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Dry eye disease (DED) is a major cause of ocular discomfort, inflammation and dysfunction worldwide. Tear film instability in DED both causes and is exacerbated by disruption of the corneal epithelium. This tandem leads to a cycle of inflammation at the corneal surface involving immune cell dysregulation and increased chemokines and cytokines, which activate mitogen-activated protein kinases in the epithelium and elevates matrix metalloproteinases (MMPs). We review evidence suggesting that corneal collagen might be highly susceptible in DED to MMP-induced disruption, digestion, and thinning. We also summarize that collagen is far from inert and contains binding sites that serve as ligands for multiple inflammatory and immune regulators. Fragmented collagen not only challenges these receptor-ligand binding relationships, but also can promote recruitment and motility of pro-inflammatory immune cells. Current physician-directed therapies for DED focus on reducing inflammation, but do not directly ameliorate the underlying corneal damage that could exacerbate surface inflammation. We argue that an important gap in practice is lack of a direct therapeutic reparative for damaged corneal collagen, which is slow to heal, and likely amplifies sight-threatening inflammation. Healing fragmented collagen in the cornea may represent a more effective means to interrupt the “vicious cycle” of inflammation in DED and other conditions that damages, sometimes irreversibly, the ocular surface.
The disease is a major chronic and progressive contributor to the collection of conditions known as ocular surface disease, which also includes blepharitis, Meibomian gland dysfunction, allergy- related irritation, and chemical and thermal injury, among other conditions.
DED is linked to instability and hyperosmolarity in the tear film. These set into motion a cycle of tissue damage involving proinflammatory mediators (cytokines and chemokines), matrix metalloproteinases, antigen-presenting receptors, and immune cell infiltration (lymphocytes, macrophages, and dendritic cells) that implicate (from human patient studies) a degree of autoimmunity in DED.
Thus, physician-guided treatment of DED typically focuses on reducing inflammation and inflammatory immune responses through the use of topical cyclosporines, corticosteroids, tetracycline derivatives, and the like.
a better approach to DED, as well as other forms of ocular surface disease, may be a therapy that addresses the structural damage to the cornea in DED as a means of suppressing or even eliminating the cycle of inflammation that is so deleterious to positive clinical outcomes. In the ensuing sections we will focus on the inflammatory etiology of DED, its influence on corneal structures, and the role that collagen plays as a mediator of inflammation at the ocular surface and in the cornea.
2. Collagen composition differs across major corneal structures
A signature feature of all collagens is their triple helical structure– a set of three polypeptide chains comprising repeating sequences of glycine-x-y triplets where x and y often (but not always) represent proline and hydroxyproline.
Collagens include both fibrillar (I, II, III, V, XI) and non-fibrillar types, which themselves form different classes (e.g., the basement membrane type IV and short chain types VIII and X). Type I collagen is the most common, representing about 90% of the collagen in the human body, followed by types II-V. The fibrillar collagens comprise triple helical monomers of either identical (homotrimer) or different (heterotrimer) subunits that form long fibers through interactions with other monomers. In contrast, non-fibrillar collagen is able to interact in different orientations to form a meshwork or lattice with other extracellular matrix proteins such as laminin.
The cornea is a highly collagenous structure in which several types of collagen distribute across five primary layers (Fig. 1). On the anterior side, the corneal epithelium consists of multiple layers of non-keratinized squamous (or flattened) epithelial cells that serve as a cellular barrier to environmental, microbial, and inflammatory insults.
They produce a basement membrane that, like all basement membranes, is a specialized extracellular matrix that provides scaffolding during migration, differentiation, and maintenance of the epithelium. In a healthy eye the basement membrane is renewed by the basal epithelial cells as they regenerate and migrate from the outside edge of the cornea towards the center on a continuous basis. This process is challenged in many diseases and conditions, including keratoconus, corneal dystrophies, and recurrent corneal erosion.
The epithelial basement membrane is comprised primarily of four components: collagens, laminins, proteoglycans, and nidogens. Of these, the non-fibrillar type IV collagen is the most abundant and provides the greatest adherence for the epithelium; laminins represent the most prevalent of the non-collagenous proteins.
Proteoglycans are known to interact with collagen fibrils in the corneal stroma and to organize the collagen stroma. Indeed, a mutation in the gene encoding the core protein of a particular dermatan sulfate proteoglycan located in the cornea (decorin) is associated with stromal corneal dystrophy.
The basement membrane also helps anchor the epithelium by forming hemidesmosome attachments to underlying Bowman layer; through these, type VII collagen-rich fibrils pass and anchor to extracellular matrix plaques within the deeper corneal stroma, as shown by a combination of studies from human and animal cornea.
Bowman layer itself is thin (about 10-15 µm) and acellular. Its collagen is not renewed by keratocytes if it is damaged, nor during the natural thinning that in vivo human imaging studies show occurs with aging possibly through collagen crosslinking.
The functional contribution of the Bowman layer remains a matter of debate, though a combination of animal and human tissues studies indicate the layer likely offers a protective barrier to the underlying stroma against injury or infection.
Sometimes called Bowman membrane, this layer consists of randomly oriented collagen fibrils spanning the basement membrane anteriorly and the collagen lamellae of the corneal stroma posteriorly. The major component is type I collagen, followed by types III, V, and XII,
Most of the thickness of the cornea, some 80-90%, is reflected in the stroma, which is nearly half a millimeter thick. The stroma comprises a series of highly organized lamellae of collagen fibrils (primarily types I and V) and non-collagenous extracellular matrix components, particularly proteoglycan aggregates. Analysis of human eye bank corneas suggests that the anterior- most lamellae may insert directly into the Bowman layer to increase the strength of the corneal surface.
The integrity and shape of the stroma is maintained by keratocytes, which are specialized fibroblasts that produce collagen, glycosaminoglycans, and matrix metalloproteinases (or MMPs).
The posterior-most layer of the cornea is the endothelium, which separates the stroma from the aqueous chamber of the eye. The endothelium contains a single layer of flat, polygonal specialized epithelial cells that maintain the relatively dehydrated state of the stroma through a series of ionic pumps.
In contrast to the basal epithelium, cell replacement in the endothelium following insult or apoptosis does not occur via mitosis, but rather through centripetal migration. The endothelium is separated from the overlying stroma by its own basement membrane called Descemet membrane. Like other basement membranes, Descemet membrane is primarily type IV collagen, but also contains type VIII collagen, which enables hexagonal lattice structures.
An innermost layer secreted by conjunctival goblet cells consists of electrolytes, water, and mucins, which are hydrophilic carbohydrates. This mucous-containing layer is important for tear film adherence to the corneal surface; it binds with connecting microvilli of the hydrophobic apical epithelial cells, thus allowing the hydrophilic aqueous layer to adhere. This layer also protects underlying epithelial cells through high levels of transmembrane mucins and galectin‐3, which in their interactions with epithelial microvilli form the corneal glycocalyx.
This reduction also depletes the tear film of critical immune regulators like transforming growth factor-β2 (TGF-β2), which inhibits activation of antigen-presenting dendritic cells on the ocular surface.
The tear film contains several other anti-inflammatory factors, including tissue inhibitor of matrix metalloproteinase (TIMP-1), a glycoprotein that counters the action of MMPs in degradation of extracellular matrix and has anti-nociceptive effects.
The degree of SPK is reflected directly in severity of symptoms that include ocular burning, scratchiness and irritation, and blurred vision. Unfortunately, many topical drugs used to treat DED contain preservatives that are cytotoxic to the epithelium.
Damage to the basement membrane of the epithelial layer allows penetration of inflammatory cytokines, chemokines and other signaling molecules (including TGF-β) to the corneal stroma that can induce the differentiation of myofibroblast precursor cells, promoting haze, opacity and surface irregularity.
For example, IL-8 in DED patients promotes the recruitment of T lymphocytes that can damage the ocular surface by producing cytokines such as IL-17 and interferon gamma (IFN-γ), both of which are linked to increased MMPs.
Also, evidence primarily from animal models of DED indicates that apoptotic thinning of the epithelial layer in DED involves activation of mitogen-activated protein kinases (MAPKs), nuclear factor κB (NFκB), and IFNγ-dependent pathways.
In the next section we will examine the ramifications of increased MMP levels and activation on collagen throughout the cornea.
4. Matrix metalloproteinase activity in DED degrades corneal architecture
The sequelae of events that disrupt the corneal surface in DED clearly are inflammatory in nature, involving increased levels of MMPs, cytokines, and chemokines, all of which have secondary pro-inflammatory effects that often cascade and amplify each other through positive feedback. These relationships are summarized in a recent review of clinical and experimental studies.
While it is widely acknowledged that inflammation in DED influences the integrity and function of cellular structures (conjunctival goblet cells, corneal epithelium, stromal keratocytes, and so on), inflammation also could directly challenge integrity of the collagen substrate that forms the backbone of the cornea and the basis of the integrity of the epithelial cell layer.
The degradation of collagens depends on an initial enzymatic break of at least one of the strands forming the collagen trimer. Such a break causes uncoiling of the triple helical structure that in turn allows for subsequent cleavage and degradation. Like elastin, collagens are resistant to most proteolytic enzymes, thus adding to their general stability, but are susceptible to cleavage by MMPs, which comprise many types.
Of the MMPs notably elevated at the corneal surface in humans and animal models of DED (MMP-1, -3, -9, -10, and -1334,70), the collagenases MMP-1 and MMP-13 cleave triple-helical fibrillar collagens, while the gelatinase MMP-9 digests unwound collagen.
The effects of MMPs released by injured corneal epithelium in DED could act immediately at the surface. The type IV collagen so critical to the integrity of the epithelial basement membrane is susceptible to digestion by MMP-9 and the stromelysins MMP-3 and -10.
While MMP-9 is important to the cleavage of basement membrane components that enables migration of new cells under normal conditions, in recurrent injury, increased MMP-9 impedes reattachment of the collagen fibrils of migrating epithelial cells to underlying Bowman layer.
Also, human tissue studies indicate that an aberrant basement membrane can prohibit migration of epithelial cells to the apical surface for shedding, leading to cystic accumulation of cellular debris that compromises adherence of the epithelium to underlying Bowman layer.
Reevaluation of corneal dystrophies of Bowman's layer and the anterior stroma (Reis-Bucklers and Thiel-Behnke types): a light and electron microscopic study of eight corneas and a review of the literature.
Type VII collagen that forms the fibrils of the epithelial basement membrane and Descemet membrane is susceptible to the collagenase MMP-1, which also cleaves type VIII collagen found in Descemet' membrane.
MMP-1 also disturbs the helical region of both type I collagen and corneal stroma and type III collagen found in the Bowman layer. This initial injury increases susceptibility of types I and V collagen to further digestion via MMP-9,
Thus, recurrent stress to the corneal epithelium layer is compounded by MMP- induced damage to the basement membrane, which in turn perpetuates injury to underlying collagen-rich tissue. These interactions likely contribute to (or even explain) thinning of the stroma, of Descemet membrane, and of the endothelial layer in DED, all of which track closely with severity of symptoms.
5. Damaged collagen in DED could amplify inflammation
Inflammation in DED likely degrades corneal structure by increasing collagen exposure to the activity of MMPs, but in turn could also be exacerbated by that very damage, leading to a destructive cycle of inflammation and structural damage to the cornea. Far from inert, collagen within the layers of the cornea – like collagen in cellular networks in other tissues – is involved in a multitude of physiological processes, including mediation of cell proliferation, migration and adhesion, and inflammation. Collagen remodeling is a crucial process in tissue homeostasis and many other constitutive physiological events, and collagen is the major extracellular matrix component that interacts with almost all cell types. These functionalities arise from collagen's natural structure.
During normal turnover, intact collagen in its stable triple-helix structure is incorporated into the extracellular matrix, presenting multiple binding sites that serve as ligands for several cell surface receptors, including integrins, discoidin domain receptors, glycoprotein VI, and proteoglycan receptors.
Upon binding, such receptors modulate other molecules and cellular pathways related to remodeling, inflammation and immune regulation. Thus, aside from their structural role as scaffolding proteins, nearly all collagens initiate cellular signaling cascades by activating specific receptors on the cell surface. Signaling events mediated by these binding interactions are necessary for proliferation, motility, adhesion and survival of overlying epithelial cells as they regenerate and form new apical tissue, as in embryonic development.
Integrins, for example, are cell adhesion structures that play critical roles in cellular signaling, migration and survival, through their interactions with extracellular matrix components and other intra- and extracellular signaling moieties (e.g.,
Intact collagen, however, also provides ligand sites for signaling pathways involved in inflammation, including the leukocyte receptor complex (LRC), which comprises a diverse group of cell surface receptors primarily expressed by immune cells. Interestingly, the most highly expressed gene in cells of human Meibomian glands encodes a member of this group, the leukocyte-associated immunoglobulin-like receptor or LAIR-1,
LAIR-1 (also called CD305) is an inhibitory receptor that attenuates activation of most immune cells, including T cells, B cells, mast cells, eosinophils, basophils and monocytes/macrophages, while suppressing proinflammatory cytokine production upon binding ligand sites on intact triple helical collagen.
Crosslinking of LAIR-1 by triple helical collagens at glycine-proline-hydroxyproline repeats (GPO10) inhibits immune cells while diminished LAIR-1 or its binding sites in disrupted collagen exacerbates immune cell activation and leads to chronic inflammation in tissue.
Degradation of collagen produces much shorter triple-helical proteins, along with various single strand fragments. Thus, collagens through binding LAIR-1 set a threshold for inhibition or activation of immune cells entering damaged tissue based on the level of intact binding sites.
In DED, MMP-induced damage to Bowman layer, stroma and both epithelial basement and Descemet membranes could disrupt available binding sites for LAIR-1 on the collagen comprising these layers, types I, III, and IV, thus compromising its inhibitory actions on immune cells at the ocular surface.
The discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that bind collagen and serve as cellular sensors of environmental cues involved in cellular invasion, migration and adhesion and in remodeling of extracellular matrix.
Functionally, DDR1 and DDR2 are best characterized for their role in various metastatic cancers, promoting an invasive phenotype for proliferating cells; however, through an E-cadherin-dependent pathway, activated DDR1 contributes to epithelial cell differentiation and spreading.
which through type IV collagen in the basement membrane could activate DDR1. Since collagen types I and III are represented in Bowman's layer and I and V in the stroma, there is ample substrate for activation of both receptors.
Inhibitors of receptor tyrosine kinases used in cancer therapies, including several targeting DDR1 and DDR2, often cause DED in patients in conjunction with corneal inflammation (keratitis), conjunctivitis, and corneal thinning with erosion of the epithelium.
That these major side effects occur indicates that collagen activation of DDR1 and DDR2 is necessary to maintain homeostasis of the immune environment of the ocular surface. In fact, neither DDR1 nor DDR2 are able to bind to degraded collagen or to individual collagen α-chains but rather only to native triple helical collagens.
Thus, MMP-induced degradation of collagenous structures in the cornea during DED could very likely reduce binding of key modulators of inflammation, which may exacerbate the cycle of damage and impede repair of the corneal surface (Fig. 2). Interestingly, type IV collagen comprising the epithelial basement and Descemet' membrane is among the highly glycosylated collagens, the fragmentation of which promotes binding of the endocytic cell-surface receptor uPARAP/Endo180 (urokinase plasminogen activator receptor-associated protein
). This receptor is a member of the macrophage mannose receptor family of endocytic transmembrane glycoproteins and is specifically involved in the uptake and lysosomal degradation of collagen fragments generated by initial MMP-mediated cleavage, as shown by studies of tumor cells.
6. Conclusion: targeting collagen in dry eye could be therapeutic
The evidence reviewed thus far indicates that collagen damage in the cornea could exacerbate the inflammatory “vicious cycle” of DED (Fig. 2). To date, there has not been a direct therapeutic reparative for damaged collagen in DED or other corneal conditions and injury. This is an important gap in pharmaceutical interventions, DED aside.
Anterior ocular surface injuries primarily involve the cornea since ocular fixation tends to direct the cornea towards sources of potential danger. These include abrasions, lacerations, ulcers, thermal injuries, chemical injuries and blunt, concussive injuries. In each of these cases, like DED, a secondary effect of the primary insult is a complex inflammatory cascade that disrupts the immune homeostasis of the ocular surface. In what we have shown here, inflammation not only could damage collagen throughout the cornea, but in turn could amplify the effects of MMP-induced digestion of collagen, which reduces critical receptor-ligand binding sites. Thus, damage to the collagen scaffold would result in slowing of the healing process (in particular, healing of the corneal epithelium) with amplification of sight-threatening inflammation that underlies aberrant healing and even scarring.
In this sense, current physician-directed therapies for DED that rely heavily on reducing inflammation at the ocular surface have a relatively narrow range of action, since they do nothing directly to reduce the underlying structural damage arising from inflammation and possible corneal neuropathy that occurs in severe DED.
As well, long-term use of these traditional therapies is not without risk. For example, ocular application of corticosteroids over time is associated with both cataract and glaucoma and tissue-specific resistance, necessitating the use of alternative and more potent antiinflammatories.
Thus, there may be considerable advantage to repairing corneal collagen directly.
As ligand binding sites on collagen are identified, so too are small biomolecules that target such sites as possible therapeutics – including sites involved in tumor progression (like DDR1 and DDR2) and in inflammatory signaling.
Such peptide strands ostensibly could be applied as topical agents to heal corneal type I collagen. Collagen peptides can also be crafted to deliver custom bioactive molecules to sites of damaged and unfolded collagen triple-helices to promote healing and reduce inflammation, as in cutaneous wound beds.
Already, synthesized collagen peptides intended to promote tear adherence to the ocular surface show promise in animal models of dry eye in reducing surface inflammation and facilitating epithelium stabilization.
These results in total suggest that healing fragmented collagen in the cornea may in the end represent a more effective means to interrupt the non-ending cycle of inflammation in DED and other conditions that damage the ocular surface.
7. Method of literature search
We conducted a search of the PubMed database for “dry eye disease” and each of the following keywords: cornea, ocular surface, Bowman's layer, stroma, Descemet's membrane, collagen, collagen fibrils, collagen ligand, matrix metalloproteinase, collagenase, collagen degradation, collagen receptor, inflammation, basement membrane, keratitis, cell migration, cell adhesion, and collagen fragment. We limited the search to articles published in English from 1990 to June 2020, when the search was conducted last. We screen all abstracts and included relevant articles in this review, along with older seminal papers published prior to 1990.
Conflict of Interest
B.O.B, E.S, B.J.D.B., S.S.D, and D.J.C. are stakeholders in Stuart Therapeutics, Inc.
The authors thank Jay S. Pepose, MD, PhD, for helpful comments on the manuscript.
The epidemiology of dry eye disease: report of the epidemiology subcommittee of the international dry eye workShop.
Reevaluation of corneal dystrophies of Bowman's layer and the anterior stroma (Reis-Bucklers and Thiel-Behnke types): a light and electron microscopic study of eight corneas and a review of the literature.