Macular Degeneration: Cai H

Sun K, Cai H, Tezel TH, Paik D, Gaillard ER, Del Priore LV. · Department of Ophthalmology, Harkness Eye Institute, Columbia University, New York, NY 10032, USA. · Mol Vis. · Pubmed #18199972 links to  free full text

Abstract: PURPOSE: We have shown previously that aging of human Bruch's membrane affects the attachment, survival and gene expression profile of the overlying retinal pigment epithelium (RPE). Herein we determine the effects of Bruch's membrane aging on RPE phagocytosis of rod outer segments. METHODS: Explants of human Bruch's membrane were prepared from cadaver donor eyes (aged 9-81years) within 48 h of death, and 6 mm punches were embedded with the basal lamina in a 96-well plate. Approximately 50,000 ARPE-19 cells per well were seeded onto the explant surface and cultured for two weeks until they reached confluence. In addition, ARPE-19 were also seeded onto RPE-derived extracellular matrix (RPE-ECM) that was unmodified or modified by nonenzymatic nitration. Bovine rod outer segments were purified by sucrose gradient centrifugation, labeled with 10 ug/ml fluorescein isothiocyanate, and added to ARPE-19 cultured on Bruch's membrane or RPE-ECM for 24 h. Phagocytic activity was quantified by flow cytometry of harvested cells. RESULTS: The ability of RPE to phagocytose rod outer segments decreased as a function of aging of Bruch's membrane; mean phagocytotic activity of ARPE-19 on younger Bruch's membrane was significantly higher than on older Bruch's membrane (129.7 +/- 34.8 versus 67.4 +/- 4.2 arbitrary units, respectively; p<0.01). Nitrite treatment of RPE-ECM decreased rod outer segment phagocytosis compared to untreated RPE-ECM and mimicked the effects of aging of human Bruch's membrane. CONCLUSIONS: Aging of human Bruch's membrane decreases rod outer segment phagocytosis by ARPE-19. This effect can be mimicked by nonenzymatic nitration of extracellular matrix in vitro. Our observations may have implications for understanding the role of aging changes within Bruch's membrane on pathogenesis of age-related macular degeneration and other disorders.

Cai H, Shin MC, Tezel TH, Kaplan HJ, Del Priore LV. · Department of Ophthalmology, Harkness Eye Institute, Columbia University, 635 W. 165th Street, New York, NY 10032, USA. · Arch Ophthalmol. · Pubmed #16966623 links to  free full text

Abstract: OBJECTIVE: To determine the gene expression profiles of primary retinal pigment epithelium (RPE) and iris pigment epithelium (IPE) using microarrays. METHODS: Primary RPE and IPE from 6 human donor eyes were collected, and total RNA was isolated. Differences in gene expression were determined using a human genechip (human U95Av2 [12 600 probes]; Affymetrix Inc, Santa Clara, Calif). RESULTS: Hierarchical cluster analysis differentiated the gene expression profiles of RPE and IPE clusters into 2 distinct groups. A mean +/- SD of 5308 +/- 416 gene probes were expressed in RPE vs 6130 +/- 205 in IPE. Sixty-eight genes were expressed only in RPE; 154 genes were expressed only in IPE. Twenty-two additional genes had greater than 3-fold increased expression in RPE vs IPE, and 147 genes had greater than 3-fold decreased expression in RPE vs IPE. CONCLUSION: There are major differences in the gene expression profiles of primary RPE vs IPE.Clinical Relevance The different gene expression profiles of primary RPE vs IPE harvested from the same donor eyes infer that it may be difficult for IPE to replace all aspects of damaged RPE function in transplantation studies.

Cai H, Del Priore LV. · Department of Ophthalmology, Harkness Eye Institute, Columbia University, New York, New York, USA. · Curr Eye Res. · Pubmed #16500769 No free full text.

Abstract: We investigated the effects of age-related changes within the Bruch membrane on the gene expression profile of the RPE. Immortalized human ARPE-19 cells were seeded onto acellular human Bruch membrane from younger and older donors and harvested 72 hr later; total RNA was isolated and the gene expression profile was determined using the Affymetrix Human Genome U95A gene chip. Twelve genes were upregulated and 8 genes were downregulated with Bruch membrane aging; RT-PCR confirms that Bruch membrane aging upregulates genes in RPE cells encoding for transforming growth factor alpha and downregulates genes for vitronectin and the membrane transporter ABCC5. The role of these changes in the pathogenesis of age-related diseases such as macular degeneration remains to be elucidated.

McLaughlin BJ, Fan W, Zheng JJ, Cai H, Del Priore LV, Bora NS, Kaplan HJ. · Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, Kentucky, USA. · Invest Ophthalmol Vis Sci. · Pubmed #12882822 links to  free full text

Abstract: PURPOSE: There is increasing evidence that the complement system may play a significant role in one of the leading diseases causing blindness in the elderly population, age-related macular degeneration. In this study, a novel role in the retina for a regulatory protein in the complement system, CD46, is proposed. METHODS: The retinal pigment epithelium (RPE) was obtained from human donor eyes as well as human immortalized RPE cell lines (ARPE19). Immunohistochemistry and confocal microscopy were used to immunolocalize CD46 and beta1 integrin. Immunoprecipitation experiments with antibodies to either CD46 or beta1 integrin were performed on RPE cell lysates. A cell adhesion assay was used to determine the proportion of RPE cells that adhere to Bruch's membrane explants from donor eyes. RESULTS: Immunohistochemistry and confocal microscopy demonstrated that CD46 was polarized to the basal surface of the RPE along with beta1 integrin, shown previously to be involved in RPE adhesion. Immunoprecipitation experiments demonstrated that CD46 and beta1 integrin coprecipitated from RPE cell lysates when either protein was used as the precipitating antibody. The adhesion assay showed that antibodies to either CD46 or beta1 integrin reduced RPE adhesion to the surface of Bruch's membrane compared with the control. CONCLUSIONS: These findings suggest that this complement regulatory protein, which protects host cells from autologous complement attack, may have a functional interaction with beta1 integrin in the eye that is related to RPE adhesion to its basement membrane and Bruch's membrane.