macrophages, monocytes, fibroblasts, and neutrophils), fibroblast proliferation, collagen production, and angiogenesis. They act on target cells to generate a specific molecular signaling response by binding to cell surface TGF-b receptors, types RI, RII, and RIII, which then initiates activation of several intracellular signaling pathways. All three TGF-b isoforms are secreted from cells as a large (125–160 kDa) latent TGF-b complex (LL-TGF-b complex), composed of an active TGF-b dimer, a latency-associated peptide (LAP), and one of four possible isoforms of the latent TGF-b-binding protein (LTBP). Overall, the LL-TGF-b complex covalently binds with high affinity to ECM, such as fibronectin, through its specific LTBP domain to create storage pools of latent TGF-b, whereas the free active TGF-b dimer binds to TGF-b cell receptors or creates more storage pools by binding to the core protein portion of dermatan sulfate and heparin sulfate protoglycans (PGs) in the ECM.

Introduction

Tissue or organs of mammalian embryos and fetuses up to the first-half of gestation heal through scar-free regenerative wound healing, whereas tissues in late gestation fetuses up to elderly adults heal more slowly (inversely related to postnatal age) and less efficiently through scarforming fibrotic wound healing. At the microscopic level, a fibrotic scar is basically defined as disorganized extracellular matrix (ECM) containing repair mesenchymal cell phenotypes as opposed to regenerative or uninjured normal tissue, which have normal ECM architecture containing quiescent mesenchymal cell phenotypes, or tissue fibrosis, which has disorganized ECM containing quiescent mesenchymal cell phenotypes. At the macroscopic level, a fibrotic scar leaves a mark or cicatrix on the skin or tissue after injury, often leading to an alternation in function or cosmesis, whereas tissue fibrosis leaves an inconspicuous mark and regenerative tissue leaves flawless tissue restoration. Thus, fibrotic scarring represents reparation failure to complete tissue fibrosis and both represent failures to achieve true tissue regeneration. Research studies on prenatal embryos and fetuses suggest that the mechanisms involved in the regenerative response are intrinsic to the tissue itself and are not caused by the sterile prenatal environment in the womb. The evolutionary reason for the transition from the scar-free regenerative response to the scar-forming fibrotic repair response in higher mammals, such as humans, is that such a variation helped an individual live longer to reproduce and

take care of their young (i.e., natural selection). The fibrotic repair response results in rapid inflammatory cell recruitment and subsequent wound contracture and filling of space, which can be organ or life saving. The main disadvantages of the fibrotic repair response are that it commonly results in the reconstitution of semifunctioning tissue or can fill the tissue with a cosmetically displeasing scar. In modern times, the result of this evolutionary mismatch is that a fibrotic scar can occur from even minor injury (e.g., sterile, simple surgical incision with suturing).

In humans, tissue fibrosis or fibrotic scarring occurs after almost any type of injury to any tissue in the adult human body, including the eye. One exception is injury in which ectodermal cells alone are damaged (e.g., firstdegree burns, corneal abrasion, skin abrasion) and heal through scar-free cellular regeneration without any longterm fibrotic effects to the underlying interstitial mesenchymal cells. In fact, in certain cases where the context of the injury and the molecular signaling pathways invoked are shifted toward embryonic mechanisms, scar-free tissue regeneration may still occur as opposed to fibrotic scarring. Thus, tissue fibrosis or fibrotic scarring is not inevitable. For example, if oral mucosa (e.g., gums, palate, and inner cheek) is superficially damaged, complete regeneration occurs. Other examples include complete regeneration of the liver even if up to two-thirds of the liver is removed or complete regeneration of the skin in specific injury contexts, such as a pin-prick, a hypodermic needle stick, or multiple needle insertions (tattooing). In contrast, fibrotic scarring results from penetrating stab incision into the liver or various more severe injuries to the skin, such as: (1) a larger incision than a needle stick, (2) an excision,

(3) suturing a wound closed with pro-inflammatory material (cat gut suture), and (4) leaving sutures in place too long or with incorrect tension. These examples illustrate the plasticity of the two wound-healing responses, fibrosis and regeneration. Differences between the desired responses (regeneration or secondarily minimal to mild tissue fibrosis) and the less desired responses (nonhealing wounds or excessive fibrotic scarring) are subtle, but fundamentally come down to three variables: (1) the molecular signaling pathways invoked, (2) the targeted responding cells, and (3) the context and degree in which the targeted responding cells are stimulated.

Central to our understanding of the pathogenesis of tissue regeneration versus fibrotic scarring (and tissue fibrosis) has been the identification of several cytokines and growth factors (GFs) involved in initiating and/or regulating the ongoing wound-healing responses. The relationship between these cytokines and GFs and their predicted cellular responses is now only being partially understood. As such, it is currently known that cytokines and GFs, their associated target cell surface receptors, and their molecular signaling pathways play a significant role

in directing cellular behavior and/or phenotype in normal, diseased, and wound-healing states. Currently, the cytokine best known to play the most crucial role in mediating either tissue regeneration or fibrotic scarring is transforming growth factor-beta (TGF-b). Three isoforms of TGF-b have been identified in mammals, TGF- b1, b2, and b3, all having major roles in wound healing. Studies comparing embryonic tissue regeneration versus adult fibrotic scarring have identified a variety of differences in their intrinsic cellular levels of expression and release. TGF-b3 elicits a regenerative response, whereas both TGF-b1 and TGF-b2 elicit a fibrotic-repair response. There are other notable disparities between the TGF b isoforms (Table 1). TGF-b is secreted extracellularly in a latent or inactive pro-peptide form, consisting of a 25 kDa disulfide-linked homodimeric TGF-b peptide, also known as the active form, that is bound to a latency-associated peptide (LAP), which itself is bound to a specific latent TGF-b-binding protein (LTBP). This 125–160 kDa TGF-b complex is known as a large latent

Table 1 Comparison of embryonic and adult wound healing characteristics

Data from 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, London B

359: 839–850.

TGF-b (LL-TGF-b) complex. It binds covalently to ECM (e.g., fibronectin) through its LTBP domain to create storage reservoirs of latent TGF-b. During wound healing or inflammation, proteolytic enzymatic cleavage (e.g., tissue plasminogen activator (TPA), plasmin, matrix metalloproteinases (MMPs), or cathepsin) or nonproteolytic dissociation (e.g., low pH, reactive oxygen species) of the active TGF-b dimer occurs from the LL-TGF-b complex, leading to TGF-b activation. Active TGF-b isoform dimer binding to its targeted cell surface receptor(s) leads to a cascade of intracellular signaling pathways, including a Smad pathway as well as other pathways, each resulting in a cell-specific set of responses.

In general, TGF-b/receptor-mediated intracellular signaling pathways stimulate the production of various ECM proteins and inhibit ECM degrading enzymes (increases inhibitors of MMPs and decreases expression of MMPs), although exceptions to these principles abound. TGF-b also modulates cellular functions such as apoptosis, motility, mitosis, and differentiation or transdifferentiation into new cellular phenotypes. With wound healing, the various cytokine or GF cascades sometimes result in overlapping specific cellular responses that are not completely well understood. For mesenchymal fibrocytes (e.g., keratocytes), this set of responses includes fibrocyte migration and proliferation and subsequent differentiation into a metabolically activated repair cell type known as a fibroblast. Fibroblast cellular behavior results in the synthesis and deposition of a provisional fibronectin matrix comprised of ECM adhesion molecules (fibronectin), ECM space-filling molecules (hyaluronic acid), and ECM signaling molecules (tenascin). The next step is the conversion to early mature ECM, comprised of collagen fibrils, sulfated protoglycans (PGs), and possibly epithelialor endothelialderived surface basement membranes (BMs). Under appropriate conditions, such as prolonged or excessive TGF-b exposure, some fibroblasts differentiate into another repair phenotype known as a myofibroblast, which is the central cellular mediators of the fibrotic repair response (Figure 1). Myofibroblasts originate by differentiation from fibrocytes or activated fibroblasts or by transdifferentiation, also known as metaplasia, from epithelial, endothelial, or local smooth muscle cells that are associated with blood vessels (Figure 1). Morphologically, myofibroblasts are larger than activated fibroblasts and uniquely express alpha-smooth muscle actin (a-SMA), an important morphologic marker identifying this cell type using immunohistochemistry techniques. Myofibroblasts are highly contractile repair cells that promote wound closure and contraction. Functionally, myofibroblasts also express and secrete greater amounts of cytokines and GFs, synthesize and deposit greater amounts of ECM, and have stronger and more focal ECM cell surface adhesion receptor sites compared to activated fibroblasts. Myofibroblasts first appear in the stroma directly under the epithelium and

Epithelial cell

Epithelial BM

Fibroblast

(activated keratocyte)

TGF-β

TGF-β

− / +

TGF-β, etc.

Fibrocyte

(keratocyte)

+

TGF-β, etc.

Macrophage

then their domain advances to progressively deeper levels. Myofibroblast-mediated ECM deposition occurs in an orderly sequence from immature (primitive) to mature (fibrotic) stages: first fibronectin and other noncollagenous provisional ECM proteins (hyaluronic acid, tenascin, etc.), then collagen type III, and lastly collagen type I and sulfated PGs (e.g., decorin, lumican, biglycan). As the scar matures, most of the provisional ECM components are replaced by collagen fibrils (type I >> type III) and sulfated PGs. As judged by collagen fibril diameter and density, ECM usually first reconstitutes to the most mature and most fibrotic stage in the portion of the wound just under the epithelium and then it advances progressively to deeper levels. Additionally, during the evolution of maturation, the hypercellularity and hypervascularity decreases due to apoptosis and tensile strength increases due to increased maturity acquired collagen fibril cross-linking. The mechanism of late disappearance of myofibroblasts is uncertain, but probably is due to a combination of apoptosis and dedifferentiation of cells back to quiescent fibrocytes.

In humans, fibrotic scarring commonly causes major medical problems in a variety of tissues. In the eye, fibrotic scarring causes visual impairment or even blindness. In the peripheral and central nervous systems, glial scarring prevents neuronal reconnections and hence blocks

reparative attempts at restoring normal neuronal function. In gastrointestinal and reproductive organs, strictures and adhesions can give rise to serious or life-threatening conditions, such as infertility, bowel obstruction, or chronic pain. In ligaments and tendons, fibrotic scarring restricts motility and decreases strength. Additionally, if the fibrotic repair response is incomplete, becomes excessive, or fails to appropriately terminate, pathologic wound healing occurs resulting in unhealed wounds, hypertrophic scars, or keloid scars, respectively. Overexpression and release of pro-fibrotic TGF-b1 and b2 cytokines, upregulation of TGF-b receptors RI and RII, and/or the formation of a positive, autoinducible feedback loop have been suggested as the primary reasons for excessive fibrotic scarring in hypertrophic or keloid scars. The potential for TGF-b autoinduction is usually self-limited in nonpathologic fibrotic repair responses through a negative feedback loop that terminates further TGF-b expression and release from that cell. In adult humans, the degree of fibrotic scarring versus tissue regeneration or tissue fibrosis depends on the following five factors: tissue site, sex, race, age, and magnitude and contamination of the wound. For tissue site, the gums, liver, and skin can either regenerate or scar, depending on the context of the injury. Most other tissues or organs in the body just repair themselves through tissue fibrosis or

fibrotic scarring, which is body site dependent. The deltoid, sternum, and conjunctiva scar more than the face, abdomen, legs, and cornea. Concerning sex, fertile females scar more than postmenopausal females and all males as estrogen has a major stimulatory effect on the fibrotic repair response. For race, darker pigmented individuals scar more than less pigmented individuals. Concerning age, young people in their teens and 20s scar more than infants and children, who have an immature immune response, and both heal better than older individuals, whose inflammatory response becomes sluggish and less effective with age and whose mesenchymal cells become senescent. Finally, the magnitude of injury and wound contamination alter the wound-healing response as larger, more inflamed, more contaminated wounds scar more than smaller, uncontaminated wounds.

Ocular Wound Healing

The ocular fibrotic wound-repair response is quite similar to that seen in other tissues in the human body. In the eye, fibrotic scarring most commonly occurs in the conjunctiva, cornea, iris, lens, retina, and choroid (Figure 2). In vascularized ocular tissues, like the conjunctiva, iris, choroid, or even the cornea or retina after having chronic ocular disease, fibrovascular scars are basically granulation tissue. Scars normally develop in response to vascular stromal injury whereby serum plasma or blood leaks into

Vascular fibrotic scarring of the conjunctiva

the wound resulting in a clot. This initiates a fibrovascular repair response predominantly through a platelet-derived growth factor (PDGF)-mediated platelet degranulation signaling pathway resulting in massive release of PDGF into the wound along with some TGF-b release. However, the eye also has several avascular transparent tissues (e.g., cornea, lens, vitreous, and portions of the retina) that also can repair themselves through fibrosis, albeit through four less intense TGF-b-mediated signaling pathways.

TGF-b-mediated wound healing occurs because TGF-b and its associated receptors are constitutively expressed in the normal state by almost all cells residing in or on ocular tissues. The external ocular surface has other factors to additionally consider in that it is bathed in a tear film composed of lacrimal gland secretions that are rich in latent TGF-b1. Similarly, internal intraocular structures are bathed in aqueous humor composed of ciliary and lens epithelial secretions that are rich in latent TGF-b2. Latent TGF-b2 is thought to be one of the principal factors promoting the normal naive immune status of intraocular tissues, including the cornea, due to its ability to maintain resident tissue immune cells in an immature state. Secondarily, latent TGF-b2 has a direct immunosuppressive effect whereby it inhibits immune cell proliferation and inflammatory cell cytokine and GF release. At the concentration levels found in the normal aqueous humor, latent TGF-b2 is also thought to participate in the inhibition of angiogenesis into transparent parts

Avascular or vascular fibrotic scarring of the cornea

Vascular fibrotic scarring of iris

Lens

of the eye, although a primary role for latent TGF-b2 in preventing vascularization in the cornea remains controversial. In various ocular diseases or after injury, TGF-b release and subsequent extracellular activation usually initiates a fibrotic repair response. The TGF-b-mediated fibrotic repair response can lead to significant visual impairment. TGF-b can cause overcorrection, undercorrection, or regression of refractive effect leading to refractive instability. TGF-b can also cause loss of refractive function by inducing irregular astigmatism or it can cause loss of transparency by inducing corneal haze, cataract, capsular fibrosis, or vitreous opacification. Tractional distortion of ocular structures, such as proliferative vitreoretinopathy, or other ocular complications, such as wound dehiscence, epithelial ingrowth, infection, hypotony (low intraocular pressure), epiretinal membranes, or retinal detachment, can also be caused by TGF-b . Thus, this molecule is a subject of intense research. Avascular transparent tissue fibrosis or fibrotic scarring in the cornea, lens, and retina is perhaps even more intriguing to researchers since it provides a unique opportunity to directly examine the cell biology during wound healing caused by the four TGF-b-mediated signaling pathways in isolation from the PDGF-mediated platelet degranulation signaling pathway.

Corneal Wound Healing

Although corneal neovascularization resulting from chronic ocular disease, such as corneal infection or chemical injury, may lead to a fibrovascular repair response (Figure 3, far-right), this type of repair response will not be focused on in this article. Instead, this article will strictly focus on the avascular corneal fibrotic repair response (Figure 3, middle). In the latter repair response, all cases have in common early inflammation that is minimally present up to 2 weeks after injury and maximally

present up to 1 month after injury, at least some transient myofibroblast differentiation that is minimally present up to 4–6 months after injury and maximally present longterm, and the long-term presence of disorganized collagenous ECM (Figure 4).

Cell–Cell Communication

Cytokines and GFs

Corneal wound healing is an exceedingly complex process mediated by autocrine (affecting the same cell), juxtacrine (affecting adjacent similar cell types), or, most commonly, paracrine (affecting other adjacent cell types) cell–cell interactions involving cytokines and GFs produced by epithelial, stromal keratocyte, endothelial, immune, lacrimal gland, and corneal nerve cells (Figure 4). The paracrine interactions usually affect cells at most 50–75 mm from one another, while the other modes of interactions operate over shorter distances. The earliest event involves cell-mediated release of several cytokines and GFs by directly injured resident tissue cells or secretion by resident tissue cells just adjacent to the injury site and their subsequent extracellular activation. In the normal, uninjured state, the participating cytokines and GFs are expressed constitutively and stored intracellularly by the corneal epithelium, corneal nerves, keratoctyes, and corneal endothelium. These cytokines play a vital role in the maintenance of normal corneal health, structure, and function as some are secreted in a limited controlled fashion, mostly in the latent or inactive form. The adjacent tear film produced by the lacrimal gland and other ocular surface epithelia and the aqueous humor produced by ciliary and lens epithelial cells’ secretions are another rich source of cytokines and GFs in the normal, uninjured state since these fluids supply various nutrients to the avascular cornea that otherwise would not be available. After wounding activates this direct resident tissue injury-induced

Persistent corneal edema

Chronic mechanical irritation to keratocytes

Persistent keratoyte activation and extracellular lipid collection=stromal crystalline-like deposits

Early provisional matrix synthesis and deposition and later replacement by prolonged or excessive collagen and sulfated PG synthesis

and deposition as well as chronoic myofibroblastic differentiation

Wound remodeling

Hypercellular

fibrotic scar

cytokine and GF cascade, supplemental exposure to additional cytokines and GFs may occur by four dynamic signaling pathways (Figure 4): epithelial– or endothelial–stromal interactions, inflammatory cell–stromal interactions, tear film– or aqueous humor–stromal interactions, and keratocyte mechanotransduction.

The major pertinent cytokines and GFs studied to date in regard to corneal wound healing include connective tissue growth factor (CTGF), epithelial growth factor (EGF), fibroblast growth factor (FGF), interleukin-1

(IL-1), nerve growth factor (NGF), PDGF, TGF-a, TGF-b, tumor necrosis factor-alpha (TNF-a), and vascular endothelial growth factor (VEGF). Currently, TGF-b is thought to be the most important one of this group in regard to stimulating a fibrotic repair response. The major resident cellular source of TGF-bs in the normal, uninjured cornea is the epithelium, which primarily expresses TGF-b2, while keratocytes and endothelial cells express only a little TGF-b1. No TGF-b3 expression has been found in the adult human cornea.

Resident cell surface TGF-b receptor sites are most numerous on keratocytes, while epithelial and endothelial cells have very little. After injury, increased expression of both TGF-b1 and -b2 and their associated receptors occurs, with TGF-b2 predominating in epithelial–stromal interactions (stroma within 75 mm from the epithelium) and TGF-b1 predominating in endothelial–stromal interactions (stroma within 75 mm from the endothelium). In contrast, stromal injury more than 75 mm from either surface invokes only slightly increased TGF-b1 expression and, most importantly, receives no paracrine supplementation from epithelial– or endothelial–stromal interactions.

Although the normal tear film contains high concentrations of latent TGF-b1, immediately following corneal epithelial injury, stimulation of the corneal nerve-lacrimal gland reflex arc causes hypersecretion of tears and increased secretion of several pro-fibrotic cytokines and GFs, such as PDGF-BB, NGF, and additional TGF-b1, all of which are normally stored intracellularly in lacrimal gland cells. The higher amounts of latent TGF-b1 as well as the newly secreted PDGF-BB and NGF are then activated by plasmin or MMPs, which are both released or secreted by injured or healing corneal epithelial cells into the wound. This corneal nerve-lacrimal gland reflex arc secretion and protease release usually returns to baseline levels within 7 days of epithelial defect closure; hence it is a transient event.

Other cytokines or GFs from the group listed above have intriguing complementary or antagonizing effects to TGF-b’s pro-fibrotic effects. As discussed in the ocular fibrovascular repair response section, PDGF’s pro-fibrotic effects are almost identical to TGF-b’s pro-fibrotic effects. In the normal, uninjured cornea, small amounts of PDGFBB are produced by the corneal epithelium that reacts with its receptors, which are most highly concentrated in the keratocytes of the stroma and the corneal endothelium. Also, a limited amount of PDGF-BB is secreted by macrophages and lacrimal gland cells. After epithelial injury, the expression, release, and activation of PDGF-BB increases in parallel to that of TGF-b. NGF, a trophic neuropeptide important in maintaining corneal epithelial health, is another GF that is complementary to TGF-b’s profibrotic effects as NGF is known to directly stimulate myofibroblastic transformation, but without the proliferative or ECM deposition effects of TGF-b. CTGF is also complementary to TGF-b as it too stimulates tissue fibrosis. However, CTGF is only potently expressed when stimulated by TGF-b, suggesting CTCF mediates several downstream actions of TGF-b. Thus, CTGF may be more important in chronic stages of fibrotic scarring as opposed to initiation or early regulation of the fibrotic repair response. Alternatively, CTGF may just synergize with TGF-b’s pro-fibrotic effects since CTGF binds to TGF-b and potentiates TGF-b binding to TGF-b type II receptors. In contrast, TNF-a directly antagonizes TGF-b’s profibrotic effects by inhibiting the TGF-b/Smad pathway.

This direct antifibrotic effect may seem counterintuitive since TNF-a is one of the major pro-inflammatory cytokines of the cornea. As can be appreciated, a correct combination and temporal sequencing of the various cytokines and GFs is required for proper healing to occur. Imbalances can result in incomplete wound repair, such as (1) persistent epithelial defects and corneal ulceration or melting, or

(2) excessive pathological tissue fibrosis, such as stromal outgrowth, fibrodegenerative pannus formation, stromal ingrowth, or retrocorneal membrane formation.

TGF-b signaling pathways

Both historical clinical anecdotal observations and recent experimental investigations have suggested that epithelial–stromal interactions are the primary mediator of the avascular fibrotic repair response in the cornea. Thus, epithelial cell-derived substances substitute for those produced by platelets in the skin and other vascular tissues. The cornea consists of three cellular layers consisting of epithelium, interstitial stroma with interspersed keratocytes, and endothelium and two acellular layers consisting of an epithelial BM/Bowman’s layer complex and an endothelial Descemet’s membrane. Although Bowman’s layer is not re-formed if damaged, both BM layers are re-formed if damaged because they are produced by epithelial or endothelial cells. Both acellular layers seem to function clinically as physiological barriers to separate the three cellular layers from direct contact or communication with one another to maintain normal corneal homeostasis. Disruption of the epithelial BM/Bowman layer complex induces maximal epithelial–stromal interactions that are intense enough even in a minimal injury state to cause a fibrotic repair response. This is seen clinically with bullous keratopathy – a pathological condition in which small blisters (vesicles or bullae) are formed in the corneal epithelium due to endothelial dysfunction, at the host–graft interface after penetrating keratoplasty (aka full-thickness corneal transplantation; a surgical procedure where a damaged or diseased cornea is replaced by full-thickness donated corneal tissue), or with Bowman’s layer breaks (a degenerative disorder of the cornea in which structural changes cause it to thin and change in shape resulting an outward bulging of the tissue) as a subepithelial fibrotic scar, stromal outgrowth, or fibrodegenerative pannus formation (fibrocollagen connective tissue that proliferates in the anterior layers of the cornea in degenerative corneal disease). In some cases of bullous keratopathy, there may even be an no obvious microscopic full-thickness Bowman’s layer break, but rather just separation of the of epithelium and its associated BM from Bowman’s layer due to severe epithelial edema causing subepithelial bullae or blisters. In this extreme example of epithelial–stromal interactions, the keratocytes apparently migrate through trigeminal nerve fiber perforation sites found in Bowman’s layer to the

subepithelial space created by the edema, where they undergo a fibrotic repair response. Separation or breaks in Descemet’s membrane provide a similar trigger for causing a fibrotic repair response in the posterior cornea through endothelial–stromal interactions. For example, with penetrating corneal injury or breaks in Descemet’s membrane, whether caused by trauma, disease (keratoconus), or surgery, endothelial–stromal interactions stimulate fibrotic scarring resulting in a supraendothelial fibrotic scar, stromal ingrowth, or retrocorneal membrane formation, depending on the degree of apposition of the broken Descemet’s membrane wound margins. Therefore, current evidence suggests that the main TGF-b pro-fibrotic signaling pathway to block or suppress to prevent or reduce the chance for fibrotic scarring is that of direct epithelial–stromal or endothelial–stromal interactions.

Three other TGF-b pro-fibrotic signaling pathways are also present in the cornea and can initiate an avascular fibrotic repair response, but more often they just sustain the response already initiated by epithelialor endothelial–stromal interactions. As already discussed earlier, a robust inflammatory cell response is part of a normal or nonpathologic fibrotic repair response. However, along with the advantages of stimulating an immediate innate immune response and thus preventing or at the very least lowering the risk for infection, leukocytes also secrete many pro-fibrotic cytokines and GFs, such as TGF-b and, to a lesser extent, PDGF. These pro-fibrotic factors are disadvantageous because they cause keratocytes in the corneal wound to undergo a higher degree of tissue fibrosis, which can lead to chronic nonpathologic scarring or even pathologic scarring. Usually after sterile clean injury, the inflammatory response in the avascular cornea is quite mild compared to that of the skin or other vascular tissues. However, dirty injuries or wounds complicated by infection or with preexisting immune-related disease may make the inflammatory response so massive that it becomes the primary pro-fibrotic signaling pathway resulting in varying degrees of severe corneal scarring. Another TGF-b pro-fibrotic signaling pathway was already discussed in the the section entitled ‘Cytokines and GFs’ when discussing the epithelial cell injury-induced corneal nerve-lacrimal gland reflex arc. These tear film–stromal interactions are usually transient (<7 days) and minor in comparison to the two already discussed. However, with persistent epithelial defects or chronic corneal ulceration, tear film–stromal interactions may become of paramount importance in causing fibrotic scarring in addition to the chronic increased release of epithelial cellderived cytokines at the peripheral edge of the epithelial defect. The aqueous humor may have a similar role with posterior stromal injury. Finally, the fourth TGF-b profibrotic signaling pathway involves keratocyte mechanotransduction interactions. Historically, wound tension has been known for years to promote a higher degree of fibrotic

scarring. This mechanotransduction pathway was confirmed experimentally using cell culture experiments where mechanical strain on fibroblasts increased pro-fibrotic TGF-b1 expression, upregulated TGF-b receptors RI and RII, and markedly increased collagen expression. Findings by other researchers indicate that fibrocyte (e.g., keratocyte) cell surface integrins, which form cell–ECM adhesions, can act as strain gauges to external mechanical stress, triggering both direct intracellular signaling pathways or indirect release of paracrine cytokine and GFs factors, which stimulates the fibroblastic phenotype and augments ECM synthesis and deposition.

Cell–ECM Communication

Suppressing or amplifying factors

The ECM, through intrinsic binding and storage properties or cellular receptor-mediated adhesive properties, collaborates with cytokines and GFs in regulating cell behavior and phenotype through bidirectional cytokine and GF–ECM and cell–ECM interactions. In the latter, ECM adhesion and surface characteristics influence cellular behavior through interaction with cell surface receptors.

Clinical anecdotal observation supports the contention that having an intact corneal BM usually prevents fibrotic repair phenotypes from forming in the stroma and return of the BM usually correlates with loss of the fibrotic repair phenotype. Apparently, an intact epithelial BM or Descemet’s membrane is the key factor in suppressing direct maximal epithelialor endothelial–stromal interactions from taking place in the normal, uninjured state by binding and storing the soluble LL-TGF-b complex. Thus, epitheial BM or Descemet’s membrane act as passive diffusion barriers to epithelialor endothelial-derived latent TGF-b, respectively, and they ultimately regulate the bioavailability of TGF-b to the underlying or overlying corneal stroma. BMs are composed mainly of collagen type IV, entactin/nidogen, different laminins, and heparan and chondroitin sulfate PGs. The LL-TGF-b complex binding and storage property of BMs is likely related to heparin binding mechanisms intrinsic to the heparin sulfate PGs found in BMs, which bind to the soluble LL-TGF-b complex through either LTBP-1 or -3 domains. Fibronectin, which is found in the provisional matrix, also has LL-TGF-b complex binding and storage properties similar to that of BMs. Some sulfated PGs in the corneal stroma, like decorin, can even bind to the active dimer form of TGF-b through their core proteins; thus, additionally antagonizing the biological affects of TGF-b. BM-bound heparin sulfate PGs may have a similar function through their core proteins too.

With injury, corneal BM barriers are breached so that normal maximal epithelialor endothelial–stroma interactions take place in addition to the acute wound-healing cascade already started by direct resident tissue injury itself. For example, with combined superficial epithelial

and stromal injury (photorefractive keratectomy (PRK), sterile corneal ulceration), this stromal wounding response is clearly seen clinically as it frequently stimulates excessive subepithelial fibrotic scarring resulting in prolonged or even permanent subepithelial haze. Apparently, removal of the epithelial BM directly exposes anterior stromal keratocytes to maximal pro-fibrotic effects of TGF-b released by the injured epithelial cells or found in the neurally stimulated tear film. Moreover, after injury, return of an intact epithelial BM suppresses normal epithelial cell-derived TGF-b2 intracellular expression and, more importantly, binds the TGF-b2 extracellularly secreted from epithelial cells. Thus, epithelial and presumably endothelial BMs represent an efficient ECM antifibrotic mechanism that goes slightly beyond just simple barrier function. However, this ECM antifibrotic mechanism is not foolproof since chronic subepithelial haze still persists long-term or episodic subepithelial haze still exacerbates after ultraviolet light overexposure, despite having an intact epithelial BM. This persistent haze seems to be best explained by TGF-b signaling pathways that work only in the stroma, such as mechanotransduction or inflammatory cell TGF-b signaling pathways, respectively. These two TGF-b signaling pathways can act directly on activated keratocytes and/or myofibroblasts in the stroma without having to send cytokines or GFs through a BM. Regarding the latter UV light example, quiescent or regressed myofibroblasts in a subepithelial fibrotic scar may have long-term upregulated TGF-b receptor sites densities, making them hypersensitive to any TGF-b, whether caused by inflammation or late resident cell injury. This may then lead to an autocrine positive feedback loop of more TGF-b production from a single myofibroblast or from a paracrine positive feedback loop due to macrophage infiltration and further TGF-b release.

Corneal cell–ECM interactions are an area that needs further study. Other disciplines have already shown that cells adhere to the ECM usually through their cell surface receptors (e.g., integrins, cell surface proteoglycans). For example, cell–ECM adhesion by integrins receptors regulates cellular homeostasis in multiple ways, including regulation through direct and indirect connections to the actin cytoskeleton, growth factor receptors, and intracellular signal transduction cascades. Integrins also sense mechanical strain arising from the ECM, thereby converting these stimuli to downstream signals modulating cell behavior. Disruption of this connection to the ECM has deleterious effects on cell survival, sometimes leading to a specific type of programmed cell death called anoikis. Anoikis is defined as apoptosis that is induced by inadequate or inappropriate cell–ECM interactions. Another direct extension of this cell–ECM interaction theme is clinical anecdotal reports and experimental animal research studies in which injuryinduced stromal surface irregularity, also known as irregular surface nanotopography, results in increased TGF-b

expression and a higher degree of corneal fibrotic scarring. Although irregular surfacenanotopography isknownto alter cell adhesion and positional information to the surface cells, such as epithelium and endothelium, it remains to be proven whether this pathway directly leads to myofibroblastic transformation since incomplete return of an intact epithelial BM has been reported as another possible cause.

Bone marrow-derived stem cells

Bone marrow-derived immune cells have been recently been detected in the corneal stroma and they may also participate in TGF-b molecular signaling pathways, especially the histiocytes (tissue macrophages). The relative contribution and importance of bone marrow-derived stem cells to regenerative or fibrotic repair responses needs to be explored further. Because of their extraordinary plasticity and their ability to secrete embryonic cytokines and GFs that promote tissue regeneration over fibrotic repair, bone marrow-derived stem cells are being explored as restorative cellular therapy in preclinical trials for myocardial infarction, neurodegenerative disorders, and osteogenesis imperfecta. However, some bone marrow-derived cells, or their progeny, may reside for prolonged periods in the stroma and could make an as yet unknown contribution to the wound-healing process.

Cellular responses

After injury-mediated induction of the TGF-b/receptor molecular signaling pathway, keratocytes undergo a series of repair phenotypic and behavioral changes that are temporally and spatially regulated during the corneal wound-repair process. Keratocytes are neural crestderived mesenchymal cells that are sandwiched between collagenous lamellae forming a closed, highly organized syncytium. Keratocytes communicate with one another through gap junctions present on their long dendritic processes. The adult human corneal stroma has approximately 2.4 million keratocytes that occupy approximately 10% of the stromal volume. In postnatal life, they remain in the corneal stroma as modified fibrocytes, where they inconspicuously maintain the ECM of the corneal stroma. Depending on the type of injury and the molecular signaling invoked, keratocytes can be stimulated to undergo cell death (apoptosis) or transform to become a migratory keratocyte, an activated keratocyte, or a myofibroblast. Within minutes after injury, exposure to cytokines and GFs, such as IL-1 and TNF-a, found in the neurally stimulated tear film or released from injured epithelial cells is thought to lead to keratocyte apoptosis. The phenotypic transition (actin-cytoskeleton rearrangement) from a keratocyte to a migratory keratocyte appears to be initiated by loss of gap junction contact and possibly loss of keratocyte ion channels. Behaviorally, migratory keratocytes serve to reconstitute the cellularity of the tissue by once again re-forming a large intercommunicating network of keratocytes. The phenotypic transition