DETECTION OF PROTEINASE INHIBITORS IN EQUINE AMNIOTIC MEMBRANE FOR DEVELOPMENT OF THERAPEUTIC AGENT FOR ULCERATIVE KERATITIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: EXTENDED
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0191480
• Project Start Date: Jan 1, 2002
• Project End Date: Dec 31, 2010
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
College of Veterinary Medicine

Non Technical Summary
The cornea is composed of three layers; the outermost tear film and epithelium, the middle stroma and the innermost endothelium. The epithelium consists of layers of cells. Loss of the epithelium is called an ulcer. The thickest layer, the corneal stroma, is composed of a few cells and a large amount of collagen. The maintenance and the repair of cornea are thought to involve a balance of collagen formation, breakdown, and repair. A large variety of both normal and disease processes involving corneal collagen remodeling have been shown to be caused by enzymes called proteases. Excessive levels of these proteases can lead to rapid breakdown of collagen and other components of the cornea. Bacteria and fungi can also produce enzymes that also contribute to corneal ulcers. Corneal ulcers occur when epithelial cells are absent, and are common causes of blindness in horses in Florida. A simple corneal scratch or ulcer can rapidly become infected. These ulcers are terrible diseases that can lead to the loss of many eyes and premature retirement of many horses. Any type of corneal ulcer can result in extensive corneal scar tissue formation, with large scars causing blindness. Treatment of ulcers in horses is directed at removing infection and reducing scarring. Therapy against enzymes in the cornea could speed healing. Sources of enzymes inhibitors are blood, and the amniotic membrane. Amniotic membrane is tissue that surrounds the fetus or baby horse. It has been used as a graft or transplant in humans to successfully repair corneal ulcers. The amniotic membrane not only fills in the corneal ulcer but helps the cornea heal as it contains enzyme inhibitors. The purpose of this project is to determine the presence of proteinase inhibitors in the equine amniotic membrane. We anticipate that the information gathered from this project will result in a better understanding of the possible mechanisms of action of this tissue when it is grafted to the diseased cornea before undertaking further studies to assess the clinical use of the amniotic membrane in equine ophthalmology. The amniotic membrane graft could represent an interesting treatment of corneal ulcerations in horses, by keeping corneal scars small. This information will greatly benefit horses and the equine industry in the USA and the State of Florida.

Animal Health Component

50%

Research Effort Categories

Basic

25%

Applied

50%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3810	1102	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1102 - Mycology;

Keywords

proteinases

amniotic

membrane

horse

equine

Goals / Objectives
Ocular surface diseases (corneal ulceration, corneal stromal abscessation) are common causes of loss of vision in horses in Florida and the USA. Corneal injuries are devastating diseases that lead to the loss of many eyes and premature retirement of racing performance and show animals. Any type of corneal damage in horses will result in corneal scar tissue formation with large scars often causing significant visual impairment. The cornea is composed of three layers; the outermost tear film and epithelium, the middle stroma and the innermost endothelium. The thickest layer, the corneal stroma, is composed of an extracellular matrix (ECM) with a few cells and a large amount of collagen fibrils. The normal maintenance and the repair of the corneal ECM involve enzymes called matrix metalloproteases (MMPs) and inhibitors of these enzymes (TIMPs, tissue inhibitors of metalloproteinases). Excessive levels of tear film and stromal enzymes (or proteinases) can lead to rapid degradation of corneal stromal collagen and other components of the corneal ECM. These enzymes are produced by the cornea leucocytes and by bacterial and fungal organisms. Therapy of deep corneal ulcers in horses consists of aggressive medical and often surgical treatment. Topically administered antimicrobials, cyclosporin, and proteinase inhibitors are utilized. Amniotic membrane obtained by caeserean section has been used successfully in humans to repair corneal ulcers of various types. The amniotic membrane transplant not only physically fills in a corneal wound, but speeds the corneal healing as it contains several proteinase inhibitors that reduce vascularization and scarring. Amniotic membrane from term deliveries may contain increased proteinases levels and it is not utilized. The goals of this project are to determine the presence of proteinases (MMP2, MMP9) and proteinase inhibitors (α2 macroglobulin, α1 antichymotrypsin, inter α-1 trypsin inhibitor, α1 antitrypsin, and α2 plasmin inhibitor) in equine amniotic membranes obtained from normal delivery as well as from caesarean section (c-section). We propose to obtain placentas from 6 mares presented to the University of Florida Veterinary Medicine Teaching Hospital. We believe that the determination of the presence of protease inhibitors in equine amniotic membrane will allow us to better understand the possible mechanisms of action of an amniotic membrane transplant or graft onto an ulcerated cornea, and will allow us to undertake an educated use of the amniotic membrane transplants in equine ophthalmology.

Project Methods
Collection of the placenta from normal delivery and caesarian section: We will obtain the placenta and amniotic membrane from mares that have a normal delivery. The placenta will be collected in a sterile manner. After cleaning the amniotic membrane will be cut in 5X5 cm pieces and place on a 0.45 micron pores nitrocellulose paper (Biorad, CA). Two different methods will then be used to store the pieces of amniotic membrane: a "dry" storage in Petri dishes and a storage in Dulbecco's modified Eagle medium and glycerol containing an antibiotic mixture. Both of them will be stored at -80 degrees C until use. Analyses will be conducted on the fresh amniotic membrane, the amniotic membrane stored in a dry form, and the one stored in the media. The amniotic membrane (AM) extracts will be analyzed using one of the methods described. To obtain the extracts, the amniotic membrane samples will be thawed and homogenized in a grinder with PBS and TritonX-100 (1 square cm of AM with 1 mL of 1% triton in PBS).The mixture will be centrifuged at 15.000 rpm during 1 minute and the supernatant will be analyzed. Electrophoresis: 10 μL of the sample (supernatant of the homogenized amniotic membrane) will be mixed with a sample buffer (1:1) and 15 μL of the mixture will be loaded in to the well of either a 10% gelatin gel, or a 12% casein gel (Novex, CA). Pro and active forms of MMP2 and MMP9 (from Oncogen, MA) as well as molecular weight protein standards will be also loaded. The electrophoresis will be done using the X-cell Sure lock apparatus. Then the electrophoresis will be followed by renaturation, incubation (digestion of substrates) and staining (Rapid coomassie blue, Diversified Biotech,MA ) of the gel. 10 μL of the sample of amniotic membrane extracts (supernatant) will be mixed with a sample buffer (1:1) and 15 μL of the mixture will be loaded in to the well of 10% Tris-glycine SDS PAGE Gels. The electrophoresis will be done using the X-cell Sure lock apparatus( from Novex, CA). The transfer to the membrane will be immediately done after the electrophoresis using the X-cell Sure lock apparatus. After blocking the membrane, the specific antigen detection will be done by incubation. Then will follow the incubation with the secondary antibody (and color reagent). The membrane will be scanned with the GS-710 Calibrated imaging densitometer for a computerized analysis (determination of the molecular weight and the concentration). The analysis of the zymogram gels and the western blots will be done using the GS-710 Calibrated imaging densitometer. Mean concentrations of proteinases will be compared between amniotic membranes from normal delivery and from c-section by using the student t-test. Mean concentrations of proteinase inhibitors will be compared between amniotic membranes from normal delivery and from c-section by using the student t-test.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: We continue to investigate the proteins present in normal frozen equine amniotic membrane (EAM). Over 300 have been detected using proteogenomic techniques. PARTICIPANTS: We are now collaborating with Dr Paula Galera of the University of Brasilia in Brazil. TARGET AUDIENCES: Scientists and veterinarians studying corneal disease will be interested in this project data. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have described a reference map from the equine amniotic protein that can be used to help explain the positive results found in horse keratoplasties using this graft. The identification of key proteins can help explain the observed corneal wound healing. The continue identification of other amniotic proteins may further our understanding of corneal healing in the horse.

Publications

• Galera P, Coleman J, Sapp H, Plummer C, Brooks D: Proteome from frozen equine amnion. American College of Veterinary Ophthalmologists 42nd Annual Meeting, Hilton Head, SC, October 28, 2011, (Abstract) p 10.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Case series Corneal disease is the most frequent presenting complaint for equine patients presenting to the ophthalmology department at the Veterinary Medical Center of the University of Florida. Over the last few years, we have been utilizing AMT as a therapy for severe keratopathy in our equine patients. The following is a description of the equine cases we have to date with follow-up greater than six months, the indication for which AMT was performed, and the cosmetic and visual outcomes thereafter. Results Fifty-eight cases were identified that had received AMT for some sort of globe or sight threatening keratopathy. Thirty cases were female and 28 were male, neuter-status undetermined. Nine breeds were represented including thoroughbred, Quarter Horse, warmblood, Standardbred, Welsh Pony, Tennessee Walking Horse, Arabian, American Paint Horse and Belgian Draft. The ages ranged from one month to 22 years of age with a median of 6.7 years. Of these 58 cases, 31 were treated for melting ulcers or bullous keratopathy, 13 had AMT performed in conjunction with penetrating keratoplasty, 12 received AMT following keratectomy for a superficial neoplasm and 2 received AMT following keratectomy for biopsy and treatment of an immune-mediated keratitis. Pre and post-operative medical therapy for all cases consisted of the systemic non-steroidal anti-inflammatory medication flunixin meglumine, topical atropine, topical autologous serum and topical antibiotics and antifungal agents. The dosage and frequency of these medications varied with the severity of the disease process and the response to therapy. At the final post-operative examination, visual status was assessed and 53 affected eyes were determined to be sighted. The five eyes that were not sighted had either severe corneal scarring preventing vision, had secondary intraocular damage resulting in phthisis bulbi with cataract formation and extensive posterior synechia or were enucleated because of endophthalmitis. The extent of corneal scarring was used to determine the cosmetic outcome. Mild corneal scarring was present in 40 eyes (69%), all of which were deemed to have excellent cosmesis, barely noticeable to the casual observer. Moderate corneal scarring was present in 14 eyes (24%), all of which were characterized as having acceptable cosmesis. Unacceptable cosmesis occurred in 4 eyes (7.0%), one of which had extensive corneal scarring that prevented sight, two of which became phthisical and one of which was enucleated secondary to endophthalmitis. The eyes that were ultimately characterized with moderate or severe scarring were the most severely affected by primary disease. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The utility of AMT in the healing of ocular surface defects is undeniable. Its uses and indications are constantly evolving and expanding in both the human and veterinary ophthalmology arenas. In many cases such as the more severely affected described above, the traditional therapy of placing a conjunctival graft upon a corneal wound or defect would render a sighted or a potentially sighted globe blind. AMT should be considered as a treatment option in cases that require ocular surface reconstruction for the treatment of corneal and conjunctival defects both organic and surgically induced.

Publications

• Plummer CE, Ollivier F, Kallberg M, Brooks D, Barrie K, Utter M, Gelatt K: The use of amniotic membrane transplantation for ocular surface reconstruction: A review and series of 58 equine clinical cases (2002-2008). Veterinary Ophthalmology 12 (Supple-ment 1: 17-24, 2009. Addendum correction: Veterinary Ophthalmology 14.2, 2011.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: IL-1 beta is a cytokine that plays an important role in the inflammatory process. It is present in the tear film of humans with inflammatory corneal and conjunctival disease and has been found in the cornea of humans, primates and laboratory animals with corneal disease. It plays a role in the activation of numerous enzymes that are elaborated by infectious agents that can affect the cornea and by the keratocytes (corneal cells) themselves. These enzymes, once activated, breakdown the corneal stromal collagens and proteins and produce a "melting" phenomenon that compromises the cornea's integrity and puts the globe at risk for rupture. Samples of tears from 25 normal horses and 25 horses with corneal ulcers have been collected and will be assayed with a commercially available quantitative sandwich ELISA for the presence of IL-1 beta. The presence and quantity of IL-1 beta will be compared between the two groups. PARTICIPANTS: Primary Investigators: Dr. Caryn E. Plummer is an Assistant Profesor in Ophthalmology at the University of Florida Veterinary Medical Teaching Hospital. She will be involved in all phases of the project, including project design, and subsequent data analysis. Dr. Dennis E. Brooks is Professor in the Ophthalmology Section of the Small Animal Clinical Sciences Department at the University of Florida, and has experience with tear film studies. He will directly supervise all aspects of the project and provide advice and guidance as needed. Dr Michala Henriksen is a Danish veterinarian that will involved in all phases of the project for her PhD project. TARGET AUDIENCES: The target audience includes ophthalmologists, and scientists working in corneal diseases. PROJECT MODIFICATIONS: None

Impacts
No studies have yet been performed prior to this one to determine if IL-1 beta is present in equine corneas or tear fluid in either normal or diseased eyes. It is our hypothesis that IL-1 beta is present in higher concentrations in the tear fluid of horses with corneal ulcerations. The tear film that covers the surface of the eye contains the inflammatory mediators that are produced at the level of the cornea and is easily sampled and assayed. This study will serve as the precursor to testing inhibitors and receptor antagonists of IL-1 beta in the search for better treatments for equine corneal disease.

Publications

• No publications reported this period

Progress 10/01/05 to 09/30/06

Outputs
Progress regarding drug and antiproteinase therapy for horses with severe eye problems continues. This project is ongoing.

Impacts
This research is changing and setting the medical standards of care for the treatment of eye disease in horses.

Publications

• Nasisse MP, Jamieson VE, Brooks, D.E.: Cornea and Sclera. In Equine Surgery, Auer, JA, Stick JA (eds), 3rd ed, W.B. Saunders, St Louis, pp 731-743, 2006.
• Dawson WW, Dawson JD, Hope GM, Brooks DE, Percicot CL: Repeat sample in-traocular pressure variance in induced and naturally ocular hypertensive monkeys. J Glaucoma 14(6): 426-431, 2005.
• Tolar EL, Hendrix DVH, Rohrbach BW, Plummer CE, Brooks DE, Gelatt KN: Evaluation of clinical characteristics and bacterial isolates in dogs with bacterial keratitis: 97 cases (1993-2003). Journal of the American Veterinary Medical Association 228(1): 80-85, 2006.
• Brooks DE: Painful ocular lesion. NAVC Clinicians Brief 4(1): 49-51, 2006.
• Plummer CE, Kallberg ME, Ollivier FJ, Brooks DE, Gelatt KN: Suspected ivermectin toxicosis in a miniature mule foal causing blindness. Veterinary Ophthalmology 9(1): 29-32, 2006.
• Ollivier FJ, Barrie KP, Mames RN, Kallberg ME, Greiner EC, Plummer CE, Gelatt KN, Strubbe DT, Brooks DE: Pars plana vitrectomy for the treatment of ophthalmomyiasis interna posterior in a dog. Veterinary Ophthalmology 9(4): 259-264, 2006.
• Brooks DE: Hypertensive uveitis and glaucoma of horses. Current Techniques in Equine Practice 4(1): 72-80, 2005.
• Brooks DE: Guest Editor. Current Techniques in Equine Practice 4(1), 2005.
• Brooks, D.E.: Orbit. In Equine Surgery, Auer, JA, Stick JA (eds), 3rd ed, W.B. Saunders, St Louis, pp 755-766, 2006.
• Brooks, D.E.: Ocular Emergencies and Trauma. In Equine Surgery, Auer, JA, Stick JA (eds), 3rd ed, W.B. Saunders, St Louis, pp 767-774, 2006.
• Scotty NC, Brooks DE, King MA, Clare-Salzer M, Schultz GS: Antigen presenting cells in the canine corneal stroma. (Abstract 2811) Association for Research in Vision and Ophthalmology Annual Meeting, Ft Lauderdale, FL May 1-5, 2005.
• Scotty NC, Brooks DE, Schuman CD, Barrie KP, Plummer CE, Kallberg ME: In vitro efficacy of an ophthalmic drug combination against common equine corneal pathogens. ACVO abstract, p 59, 2006.

Progress 10/01/04 to 09/30/05

Outputs
No progress reported this period

Impacts
Great progress studying the use of antiprotease medications and amniotic membrane transplantation have been made. We now understand horse eye problems better and are better able to treat diseases of the horse cornea. This project is ongoing.

Publications

• Ollivier FJ, Brooks DE, Kallberg ME, Komaromy AM, Lassaline ME, Andrew SE, Ge-latt KN, Stevens SR, Blalock TD, van Setten G, Schultz GS: Evaluation of various com-pounds to inhibit activity of matrix metalloproteinases in the tear film of horses with ul-cerative keratitis. American Journal of Veterinary Research 64(9): 1081-1087, 2003.
• Ollivier FJ, Brooks DE, Schultz GS, Blalock TD, Andrew SE, Komaromy AM, Cutler TJ, Lassaline ME, Kallberg ME, Van Setten GB: Connective tissue growth factor in tear film of the horse: detection, identification and origin. Graefes Arch Clin Exp Ophthalmology 242(2): 165-171, 2004.
• Ollivier FJ, Brooks DE, Van Setten GB, Schultz GS, Gelatt KN, Stevens GR, Blalock TD, Andrew SE, Komaromy AM, Lassaline ME, Kallberg ME, Cutler TJ: Profiles of matrix metalloproteinase activity in equine tear fluid during corneal healing in 10 horses with ulcerative keratitis. Veterinary Ophthalmology 7(6): 397-406, 2004.
• Barros PSM, Safatle AMV, Godoy CA, Souza MSB, Barros LFM, Brooks DE: Amniotic membrane transplantation for the ocular reconstruction of the ocular surface in three cases. Veterinary Ophthalmology 8(3): 189-192, 2005.
• Andrew SE, Nguyen A, Jones GL, Brooks DE: Seasonal effects on the aerobic bac-terial and fungal conjunctival flora of normal thoroughbred brood mares in Florida. Vet Ophthalmol 6(1): 45-50, 2003.
• Lassaline ME, Brooks DE, Ollivier FJ, Komaromy AM, Kallberg ME, Gelatt KN: Equine amniotic membrane transplantation for corneal ulceration and keratomalacia in three horses. Veterinary Ophthalmology 8(5): 311-318, 2005.
• Brooks DE: Corneal/Scleral Lacerations. THE 5-MINUTE VETERINARY CONSULT Equine. Brown CM, Bertone J (eds). Lippincott, Williams & Wilkins, Philadelphia, pp 280-281, 2002.
• Brooks DE: Corneal Stromal Abscesses. THE 5-MINUTE VETERINARY CONSULT Equine. Brown CM, Bertone J (eds). Lippincott, Williams & Wilkins, Philadelphia, pp 282-283, 2002.
• Brooks DE: Corneal Ulceration (Expanded). THE 5-MINUTE VETERINARY CONSULT Equine. Brown CM, Bertone J (eds). Lippincott, Williams & Wilkins, Philadel-phia, pp 284-285, 2002.
• Brooks DE: Ulcerative Keratomycosis. THE 5-MINUTE VETERINARY CONSULT Equine. Brown CM, Bertone J (eds). Lippincott, Williams & Wilkins, Philadelphia, pp 1086-1087, 2002.
• Brooks DE: Equine Ophthalmology-Made Easy Book. Teton NewMedia, Jackson Hole, WY, 2002.
• Brooks DE, Ollivier FJ: Matrix Metalloproteinase Inhibition in Corneal Ulceration. Veterinary Clinics of North America-Ocular Therapeutics 34(3): 611-622, 2004.
• Brooks DE: Inflammatory Stromal Keratopathies: Medical Management of Stromal Keratomalacia, Stromal Abscesses, Eosinophilic Keratitis, and Band Keratopathy in the Horse. Veterinary Clinics of North America-Equine Practice 20(2):345-360, 2004.
• Brooks DE:Penetrating keratoplasty, deep lamellar endothelial keratoplasty, and posterior lamellar keratoplasty in the horse. Current Techniques in Equine Practice 4(1): 37-49, 2005.