Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

FIGURE 15.1 Glaucoma surgery failed due to scarring – the processes involved in scarring play a part in causing failure of treatment of most major blinding conditions

progression, and similar scarring restricts the predictability of strabismus surgery and contributes to motility problems in thyroid eye disease. Retinal scarring in proliferative vitreoretinopathy, and specifically age related macular degeneration, results in more loss of vision than any other disease in the developed world (see Figure 15.1).

Treatments and surgical approaches have been developed to successfully modulate the wound healing response. Anticancer agents such as mitomycin C inhibit fibroblast function and survival when applied locally. Steroids applied topically and systemically reduce inflammation and fibrosis. However, both these agents have significant side effects. In this chapter we will review the agents used to modulate healing scarring. We will then take a more clinical disease/tissue oriented approach to the modulation of scarring in the eye and surrounding tissues, reviewing clinical data.

II. BASIC HEALING AND

SCARRING PROCESSES

A great deal of our knowledge on wound healing is based on skin repair (Martin, 1997). Most of the principles of healing of skin wounds also apply to healing and fibrosis of ocular tissues. The processes of wound healing after injury or disease are

multiple, complex and overlap. However, these events can be grouped into sequential phases. The early phase is characterized by coagulative and inflammatory events. The phase of wound repair and closure is dominated by fibroblast activities (proliferation, secretion of extracellular matrix proteins, matrix remodeling, and contraction of wound margins). There is the phase of continuous matrix remodeling and scar consolidation, and then the final stage characterized by cellular apoptosis, which leads to an acellular, fibrous scar.

III. MODULATING THE

DIFFERENT STAGES OF WOUND HEALING AND SCARRING

Alarge number of pharmacological agents have been used to modulate wound healing and scarring. For this chapter the events in wound healing have been separated (see Table 15.1, page 340) and the different agents classified under the process the evidence suggests is the main mechanism of action. Table 15.1 gives an overview, including what we currently understand to be the main mechanism of action, although there is considerable overlap. The different agents are used in a different context in each subspecialty, from the front to the back of the eye.

A. Cornea

The processes leading to corneal scarring are a leading cause of blindness in the world. The underlying pathologies are mainly infectious and inflammatory diseases (trachoma, trauma, ulceration, and xerophthalmia; see Figure 15.2), all of which are more successfully targeted by prevention rather than treatment of established disease. All these pathologies lead to a decrease in corneal transparency. The great increase in corneal refractive surgery has led to a great deal of research and an increased understanding of the biology of corneal wound

healing. The components of corneal transparency include:

1.A regular arrangement of collagen lamellae

2.A relatively uniform refractive index of the cornea

3.Quiescent keratocytes in the stroma

4.Avascularity.

Corneal oedema reduces light transmission as a result of an increased mismatch in the refractive index of collagen fibrils and hydrated matrix, interlamellar disruption in the spatial arrangement of fibrils and increased stromal thickness (Meek et al., 2003). Healing after photorefractive keratectomy (PRK) is associated with the deposition of disorganized and irregular extracellular matrix (ECM). Corneal keratocytes are normally “invisible”, as they have a similar refractive index as the surrounding stroma. This is achieved by crystalline proteins which form 28% of the total protein in the keratocyte cytoplasm (Jester et al., 1999). Once a keratocyte is activated, the relative proportion of these proteins is reduced, making the keratocyte “visible” in the stroma.

The interaction between epithelium and stroma is a critical component of corneal wound healing (Wilson et al., 1999). The earliest response to epithelial injury is apoptosis of the anterior stromal keratocytes. Interleukin 1 (IL-1) and tumour necrosis factor alpha (TNFα) derived from the injury

mediate this response. Together with chemokines released by dying keratocytes they recruit inflammatory cells into the stroma within hours of injury (Hu et al., 2000). Subsequently, mitosis and migration of keratocytes from deeper stromal layers replenish the anterior stroma (You and Fang, 2001). Platelet-derived growth factor secreted by epithelial injury induces keratocyte mitosis and migration, while transforming growth factor-β(TGF-β) induces myofibroblastic transformation, resulting in new ECM production in the subepithelial layer. There is some evidence that when the basement membrane is disrupted, fibroblasts become activated and transform into myofibroblasts due to the release of transforming growth factor-β2 (TGF-β2) (Stramer et al., 2003). Newly formed ECM and activated keratocytes are probably responsible for clinically visible “haze”. Over the following months, the new ECM is remodeled and “normalized”, while the activated keratocytes undergo apoptosis and disappear (Figure 15.3).

1. Surgical techniques

The use of the excimer laser and the way it is applied has revolutionized refractive surgery. It is a good example of how a change in surgical technique can modify wound healing. Laser in situ keratomileusis (LASIK) is now the most commonly used technique, as it causes less pain, haze, and regression, and is associated with faster

recovery than surface treatments such as PRK, laser subepithelial keratomileusis (LASEK), or epi-LASIK. LASIK has the advantage of avoiding epithelial injury and hence the epithelial–stromal wound healing response. Surface treatment with PRK tends to cause more haze than LASEK and LASIK. The loss of the epithelial basement membrane potentially increases levels of epithelial TGF-β2 reaching the stroma, and hence increases transformation of keratocytes (Stramer et al., 2003). This may help explain the reduced haze in LASIK or LASEK where the epithelium is split from Bowman’s layer compared with PRK.

2. Amniotic membrane

Amniotic membrane applied soon after laser ablation has significant antiinflammatory, anti-apoptotic and anti- TGF-β effects and reduces haze after PRK (Lee et al., 2003). However, increased surgical time, and the need to remove the amniotic membrane, make its use practical only in patients with severe haze undergoing PRK retreatment. Amniotic membrane has been successfully used to enhance re-epithelialization in non-healing ulcers and prevents corneal perforation after acute fungal keratitis (Chen et al., 2006). Furthermore, transplantation of rebuilt corneal epithelium and fibroblasts on a lyophilized amniotic membrane hastened the recovery of alkali-injured epithelium in an animal model (Jang et al., 2006). Amniotic membrane transplantation combined with cultivated limbal epithelial cells (autografts or allografts) in severe ocular surface disease can enhance the improvement of vision and tissue remodeling.

3. Anti-inflammatory agents

Steroids and NSAIDs are routinely used after refractive surgery to reduce inflammation. However, their effect on the prevention of haze remains controversial. In two human studies, fluorometholone 0.1% and dexamethasone 0.1% failed to show significant haze reduction (Gartry et al., 1992), while in an animal model of PRK, beclamethasone

0.1% significantly reduced haze and collagen deposition (Kaji et al., 2000). In the same animal study, diclofenac 0.1% did not significantly reduce haze (Kaji et al., 2000).

4. Anti-proliferative agents

In patients with subepithelial fibrosis from previous refractive surgery, mechanical debridement and a single intraoperative dose of MMC 0.02% for 2 minutes effectively prevents recurrences of fibrosis, in the absence of complications, for more than a year (Majmudar et al., 2000). Similarly, MMC markedly reduces corneal haze after PRK for high myopia (Gambato et al., 2005). No complications were encountered with a follow-up period of up to 3 years. In animal models, increased apoptosis and reduced repopulation of stromal keratocytes after MMC treatment following PRK have been described, but the potentially serious long-term deleterious effects, if any, of these keratocyte changes is not known. In LASIK treatment as well, application of MMC 0.02% for 2 minutes after the laser ablation resulted in significantly lower haze rate than the control group at the 6 month follow-up, and almost double the number of patients in the MMC treated erosion group achieved the targeted refractive outcome compared with the control group (Carones et al., 2002). The application of 0.01% MMC after LASEK also results in less subepithelial haze, although it is of note that a higher percentage of complications was noted (Camellin, 2004). This note of caution is further emphasized in a PRK animal model which indicated a continuing reduction of the density of keratinocytes after MMC application. A lower concentration of MMC (0.002%) for less time has been shown to have the same efficacy as a higher concentration for a longer period (Netto et al., 2006).

5. Growth factor modulators

The effect of TGF-β on keratocyte activation, myofibroblastic transformation and deposition of extracellular matrix is well recognized. TGF-β2 induces in corneal

fibroblasts both the expression of insulin growth factor-I that stimulates the proliferation of myofibroblasts and insulin growth factor binding protein-3 (IGFBP-3). This may eventually block DNA synthesis in corneal fibroblasts, and the expansion of the myofibroblasts in corneal wounds (Izumi et al., 2006). It is possible that the triggering of IGFBP-3 expression could be one of the future therapeutic targets in the attempt to reduce opacity after corneal wound healing. TGF-β 1 also promotes the expression of connective tissue growth factor CTGF, a factor that influences the ECM production and subsequent scar formation and fibrosis. CTGF is also necessary for TGF-β stimulation of myofibroblast differentiation and collagen contraction (Garrett et al., 2004), and inhibition of this factor could be a possible future therapeutic target (Wu et al., 2006). Myofibroblasts express a PAF nuclear receptor and TNF-α receptors, and PAF and TNF-α cause time-dependent myofibroblast apoptosis. Future therapeutic approaches of fibrosis will possibly take advantage of the expression of these receptors (He and Bazan, 2006).

Neutralizing pan-antibody to TGF-β significantly reduced anterior stromal fibrosis, haze and extracellular matrix deposition in a rabbit model of PRK. However, haze reduction is not associated with an effect on post-treatment regression, indicating that haze and stromal regeneration might be controlled by different mechanisms (MollerPedersen et al., 1998). The issue of which isoform to neutralize has not been resolved. SiRNA against TGF-β receptor II reduced the production and deposition of fibronectin from fibroblasts and delayed the migration of cultured human fibroblastsindicating a potential therapeutic effect against corneal scarring (Nakamura et al., 2004). Specific inhibition of TGF-β1 isoform may or may not be more effective, and the finding that TGF-β3 reduces fibrosis in rat skin (Ferguson and O’Kane, 2004) and reduces the keratinocyte population in the site around the wound of corneal cultures is replicated in vivo (Carrington et al., 2006).

It has also been proposed that high concentrations of hepatocyte growth factor (HGF) which are expressed following a wound may be responsible for abnormal epithelial and stromal healing. HGF treated corneas resulted in mass differentiation of keratocytes into myofibroblasts, suggesting HGF as a target for fibrosis inhibition (Carrington and Boulton, 2005). Apart from inhibitory strategies to prevent fibrosis, it may be possible that the use of agonists could achieve improved therapeutic results. The use of EGF in the past is an example of where increased epithelialization could reduce scarring. Topical application of nerve growth factor (NGF) after cataract surgery has resulted in repair of the corneal wound – normal thickness and transparency – within 3 weeks. It has been found that NGF plays this active role in normal wound healing and the restoration of the transparency of the cornea by facilitating the receding of corneal edema, promoting the fibroblast migration and inhibiting the cellular apoptosis only at the first stages of wound healing (Cellini et al., 2006).

B. Nasolacrimal System

Blockage of the tear draining apparatus is common. The treatment of choice for nasolacrimal duct obstruction is dacryocystorhinostomy (DCR), but granulation tissue and fibrosis can effectively close the osteotomy in the lacrimal bone. 5-Fluorouracil and

MMC have been shown to be safe and effective in preventing osteotomy closure. In vitro, MMC inhibits the proliferation of cultured human nasal mucosa fibroblasts and induces apoptosis (Hu et al., 2000). In vivo, MMC and 5-FU, applied to the bone surrounding the osteotomy and to the mucosal flaps, reduce fibrosis, resulting in larger osteotomy size and higher patency rates. In external DCRs, patency at a mean followup of 3 years has been reported to be 94– 100% in MMC treated patients versus 83% in the control group (You and Fang, 2001). In endonasal DCRs, 5-FU improved patency from 63 to 76%, and MMC from 89.6 to 99.2% (Camara et al., 2000; Bakri et al., 2003).

C. Orbit and Optic Nerve

1. Thyroid-associated orbitopathy (TAO)

Orbital fibroblasts can express receptors for, and become activated by, the circulating thyroid-stimulating autoantibodies (Wiersinga and Prummel, 2001). In addition, the orbital connective tissue is infiltrated by inflammatory cells which secrete cytokines, inducing fibroblast proliferation, production of extracellular matrix proteins, and fibroblast transformation into adipogenic cells and myofibroblasts (Koumas et al., 2003). The treatment of TAO is still largely based on systemic corticosteroids and surgery. Orbital radiotherapy is controversial, as most of the evidence in its favor is based on uncontrolled studies, while recent randomized, controlled trials have delivered conflicting results (Behbehani et al., 2004). Due to the difficulty and potential side effects of targeted delivery of soluble antifibrotic agents such as 5-FU and MMC, these are not currently used to modulate orbital fibroblasts. However, other systemic treatments have shown promise in the treatment of TAO, in particular synthetic somatostatin analogs and retinoids. Somatostatin analogs, such as octreotide and lanreotide, efficiently control

inflammation, but they are less efficient than glucocorticoids in reducing extraocular muscle size (Kung et al., 1996). Retinoids inhibit proliferation and induce apoptosis of orbital fibroblasts from patients with TED in vitro, but have not yet been investigated in clinical trials (Pasquali et al., 2003). Retrobulbar triamcinolone injections may be of benefit in patients with thyroid associated orbitopathy, but long-term studies are needed in this chronic condition (Poonyathalang et al., 2005).

2. Orbital implants

Orbital implants are used to replace lost volume in anophthalmic orbital sockets. The cosmetic outcome depends on the appearance and motility of the overlying ocular prosthesis. To maximize motility, ocular prostheses are attached to the orbital implant via a motility peg. However, problems with peg extrusion and hole occlusion by fibrovascular tissue occur in a significant number of cases (25–30%). In an animal study on albino rabbits, MMC applied to the holes freshly drilled into hydroxyapatite implants reduced peg extrusion and hole exclusion from 100% to 0% (Lew et al., 2001), but to date no study on human subjects has been published.

3. Optic nerve decompression surgery

Scarring and fibrosis affect the long-term outcome of optic nerve sheath decompression for idiopathic intracranial hypertension in about a third of cases. A small case series on 6 patients has shown that the pretreatment of the optic nerve sheath with MMC before incision was safe and effective at reducing scarring (Spoor et al., 1995). Due to the potential risks it is not widely used as an adjunct in this type of surgery. It is of interest that a single intraoperative application of 5FU has been shown to be of use in preventing experimental dural scarring without nervous system damage (Spinks et al., 2003).

D. Strabismus Surgery

Extraocular muscle adhesion to sclera, Tenon’s capsule, and conjunctiva following strabismus surgery can lead to significant restrictions in eye movement. This is one of the main reasons for poor results following strabismus surgery. The risks of adherence syndromes are higher with multiple operations, bleeding, excessive cautery and improper tissue handling. Delayed muscle adjustment after placement of adjustable sutures can increase the longterm success of binocular alignment. Many interventions have been tested in animal models, but at present none is in routine clinical use.

1. Physical barriers

To prevent muscle adhesion to the surrounding tissue, a physical barrier in the form of a biocompatible material can be used to separate the tissues. Polyurethane sheets, with or without a dexamethasone sustained drug delivery system, significantly reduce adhesions (Kim et al., 2004). Polytetrafluoroethylene (PTFE), Seprafilm (a biodegradable membrane made of sodium hyaluronate and carboxymethylcellulose),ADCON-L(a polyglycan ester), Interceed (a cellulose matrix), sodium hyaluronate and polyglactin mesh all have been found to be of some, albeit limited, value.

2. Antiproliferative agents

In experimental rabbit strabismus surgery, MMC, but not 5FU, inhibited extraocular muscle adhesions (Hwang and Chang, 2000; Esme et al., 2004). No complications, other than avascularity of the conjunctiva in some cases, were reported.

success of GFS, and just as importantly determines the percentage of patients achieving final intraocular pressures that are associated with virtually no glaucoma progression. A considerable amount of research into the basic processes of ophthalmic wound healing and scarring and the use of antifibrotic agents has been performed in the context of healing after glaucoma filtration surgery (GFS). Recent years have seen the establishment of proven protocols for the use of antifibrotic agents to inhibit scarring of trabeculectomy blebs. However, the modulation of the healing response after GFS is a fine line: too much suppression of healing and scarring results in severe complications such as leakage, infection, and, potentially, hypotony, endophthalmitis and complete loss of vision (Figure 15.4). There is a great need to develop gentle but effective treatment approaches, and promising new agents are in the stages of clinical evaluation and in vitro assessment. Most of the agents listed in Table 15.1 have been evaluated as scarring-inhibiting agents in experimental models of GFS. However, only four treatment modalities are in common clinical use: anti-inflammatories; 5-fluorouracil (5-FU); mitomycin C (MMC); and beta-irradiation.

1. Surgical technique

Equally important, simple changes in surgical and application technique can radically reduce side effects even when the same

Urokinase or single-chain urokinase-type plasminogen activator

Historically, urokinase was first purified and concentrated from urine. Thrombolytic (fibrinolytic) agent, plasminogen activator (WuDunn, 1997)

Non-steroidal antiinflammatory drugs (NSAIDs)

Cyclosporine A

Amniotic membrane

Cytokine release and action

Tranilast ((N-3 ,4 - dimethoxycinnamoyl) anthranilic acid) – first described as inhibitor of histamine release from mast cells

Genistein – isoflavone from soy products

Inhibition of cyclooxygenase, resulting in a reduction of prostaglandins, prostacyclin, and thromboxane A. Also inhibit platelet aggregation and function. Direct antiproliferative effect on human ocular

fibroblasts

First isolated from the fungus Tolyplocadium inflatum.

Inhibits lymphocyte-mediated immune responses (Turacli et al., 1996) Innermost of the three fetal membranes, first used therapeutically as skin graft material. Potent anti-inflammatory properties, maintenance of oxygenation and moisture and mechanical protection of covered tissues

Inhibition of TGF-β activity (Chihara et al., 2002)

Inhibition of TGF-β activity, tyrosine kinases, matrix metalloproteinases, and angiogenesis (Kim et al., 1998)

(continued)

Anti-TGF-β antibody – recombinant human monoclonal antibody specific to the active form of TGF-β 2

Inhibition of TGF-β activity (Siriwardena et al., 2002)

Anti-TGF-β oligonucleotides – synthetic molecules which bind to specific intracellular messenger RNA strands

Inhibition of transcription of the mRNA with subsequent inhibition of synthesis of the protein TGF-β (Cordeiro et al., 2003)

Other anti-TGF-β strategies

D( )-glucosamine and D( )-glucosamine 6-sulfate dendrimers

Simvastatin – inhibitor of the enzyme HMG-CoA reductase, isolated from a strain

of the bacterium, Penicillium

Ribozymes: RNA molecules which can cleave specific bonds in other RNA molecules. Cleavage of TGF-β-mRNA with subsequent inhibition of synthesis of the protein TGF-β

Anti-TGF-β siRNA: RNA sequence complementary to messenger RNA (mRNA) for TGF-β. The silencing RNA (antisense RNA) and the target mRNA hybridize and block translation and the production of the TGF-β protein (Nakamura et al., 2004)

D( )-glucosamine and D( )-glucosamine 6-sulfate dendrimers have immunomodulatory and anti-angiogenic properties, respectively (Shaunak et al., 2004)

First isolated and used for its lipid-lowering properties. Now additional, different mechanisms are emerging. Inhibition of the connective tissue growth factor gene and protein expression, a downstream mediator of TGF-β (Watts et al., 2005)

β-irradiation – ionizing radiation with minimal tissue penetration

Inhibits cell proliferation (Miller and Rice, 1991; Kirwan et al., 2003)

Mitomycin C – isolated from the soil fungus, Streptomyces caespitosus

Photodynamic therapy

Undergoes metabolic activation via reduction into an alkylating agent that cross-links DNA. Affects all phases of the cell cycle: DNA replication, mitosis, and protein synthesis. Inhibits fibroblast and endothelial cell growth and proliferation

2 ,7 -bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECFAM) is a fluorescent probe and is an intracellularly acting photosensitizer. It is applied locally in its inactive form, diffuses into adjacent cells, and is then cleaved and rendered fluorescent by intracellular esterases. After illumination (activation) with blue light, it exerts a photo-oxidative effect that is only cell destructive within the targeted cells ( Jordan et al., 2003)

(continued)

Retinoid acid and its derivatives – vitamin A derivative

Retinoic acid regulates gene expression by binding to nuclear transcription factors

Interferon alpha (IFN-alpha) – recombinant protein mimicking the effects of natural IFN-alpha

Lectins (phytoagglutinins) – proteins that agglutinate erythrocytes and other cells

Saporin – derived from the plant Saponaria officinalis

Interferon alpha regulates cell proliferation and differentiation by affecting several cellular communication and signal transduction pathways (Gillies et al., 1999)

The mushroom lectin from Agaricus bisporus binds to galactosyl-β-1, 3-N-acetyl-galactosamine-alpha (Gal-Gal-NAc) and has a strong antiproliferative effect. The exact mechanism of action is unknown (Batterbury et al., 2002)

Ribosome inactivating protein → cell proliferation inhibitor

Antiproliferative gene insertion – antiproliferative gene p21(WAF-1/Cip-1)

p21(WAF-1/Cip-1) is a transcription factor that mediates cell cyle arrest in response to cellular stress. Transfection resulted in inhibition of scarring (after Perkins et al., 2002)

Paclitaxel (Taxol) – first isolated from the bark of the Pacific yew tree, Taxus breviofolia, L., Taxaceae

Vincristine – vinca alkaloid from the leaves of the periwinkle plant

EDTA (ethylendiaminetetraacetic acid) – EDTA chelation therapy was first introduced to treat lead poisoning

Antileukemic and antitumour agent; promotes the assembly of microtubules and inhibits the tubulin disassembly process (Jampel et al., 1993; Jampel and Moon, 1998)

Binds to tubulin, which are then unable to aggregate to form microtubules

Calcium-chelating agent used to dissociate epithelial cells from basement membranes in vitro

(continued)

FIGURE 15.5 Change in bleb morphology with different surgical technique. The patients’ left cyctic bleb was created using a small surface area of MMC treatment, the right a large surface area of treatment

concentrations of antimetabolites may be used (Wells et al., 2003, 2004) (Figure 15.5). This principle is also very important in other situations where drug treatments have to be used in tandem with an understanding of surgical and anatomical principles. Minimizing tissue damage is of obvious relevance. Either perfluoropropane gas or sodium hyaluronate 2.3%, which was injected after trabeculectomy in the subconjunctival space, was linked with the creation of more diffused blebs, but with no long-term data on bleb survival (Wong et al., 1999; Lopes et al., 2006).