The Cornea: New Biologic Research

C. Stephen Foster, MD

Disclosures

June 02, 2003

Editorial Collaboration

Medscape &

The 2003 Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO) was held in Ft. Lauderdale, Florida, May 4-9, 2003. There were 5246 presentations -- over 1000 more than were presented a year earlier. Presentations relevant to cornea research included 8 oral paper presentation sessions and 20 poster sessions. The general topic areas broadly broke down into: wound healing (epithelial and stromal), neovascularization, development and differentiation, gene expression, dystrophies and degenerations, inflammation, dry eye, allergy, contact lens, and corneal transplantation. This paper discusses some of the early and groundbreaking work that is currently ongoing, in particular, in the ocular wound healing process and corneal neovascularization, where a better understanding of the biology involved should help researchers find successful treatments. Such approaches as stem cell therapy, antiangiogenesis, and growth factor inhibitors were explored at the meeting and are discussed here.

Ocular Wound Healing

A symposium dedicated to ocular wound healing began with a tribute[1] to a colleague before a presentation by Dr. Steven Wilson,[2] from the University of Washington School of Medicine in Seattle, Washington, on the basic biology of corneal wound healing. Dr. Wilson emphasized the complicated interactions that occur after corneal wounding, both between cells and among cytokines, cells, and nerves. An area of increasing interest that has emerged in recent years involves the development of myofibroblasts. These cells -- which develop from keratocytes as a consequence of signals inducing transdifferentiation after injury, and then diminish in numbers much later after the injury -- are unequivocally involved in the scarring process. Dr. Wilson remarked that a relatively recent hypothesis is being considered regarding the generation of "keratoclast" cells from invading inflammatory cells (monocytes), in a process imagined to be similar to that which occurs in bone when cytokine-inflammatory cell interactions result in new osteoclasts.

Cornea stem cells appear to play a role in wound repair, and Dr. James Zieske,[3] of the Schepens Eye Research Institute in Boston, Massachusetts, provided an overview of the biology of corneal stem cells. Questions Dr. Zieske addressed included whether all limbal basal cells are stem cells, and how stem cells respond to corneal wound healing. He stated that it is clear that the limbal stem cells reside in the region of the peripheral cornea, at the corneoscleral limbus, and that they specifically reside in the basal cell layer. But it is equally clear that not all basal peripheral corneal cells are "stem cells." The stem cells respond to injury by proliferating, and undergoing transdifferentiation to so-called transient amplifying cells, which then themselves continue to amplify the proliferative response and to become terminally differentiated into corneal epithelial cells. His final question -- "what are the markers of corneal stem cells?" -- was a little more difficult to address, and was the subject of additional presentations throughout the rest of the meeting (further discussed below).

F.E. Kruse,[4] from the University of Heidelberg in Germany, presented an update overview on "Neuronal Growth Factors in the Cornea" that reminded us that the cornea contains both sensory as well as cholinergic and parasympathetic fibers interwoven into the most densely innervated tissue outside the central nervous system. He emphasized the fact that the corneal epithelium, in particular, is highly dependent upon soluble neurotrophic growth factors and neurotrophins, opioid growth factors, and neuropeptides and neurotransmitters, which are released by neurons in the cornea. Neurotrophins play a particularly special role in regulating corneal epithelial wound healing. Additionally, nerve growth factor and glial cell-derived neurotrophic factor are delivered to the epithelium via the nerves in the cornea -- and also promote epithelial migration and healing. Substance P, calcitonin gene-related peptide, acetylcholine, and norepinephrine round out this extraordinary panoply of neurotransmitters and neural factors that help regulate the general health and integrity of the ocular surface. Jonas Friedenwald was one of the first to hypothesize this more than a half century ago, and, of course, it has been clinically well-recognized that fifth nerve ablation has extraordinary deleterious clinical consequences on the corneal epithelium. Exogenous addition of nerve growth factor and other neuronally derived mediators can have a therapeutic effect on persistent epithelial defects, but their widespread availability and clinical applicability require further development.

Dr. P. Khaw,[5] from the Moorfields Eye Hospital in London, United Kingdom, addressed the matter of conjunctival subepithelial fibrosis, which is a consequence of ocular cicatricial pemphigoid, chemical burns, trachoma, and some glaucoma surgeries. Dr. Khaw pointed out that we have made a start in the prevention of such scarring through the use of 5-fluorouracil and mitomycin-C. However, much more needs to be learned about the basic biology of scarring in order to be more effective and selective in preventing or treating it. It is now known, for example, that transforming growth factor beta (TGF-beta) is an especially potent promoter of fibrosis, and anti-TGF-beta2 antibody therapy, which neutralizes TGF-beta activity, can reduce subepithelial fibrosis in, for example, patients undergoing glaucoma filtering surgery. Additionally, in an animal model of postsurgical conjunctival scarring, very substantial reduction in scarring can be achieved through the use of strategies that inhibit the activity of one or more matrix metalloproteinases, particularly those that appear to be essential for fibroblast-mediated collagen contraction.

In a presentation entitled "Human Corneal Epithelial Cells Proliferate in Response to Exogenous CTGF and Release CTGF During Differentiation in Multi-layered Epithelium," J.T. Daniels and associates,[6] from the Institute of Ophthalmology, London, England, indicated that transforming growth factor beta induces expression of the gene encoding for the cysteine-rich heparin binding protein known as connective tissue growth factor (CTGF). They discovered that human corneal epithelial cells, in culture, release CTGF during proliferation and differentiation into a multilayered epithelium. The authors concluded that sources for CTGF may be not only fibroblasts stimulated by TGF-beta, but also proliferating and differentiating corneal cells participating in the wound healing response. CTGF promotes stromal scarring; hence, its artificial modulation in certain clinical settings may be of benefit with respect to reducing corneal stromal scar formation.

Stem Cell Research

With the potential to restore damaged cornea, corneal epithelial stem cells are the subject of much research. In their work, Li and associates,[7] from the Third Hospital of Hebei Medical University, Shijazhuang, China, sought to isolate corneal epithelial stem cells. They examined a number of cell and cell differentiation markers; the nuclear protein P63 was the pre-eminent marker in their studies for limbal stem cells. Integrin-beta1, cytokeratin 19 (K19), EGF receptor (EGFr), cytokeratin 3 (K3), and involucrin were also studied in the experiments designed to attempt isolation of corneal epithelial stem cells by having them adhere to sheets of type IV collagen. These researchers found that the expression of P63 protein and its transcripts were located only in the nuclei of limbal epithelial basal cells. They showed that K19 (as well as integrin-beta1 and EGFr) were present in both limbal basal cells and also in the superficial cells, but to a much greater degree in the basal cells than in the superficial cells. Cells that adhered very rapidly to the type IV collagen (within 20 minutes) had a very strong representation of limbal stem cells as detected by their P63 positivity, and these cells, thus isolated, had by far the greatest clonal growth capacity when transferred to 3T3 fibroblast feeder layers. These findings provide very strong putative evidence that these cells represented the immortal limbal stem cell. Indeed, this same group[8] went on to show that this cell population could be expanded in tissue culture in vitro, with the cultures becoming confluent in 10-12 days.

A group from Hopital Huriez Chru Lille, Lille, France, headed by M. Bonne,[9] examined the optimal conditions under which to expand, in vitro, cultured limbal epithelial stem cells. They determined that serum-free medium was superior for promotion of the epithelial cell growth, compared with 3T3 fibroblast feeder layers in plastic dishes for expanding limbal epithelial cell numbers in vitro.

Yiu and associates,[10] from the Doheny Eye Institute in Los Angeles, California, have been impressed with the high expression level of beta1 integrin in limbal cell populations, and have hypothesized that this integrin is a marker for corneal limbal stem cells. The researchers used human corneal donor rims obtained at the time of corneal transplant surgery. Using explants of 4 mm2 from the corneolimbal area, they cultured the epithelial cells as outgrowths from these explants, growing the epithelial cells to confluence and then studying them for the expression of both the alpha6 and the beta1 peptide chains, which comprise the alpha6beta1 integrin. They demonstrated high expression of both these peptides in the cells emerging from these explants.

Corneal Neovascularization

Antiangiogenesis and other strategies aimed at inhibiting corneal neovascularization were also subjects of great interest at this ARVO meeting. It has been known for many years that both vascular endothelial growth factor (VEGF) and interleukin (IL)-1 are potent stimulators of corneal neovascularization. A team[11] from Regeneron Pharmaceuticals, Inc., of Tarrytown, New York, has produced a novel IL-1 inhibitor, murine IL-1 trap, which they used in a mouse model of corneal injury and subsequent corneal neovascularization. They showed that inhibition of IL-1 blocks corneal inflammation and neovascularization. The IL-1 trap is a chimeric protein comprising the ligand binding domains of murine IL-1r1 and IL-1RacP and mouse Fc. This chimeric protein was incorporated into an E1E3 depleted adenovirus, and mice were injected intravenously with this material 24 hours after corneal injury. Subsequent development of corneal inflammation and neovascularization was assessed in mice receiving the active material as well as in mice receiving a placebo. Treatment of the mice with Murine-IL-1 Trap significantly inhibited corneal edema and neovascularization in this suture-induced corneal injury model, dramatically inhibiting infiltration of neutrophils and macrophages into the damaged cornea. These findings have profound therapeutic implications for the treatment of keratitis and corneal neovascularization.

A strikingly different approach was taken by the research group from the Rothschild Foundation in Paris, France. Berdugo Polak and associates[12] employed an antisense oligonucleotide designed to inhibit VEGF, and they examined the potential of iontophoresis to deliver the anti-VEGF R2 ODN oligonucleotide into the anterior segment eye tissues. They employed CY5-labeled ODNs by corneoscleral iontophoresis (employing 300 micro-amps for 5 minutes) in a rat corneal neovascularization model. Controls received the oligonucleotide without any accompanying current. As opposed to the topically applied oligonucleotides with no current (without iontophoresis), which did not penetrate into the cornea, iontophoresis resulted in penetration of the material not only into all parts of the cornea but also into the iris as well. Delivery into the vascular endothelium of neovascularized corneas was documented, with obvious implications with respect to the potential for this technique for treating corneal neovascularization.

The group from Regeron Pharmaceutical of Tarrytown, New York, headed by Wiegand,[13] again using the trap strategy (this time with VEGF trap) also demonstrated long-lasting inhibition of corneal neovascularization following systemic administration, in mice, of the protein fusion comprising the ligand binding domain of VEGF receptors 1 and 2 and human Fc (VEGF trap) subcutaneously after corneal injury. The treatment with the VEGF trap inhibited neovascularization not only during the period of active treatment, but also 2 and 4 weeks following treatment cessation. The authors concluded that the acute inhibition of VEGF following corneal injury may have long-term and long-lasting effects and benefits.

Finally, Charukamnoetkanok and associates,[14] from the Massachusetts Eye and Ear Infirmary and the Schepens Eye Research Institute in Boston, Massachusetts, reported on their discoveries of the proangiogenic role of matrix metalloproteinase 14 (MMP-14) in corneal neovascularization. MMP-14 knockout mice do not develop corneal neovascularization following corneal implantation of a basic fibroblast growth factor (BFGF) pellet. Moreover, the Massachusetts Eye and Ear Infirmary group has shown, by Western blot analysis, enhanced production of MMP-14 in the corneas of mice implanted with BFGF pellets. Additionally, this group has shown that MMP-14 naked DNA intrastromal injections elicit significant corneal neovascularization following hemilimbal injury. The group concluded that MMP-14 may play an important role in corneal neovascularization.

References
  1. Klyce S. A tribute to David Maurice. Program and abstracts of the Association for Research in Vision and Ophthalmology 2003 Annual Meeting; May 4-9, 2003; Fort Lauderdale, Florida. Abstract 15.

  2. Wilson S. Biology of corneal healing. Program and abstracts of the Association for Research in Vision and Ophthalmology 2003 Annual Meeting; May 4-9, 2003; Fort Lauderdale, Florida. Abstract 16.

  3. Zieske J. Corneal stem cell biology. Program and abstracts of the Association for Research in Vision and Ophthalmology 2003 Annual Meeting; May 4-9, 2003; Fort Lauderdale, Florida. Abstract 17.

  4. Kruse FE. Neuronal growth factors in the cornea. Program and abstracts of the Association for Research in Vision and Ophthalmology 2003 Annual Meeting; May 4-9, 2003; Fort Lauderdale, Florida. Abstract 19.

  5. Khaw P. New treatments for conjunctival scarring. Program and abstracts of the Association for Research in Vision and Ophthalmology 2003 Annual Meeting; May 4-9, 2003; Fort Lauderdale, Florida. Abstract 20.

  6. Daniels JT, Garrett Q, Blalock TD, Grotendorst GR, Khaw PT, Schultz GS. Human corneal epithelial cells proliferate in response to exogenous CTGF and release CTGF during differentiation into multilayered epithelium. Invest Ophthalmol Vis Sci. 2003;44: E-Abstract 3805. Abstract

  7. Li DQ, Song X, Chen Z, de Paiva CS, Kim HS, Pflugfelder SC. Partial isolation of corneal epithelial stem cells by adhesion to collagen IV. Invest Ophthalmol Vis Sci. 2003;44: E-Abstract 1348. Abstract

  8. Kim HS, Li DQ, Song X, de Paiva CS, Pflugfelder SC. Ex vivo expansion and phenotypes of human limbal epithelial cells. Invest Ophthalmol Vis Sci. 2003;44: E-Abstract 1349. Abstract

  9. Bonne M, Dedes V, Labalette P, et al. Comparative study of cultured limbal epithelial cells on a 3T3 feeder layer or in defined medium. Invest Ophthalmol Vis Sci. 2003;44: E-Abstract 1350. Abstract

  10. Yiu SC, Wasilewski D, Stevenson D, et al. High expression level of beta-1 integrins in expansion of human corneolimbal explants. Invest Ophthalmol Vis Sci. 2003;44: E-Abstract 1351. Abstract

  11. Cao J, Renard R, Song H, et al. Inhibition of IL-1 blocks corneal inflammation and neovascularization. Invest Ophthalmol Vis Sci. 2003;44: E-Abstract 823. Abstract

  12. Berdugo Polak M, Valamanash F, Courtois Y, Behar-Cohen F. Safe and efficient intracorneal delivery of an antisense oligonucleotide using iontophoresis. Invest Ophthalmol Vis Sci. 2003;44: E-Abstract 828. Abstract

  13. Wiegand SJ, Cao J, Renard R, Rudge JS, Yancopoulos GD. Long-lasting inhibition of corneal neovascularization following systemic administration of the VEGF Trap. Invest Ophthalmol Vis Sci. 2003;44: E-Abstract 829. Abstract

  14. Charukamnoetkanok P, Mian S, Javier J, Oliveira H, Chang JH, Azar DT. Proangiogenic role of matrix metalloproteinase (MMP) 14 in cornea. Invest Ophthalmol Vis Sci. 2003;44: E-Abstract 833. Abstract

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