Evidence of MMPs Being Removed by Cysteine, EDTA and N Acetyl  Cysteine

	AUTHOR INFORMATION	Section 1 of 10
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Author: C Stephen Foster, MD, FACS, Director of Immunology and Uveitis Service, Professor, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School Coauthor(s): Joseph JK Ma, MD, Staff Physician, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary

C Stephen Foster, MD, FACS, is a member of the following medical societies: American Academy of Ophthalmology, American College of Rheumatology, American College of Surgeons, American Medical Association, American Society for Microbiology, Association for Research in Vision and Ophthalmology, and Royal Society of Medicine

Editor(s): Fernando H Murillo-Lopez, MD, Instructor, Department of Ophthalmology, Bolivian National Institute of Ophthalmology; Francisco Talavera, PharmD, PhD, Senior Pharmacy Editor, eMedicine; Christopher J Rapuano, MD, Co-Chairman of Refractive Surgery Department, Associate Professor, Cornea Service, Wills Eye Hospital, Jefferson Medical College; Ralph Garzia, OD, Assistant Dean for Clinical Programs, Associate Professor, School of Optometry, University of Missouri at St Louis; and Hampton Roy, Sr, MD, Clinical Associate Professor, Department of Ophthalmology, University of Arkansas for Medical Sciences

	INTRODUCTION	Section 2 of 10
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Background: A corneal ulcer is defined as a lesion that involves degradation of the corneal stroma. This condition is associated with inflammation, either sterile or infectious. The primary purpose of this article is to highlight the pathogenesis of noninfectious stromal ulceration. The immune mechanisms of autoimmune ulcerative keratitis, particularly peripheral, are not included within this article.

Pathophysiology: An understanding of the pathophysiology of sterile corneal ulceration requires a review of the processes involved in epithelial and stromal wound healing, as well as an examination of the role of precorneal tear film, corneal nerves, proteolytic enzymes, and cytokines.

Epithelial wound healing

Corneal ulceration always begins with an epithelial defect. A persistent epithelial defect allows the corneal stroma to be exposed to the external environment and permits the process of stromal degradation.

Epithelial cell migration occurs centripetally until a defect is covered completely. Epithelial cells adjacent to the area of the defect flatten, lose their hemidesmosome attachments, and migrate on transient focal contact zones that are formed between cytoplasmic actin filaments and extracellular matrix proteins. Vinculin, integrin, fibronectin, fibrinogen, and fibrin are found in the region of these contact zones, which are formed continuously and cleaved to allow for cell migration. Plasmin is the protease responsible for cleaving fibrinogen and fibrin at these focal contact zones. The basement membrane is also important for epithelial migration, and abnormalities in basement membrane structure, whether due to trauma (eg, recurrent erosion syndrome) or dystrophy (eg, basement membrane dystrophy), can lead to persistence of corneal epithelial defects and stromal ulceration.

A sufficient supply of progenitor stem cells to facilitate epithelial cell proliferation is important for the cornea. A deficiency of limbal stem cells, either from disease (eg, aniridia) or trauma (eg, chemical burn), can preclude adequate epithelial wound healing, resulting in a persistent epithelial defect and allowing for stromal ulceration. Limbal stem cell transplantation (autograft, allograft, or ex vivo expansion) may be necessary in these cases.

Stromal wound healing

Stromal wound healing occurs via stromal keratocyte migration, proliferation, and deposition of extracellular matrix molecules, including collagen (specifically type III), adhesion proteins (eg, fibronectin, laminin), and glycosaminoglycans. These processes are facilitated by a phenotypic change among quiescent keratocytes to become active myofibroblasts, a task mediated by transforming growth factor beta (of presumptive epithelial origin).

Stromal necrosis and degradation

Matrix metalloproteinases (MMPs) are a group of structurally related endopeptidases that require a metal cofactor. To date, 20 such enzymes have been identified and are categorized according to their substrate specificity. The main function of metalloproteinases is to degrade extracellular matrix and basement membrane components. With respect to corneal wound healing and ulceration, MMP-1, MMP-2, MMP-8, and MMP-9 appear to be the most important. MMP-2 and MMP-9 are known as gelatinases and are involved in cleaving collagen types IV, V, VII, and X, as well as fibronectin, laminin, elastin, and gelatins. MMP-1 and MMP-8 are involved in cleaving collagen types I, II, and III. Metalloproteinases are secreted as proenzymes by neutrophils, injured epithelial cells, and keratocytes. They are activated by proteolytic cleavage of the N-terminal region in the extracellular compartment. In vivo, tissue inhibitors of metalloproteinases (TIMPs) inhibit collagenase activity.

MMP-9 is expressed by basal (replicating) epithelium and is thought to be important in the degradation of the basement lamina. In chemical injuries, this step always precedes the degradation of stromal extracellular matrix by MMP-1 and MMP-8. The collagenolytic activity of these latter enzymes reach a nadir of activity at approximately 3 weeks following injury, a time frame that parallels the peak of collagen synthesis activity in an alkali burn animal model. A relatively higher degree of collagenolysis relative to synthesis is thought to result in degradation, progressive corneal thinning, and, hence, ulceration of the corneal stroma. In vivo, this balance is moderated by cytokines secreted by the epithelium, stromal keratocytes, and inflammatory cells.

Since all metalloproteinase enzymes require metal cofactors Ca2+ and Zn2+, such chelating agents as ethylenediaminetetraacetic acid (EDTA), acetylcysteine, and penicillamine inhibit collagenase activity. Tetracyclines also possess anticollagenolytic activity. Endogenous TIMPs and alpha2-macroglobulin have metalloproteinase inhibitory activity and are probably the main inhibitors of MMPs in vivo.

The role of corneal nerves

The cornea is densely innervated by fibers of the ophthalmic division of the trigeminal nerve and sympathetic nerve fibers from the superior cervical ganglion. The corneal epithelium is supplied by approximately 1000 small axons. Decreased corneal sensation from denervation can result in stromal ulceration and perforation. A decrease in tearing, protective reflexes, and blink rate are associated with decreased corneal sensation.

In 1954, the classic experiment by Sigelman et al demonstrated that ocular surface changes associated with neurotropic keratitis in denervated animals persist despite tarsorrhaphy, suggesting a trophic effect of corneal nerves. The exact mechanism of this trophic effect is not definitively known. Evidence suggests that sensory neuron loss leads to a severe depletion of acetylcholine in an otherwise acetylcholine rich tissue, resulting in a relative decrease in epithelial cell growth. Other studies attributed the depletion of substance P associated with sensory denervation as the cause of the changes associated with neurotrophic keratitis. Recent clinical trials of nerve growth factor (NGF) by Bonini et al demonstrated a beneficial effect in promoting corneal epithelial wound healing and possibly improving sensitivity in patients with neurotrophic keratitis.

The role of the precorneal tear film in ulceration

The exposure of the bare corneal stroma to its environment secondary to deficient or impaired epithelial wound healing is thought to contribute to stromal degradation through environmental factors, cytokines, lytic enzymes, and neutrophils in the tear film. Direct neutrophil adhesion to the corneal stroma theoretically allows hydrolytic and collagenolytic enzymes, including MMP-8 (neutrophil collagenase), to contribute to the degradation of the corneal stromal extracellular matrix. Dohlman et al and subsequently Kenyon et al demonstrated that a glued on methylacrylate lens applied to a rabbit alkali burn model of corneal ulceration protected the stroma from collagenolysis by neutrophils and injured epithelial cells. Keratocyte fibroblasts also may contribute to this milieu. The prevention of neutrophil infiltration and promotion of epithelialization is thought to be at least one of the mechanisms responsible for the beneficial effect of amniotic membrane graft use in preventing stromal ulceration.

In addition, cytokines, such as hepatocyte growth factor (HGF), keratocyte growth factor (KGF), and epidermal growth factor (EGF), are produced by the lacrimal gland and, thus, are present in tears. HGF is upregulated in response to corneal injury in parallel with increased aqueous tear production. In the wounded cornea, these cytokines may play an important role in regulating epithelial healing. Inflammatory cytokines, including interleukin 1 (IL-1) alpha, are detectable in normal human tears and may be important in causing further degradation of the corneal stroma either directly by inducing keratocyte apoptosis or by recruiting inflammatory cells via their chemotactic properties. In addition, an irregular tear film and a decreased tear film breakup time over the area of the bare stroma can cause a delle effect that may contribute to an unfavorable cellular environment for the viability and proliferation of stromal keratocytes.

The role of cytokines

The complex autocrine and paracrine functions of the cytokines involved in the interactions between the corneal epithelium and stromal keratocytes are important in achieving the appropriate responses to corneal wound healing. These responses are orchestrated by complex interactions between the cytokines secreted by each of these cell types. While their precise triggers and interactions are still being elucidated, cytokines can induce and mediate many of the fundamental steps involved in wound healing.

Epithelial cell migration, proliferation, and differentiation are influenced by the stromal keratocyte cytokines, KGF and HGF. The cornea is not unique with respect to the stromal-epithelial interactions of these 2 cytokines, which are mediators of similar interactions in the breast, skin, and lung. Although the expression profiles of these cytokines lend themselves toward a linear interpretation of their stromal-epithelial interactions, these cytokines clearly are modulated further in vivo by the effects of other cytokines and truncated receptors of these molecules.

In what is likely to be merely the tip of the iceberg with respect to the understanding of cytokine-cytokine interactions, both KGF and HGF mRNA production are altered by the fibroblast cytokines, EGF, transforming growth factor alpha, platelet-derived growth factor (PDGF), and IL-1. In addition, EGF, PDGF, IL-1 alpha, interleukin 6 (IL-6), and tumor necrosis factor (TNF) at low concentrations appear to enhance fibronectin (FN)-induced epithelial cell migration.

Not to be eclipsed by stromal influences, epithelial cells modulate important keratocyte responses to epithelial cell injury. Keratocyte wound healing processes, including MMP production and regulation, HGF and KGF production, and keratocyte apoptosis, are mediated via various cytokines, including IL-1 and soluble Fas ligand. Anterior stromal keratocyte cell death is an important feature of corneal wounding and stromal degradation.

Beyond keratocyte cell death caused by mechanical injury or necrosis associated with neutrophil infiltration, IL-1– and Fas ligand–mediated apoptosis is an important stromal response to epithelial injury. Since both of these cytokines can be produced by keratocytes, autocrine modulation of these responses may occur. IL-1 and PDGF also regulate MMP expression in stromal keratocytes. The exact keratocyte response to IL-1 is likely to be determined by the cytokine milieu in which the targeted keratocyte resides. Other cytokine systems that have demonstrated fibroblast apoptosis include TNF and bone morphogenic protein (BMP).

Meticulous control of these cytokines conceivably allows for more predictable corneal wound healing. Topical KGF has been shown to accelerate epithelial wound healing in a rabbit model of corneal ulceration. Since its effects are mediated through a paracrine pathway, topical use of cytokines (eg, KGF) may prove to be especially effective in ocular disorders accompanied by loss of epithelium that require corneal limbal stem cell proliferation.

Cytokines and trophic factors from corneal nerves, the tear film, the conjunctiva, conjunctival vessels, the endothelium, and the anterior chamber may have important modulating effects on corneal epithelial and stromal healing responses and, thus, corneal ulceration.

Frequency:
 

• In the US: The incidence rate depends on the etiology of the corneal ulcer.

Mortality/Morbidity: Corneal scarring, decreased vision, neovascularization, perforation, and blindness are associated with this condition.

Sex: Because of an increased incidence of injuries, this condition may be seen more frequently in males than females.

	CLINICAL	Section 3 of 10
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History:

• In diagnosing this condition, differentiating between infectious and noninfectious etiologies is crucial. Since the clinical management of any corneal ulcer is dependent on its etiology, obtaining all of the salient factors (eg, endogenous, exogenous, local) is important. Therapies for sterile persistent ulcerations should be considered only after adequately addressing infectious and systemic factors.

• Key points to assess in obtaining the history of a patient with a corneal ulcer include the following:

• Prior ocular history - Prior ocular and adnexal surgery, recurrent episodes or infections (eg, herpes), and corneal dystrophy

• Past medical history - Immune status, collagen vascular diseases, systemic infections, diabetes, malnutrition, alcoholism, and chronic debilitating diseases

• History of trauma - Foreign bodies and their origin (eg, soil, vegetation, water), chemical splashes, and lid lacerations

• Contact lens use - Type, frequency, duration, overnight use, and hygiene

• Medications - Ocular and otherwise

• History of present illness - Duration, ocular symptoms (eg, degree of pain vs clinical impression), and chronicity

• Social history - Patient from an area endemic for certain infectious processes, nutritional status, and any alcohol abuse

• The etiology of a sterile ulcer is often multifactorial; in this setting, identifying the coconspirators in this process is important. A thorough evaluation to identify potential factors, including medications (medicamentosa), impaired corneal sensation (neurotrophic), exposure (eg, lagophthalmos), and reduced tear production (sicca), is necessary in most cases of persistent noninfectious ulceration.

Physical:

• The physical examination should begin with a gestalt impression of the entire patient, with attention to the following:

• General health of the patient - Skin lesions, skeletal abnormalities, mental status, degree of discomfort, hearing aids, scars, and limitations to ambulation that may indicate a systemic illness

• Local ocular adnexa and related organs - Eyelids, lacrimal system, blink rate, scars, mucous membranes (eg, lips/mouth, conjunctiva), orbit, symmetry, and evidence of inflammation or infection

• Palpation - If indicated for orbital resiliency (thyroid/exposure), lymphadenopathy, and lacrimal or other adnexal masses

• Observation - Lagophthalmos and blink rate

• Assessment of vital signs of the eye - Visual function, corneal sensation, tonometry, pupil function, and motility of the eye (Corneal sensation should be checked prior to tonometry.)

• On slit lamp examination of the cornea, note the appearance of and evaluate the following:

• Conjunctiva, sclera, and lids - Erythema, pattern of injection (ciliary flush, diffuse or deep), perilimbal nodules, discharge, lid closure, lid margin disease, and flipped upper lid to exclude foreign body and floppy eyelid syndrome

• Tear film - Degree, symmetry, regularity, and presence of debris

• Epithelium - Location of epithelial defect (localized or diffuse), regularity, and microcysts

• Stroma - Thinning and presence/pattern of infiltrates (eg, ring, feathery, radial)

• Endothelium - Keratic precipitates

• Anterior chamber - Hypopyon and inflammation

• Corneal sensation

• Symmetry between the eyes

• Fluorescein examination

• Dilated examination (if necessary)

Causes: A thorough history and physical examination should allow a clinician to narrow down the differential diagnosis.

• Infectious causes (which need to be ruled out first) include the following:

• Bacterial (focal infiltrate)

   

• Fungal (vegetable matter, eg, branch; appearance of satellite lesions; feathery borders to infiltrate; chronic)

   

• Acanthamoeba (contact lens wear, tap water, soil, severe pain out of proportion to the appearance, radial keratitis, ring ulcer)

   

• Herpes simplex virus (history, dendrites, decreased sensation, disciform keratitis, increased intraocular pressure)

• Herpes zoster virus (vesicles over dermatome; pseudodendrites, no true terminal bulbs; decreased sensation; increased intraocular pressure)

   

• Contact lens related (infectious or noninfectious)

• Noninfectious causes include the following:

• Chemical burns, including alkali/acid burn (check pH)

   

• Thermal/radiation burns (history)

   

• Sicca (filaments, Sjögren syndrome)

   

• Neurotrophic (decreased sensation, may have minimal pain, rolled edges, oval, lower one half of cornea, may be quite thin, herpes zoster virus/herpes simplex virus, postsurgery)

• Exposure (lagophthalmos, lid abnormalities, inadequate blink, facial palsy, proptosis, thyroid disease)

   

• Medicamentosa (drops)

   

• Atopic (history, follicles/papillae)

   

• Basement membrane abnormalities (microcysts, evidence of map-dot-fingerprint or anterior stromal dystrophies, history of trauma, other dystrophy)

   

• Factitious

• Immune-related causes (usually peripheral) include the following:

• Wegener granulomatosis

• Rheumatoid arthritis

• Other collagen vascular diseases (indicated from history and associated systemic findings)

	DIFFERENTIALS	Section 4 of 10
Author Information Introduction Clinical Differentials Workup Treatment Medication Follow-up Miscellaneous Bibliography

Corneal Abrasion
Corneal Erosion, Recurrent
Corneal Melt, Postoperative
Dry Eye Syndrome
Herpes Simplex
Herpes Zoster
Keratitis, Bacterial
Keratopathy, Neurotrophic
Sjogren Syndrome
Ulcer, Corneal