Macular Degeneration: Hollyfield JG

Hollyfield JG, Salomon RG, Crabb JW. · Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA. · Adv Exp Med Biol. · Pubmed #15180251 No free full text.

Abstract: Microdissection methods have been developed for isolating drusen and Bruch's membrane from human eyes. Comparative proteomic studies of these isolates from normal and AMD donors were pursued for clues to the biochemical pathways involved in the pathogenesis of AMD. A total of 129 potential drusen proteins were identified by LC MS/MS and immunocytochemical analyses have confirmed drusen localization for approximately 16% of the proteins. The most common drusen proteins appear to be TIMP-3, clusterin, vitronectin and serum albumin. Western blot analysis suggests that carboxyethyl pyrrole-protein adducts derived from docosahexaenoate-containing lipids are more abundant in AMD than in normal tissues. Abnormal protein cross-links and advanced glycation end products were also observed in drusen and Bruch's membrane. Lipid oxidation products and oxidative protein modifications may be causally involved in drusen formation and Bruch's membrane thickening.

Bonilha VL, Trzupek KM, Li Y, Francis PJ, Hollyfield JG, Rayborn ME, Smaoui N, Weleber RG. · The Cleveland Clinic Foundation, The Cole Eye Institute, Cleveland, Ohio 44195, USA. · Ophthalmic Genet. · Pubmed #18766988 No free full text.

Abstract: PURPOSE: To define the retinal pathology in a 91 year-old affected matriarch of a three-generation choroideremia family with multiple manifesting carriers. METHODS: Tissue from three different retinal areas was processed for immunohistochemistry. The macular area was processed for transmission electron microscopy. Cryosections were studied by indirect immunofluorescence, using well-characterized antibodies to cone cytoplasm, rhodopsin and cone opsins. The affected donor eyes were compared to a postmortem matched normal eye. RESULTS: The retina displayed areas of severe degeneration, with no photoreceptor outer segments, photoreceptor nuclear atrophy, and atrophy of the inner retina. Other retinal areas were near to normal. The RPE was severely degenerated, with thinning, pigment clumping and sub-epithelial debris deposition in all the areas examined. The choroid displayed depigmentation. Labeling with cone opsin antibodies revealed that cones were drastically affected: blue opsin was almost completely absent, while red/green opsins were distributed along the entire plasma membrane of the cell. Rhodopsin was also distributed along the entire rod plasma membrane. Ultrastructural analysis of the affected macula revealed the absence of RPE apical microvilli and basal infoldings. Instead, RPE's basal surface and choroid displayed the presence of banded fibers composed of clumps of wide-spacing collagen. Bruch's membrane was filled with vesicular structures, some smooth and others with bristle-like projections. CONCLUSIONS: The histological data suggests that the clinical manifestation in this donor is related to degenerative changes in the retina, RPE, and choroid.

Ng KP, Gugiu B, Renganathan K, Davies MW, Gu X, Crabb JS, Kim SR, Rózanowska MB, Bonilha VL, Rayborn ME, Salomon RG, Sparrow JR, Boulton ME, Hollyfield JG, Crabb JW. · Cole Eye Institute and Lerner Research Institute, Cleveland Clinic Foundation, Case Western Reserve University, Cleveland, Ohio 44195, USA. · Mol Cell Proteomics. · Pubmed #18436525 links to  free full text

Abstract: Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

Bhattacharya SK, Sinicrope B, Rayborn ME, Hollyfield JG, Bonilha VL. · Bascom Palmer Eye Institute, 1638 NW 10th Avenue, 706, University of Miami, Miami, FL 33136, USA. · Aging Cell. · Pubmed #18248664 No free full text.

Abstract: Increased deimination and peptidyl arginine deiminase type 2 (PAD2) expression has been observed in age-related neurodegenerative diseases without discrimination between their aging and disease component. Here, we describe reduced levels of deimination commensurate with reduced protein, mRNA and activity of peptidylarginine deiminase type 2 in the retina, optic nerve and plasma of aged rats when compared to young rats. The decrease was significant in the ganglion cell layer, inner plexiform layer and inner nuclear layer. Because our observations suggest reduced deimination is a consequence of aging, we conclude that increased deimination must be a consequence of disease. Our findings are important to understand late-onset and progressive diseases such as glaucoma, pseudoexfoliation syndrome, age-related macular degeneration and Oguchi's disease.

Hollyfield JG, Bonilha VL, Rayborn ME, Yang X, Shadrach KG, Lu L, Ufret RL, Salomon RG, Perez VL. · Cole Eye Institute (i31), Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195, USA. · Nat Med. · Pubmed #18223656 No free full text.

Abstract: Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues and in plasma samples from individuals with AMD. Immunized mice develop antibodies to this hapten, fix complement component-3 in Bruch's membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these mice are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.

Bonilha VL, Rayborn ME, Shadrach KG, Li Y, Lundwall A, Malm J, Hollyfield JG. · Department of Ophthalmology, The Cole Eye Institute, Cleveland Clinic Lerner College of Medicine, 9500 Euclid Avenue, Cleveland, OH 44195, USA. · Exp Eye Res. · Pubmed #18036592 links to  free full text

Abstract: The two cellular targets of interest in age-related macular degeneration (AMD) are the photoreceptors and the RPE. However, the mechanisms involved in AMD pathology are not yet fully understood. In the present report, we extend our previous studies on semenogelin proteins (Sgs) in normal human retina and compare these with the distribution in retinas from AMD donor eyes. Semenogelins I (SgI) and II (SgII) are the major structural protein components of semen coagulum, but have been recently found in non-genital tissues as well. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with a specific antibody. The presence of SgI was analyzed in retina and RPE total lysates and SgI was detected by western blot in human retina and RPE. The intensity of immunoreactivity was significantly reduced in the AMD eyes. SgI is expressed in the normal human retina and in the retina of AMD donor eyes, where localization was detected in the photoreceptors and in a few ganglion cells. We find the distribution of SgI in the AMD retinas substantially lower than observed in normal retina. SgI localization to photoreceptors and the RPE suggests a possible function related to the ability of these cells to sequester zinc.

Sturgill GM, Pauer GJ, Bala E, Simpson E, Yaniglos SS, Crabb JW, Hollyfield JG, Lewis H, Peachey NS, Hagstrom SA. · Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH, USA. · Ophthalmic Genet. · Pubmed #17148042 No free full text.

Abstract: Clusterin is a secreted glycoprotein expressed ubiquitously in many tissues that appears to function as a molecular chaperone capable of protecting stressed proteins. It is upregulated in many different forms of neurodegeneration and is thought to represent a defense response against neuronal damage. Clusterin has been found to be a common protein identified in drusen preparations isolated from the retina of donor eyes of patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population of developed countries. A retina-specific clusterin-like protein (CLUL1) showing nearly 25% identity to clusterin at the protein level was recently cloned and shown to be expressed specifically in cone photoreceptor cells. For these reasons, we investigated CLUL1 as a candidate gene for AMD. A mutation screen of the entire coding region of the CLUL1 gene in 376 unrelated patients with AMD uncovered three sequence variations, one isocoding change and two intronic changes. One intronic change appears significantly less frequent in patients with the more severe forms of AMD than in control subjects, suggesting that this variant may reduce the risk for AMD or may be linked to a nearby variant that may reduce AMD risk. Variant alleles of the CLUL1 gene were found; however, none are considered pathogenic. None of the variants identified are predicted to create or destroy splice donor or acceptor sites based on splice-site prediction software.

Bando H, Shadrach KG, Rayborn ME, Crabb JW, Hollyfield JG. · Cole Eye Institute (i-31), Department of Ophthalmology, Cleveland Clinic Foundation Lerner College of Medicine of Case Western Reserve University, 9500 Euclid Avenue, Cleveland, OH 44195, USA. · Exp Eye Res. · Pubmed #17097084 No free full text.

Abstract: Clathrin was identified in a recent proteomic analysis of Bruch's membrane from age-related macular degeneration (AMD) donor eyes. The present study was conducted to determine the localization of clathrin in AMD tissues and to compare this distribution and relative content with that in non-AMD control tissues. The distribution of adaptin, which is functionally linked to clathrin, was also evaluated. Human eyes were from donors between 66 and 94 years of age; 13 eyes were from donors with AMD and 13 from non-AMD donors. Bruch's membrane and choroid from the macula of each donor eye were prepared for immunohistochemistry and Western blotting. Differences in immunoreactivity were quantitated. Drusen, Bruch's membrane and choroid from AMD tissues showed greater immunoreactivity for clathrin and adaptin than did non-AMD tissues. Western blots also showed more intense clathrin and adaptin immunoreactivity in AMD tissues than were present in non-AMD samples. This study suggests that accumulation of clathrin and adaptin in drusen, Bruch's membrane and choroid may reflect a higher rate of clathrin mediated endocytosis in AMD tissues. Alternatively, the accumulation of these proteins in these extracellular compartments may reflect a higher susceptibility to oxidative damage.

Bonilha VL, Hollyfield JG, Grover S, Fishman GA. · Cole Eye Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue i31, Cleveland, OH 44195, USA. · Ophthalmic Genet. · Pubmed #16020309 No free full text.

Abstract: PURPOSE: To define the distribution of the red/green and blue opsins in cones from donor eyes from an affected member of a clinically well-characterized family with an autosomal dominant form of cone dystrophy. METHODS: Tissue was fixed and processed for immunohistochemistry. Cryosections were studied by indirect immunofluorescence, using well-characterized antibodies to cone cytoplasm, rhodopsin, and cone opsins. The cone-associated matrix was also labeled with the lectin PNA. The affected donor eyes were compared to a postmortem matched normal eye. RESULTS: Electroretinogram (ERG) testing three years prior to the affected member's death showed normal rod function, while the cone b-wave amplitude was reduced 40% below the lower limit of normal. Fundus exam showed only isolated drusen within the macula. Either a normal-appearing or only nonspecific macular findings were noted in the other affected family members who were examined. Immunofluorescence studies showed that blue cone opsin was restricted to the outer segments of blue cones in the affected retina. Red/green opsins were distributed along the entire plasma membrane of these cone types, from the tip of the outer segment to the synaptic base. Cone-associated matrix displayed a heterogeneous distribution. These patterns were observed both in the macula and in the periphery of the affected retina. Cone pedicles appeared larger than normal. In contrast, rhodopsin staining appeared normal. CONCLUSIONS: The immunocytochemical data obtained suggest that the clinical manifestation of this dystrophy is associated with an abnormal distribution of cone red/green opsins. Additionally, changes in the cone pedicles could have contributed to the abnormal cone ERG in this patient.

Nakata K, Crabb JW, Hollyfield JG. · Department of Ophthalmic Research, Cole Eye Institute, i-31, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. · Exp Eye Res. · Pubmed #15939038 No free full text.

Abstract: Crystallins were consistently found in a recent proteomic analysis of drusen from age-related macular degeneration (AMD) donor eyes. Here we compare the distribution of several crystallins in drusen, Bruch's membrane and choroid from AMD and non-AMD age-matched control eyes. Immunohistochemistry and Western blots of tissue samples were performed using antibodies to alphaA- and alphaB-crystallins. Bruch's membrane, drusen and the subjacent choroidal connective tissue from AMD tissues showed greater immunoreactivity for alphaA- and alphaB-crystallins than were observed in normal age-matched control tissues. Western blots also demonstrated more intense alphaA- and alphaB-crystallin signals from AMD tissues than were present in age-matched controls. These data indicate that alphaA- and alphaB-crystallins accumulate in Bruch's membrane and choroidal connective tissues to a greater degree in AMD than in normal aging. These findings suggest that the accumulation of these small heat shock proteins at this critical interface below the RPE reflects a disease-related stress response manifested during the progression of AMD.

Gu X, Meer SG, Miyagi M, Rayborn ME, Hollyfield JG, Crabb JW, Salomon RG. · Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA. · J Biol Chem. · Pubmed #12923198 links to  free full text

Abstract: Age-related macular degeneration (AMD) is a slow, progressive disease with both genetic and environmental risk factors. Free radical-induced oxidation of docosahexaenoate (DHA)-containing lipids generates omega-(2-carboxyethyl)pyrrole (CEP) protein adducts that are more abundant in ocular tissues from AMD than normal human donors. To understand better the role of oxidative damage in AMD, we have synthesized CEP-modified proteins, produced anti-CEP antibodies, and initiated analysis of CEP immunoreactivity and autoantibodies in human plasma. A highly selective rabbit polyclonal anti-CEP antibody was raised that binds CEP 1000 times more strongly than carboxypropylpyrrole, a close structural analogue. The CEP adduct uniquely indicates oxidative modification from DHA derivatives because CEP protein modifications cannot arise from any other common polyunsaturated fatty acid. Immunocytochemistry localized CEP to photoreceptor rod outer segments and retinal pigment epithelium in mouse retina and demonstrated more intense CEP immunoreactivity in photoreceptors from a human AMD donor compared with healthy human retina. The mean level of anti-CEP immunoreactivity in AMD human plasma (n = 19 donors) was 1.5-fold higher (p = 0.004) than in age-matched controls (n = 19 donors). Sera from AMD patients demonstrated mean titers of anti-CEP autoantibody 2.3-fold higher than controls (p = 0.02). Of individuals (n = 13) exhibiting both antigen and autoantibody levels above the mean for non-AMD controls, 92% had AMD. These results suggest that together CEP immunoreactivity and autoantibody titer may have diagnostic utility in predicting AMD susceptibility.

Crabb JW, Miyagi M, Gu X, Shadrach K, West KA, Sakaguchi H, Kamei M, Hasan A, Yan L, Rayborn ME, Salomon RG, Hollyfield JG. · Cole Eye Institute and Lerner Research Institute, Cleveland Clinic Foundation, and Department of Chemistry, Case Western Reserve University, OH 44195, USA. · Proc Natl Acad Sci U S A. · Pubmed #12391305 links to  free full text

Abstract: Drusen are extracellular deposits that accumulate below the retinal pigment epithelium on Bruch's membrane and are risk factors for developing age-related macular degeneration (AMD). The progression of AMD might be slowed or halted if the formation of drusen could be modulated. To work toward a molecular understanding of drusen formation, we have developed a method for isolating microgram quantities of drusen and Bruch's membrane for proteome analysis. Liquid chromatography tandem MS analyses of drusen preparations from 18 normal donors and five AMD donors identified 129 proteins. Immunocytochemical studies have thus far localized approximately 16% of these proteins in drusen. Tissue metalloproteinase inhibitor 3, clusterin, vitronectin, and serum albumin were the most common proteins observed in normal donor drusen whereas crystallin was detected more frequently in AMD donor drusen. Up to 65% of the proteins identified were found in drusen from both AMD and normal donors. However, oxidative protein modifications were also observed, including apparent crosslinked species of tissue metalloproteinase inhibitor 3 and vitronectin, and carboxyethyl pyrrole protein adducts. Carboxyethyl pyrrole adducts are uniquely generated from the oxidation of docosahexaenoate-containing lipids. By Western analysis they were found to be more abundant in AMD than in normal Bruch's membrane and were found associated with drusen proteins. Carboxymethyl lysine, another oxidative modification, was also detected in drusen. These data strongly support the hypothesis that oxidative injury contributes to the pathogenesis of AMD and suggest that oxidative protein modifications may have a critical role in drusen formation.

Marmorstein AD, Marmorstein LY, Sakaguchi H, Hollyfield JG. · Department of Ophthalmic Research, Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. · Invest Ophthalmol Vis Sci. · Pubmed #12091448 links to  free full text

Abstract: PURPOSE: To compare the autofluorescence spectra of retinal pigment epithelium (RPE)-associated lipofuscin, Bruch's membrane, and sub-RPE deposits (drusen and basal laminar-linear deposits) in eyes of donors with age-related macular degeneration (AMD) against eyes of age-matched control donors. METHODS: Cryosections were cut from the maculae of unfixed human donor eyes with AMD or from age-matched control eyes. Tissues were excited at wavelengths of 364, 488, 568, and 633 nm. Emission spectra were collected with a confocal microscope equipped with a spectrophotometric detector at 10-nm wavelength intervals between 400 and 800 nm. RESULTS: RPE lipofuscin had strong autofluorescent emissions that were excited at all wavelengths. Bruch's membrane exhibited strong autofluorescence with an emission peak of 485 +/- 5 nm when excited with 364-nm light. At 488-, 568-, and 633-nm excitations, Bruch's membrane and sub-RPE deposits in normal eyes exhibited minimal autofluorescence. In AMD eyes, however, both the 364- and 488-nm excitation wavelengths stimulated substantial blue-green emissions from sub-RPE deposits and Bruch's membrane, with average pixel intensities substantially exceeding that elicited in the yellow-orange range by RPE lipofuscin. CONCLUSIONS: These data suggest that an increase in blue-green autofluorescence of Bruch's membrane relative to the yellow-orange autofluorescence of RPE-associated lipofuscin is associated with AMD. Knowledge of these spectra will be useful in evaluating animal models of macular degenerative disease and in diagnosis of AMD, and will provide a novel signature for further analysis of the molecular entities emitting these fluorescent signatures.

Marmorstein AD, Marmorstein LY, Rayborn M, Wang X, Hollyfield JG, Petrukhin K. · Department of Ophthalmic Research, Cole Eye Institute, and Department of Cell Biology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland OH 44195, USA. · Proc Natl Acad Sci U S A. · Pubmed #11050159 links to  free full text

Abstract: Best vitelliform macular dystrophy is a dominantly inherited, early onset, macular degenerative disease that exhibits some histopathologic similarities to age-related macular degeneration. Although the vitelliform lesion is common in the fundus of individuals with Best disease, diagnosis is based on a reduced ratio of the light peak to dark trough in the electrooculogram. Recently, the VMD2 gene on chromosome 11q13, encoding the protein bestrophin, was identified. The function of bestrophin is unknown. To facilitate studies of bestrophin, we produced both rabbit polyclonal and mouse monoclonal antibodies that proved useful for Western blotting, immunoprecipitation, and immunocytochemistry. To characterize bestrophin, we initially probed the retinal pigment epithelium (RPE)-derived cell lines ARPE-19, D407, and RPE-J. All of the cell lines expressed bestrophin mRNA by reverse transcription-PCR, but not on Western blots. Bestrophin in human RPE partitioned in the detergent phase during Triton X-114 extraction and could be modified by biotin in intact cells, indicative of a plasma membrane localization. Immunocytochemical staining of macaque and porcine eyes indicated that bestrophin is localized at the basolateral plasma membrane of RPE cells. When expressed in RPE-J cells by adenovirus-mediated gene transfer, bestrophin again was determined by confocal microscopy and cell surface biotinylation to be a basolateral plasma membrane protein. The basolateral plasma membrane localization of bestrophin suggests the possibility that bestrophin plays a role in generating the altered electrooculogram of individuals with Best disease.

Kamei M, Hollyfield JG. · The Eye Institute, The Cleveland Clinic Foundation, Ohio 44195, USA. · Invest Ophthalmol Vis Sci. · Pubmed #10476804 links to  free full text

Abstract: PURPOSE: To assess the distribution, content, and function of tissue inhibitor of metalloproteinases (TIMP)-3 during aging in normal eyes for comparison with the levels observed in eyes with age-related macular degeneration (AMD). METHODS: Donor tissues analyzed included 36 normal eyes (14-96 years old) and 15 AMD eyes (74 -98 years old). A tissue strip including the fovea was used for immunohistochemistry. Western blot analysis was performed on extracts of the retinal pigment epithelium (RPE)- choroid complex from the posterior part of each eye. Immunoreactivity of TIMP-3 bands in each western blot was densitometrically quantitated. The inhibitory function of TIMP-3 was evaluated with reverse zymography. RESULTS: TIMP-3 was present uniformly across Bruch's membrane in the normal samples. In samples from donors more than 50 years of age, immunostaining was intense. TIMP-3 content ranged from 92 to 1061 ng/cm2 and increased with age (r = 0.66). In AMD eyes, TIMP-3 distribution in Bruch's membrane was abundant in areas of continuous soft drusen but absent in areas below RPE atrophy. TIMP-3 levels in AMD eyes were significantly higher than in age-matched normal eyes (577 versus 877 ng/cm2; P = 0.009). Inhibitory activity correlated well with TIMP-3 content (r = 0.82) and was also significantly higher in AMD eyes than in age-matched normal eyes (P < 0.001). CONCLUSIONS: During normal aging, TIMP-3 content in Bruch's membrane of the macula shows a significant increase. TIMP-3 content in AMD eyes was elevated relative to that of age-matched normal eyes. Higher levels of TIMP-3 may contribute to the thickening of Bruch's membrane observed in AMD.

Sakaguchi H, Miyagi M, Shadrach KG, Rayborn ME, Crabb JW, Hollyfield JG. · No affiliation provided · Exp Eye Res. · Pubmed #12076098 No free full text.

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