Ординатура / Офтальмология / Английские материалы / Ocular Disease Mechanisms and Management_Levin, Albert_2010

Advances in the treatment of diseases involving the ocular anterior segment, particularly the lens and cornea, have greatly decreased the prevalence of visual impairment caused by dysfunction of these structures. Unfortunately, treatment of diseases impacting structures of the posterior segment, particularly the retina and optic nerve, have not advanced to the same extent and as a consequence these conditions are now the major source of incurable blindness in the developed world (Box 77.1). The reasons for this are not hard to discern in that both the retina and optic nerve are components of the central nervous system (CNS) and it has long been appreciated that the mammalian CNS exhibits a very restricted capacity for endogenous regeneration. Furthermore, what little capacity exists diminishes further with postnatal maturation. Additional barriers to the development of effective restorative treatments for the retina and its central projections are numerous and include the complex phenotypes of retinal cells, particularly photoreceptors and ganglion cells, as well as the need for an unusually precise cytoarchitecture. This is true both in terms of outer segment packing and the topographic organization of output fibers projecting to the visual centers of the brain.

As a consequence of the many challenges involved, there are at present no restorative treatments for retinal cell loss. This situation may change; however, a growing body of experimental data suggests that many of the barriers to retinal repair in mammals are not insurmountable. In particular, stem cell transplantation and tissue engineering have recently emerged as promising strategies for repopulating the cellular constituents of the retina.1 Alternatively, it is conceivable that an electronic prosthesis could bypass the damaged components of the retina and convey visual information directly to downstream visual neurons, although this fascinating strategy will need to overcome difficulties faced when attempting to use artificial constructs to stimulate high-resolution visual acuity. For now, it appears that the most straightforward method of restoring retinal function resulting from cell loss is to replace those cells through transplantation.

Work in animal models and limited studies in human subjects have shown that a number of different tissues and cell types can survive as allografts in the ocular posterior segment, in either the vitreous cavity or subretinal space.2–11 The general facility of graft survival seen likely results in part from the degree of immune privilege afforded in these loca- tions.12–16 Vitreal delivery may be adequate for some applications and cell types; however, subretinal placement is preferable for restoration of photoreceptors and the retinal pigment epithelium (RPE), as is needed in retinitis pigmentosa (RP) and to varying extents in retinal detachment and age-related macular degeneration. In terms of relevant cell types for outer retinal repopulation, both photoreceptors17 and RPE cells18 survive transplantation beneath the retina; however, the reluctance of donor photoreceptors to make functional connections with the surviving host circuitry19 and failure of donor RPE cells to reform a polarized monolayer on Bruch’s membrane20–22 have frustrated attempts to achieve functional repair of the outer retina using grafts of freshly isolated cells alone. Here we will focus on the challenges facing photoreceptor replacement and consider the advantages of using cultured allogeneic retinal progenitor cells (RPCs) as donor material.

To repopulate the outer nuclear layer with functional photoreceptors, there is a fundamental problem to overcome. This is the physical barrier to neurite outgrowth presented by hypertrophy of the outer limiting membrane (OLM) following photoreceptor loss. The OLM is not in fact a membrane per se but rather is an emergent structural element formed by the joined outer ends of retinal Müller cells. In the setting of photoreceptor degeneration, the OLM undergoes thickening in association with upregulation of the markers neurocan and CD44. Regenerating neurites originating from either above or below have great difficulty crossing this barrier.23 In fact, the phenomena of glial hypertrophy and scar formation are common in the setting of CNS disease and injury and have frequently been implicated in the failure of endogenous regenerative mechanisms to bridge a lesion.24

Box 77.1  Impact of retinal degenerative disease

Box 77.2  Strategies for retinal transplantation

•  Two promising approaches for targeted repopulation of the retina following injury/disease include transplantation of stem/progenitor cells and transplantation of intact retinal sheets

We have shown that grafted CNS progenitor cells are not impeded by a hypertrophied OLM and can migrate across this barrier in large numbers.10,11 The ability to migrate into the mature, diseased retina is one remarkable characteristic of CNS progenitor cells that recommends them as a potential tool for use in retinal repopulation. Moreover, these cells not only migrate into the retina, but also exhibit widespread integration into the local cytoarchitecture, with tropism for regions of injury or disease. In the case of RPCs, there is also the potential to differentiate into cells with morphological features and marker expression characteristic of photoreceptors.

Retinal transplantation

To achieve functional repair in patients afflicted with retinal degenerative disorders, there is an urgent need to reconstruct, via cellular replacement, the damaged or lost layers of the retina. While restorative repair of the retina is a daunting challenge, a range of data suggests that such a goal is now feasible. Two approaches for targeted repopulation of the retina are considered below and include stem/progenitor cell transplantation and retinal sheet transplantation, both of which pose a variety of advantages and disadvantages (Box 77.2).

Stem/progenitor cell transplantation

Over the past decade, stem/progenitor cell transplantation as a means of inducing tissue reconstruction and functional regeneration has garnered extensive interest in the field of regenerative medicine. Within the retina in particular, many exciting advances have been made. One significant achievement came in 2004 when we were able to show that a subset of transplanted RPCs developed into a variety of mature retinal neurons, including retinal ganglion and photoreceptor cells.10 Since then, numerous studies reporting varying degrees of success have utilized an assortment of different cell types ranging from the fate-restricted photoreceptor precursor to the pluripotent embryonic stem (ES) cell.25–28 ES cells are of particular interest due to their ability to undergo unlimited expansion and subsequent tissue-specific differentiation. These inherent properties may allow one to generate

a sufficiently large number of cells in order to perform clinical transplantation from single isolations rather than requiring multiple new donations, as is potentially the problem when using more terminally differentiated cell types. However, as cited above, these cells are pluripotent, meaning they can be induced to generate cell types for each of the three germ layers and as such, are not retina-specific. Thus, protocols for retina/cell type-specific differentiation are required. In light of this, many labs have been aggressively searching for the proper method of retinal cell induction. One of the first published reports of RPC, and subsequent retinal cell generation, from human ES cells came from Lamba and collegues in 2006.27 In this publication, the group was able to show that they could reliably produce healthy functioning photoreceptors, albeit at low levels, using a relatively simple induction protocol.27 Since then, similar studies using variations of Reh’s methods have reported an increased generation of retina-specific cell types, photoreceptor cells in particular, in a range of organisms including primate and human.25

Retinal sheet transplantation

Like stem cells, retinal sheets, including full-thickness and photoreceptor only, have also been used in an attempt to achieve retinal reconstruction and visual restoration following injury and disease. The goal of these techniques is to deliver retinal tissue with proper laminar structure and cellular organization directly to the site of injury in an attempt to form new functional connections between remaining host tissue and healthy donor material. Although extensive connections and subsequent visual restoration have not yet been achieved by using this technique, significant progress, particularly when using developing tissue, has been made.2,29–34 For instance, when embryonic rat retina was transplanted into the subretinal space of degenerating animals, the tissue was shown to survive without immune rejection, develop normally, continue to respond to light for up to 3 months after transplantation, and form limited functional connections with the host.31 Apart from functional integration, similar results have also been reported in a pig model of RP. For example, Ghosh et al35 have shown that transplanted retinal tissue isolated from healthy fetal donors can survive and maintain proper laminar structure and cellular organization within the subretinal space of the host for up 6 months post-transplantation. However, an issue that remains when using full-thickness retinal grafts is the introduction of redundancy into the system. For instance, in diseases such as RP where there is a progressive loss of photoreceptor cells with sparing of the inner retinal circuitry (albeit temporarily), full-thickness transplants would result in replication of a majority of the retinal cell types. Thus, in a situation such as this, transplantation and subsequent integration of photoreceptor sheets, void of all other retinal structures, would be beneficial. Although extensive research has yet to be carried out using this approach, studies with promising findings have been reported. For instance, as with full-thickness retinal grafts, transplantation of photoreceptor sheets has enjoyed prolonged survival and structural preservation.36,37

As promising as the abovementioned studies may be, issues such as inadequate cellular/axonal integration following stem/progenitor cell and retinal sheet transplantation

remain. Modest functional integration may be accounted for by a variety of factors, including the lack of cellular support and survival following bolus stem cell injection, and inadequate growth responses of retinal tissue transplantation. Most important, however, is the presence of a postinjury inhibitory extracellular CNS environment.

Pathophysiology

Glial scar formation

Unlike the peripheral nervous system (PNS), the regenerative capacity of the CNS following injury is extremely limited. Amongst other reasons, the paucity of regeneration can be attributed to the presence of an inhospitable extracellular environment (Box 77.3). Unlike the PNS, the CNS is plagued by an abundance of myelin-associated extracellular matrix (ECM) proteins such as myelin-associated glycoprotein (MAG), Nogo, and Omgp that are well known for their ability to inhibit axonal extension and cellular migration.38–40 For instance, in 1988, Caroni and Schwab identified the first of these myelin-associated molecules, later termed Nogo, as being a potent inhibitor of fibroblast cell migration and neurite extension.38

The abovementioned ECM molecules typically exert their action by binding a common complex of cell surface receptors, in the case of Nogo consisting of the Nogo receptor (NgR) in conjunction with p75 (low-affinity neurotrophin receptor), Lingo, and TROY. Collectively, the binding of these molecules stimulates an inhibitory intracellular signaling cascade which utilizes the small GTPase-dependent enzyme RhoA.41–45 Activation of RhoA and its downstream effectors ultimately stimulates growth cone collapse, axon retraction, and cellular repulsion by negative regulation of the actin cytoskeleton.46–49 Thus, chemical and/or enzymatic inhibition/neutralization of these ECM molecules or their downstream targets has been shown to alleviate inhibitory myelin-associated growth inhibition in a variety of CNS compartments, including the optic nerve. For instance, in the absence of Nogo-induced RhoA activation, by using either AAV-induced dominant negative NgR expression or NgR-null mice, significantly enhanced retinal ganglion cell axon extension was observed.50,51 Similarly, animals vaccinated with spinal cord homogenates rich in myelinassociated proteins could extend axons significantly further than control animals following optic nerve injury.52 Likewise, when serum from vaccinated animals was used to treat purified cultures of retinal ganglion cells in vitro, it was found that MAG-induced growth cone collapse and axon retraction were alleviated.52 In light of these findings, regen-

Box 77.3  The inhibitory CNS extracellular

environment

•  The inability of the injured central nervous system to regenerate can in part be attributed to the presence of an inhospitable extracellular environment, predominated by the presence of inhibitory glial scar/myelin-related extracellular matrix proteins that prevent axon extension and transplant integration

Pathophysiology

eration of the retinal ganglion cell layer following injury could potentially benefit from negative regulation of the aforementioned molecules.

As initially suggested, retinal degenerative diseases such as age-related macular degeneration and RP predominantly affect the photoreceptor layer of the retina which, unlike most other CNS tissues, is void of myelin and the associated oligodendrocytes. Thus, the environmental inhibitors mentioned above which impede ganglion cell and optic nerve regeneration are not a factor. Why then are attempts at stimulating retinal regeneration via photoreceptor or stem cell transplantation still largely unsuccessful? The lack of functional integration following transplantation is in large part due to the presence of injury-induced glial scar formation. The retina, like other nervous system compartments, undergoes a process known as reactive gliosis. This is an injuryinitiated event that involves the infiltration of a variety of cell types, with the most prominent being activated astroglia. Activation, a process that results in the upregulation of the intermediate filament proteins glial fibrillary acidic protein and vimentin, is crucial for glial scar formation.53

In the retina, the major glial cell type responsible for reactive scarring following disease-induced retinal degeneration is the Müller glial cell. During retinal reactive gliosis, hypertrophic Müller glia undergo the activation and upregulation of the above-mentioned intermediate filament proteins (Figure 77.1A), after which they respond by extending projections from their original location (forming the OLM) into the subretinal space.54,55 Here, these projections proceed to form a dense fibrotic barrier that contains a variety of growthinhibitory extracellular matrix/adhesion molecules, including the chondroitin sulfate proteoglycan (CSPG) Neurocan and the hyaluronan-binding glycoprotein CD44 (Figure 77.1B and C). Both Neurocan and CD44 have previously been shown to function as chemical inhibitors to axon growth and cellular migration, thus preventing regeneration and functional synapse formation.1,56–61 For instance, we have previously shown that an abundance of these molecules, deposited by reactive glial cells at the outer limits of the degenerative mouse retina, prevent neurite extension and subsequent integration following retinal transplantation (Box 77.4).1

Although the exact cell surface receptors for these inhibitory glial scar-related proteins are not well characterized (as with the myelin inhibitors), their actions are exerted via activation of the RhoA signaling pathway.62–64 Thus, RhoA inhibition or the removal of the inhibitory glial scar-related proteins could potentially act to enhance axonal extension and cellular migration following retinal transplantation. A variety of approaches have been taken in an attempt to remove these inhibitory ECM molecules, including enzymatic degradation of the proteins themselves. Two enzymes that have been utilized for such a purpose are chondroitinase ABC and the matrix metalloproteinase MMP2. Chondroitinase ABC, which takes advantage of the native structure of CSPGs by cleaving the glycosaminoglycan (GAG) side chains from the CSPG protein core, has been shown to enhance the integration of grafted Müller stem cells into the degenerating retina following transplantation.65 A drawback when using chondroitinase is that it only reduces CSPG activity related with GAG side chain removal. Thus, the remaining inhibitory core proteins, such as that of Neuro-

Box 77.4  Injury-induced glial cell reaction

610

can, can still function to inhibit axonal regeneration and cellular migration.66 Similarly, chondroitinase functions on the CSPG family of proteins only and therefore the inhibitory CD44 component of the glial barrier would remain even after chondroitinase treatment. MMPs, unlike chondroitinases, do not confer their function via GAG side-chain removal and, as such, are not restricted to CSPG degradation. Rather, this family of molecules are well known for their ability to degrade a variety of ECM and cell adhesion proteins, including CD44, the CSPGs and the aforemen-

Box 77.5  Combating inhibitory ECM molecules

•  Matrix metalloproteinase 2 (MMP2) has the ability to degrade the inhibitory glial barrier-associated proteins CD44 and Neurocan, thus abrogating their inhibitory influence on the scarred retina. This in turn allows for integration and synapse formation following transplantation

tioned myelin-associated inhibitors.67–69 MMP2 in particular has been shown to cleave both CD44 and Neurocan, thus releasing their negative hold on axonal extension and cellular migration.1,70–73 For instance, in a recent study, we discovered that endogenous MMP2 induction resulted in glial barrier-associated CD44 and Neurocan degradation at the outer limits of heavily scarred degenerating retina, subsequently stimulating integration and synapse formation between the host and healthy transplanted tissue grafts.1 Conversely, chemical and/or genetic inhibition/removal of MMP2 was shown to abolish cellular migration and axonal extension completely in this model (Box 77.5).1,72 As a result, there now exists a strategy of removing inhibitory

Key references

barriers in the degenerated retina, permitting the establishment of new connections from retinal transplants, be they of stem cell origin, sheets of retinal tissue, or other yet-to- be-developed techniques.

Conclusions

Here we have described several approaches to repopulating the mature, diseased retina with new cells. The goal of all of these studies is the functional reconstruction of the CNS, restoring sight to the blinded eye. At present, this remains a dream. It is, however, a dream shared by many talented scientists and clinicians, not to mention millions of patients and their families throughout the world. Difficult challenges such as these must be solved one step at a time. It is heartening to realize that we have made tremendous progress in the last 10 years in this field. While much remains to be done, we are now on the brink of achieving significant restoration of function in large-animal models of retinal degeneration. Although the precise pathway to clinical application is not yet clear, the present rate of progress bodes well for the ultimate success of retinal repopulation.

1.Zhang Y, Klassen HJ, Tucker BA, et al. CNS progenitor cells promote a permissive environment for neurite outgrowth via a matrix metalloproteinase-2-dependent mechanism. J Neurosci 2007;27:4499– 4506.

4.Klassen H, Kiilgaard JF, Zahir T, et al. Progenitor cells from the porcine neural retina express photoreceptor markers after transplantation to the subretinal space of allorecipients. Stem Cells 2007;25:1222–1230.

7.Tao S, Young C, Redenti S, et al. Survival, migration and differentiation of retinal progenitor cells transplanted on micro-machined poly(methyl methacrylate) scaffolds to the subretinal space. Lab Chip 2007;7:695–701.

8.Tomita M, Lavik E, Klassen H, et al. Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells. Stem Cells 2005;23:1579–1588.

10.Klassen HJ, Ng TF, Kurimoto Y, et al. Multipotent retinal progenitors express developmental markers, differentiate into

retinal neurons, and preserve lightmediated behavior. Invest Ophthalmol Vis Sci 2004;45:4167–4173.

11.Young MJ, Ray J, Whiteley SJ, et al. Neuronal differentiation and morphological integration of hippocampal progenitor cells transplanted to the retina of immature and mature dystrophic rats. Mol Cell Neurosci 2000;16:197–205.

12.Streilein JW, Ma N, Wenkel H, et al. Immunobiology and privilege of neuronal retina and pigment epithelium transplants. Vision Res 2002;42:487–495.

23.Zhang Y, Kardaszewska AK, van Veen T, et al. Integration between abutting retinas: role of glial structures and associated molecules at the interface. Invest Ophthalmol Vis Sci 2004;45: 4440–4449.

24.Silver J, Miller JH. Regeneration beyond the glial scar. Nat Rev Neurosci 2004;5: 146–156.

26.MacLaren RE, Pearson RA, MacNeil A, et al. Retinal repair by transplantation of photoreceptor precursors. Nature 2006;444:203–207.

27.Lamba DA, Karl MO, Ware CB, et al. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci USA 2006;103: 12769–12774.

35.Ghosh F, Engelsberg K, English RV, et al. Long-term neuroretinal full-thickness transplants in a large animal model of severe retinitis pigmentosa. Graefes Arch Clin Exp Ophthalmol 2007;245:835– 846.

56.Busch SA, Silver J. The role of extracellular matrix in CNS regeneration. Curr Opin Neurobiol 2007;17:120–127.

69.Yong VW. Metalloproteinases: mediators of pathology and regeneration in the CNS. Nat Rev Neurosci 2005;6:931– 944.

73.Tucker B, Klassen H, Yang L, et al. Elevated MMP expression in the MRL mouse retina creates a permissive environment for retinal regeneration. Invest Ophthalmol Vis Sci 2008;49: 1686–1695.

Clinical background

Proliferative vitreoretinopathy (PVR) is defined as the “growth of membranes on both surfaces of the detached retina and on the posterior surface of the detached vitreous gel.” The name was introduced in 1983 by the Retina Society Terminology Committee1 as part of a classification scheme for a group of intraocular complications previously known by more descriptive terms, including “massive vitreous retraction,” “massive preretinal retraction,” and “massive periretinal proliferation.” PVR is not a distinct disease per se, but is instead a complication common to a variety of clinical disorders. It is most prevalent as a clinical complication of surgical procedures to correct rhegmatogenous retinal detachments, which are detachments that follow formation of a retinal tear or hole. Tractional forces generated within the scar tissue-like PVR membranes can be transmitted to the retina and cause complete retinal detachment, retinal degeneration, and permanent blindness.

Pathology

Studies of PVR chronology report average development times between the initial symptoms and retinal detachment ranging between 1 and 2 months and which vary with the type of initiating event and disease severity.2 Tissue changes associated with PVR can vary widely in both severity and location at initial diagnosis. The classification scheme used to describe these two features was suggested by the Retina Society Terminology Committee1 in 1983. Stage A PVR refers to minimal or earliest signs of potential disease, including vitreous haze, protein flare, or the presence of pigmented cell clumps thought to be derived from the retinal pigmented epithelium (RPE: Figure 78.1A). PVR stage B describes moderate, but nonetheless overt, signs of PVR, including traction or wrinkling of the retinal surface, rolled edges of a retinal break, or blood vessel distortion (Figure 78.1B). PVR stage C was originally proposed to describe marked and then massive, full-thickness retinal folds that involved one to three quadrants of the eye (Figure 78.1C) and stage D indicates complete retinal detachment into a funnel shape. However, this portion of the classification scheme was later modified to include a single stage C that was subdivided into

six categories providing specific information about the severity and location of advanced PVR (Table 78.1).3

Etiology

PVR development is most often associated with the formation of retinal holes or tears and its prevalence under these circumstances correlates with the size and/or number of retinal defects. The ultimate success rate of surgical procedures to close retinal defects and correct the rhegmatogenous retinal detachments is now extremely high, exceeding 90%.4 However, PVR develops in 5–10% of these cases and remains the leading cause of surgical failure.1 Depending on the severity or location, penetrating ocular injuries and other ocular trauma also have a high risk of developing PVR.5 This is also true for conditions that lead to retinal or vitreous hemorrhage.6 PVR is also associated with seemingly unrelated conditions such as aphakia, which is the absence of the natural crystalline lens, and pseudophakia, which indicates the presence of a synthetic lens. Genetic predisposition per se does not appear to be a major factor in the development of PVR. However, PVR may be more common in genetic diseases in which risk factors such as the formation of retinal holes, tears, and detachments are more prevalent, such as severe myopia and connective tissue disorders such as Stickler and Marfan syndromes.7

Treatment

Treatments for PVR vary according to disease severity and the perceived risk of developing more aggressive disease.6,8 Early PVR that does not involve new retinal holes or tears or otherwise alter visual acuity might be monitored in the hope that it will remain asymptomatic. Light to moderate PVR might be treated with an encircling scleral band designed to close the retinal break through external deformation of the globe. More severe disease may require vitrectomy which involves surgical removal of the vitreous gel and replacement with a buffered saline solution. It may also be necessary to dissect and peel the epiretinal scar tissues off to lessen traction on the retina. Severe cases involving large expanses of retinal detachment under traction may require even more aggressive procedures such as temporary replacement of vit-

reous fluid with gas or silicone oil to encourage retinal reattachment and tamponade the retinal defects. In the most advanced cases it may be necessary to remove retinal tissue in relaxing retinectomies. The extraordinary skill with which these techniques are applied has resulted in a surprisingly high rate of surgical success. Anatomic correction, defined by successful retinal reattachment, is accomplished in 60– 80% of PVR cases. This is somewhat lower in cases of extremely severe or advanced disease. Unfortunately, high surgical success rates are not necessarily indicative of visual success. The risk of developing recurrent PVR is extremely high (approximately 40%) and often requires revision surgeries. Also, the more aggressive treatment options like gas or silicone oil tamponade, while essential to a successful surgical outcome, can lead to other unrelated complications

Scar tissue with rolled edge of retina

Figure 78.1  An illustration of the three stages of proliferative vitreoretinopathy-related change in the retina. (A) A moderate-sized retinal detachment with a break and clumps of pigmented cells dispersed/ suspended within the vitreous gel. (B) A larger area of retinal detachment with fibrous scar tissue rolling one of the edges of the retinal break. (C) An extensive retinal detachment with the retina held in a full-thickness fold by scar tissue.

such as cataractous changes or glaucomatous increases in intraocular pressure. Finally, and perhaps most importantly, even relatively brief periods of retinal detachment can lead to significant loss of retinal function. Successful, uncomplicated surgical correction of retinal detachments within 7 days allows more than 80% of patients to recover ambulatory vision of 20/200 or better.9 However, when recurrent disease and other complications are considered, these percentages fall to between 40 and 80%.6

Pathophysiology

While the initiating events and disease course can be highly variable, PVR is ultimately a cellular disorder in that it is dependent upon the combined actions of individual cells.10 Minimally, the intravitreal form of PVR requires that the pathogenic cells gain access to the vitreal space through avenues which vary according to cell type. The pathogenic cells must then proliferate to achieve the required critical mass and generate the tractional forces that ultimately cause traction retinal detachment. The ability to arrest any of these critical activities would result in control of PVR and prevent its recurrence. As a result, much of the research into PVR pathogenic mechanisms has focused on identifying the cells involved (Box 78.1) and on the critical pathogenic activities (Box 78.2).

Pathogenic cells

Cells derived from the RPE have long been considered key players in the pathogenesis of PVR.11 The RPE is a monolayer of darkly pigmented cells that underlies and provides physiologic support to the attached neural retina (Figure 78.2). Studies of PVR epiretinal tissues originally identified RPE based on pigment content and ultrastructural morphology. With the advent of immunochemical labeling techniques for microscopy, RPE have since been positively identified in

Box 78.1  Cell types associated with proliferative

vitreoretinopathy

•Retinal pigmented epithelial cells

•Müller glia

•Retinal astrocytes

•Immune cells (macrophages, lymphocytes, hyalocytes)

•Fibroblasts of unknown origin

Box 78.2  Pathogenic cellular activities in

proliferative vitreoretinopathy

•Cell migration or dispersion into vitreous

•Cell proliferation

•Tractional force generation

these tissues using antibodies raised against cytokeratins present in normal RPE and other proteins with limited ocular distribution such as cellular retinaldehyde-binding protein. RPE can be detected in nearly all PVR epiretinal membranes.12–14 However, in studies in which cell populations were actually quantified, RPE usually represents less than 25% of the total population.12,14 Under normal conditions RPE cells are not in direct physical contact with the vitreous and so their involvement in PVR is thought to require the creation of a retinal defect such as a hole or tear. In addition to migration through retinal defects, there is evidence that RPE can be physically dispersed into the vitreous if attached to a large, horseshoe-shaped retinal tear or even during retinal detachment surgeries that involve physical manipulation of the external globe wall.

Evidence of glial involvement in the pathogenesis of PVR is similar to RPE except that the glia are potentially derived from two retinal cell types. Retinal astrocytes are derived from the nerve fiber layer near the vitreoretinal interface (Figure 78.3). Müller cells are radially oriented, transretinal glia whose broad endfeet join and comprise the vitreoretinal interface (Figure 78.4). Early light microscopic studies of PVR scar tissues tentatively identified glia in these tissues by size and morphology. The immunochemical studies that followed confirmed these findings by detecting cells positive for glial fibrillary acid protein (GFAP) in most of the PVR epiretinal membranes examined.12–16 However, when quantified, glia detected using this antigen consistently represented a minority of the overall cell population.14,16 At least two studies distinguished between astrocytes and Müller glia using proteins specific to the latter cell type, including cellular retinaldehyde-binding protein, carbonic anhydrase, and glutamine synthetase. Cells positive for these antigens were detected, indicating that some of the glia present in PVR epiretinal membranes are derived from Müller cells.15,17 Recent studies have now provided direct evidence of retinal glial cell migration on to the retinal surface in human and animal studies.18 In this case, vitreal Müller cell migration was induced by retinal detachment and then reattachment.

There are also reports identifying other cell types in epiretinal membranes including lymphocytes,19,20 macrophages,19,21 and hyalocytes,22 a vitreous cell with macrophage-like characteristics23 leading to speculation

614

Figure 78.2  Light micrograph of posterior pole section pointing out retinal pigmented epithelium monolayer beneath retina. (Courtesy of Dr. Christine Curcio, University of Alabama School of Medicine.)

Figure 78.3  Glial fibrillary acid protein localization to astrocytes and Müller glia in porcine retina.

about the role of inflammation as an associated process in PVR. Other important cell types consistently reported in epiretinal membranes are fibroblast-like cells, also described as fibrocytes or myofibroblasts.12–14 In addition to cell morphology, immunochemical identification of these cells is primarily based on the absence of other ocular cell marker proteins and the presence of the myoid protein α-smooth- muscle actin.24 As with RPE cells, fibroblasts are reported in virtually all PVR epiretinal membranes examined and, when

quantified, represent the most abundant cell population in these tissues. The origins of fibroblasts in PVR epiretinal membranes are uncertain and, while it has been suggested that they originate from local connective tissues, there is no direct evidence of fibroblast translocation into the vitreal space. There is, however, evidence suggesting that fibroblastlike cells can arise from local ocular populations. Within days of isolation and introduction into tissue culture, RPE and Müller cells express α-smooth-muscle actin de novo and reduce expression of their respective marker proteins (cytokeratin 18 and GFAP), yielding a cell type that is immunochemically indistinguishable from the fibroblasts detected in epiretinal membranes.25–27

Cell proliferation and apoptosis

Of the pathogenic activities associated with PVR, cell proliferation has received the most attention. Cell division as a pathogenic mechanism in PVR epiretinal membranes has been confirmed directly in studies using antibodies against the Ki67 nuclear protein, which is detectable throughout the mitotic cell cycle and absent in G0, and the proliferation cell nuclear antigen (PCNA), which is abundant during early S-phase.28–31 With this approach, evidence of proliferation can be detected in the majority of PVR specimens examined. Interestingly, in two studies in which the positive populations were quantified, the percentages varied widely from as low as 0% to as high as 99%.28,31 Surprisingly, in neither study did the percentage of actively proliferating cells correlate with disease duration or stage, suggesting that the variance is a reflection of the heterogeneous origins and pathogenesis of this disorder. On the opposite end of the proliferation spectrum, there is also compelling evidence of cell apoptosis in PVR epiretinal membranes. Two of the Ki67

studies mentioned above also quantified apoptotic cells in the same specimens using terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).28,31 In this case, the results were more consistent in that the majority of the samples examined were positive for apoptosis and the proportions of positive cells were consistently lower, ranging from 0 to 10% in one study. Dual-label immunochemical experiments to identify the apoptotic cell types revealed that many were derived from RPE rather than glia. Finally, both studies reported a correlation with disease duration in that the proportion of apoptotic cells increases with long-term detachments.

Nearly two decades of immunochemical studies performed on PVR vitreous fluids and epiretinal membranes have implicated a relatively long list of growth factors, cytokines, and chemokines as potentially involved in modulating cell growth in PVR.32,33 The list of growth factors includes, but is not limited to, members of the transforming growth factor,28,34,35 plateletderived growth factor,36–39 epidermal growth factor,37,40–44 fibroblast growth factor,45–47 insulin-like growth factor,44,48,49 tumor necrosis factor,50,51 hepatocyte growth factor,52–55 and connective tissue growth factor families.56–58 In most cases there is also evidence of production by local cells, most often RPE or Müller cells. These growth factor families have also been demonstrated to modulate RPE or glial cell growth, migration, or apoptosis in tissue culture models, providing some indirect evidence of their potential effects in PVR. Unfortunately, there is limited direct evidence about which mitogens actually drive RPE or glial cell proliferation in situ. In light of the complex effects on cell behavior and growth factor interactions reported, it seems likely that the causal growth factors, like the disease, will also vary with the inductive events and disease stage.

Another important area in PVR research is the potential use of antineoplastic agents to control cell growth. The list of drugs tested is extensive, with 5-fluorouracil, a cell cycledependent pyrimidine analog, successfully used to control scarring in glaucoma filtration surgeries, probably being the most extensively studied drug of this type.59 Studies using tissue culture models demonstrated the capacity of this drug to control cell growth and traction retinal detachment in simplified animal models designed to mimic key features of PVR. However, the outcomes in clinical trials dating back more than 25 years have been mixed. As a result, antineoplastic drugs are not routinely used as adjunctive therapies in retinal detachment surgery. The major hurdles seem to be the relatively short half-life of the drugs following introduction into the vitreal cavity and an understandable reluctance to perform repeated intraocular injections which in and of themselves are an added risk factor for PVR development. Investigators are currently exploring alternative drug delivery systems designed to maintain therapeutic drug levels for longer periods60 and antineoplasmic agents as combined therapies4 to improve drug effectiveness when applied at the time of surgery.

Tractional force generation

Cell-generated tractional forces applied to the vitreous matrix or retinal surface cause retinal detachment and represent another potential target for intervention. While research in this field is less abundant, our understanding of this activity in PVR is comparable to that of cell proliferation. The capacity for significant tractional force generation is present in a minority of cell types and is mechanistically limited to cells expressing α-smooth-muscle actin61 and the necessary collagen-binding integrin receptors.62,63In PVR epiretinal membranes, α-smooth-muscle actin expression is detected in the fibroblast-like cells mentioned previously.24 As also mentioned previously, both RPE and Müller cells express α-smooth-muscle actin de novo following isolation and, with expression, acquire the capacity to generate tractional forces on collagen matrices in vitro.25,64 With these observations in mind, it seems most likely that fibroblast-

Key references

like cells derived from RPE and/or Müller cells are the sources of tractional forces generated in PVR. Tractional force generation, like cell proliferation, is not a constitutive behavior but is instead stimulated by exogenous growth factors. Thus far, the list of growth factors capable of stimulating RPE and Müller cell tractional force generation is fairly short, and includes members of the transforming growth factor-β, platelet-derived growth factor, and insulin-like growth factor families.25,64–66 PVR vitreous is able to stimulate tractional force generation in tissue culture models in vitro and neutralizing antibodies against insulin-like growth factors and platelet-derived growth factors are able to attenuate the majority of this activity, suggesting that these growth factor families represent the major stimuli in PVR.49

Conclusions

PVR is a cellular disorder that develops in response to several types of retinal defects and results in the introduction or migration of extravitreal cells into the vitreal cavity. Untreated, PVR can result in traction retinal detachment and blindness. The principal effector cells in PVR are most likely derived from the RPE and retinal glia and the major pathogenic activities, cell growth and extracellular matrix contraction, are potentially driven by multiple growth factors produced by local cells and/or derived from blood. At present, the most successful treatments for PVR involve surgical correction of the retinal defect and removal of the proliferating tissues. The major challenge is to find more effective avenues through which these essential pathogenic activities can be arrested in the early stages to prevent disease progression and/or recurrence.

Acknowledgments

The author is indebted to John O Mason III, MD and Sudipto Mukherjee, MD, PhD for their helpful comments during manuscript preparation.

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