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"Changes in Retinal Pigment Epithelium Trophic Factor Secretion Affect Retinal Preservation in Vitro"

by
Anton M. Kolomeyer
MD/PhD Program
B.A. 2003, Rutgers University (New Brunswick, NJ)

Thesis Advisor: Marco A. Zarbin, Ph.D., M.D., FACS
Professor
Department of Ophthalmology and Visual Science

Thursday, April 29, 2010
MSB C-600, 4:00 p.m.


Abstract

Introduction: Age-related macular degeneration (AMD) is the leading cause of blindness among persons over age 50 in the industrial world. The early form of AMD is associated with retinal pigment epithelial (RPE) and photoreceptor cell dysfunction/death, and loss of vision. It was hypothesized that: 1) there might be abnormal trophic factor production in AMD eyes that could compromise photoreceptor and retinal survival; and 2) fetal RPE cells may be better for transplantation purposes than aged-adult RPE cells, in part, due to differential expression of selected trophic factors.

Methods: The levels of neurotrophic factors in various conditioned media (CM) were quantified by multiplex ELISA. Trophic factor receptor occupancy was calculated. The expression of trophic factor mRNA was quantified by real-time PCR. The degree of porcine and aged-human retinal preservation was assessed by measuring LDH and DNA fragmentation. iTRAQ was used to identify molecules that may have affected porcine retinal viability in vitro. Confocal microscopy was used to assess retinal morphology.

Results: Multiplex ELISA and real-time PCR detected significant differences in trophic factor production by AMD vs. control adult eye-cups. However, these differences did not affect porcine retinal preservation in in vitro assays (consistent with trophic factor receptor occupancy data). CM isolated from cultured adult RPE cells did not support porcine retinal viability in vitro. Fetal eye-cups produced significantly higher protein and mRNA levels of selected trophic factors than adult eye-cups. Conditioned media from these eye-cups also displayed significantly better preservation of porcine retina in vitro compared to CM from adult eye-cups. In contrast to adult RPE, culturing fetal RPE cells did not decrease their ability to improve porcine retinal survival in vitro. In fact, co-culturing porcine retina with fetal RPE cells was even more beneficial for preservation of retinal function than CM from cultured fetal RPE cells alone. Compared to control medium, cultured fetal RPE-CM increased outer nuclear layer thickness and stratification and decreased photoreceptor axon and terminal retraction. Additionally, this medium had a similar effect on aged-human retinae obtained from control, AMD, and African American eyes. Finally, iTRAQ identified five candidate molecules that are known to regulate apoptosis.

Conclusions: Media isolated from AMD vs. control posterior segments were equally effective at promoting retinal preservation and exhibited similar trophic factor receptor occupancy despite some statistically significant differences in trophic factor concentration. Therefore, abnormal trophic factor production may not be responsible for the pathological changes in the photoreceptors and the retina in AMD eyes. Adult RPE cells may not be as useful for cell transplantation treatment of AMD retina as fetal RPE cells since cultured adult RPE-CM is less able to preserve the viability of porcine retina in vitro. Some experiments reported here indicate that subretinal transplantation of fetal RPE cells might be more effective than intraocular injections of retina-preserving factors at maintaining long-term retinal viability.


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