Macular Degeneration: Gaillard ER

Schweitzer D, Hammer M, Schweitzer F, Anders R, Doebbecke T, Schenke S, Gaillard ER, Gaillard ER. · University of Jena, Department of Experimental Ophthalmology, Bachstr. 18, 07740 Jena, Germany. · J Biomed Opt. · Pubmed #15568942 No free full text.

Abstract: An experimental setup for measurement of time-resolved autofluorescence of the human eye fundus is demonstrated. The method combines laser scanning technique and time-correlated single photon counting. The light source is a laser diode, delivering pulses of about 100 ps duration at a repetition rate of 40 MHz. The excitation wavelength is 446 nm and the cutoff wavelength of fluorescence detection is at 475 nm. The autofluorescence can be determined with a spatial resolution of 80 x 80 microm2 and 25 ps time resolution. The fluorescence decay is optimally approximated by a biexponential model. The dominating lifetime tau1 is shortest in the macula (320 to 380 ps) and reaches 1500 ps in the optic disk. The lifetime tau2 varies between 2 ns and 5 ns, but the spatial distribution is more homogeneous. Respiration of 100% oxygen for 6 min leads to changes in the fluorescence lifetime pointing to detection of coenzymes. Diagrams of lifetime tau2 versus tau1 are well suited for comparison of substances. Such lifetime clusters of a 20 deg macular field of a young healthy subject and of a patient suffering from dry age-related macular degeneration overlap only partially with tau2-tau1 clusters of lipofuscin.

Schweitzer D, Hammer M, Schweitzer F, Anders R, Doebbecke T, Schenke S, Gaillard ER, Gaillard ER. · University of Jena, Department of Experimental Ophthalmology, Bachstr. 18, 07740 Jena, Germany. · J Biomed Opt. · Pubmed #15568942 No free full text.

Abstract: An experimental setup for measurement of time-resolved autofluorescence of the human eye fundus is demonstrated. The method combines laser scanning technique and time-correlated single photon counting. The light source is a laser diode, delivering pulses of about 100 ps duration at a repetition rate of 40 MHz. The excitation wavelength is 446 nm and the cutoff wavelength of fluorescence detection is at 475 nm. The autofluorescence can be determined with a spatial resolution of 80 x 80 microm2 and 25 ps time resolution. The fluorescence decay is optimally approximated by a biexponential model. The dominating lifetime tau1 is shortest in the macula (320 to 380 ps) and reaches 1500 ps in the optic disk. The lifetime tau2 varies between 2 ns and 5 ns, but the spatial distribution is more homogeneous. Respiration of 100% oxygen for 6 min leads to changes in the fluorescence lifetime pointing to detection of coenzymes. Diagrams of lifetime tau2 versus tau1 are well suited for comparison of substances. Such lifetime clusters of a 20 deg macular field of a young healthy subject and of a patient suffering from dry age-related macular degeneration overlap only partially with tau2-tau1 clusters of lipofuscin.

Liggett TE, Griffiths TD, Gaillard ER. · Department of Biological Sciences, Northern Illinois University, DeKalb, IL, USA. · BMC Cell Biol. · Pubmed #19413901 links to  free full text

Abstract: BACKGROUND: The Retinal Pigmented Epithelium (RPE) is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa (RP), Fundus Flavimaculatus (FFM), Best's disease and Age-related Macular Degeneration (AMD). We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases. RESULTS: The tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency. CONCLUSION: The tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal maculopathies.

Murdaugh LS, Dillon J, Gaillard ER. · Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA. · Exp Eye Res. · Pubmed #19358843 No free full text.

Abstract: In a variety of retinal diseases, including age-related macular degeneration (AMD); basement membranes are susceptible to alterations in structure and function. Chemical modifications to basement membrane proteins may deleteriously affect Bruch's membrane leading to the development of AMD. The purpose of this study was to investigate modifications from glycolaldehyde and A2E, which are present in the retinal pigment epithelium (RPE), on the membrane like protein fragment, laminin, as a model for aging of Bruch's membrane in age related eye diseases. Laminin was allowed to react with either glycolaldehyde or A2E during irradiation of A2E and then tryptically digested before analysis with electrospray ionization mass spectrometry (ESI-MS). Modifications to laminin occurred preferentially on lysine or arginine residues. The A2E modified laminin fragments are consistent with additions of A2E derived aldehydes resulting from cleavages closest to the pyridinium ring in A2E and oxidized A2E. These results provide evidence that A2E and advanced glycation endproducts (AGE) may be involved in modifications to essential basement membrane proteins leading to deleterious changes in the retinal pigment epithelium extracellular matrix (RPE-ECM) environment. These preliminary experiments are essential for the identification of these modifications in vivo.

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.

Wang Z, Paik DC, Dillon JP, Gaillard ER. · Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA. · Exp Mol Med. · Pubmed #17334231 links to  free full text

Abstract: Non-enzymatic nitrite induced collagen cross-linking results in changes reminiscent of age-related damage and parallels the well-known model system, non-enzymatic glycation. We have recently observed that nitrite modification of basement membrane proteins can induce deleterious effects on overlying retinal pigment epithelial cells in studies relevant to age-related macular degeneration. The present work was undertaken in order to confirm 3-nitro-tyrosine (3-NT) as a product of the reaction and to identify the site specificity of nitration in collagen IV, a major component of basement membranes. Human collagen type IV was modified via incubation with 200 mM NaNO(2) (pH=7.38) for one week at 37(o)C. The modified protein was prepared in 2 different ways, including acid hydrolysis and trypsin digestion for site specificity determination. The samples were analyzed by LC/MS using a C(12) RP column. Site specificity was determined from tandem MS/MS data utilizing TurboSEQUEST software and the Swiss-Prot sequence database. 3-NT was detected in protein digests and acid hydrolysates of nitrite modified collagen IV. Positive identification with standard 3-NT was confirmed by identical R(t), lambda(max)=279 nm and 355 nm, and m/z=227. Analyses of tryptic digests identified four sites of tyrosine nitration, alpha1(IV)Y348, alpha1(IV)Y534, alpha2(IV)Y327, and alpha2(IV)Y1081. These sites are located in the triple-helical region of the protein and provide clues regarding potential sites for nitrite modification in collagen type IV.

Wang Z, Keller LM, Dillon J, Gaillard ER. · Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, USA. · Photochem Photobiol. · Pubmed #16813456 No free full text.

Abstract: It has been reported that the photo-oxidation of A2E, a component of human retinal lipofuscin, leads to products that are toxic to cells via dark reactions. Because these compounds have been implicated in the development of various maculopathies such as age-related macular degeneration (AMD), it is important to determine the structures of those deleterious compounds. Both the photo-oxidation and auto-oxidation of A2E lead to the same complex mixture of products, some of which have lower molecular weights than the staring material. Because A2E is homologous to beta-carotene, it was hypothesized that its oxidation would lead to products analogous to those found in oxidized beta-carotene, namely, a series of cleavage products along the acyclic chain with the concomitant formation of aldehydes. This was found to be the case based upon 1) the formation of all of the aldehydes predicted from the oxidation of beta-carotene, 2) the loss of 28 amu (carbonyl moiety) from the molecular ion, 3) the facile reaction of the aldehydes with nitrophenylhydrazines to form nitrophenylhydrazones and 4) the subsequent MS/MS cleavage of those derivatives at the N-N bond. If formed in vivo, these aldehydes would have toxic effects on any cell. Finally, the similarity in product mixtures from both the photo-oxidation and auto-oxidation strongly suggests that the intermolecular photo-oxidation of A2E results primarily from a radical process without the involvement of singlet oxygen. Any formation of singlet oxygen most likely arises from sensitization by the aldehyde oxidation products, as this process is well known for aldehydes, in general, and retinal, specifically.

Wang Z, Dillon J, Gaillard ER. · Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, USA. · Photochem Photobiol. · Pubmed #16613501 No free full text.

Abstract: The retinal pigment epithelium (RPE) is a monolayer of highly pigmented cells lining the inner aspect of Bruch's membrane. This pigmentation is due to eumelanin and a possible antioxidant role of melanin is reported here. The photo-oxidation of A2E, a constituent of RPE lipofuscin, leads to the sequential addition of up to nine oxygen atoms and/or the addition or loss of two hydrogen atoms. These photo-oxidations were investigated in the presence and absence of either calf or human RPE melanin in A2E-laden RPE cells. It was found that calf melanin was protective against the photo-oxidation of A2E, with an inhibition of oxidation of up to 50% in the case of the addition of two oxygen atoms. Calf melanin was also protective against blue light-induced damage to RPE cells. In addition this ability appears to decrease in humans as they grow older. With aging, a melanin-lipofuscin complex called melanolipofuscin forms. It is suggested that the oxidation or photo-oxidation of A2E in vivo may contribute to the age-related deterioration of the anti-oxidant role of RPE melanin and lead to various retinal disorders, such as age-related macular degeneration.

Wang Z, Paik DC, Del Priore LV, Burch RL, Gaillard ER. · Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, USA. · Curr Eye Res. · Pubmed #16109650 No free full text.

Abstract: PURPOSE: Extracellular matrix (ECM) plays an important role in the regulation of cell function. The aging process may involve chemical modifications to ECM proteins, which may contribute to the aging of the Bruch membrane and pathogenesis of age-related macular degeneration (AMD). The purpose of this study is to investigate nitrite modification of basement membrane-like proteins on RPE cell behavior as a model for the aging of the Bruch membrane in age-related eye diseases. As a comparison, retinal pigment epithelium (RPE) cell behavior on glycolaldehyde-modified matrices (GMM) was also studied. METHODS: Growth factor reduced Matrigel was reacted with nitrite or glycolaldehyde for 1 week or 12 hr, respectively. Calf RPE cells were plated on the modified matrices and examined in several ways. Attachment rates, proliferation rates, apoptosis, and necrosis were determined. Cell morphology and cell susceptibility to A2E-mediated damage was also monitored. RESULTS: Nitrite-modified matrices (NMMs) inhibited cell attachment by 65% and proliferation by 33.7% compared to 69.6% and 21.7%, respectively, by GMM. Proliferation inhibition was not significant when cells were plated at high density on GMM (3.47%) but significant on NMM (20.9%). NMM induced cell apoptosis and necrosis, but GMM induced cell apoptosis only. Both modifications inhibited RPE differentiation. RPE cells on both matrices were more susceptible to blue light mediated damage by A2E, but damage was greater on NMM. CONCLUSIONS: NMM has significant damaging effects on RPE cell function and viability that is similar to the damaging effects of GMM. These studies may have relevance to the RPE dysfunction observed during the progression of AMD.

Dillon J, Zheng L, Merriam JC, Gaillard ER. · Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA. · Exp Eye Res. · Pubmed #15642312 No free full text.

Abstract: The purpose of this study is to determine the transmission properties of the anterior segment of the human eye as a function of age and relate those changes to possible consequences for retinal disorders. For this a new method has been developed. This consists of a probe which is inserted into the posterior sclera and detects light passing through the anterior segment. The probe is connected to a CCD spectrophotometer via a fibre optic bundle. Using this, the transmission properties of human cadaver eyes were determined. A young primate anterior segment has a maximum absorption of 365 nm due to the O-beta-glucoside of 3-hydroxykynurenine (3-HKG) in the lens. There is a steep increase in transmission of the human anterior segment at wavelengths longer than 400 nm. With aging there is an increase in absorption throughout the visible such that by the sixth decade only 20% of blue light is transmitted to the retina compared to the young primate eye. The rate of decrease of blue light was similar to the age related change of the ratio of absorbance at 365/320 nm of the lens. (IOVS 41:1454;1999). The age related rate of decrease in the transmission of blue light to the retina was similar to the rate of increase of lipofuscin formation in the retina, and the amount of light absorbed by A2E in the RPE is constant from the second to seventh decade. Although this yellowing is thought to be detrimental to the lens, it would appear to be beneficial to the retina. It was determined that the implantation of a standard IOL after cataract surgery increased the amount of light absorbed by A2-E by approximately a factor of five.

Avalle LB, Wang Z, Dillon JP, Gaillard ER. · No affiliation provided · Exp Eye Res. · Pubmed #15037123 No free full text.

Abstract: Several retinal dystrophies are associated with the accumulation of lipofuscin in the retinal pigment epithelium (RPE). The only structurally characterized component of human retinal lipofuscin is the bis-retinoid pyridinium compound A2E. We report here on the observation of a monooxygenated product of A2E in the organic soluble portion of human retinal lipofuscin and in the organic extract of bovine RPE cells that have been fed A2E and irradiated. Liquid chromatography mass spectrometry confirms that the products are identical. This is the first observation of a photoproduct of A2E in human retinal lipofuscin.