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hat is the macula, what is AMD?
The macula lutea is an area of the retina that is about 5000 μm in diameter. The center of the macula, the fovea, contains specialized photoreceptors and provides high acuity vision necessary for reading, driving, and recognizing faces. In order for light-sensing photoreceptors to function properly, they must be in intimate contact with a cell layer called the retinal pigment epithelium (RPE). The photoreceptors and RPE exchange nutrients and other materials. The choroid is a vascular layer of the eye wall interposed between the sclera and RPE, and its capillaries, termed the choriocapillaris, provide the blood supply to the RPE and photoreceptors. The RPE is separated from the choriocapillaris by a thin layer of collagenous tissue called Bruch’s membrane.
Age-related macular degeneration (AMD) is the most important cause of new cases of blindness in patients older than 55 years of age in the industrialized world. RPE cells may be the target of the pathological processes that cause AMD. Approximately 10% of patients with AMD lose central vision. Among the ~75% of AMD patients with central visual loss, abnormal blood vessels, termed choroidal new vessels (CNVs), grow from the choriocapillaris and leak fluid and blood under the RPE and macula (exudative or “wet” AMD), which causes visual loss. The stimulus for CNV growth in AMD is complex, and the biochemical pathways are now being identified. One critical element is vascular endothelial growth factor (VEGF), which is involved in CNV growth and leakage. Among ~25% of AMD patients with severe central visual loss, the RPE and foveal photoreceptors die in the absence of CNVs (atrophic or “dry” AMD).
Pathway-Based Pharmacological Therapy
Pharmacological therapies (e.g., Avastin and Lucentis, both of which block the action of VEGF) that are pathway-based have provided the best treatment results for AMD patients that have ever been reported. Nonetheless, a need for improved therapy remains. Although Lucentis treatment is associated with moderate visual improvement in ~30% of patients according to the results of two randomized studies, the remaining ~70% of patients are in urgent need of an alternative approach. Also, these medications currently are administered via repeated intravitreal injection, which entails some risk and inconvenience for the patient.
Ilene Sugino, MS, director, Ocular Cell Transplantation Laboratory, and Marco Zarbin, MD, PhD, chair, Ophthalmology Department, NJMS
Cell-based therapy may offer advantages over pharmacological therapy. Pharmacological therapy, for example, involves administration of a finite number of compounds and usually involves fluctuations in drug levels above and below the desired level. In situ, cells express a plethora of molecules (e.g., neurotrophic factors, cytokines) that can inhibit pathological processes and rescue neurons that are damaged by disease. Moreover, they can express these molecules in amounts, combinations and frequencies that are tailored precisely to molecular changes that occur from moment to moment. Thus, cells have the capacity to function as “factories” that produce many more substances at appropriate doses and times than can be managed with conventional pharmacological therapy. This pharmacological salutary capacity of cell-based therapy is termed “rescue”. Another capacity of cell-based therapy is “replacement,” which refers to the ability of transplanted cells to replace native cells that have died. In diseases such as AMD, RPE and photoreceptor cell death constitutes a component of “irreversible” visual loss in many patients. Among AMD patients with evolving atrophy, RPE transplantation could be curative. These advantages of cell-based therapy are compelling. Thus, the era of pathway-based therapy probably will be eclipsed by the era of cell-based therapy.
RPE Transplants: Cell-Based Therapy for AMD
The first efforts to develop cell-based therapy for AMD involved RPE transplantation after CNV excision. Before current pharmacological therapy was available, CNV excision was proposed as a treatment for CNVs. In most AMD patients, CNV excision is associated with iatrogenic RPE defects due to the intimate association of RPE cells and the CNV. Combined RPE transplantation and CNV excision has been attempted in AMD eyes, but it has not yet led to significant visual improvement in most patients. In contrast, RPE transplantation in animal models of retinal degeneration has been proved to rescue photoreceptors and preserve visual acuity. Although animal studies validate cell transplantation as a means of achieving photoreceptor rescue, an important distinction between humans with AMD and laboratory animals in which RPE transplantation has been successful is the age-related modification of Bruch’s membrane in human eyes, which may have a significant effect on RPE graft survival.
Hypothesis: Changes associated with aging mask extracellular matrix (ECM) ligand availability to transplanted RPE cells, leading to cell death or inability of the cells to differentiate.
With normal aging, human Bruch’s membrane, especially in the submacular region, undergoes numerous changes (e.g., increased thickness, deposition of ECM and lipids, cross-linking of protein, non-enzymatic formation of advanced glycation end products). These changes and additional changes due to AMD could decrease the bioavailability of ECM ligands (e.g., laminin, fibronectin, and collagen IV) and cause the extremely poor survival of RPE cells in eyes with AMD. Thus, although human RPE cells express the integrins needed to attach to these ECM molecules, RPE cell survival on aged submacular human Bruch’s membrane is impaired (see Figure 1)
Because the changes in Bruch’s membrane from aging and AMD are complex and may not be fully reversible, one approach is to establish a new ECM over Bruch’s membrane. Adding exogenous ECM ligands (e.g., combinations of laminin, fibronectin, vitronectin, and collagen IV) can improve RPE attachment to aged Bruch’s membrane to a limited degree (by ~15%). These results are consistent with the hypotheses that ECM ligand availability may decrease with Bruch’s membrane aging and that it is possible to increase ligand density on this surface.
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| Figure 1. Morphology of cultured human RPE at day 7 in culture
A. Scanning electron microscopy (SEM) of RPE on aged (68 years) submacular human Bruch’s membrane shows that cells are irregular in shape, with some being very large (arrow). There are small (asterisk) and large (arrowhead) gaps in Bruch’s membrane coverage.
B. Light micrograph of same specimen in A. showing fragmented RPE (arrows) on Bruch’s membrane (arrowheads).
C, D. Note greater uniformity and hexagonal nature of cell shape when the same cells are cultured for seven days on BCE-ECM-coated plastic culture dishes (C, SEM; D, light microscopy). From Gullapalli et al. |
Our goal is to provide aged Bruch’s membrane with biologically synthesized ECM. The novelty of this approach is that we are not trying to provide individual ECM ligand components. We doubt that attention to individual ECM ligands without attention to their 3-dimensional organization will be highly effective (as indicated by the results of previous studies).Our results demonstrate that bovine corneal endothelial cells (BCE) can attach to Bruch’s membrane and, more importantly, lay down ECM. Thus, we can resurface Bruch’s membrane with a complex ECM that is known to support excellent RPE growth and differentiation and that is well-defined biologically.
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Figure 2. Morphology of cultured human RPE cells on BCE-ECM-resurfaced submacular Bruch’s membrane of an 82 year-old donor eye at day-7 in organ culture.
A. SEM shows cells seeded onto the BCE-exposed Bruch’s membrane uniformly resurface the submacular explants with small, compact cells of variable shape. Compared to RPE seeded onto untreated explants (Fig. 1A), cells are smaller and more uniform in size, and apical processes are more developed. Although variable in shape, RPE size approached that of cells seeded onto BCE-ECM-coated culture dishes (Fig. 1C).
B, C. Cells on the BCE-exposed explants are compact and flat with flattened nuclei, similar to those seen on day-7 BCE-ECM-coated culture dishes (Fig. 1D). The cells on the BCE-exposed explants also show stained material between the basal cell surface and Bruch’s membrane (large arrowheads).
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We have found that RPE focal adhesion formation on aged submacular Bruch’s membrane is abnormal compared to that seen on BCE-ECM-coated culture dishes. We suspect that this early event, probably resulting from poor ECM ligand availability, underlies later degenerative changes in RPE cells on aged Bruch’s membrane after they attach. RPE focal adhesion formation is markedly improved on BCE-ECM-coated aged submacular Bruch’s membrane six hours after seeding. RPE cells seeded onto the BCE-ECM-coated Bruch’s membrane uniformly resurface the submacular explants with small, compact cells of variable shape. Preliminary data suggest that resurfacing by BCE-ECM may enhance RPE cell survival on aged submacular human Bruch’s membrane by 150% (see Figure 2), which is in marked contrast to previous studies in which only modest improvement was seen following treatment with soluble ECM ligands. If additional experiments confirm that RPE survival and differentiation are enhanced via this approach, reductionist studies will identify critical components and features of BCE-ECM, which could form the basis for manufacture of an effective ECM and clinical treatment of Bruch’s membrane to prevent RPE graft failure in AMD patients.
Our efforts to develop RPE transplantation into a clinically useful surgical procedure involves novel application of established biological principles and techniques to the solution of a clinically important problem. Cell-based therapy will one day provide sight-restoring treatment to many patients who are blind due to retinal degeneration of various etiologies. RPE transplantation is an attractive starting point for this sort of therapy since RPE cells are able to integrate with host retina easily. We expect that reconstruction of the ECM will prove to be an important step in assuring the survival and differentiation of the RPE graft. The concept of providing carefully structured ECM also might apply to improving cell-based therapy for other ocular diseases (e.g., endothelial cell grafting to the trabecular meshwork as a treatment for glaucoma). Thus, the lessons we learn from our efforts to complete the work in RPE transplantation might be useful for other types of cell-based therapy.
Marco Attilio Eugenio Zarbin graduated from the Johns Hopkins University School of Medicine with an MD, and a PhD in pharmacology. He completed an ophthalmology residency and fellowships in vitreoretinal surgery and medical retinal disease at the Wilmer Ophthalmological Institute. He was appointed chair of the Institute of Ophthalmology and Visual Science at NJMS and chief of ophthalmology at University Hospital in 1994. He is a professor of ophthalmology and neuroscience at NJMS. Dr. Zarbin's research is supported by grants from the National Eye Institute, the Department of Veterans Affairs, and the Foundation Fighting Blindness. He serves on the Scientific Advisory Board of the Foundation Fighting Blindness, Inc., the Board of Governors of the New Jersey Academy of Ophthalmology, the National Advisory Council of the National Eye Institute, the Professional Development and Education Committee of the Association of Research in Vision and Ophthalmology, and is a member of the American Ophthalmological Society, the Retina Society, the Macula Society, and the Vitreous Society. Dr. Zarbin's clinical interests include medical and surgical diseases of the retina and vitreous with special expertise in age-related macular degeneration, trauma management, complex retinal detachment, and surgery of the macula. He has been listed among the Top Doctors in America.
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