Advanced glycation endproduct-induced aging of the retinal pigment epithelium and choroid: A comprehensive transcriptional response

Jane Tian*, Kazuki Ishibashi*, Kazuko Ishibashi*, Karen Reiser†, Rhonda Grebe*, Shyam Biswal‡, Peter Gehlbach*, and James T. Handa*§

*Michael Panitch Macular Degeneration Laboratory, Wilmer Eye Institute, and ‡Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins Medical Institutions, Baltimore, MD 21287; and †Department of Neurosurgery, University of California, Davis, CA 95616

Communicated by Paul Talalay, Johns Hopkins University School of Medicine, Baltimore, MD, June 7, 2005 (received for review November 28, 2004) Advanced glycation endproduct (AGE) formation is a trigger for the and ultrastructural aging in the RPE–choroid of mice treated onset of age-related disease. To evaluate AGE-induced change in with low-dose D-galactose (D-gal) (12). Using this stimulus, we the ocular fundus, 5-mo-old C57BL͞6 mice were given low-dose seek to understand the AGE-induced molecular events that D-galactose (D-gal) for 8 wk and evaluated by AGE fluorescence, contribute to aging of the RPE–BM–choroid. After AGE in- electroretinography (ERG), electron microscopy, and microarray duction, we assessed the transcriptional response in the RPE– analysis for 20 wk. Although AGE fluorescence was increased in choroid and compared it to known responses by nonocular D-gal-treated retinal pigment epithelium (RPE)–choroid compared tissues. with controls at all time points, ERG showed no AGE-induced Our findings indicate that temporal transcriptional changes functional toxicity. Progressive ultrastructural aging in the RPE– after AGE stimulus in the RPE–BM–choroid have substantial choroid was associated temporally with a transcriptional response overlap with other general aging transcriptional changes. Nota- of early inflammation, matrix expansion, and aberrant lipid pro- bly, a subset of had expression changes in a pattern cessing and, later, down-regulation of energy metabolism genes, observed with , a prototypical age-related dis- up-regulation of crystallin genes, and altered expression of cell ease. Insights into AGE biology resulting from this study of the structure genes. The overall transcriptome is similar to the gener- RPE–BM–choroid microenvironment may apply broadly to alized aging response of unrelated cell types. A subset of tran- situations in which specific aging factors combined with other scriptional changes is similar to early atherosclerosis, a chronic known risk factors, exaggerate aging, and facilitate the transition inflammatory disease characterized by matrix expansion and lipid to clinically apparent, age-related disease such as atherosclerosis deposition. These changes suggest an important contribution of a or AMD. single environmental stimulus to the complex aging response. Methods transcriptome ͉ basal deposit ͉ Bruch’s membrane Mice. All experiments were conducted according to the Associ- ation for Research in Vision and Ophthalmology Statement for ging is a multifactorial process associated with physiological the Use of Animals in Ophthalmic and Vision Research, and the Adecline. Prior efforts to comprehensively identify and eval- research was approved by the institutional research board at uate contributory factors have benefited from global assessment Johns Hopkins Medical Institutions. C57BL͞6 mice purchased strategies such as microarray analysis. Although some general from the National Cancer Institute (Bethesda) were fed standard trends in the transcriptional responses to aging have been rodent chow and water ad libitum and kept in a 12-h light–dark identified in diverse tissues such as skeletal muscle, cardiac cycle. Mice (5 mo old; n ϭ 10͞group per time point) were given muscle, and brain, the aging kidney is without modification daily s.c. injections for 8 wk of either PBS or D-gal (50 mg͞kg, (1–4). Sigma) (12). Mice were killed at 4, 8, 12, and 20 wk. Normal vision requires a functional retinal pigment epithe- lium (RPE)–Bruch’s membrane (BM)–choroid to sustain the Electroretinography. With the investigator masked to the treat- neurosensory retina. Photooxidative stress, photoreceptor outer ment group, mice (n ϭ 8 per group) were dark-adapted for a segment shedding, lipid peroxidation, high metabolic require- standardized 12-h period, and a- and b-wave recordings were ments, and increased oxygen demand result in high levels of obtained from both eyes for 11 intensity levels (Ϫ3 to 1.40 log oxidative stress to the fundus. As a result, with aging, the RPE cd⅐s͞m2) by using an Espion ERG Diagnosys (Diagnosys, Little- and choriocapillaris endothelium dedifferentiate (5, 6). The ton, MA). The data were log-transformed for normality, and most significant changes are basal deposits that develop within a-wave and b-wave amplitudes were compared by ANOVA. BM, a pentalaminar matrix that includes the RPE and chorio- capillaris endothelial basement membrane (5, 6). The location Tissue Preparation. After mice were killed and eyes were enucle- and composition of the deposits correlates with the development ated, one eye was fixed in 2% paraformaldehyde and 2.5% of age-related disease (5, 6). The molecular events surrounding glutaraldehyde in 0.08 M cacodylate buffer for electron micros- basal deposit formation, however, are poorly characterized. copy. The RPE–choroid of the contralateral eye was dissected Advanced glycation endproducts (AGEs) are a heterogeneous group of reaction products that form between a ’s primary amino group and a carbohydrate-derived aldehyde Freely available online through the PNAS open access option. group. A substantial amount of literature indicates that AGEs Abbreviations: AGE, advanced glycation endproduct; RPE, retinal pigment epithelium; exacerbate and accelerate aging change and contribute to the D-gal, D-galactose; BM, Bruch’s membrane; AMD, age-related macular degeneration; ECM, extracellular matrix; ex, excitation; em, emission; LPL, ; FABP, fatty early phases of age-related disease, including atherosclerosis, acid-binding protein. cataract, neurodegenerative disease, renal failure, arthritis, and §To whom correspondence should be addressed at: Johns Hopkins Medical Institutions, age-related macular degeneration (AMD) (7, 8). We found that 3-109 Jefferson Street Building, 600 North Wolfe Street, Baltimore, MD 21287. E-mail: AGEs accumulate with age in human BM and immunolocalize [email protected]. to basal deposits (9–11). We recently identified AGE formation © 2005 by The National Academy of Sciences of the USA

11846–11851 ͉ PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504759102 Downloaded by guest on October 2, 2021 and cryopreserved at Ϫ80°C for AGE and lipid peroxide assess- ment or placed in 600 ␮l of RLT lysis buffer (Qiagen, Valencia, CA) for transcriptional analysis.

AGE Fluorescence and Lipid Peroxide Production. Fluorescence of RPE–choroid samples was measured at excitation (ex) ϭ 370 nm͞emission (em) ϭ 440 nm and cross-checked at ex ϭ 330 nm͞em ϭ 390 nm to eliminate lipofuscin-related fluorescence, as described in ref. 12. Lipid peroxide content of hydrolysates at 8 wk was determined by using the xylenol orange method (PeroXoquant Quantitative Peroxide Assay Kit, Pierce) using an ELISA plate reader (Bio-Tek, Burlington, VT) at 595 nm. Fluorescence and peroxide concentration were normalized to hydroxyproline content, as determined with the Woessner assay.

Electron Microscopy. The central 2 ϫ 2-mm tissue temporal to the optic nerve was postfixed with 1% osmium tetroxide and dehy- drated and embedded in Poly͞Bed 812 resin (Polysciences). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a JEM-100 CX electron microscope (JEOL).

Semiquantitative Grading System. The average BM thickness was determined from the thinnest and thickest measurements by a masked observer (13). The Wilcoxon rank-sum test was used to Fig. 1. AGEs increase with D-gal treatment. AGE fluorescence measured at compare the mean BM thickness in different groups. RPE and ex ϭ 370͞em ϭ 440 (Upper) and ex ϭ 330͞em ϭ 390 (Lower) in the RPE– choriocapillaris ultrastructural changes and BM basal deposits choroid was increased in D-gal-treated eyes (red) compared with controls (criteria including location, thickness, continuity, and content) (blue) at all time points. were graded for severity and frequency by a masked observer MEDICAL SCIENCES using a modified protocol established by Cousins et al. (12, 14). cyclophilin A mRNA expression. Experiments were repeated RNA Isolation. Tissue was homogenized with a QIAshredder spin once and are reported as the average differential expression column (Qiagen), and total RNA from the RPE–choroid was change. extracted by using the RNeasy Minikit (Qiagen). RNA quality Results was assessed with the Agilent Bioanalyzer (Agilent Technolo- gies, Palo Alto, CA). AGEs and Lipid Peroxidation Products Form in the RPE–Choroid Without Functional Toxicity. Compared with controls, AGE fluo- ϭ ͞ ϭ ϭ ͞ ϭ Transcriptional Profiling by Oligonucleotide Microarray. Total RNA rescence at ex 370 nm em 440 nm and ex 330 nm em (50 ng) was reverse-transcribed with SuperScript II (0.5 ␮l, 390 nm was increased in D-gal-treated RPE–choroid (Fig. 1). Invitrogen). Two cycles of T7 amplification were performed by D-gal treatment for 8 wk induced lipid peroxidation of the using the Small Sample Labeling Assay, Version II (Affymetrix), RPE–choroid (249.1 Ϯ 68.8 ␮mol͞␮g) compared with controls and cRNA probe was biotinylated with the Affymetrix ENZO (115.1 Ϯ 30.1 ␮mol͞␮g; P ϭ 0.039). Early AMD does not induce BioArray HighYield RNA Transcript Labeling Kit. Fragmented full-field electroretinography (ERG) changes (15). To assure cRNA (10 ␮g) was hybridized onto a mouse MOE430A array that D-gal did not cause retinal toxicity, full-field ERGs showed containing 14,484 full-length genes (Affymetrix), and each array no a- or b-wave amplitude or a- or b-wave implicit time abnor- was scanned with the GeneArray scanner (Agilent Technolo- malities at all time points (data not shown). gies). Scanned output files were analyzed with Affymetrix MICROARRAY SUITE 5.0 and normalized to an average intensity of D-gal Induces Ultrastructural Aging to the RPE–Choroid. The mean 500 independently before comparison. To identify differentially BM of D-gal-treated eyes increased in thickness over the study expressed transcripts, pairwise comparison analyses were carried (0.24 Ϯ 0.11 to 0.50 Ϯ 0.036 ␮m) and was thicker than controls out with DATA MINING TOOL 3.0 (Affymetrix). Nine pairwise at all time points (P Յ 0.18; see the supporting information). comparisons for each (experimental, n ϭ 3; control, n ϭ 3) were performed. Transcripts with altered expression in at least seven of nine comparisons with Ն1.5-fold change were desig- nated as differentially expressed and are reported as the average fold change in expression.

Real-Time RT-PCR. Total RNA (100 ng) was reverse-transcribed with Sensiscript (1 ␮l, Qiagen). Primer sequences were designed by using PRIMER 3 (Whitehead Institute, Cambridge, MA), and sequences were verified by using National Center for Biotech- nology Information UniGene cluster IDs (see the supporting information, which is published on the PNAS web site). Ther- mocycling (LightCycler, Roche Diagnostics) was performed in a ␮ ␮ 20- l volume containing SYBR Green PCR Master Mix (10 l, Fig. 2. Early ultrastructural changes after D-gal treatment. At 4 wk, controls ␮ ␮ Qiagen), Primer A and B (0.5 M each), and 2 l of cDNA. PCR (Left) had normal RPE, BM, and choriocapillaris (cc), whereas D-gal-treated products were quantified by using LIGHTCYCLER analysis soft- RPE–choroid (Right) had fewer and dilated basolateral infoldings and outer ware, checked by melting point analysis, and normalized to collagenous deposits (*). (Scale bar, 1 ␮m.)

Tian et al. PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 ͉ 11847 Downloaded by guest on October 2, 2021 Fig. 3. Ultrastructural aging after D-gal treatment. At 8 wk, control RPE– choroid appears unchanged in controls (Left), whereas D-gal-treated eyes (Right) had exaggerated dilation and loss of basolateral infoldings and basal laminar deposits (arrows) that contain long-spacing collagen (*). Choriocap- illaris (CC) endothelium is without fenestrations. (Inset) A magnified image of long-spacing collagen. (Scale bar, 1 ␮m.)

Control eyes at 4 wk showed a rare outer collagenous layer deposit, whereas D-gal-treated eyes had RPE with dilated and fewer basolateral infoldings and small outer collagenous layer Fig. 4. Continued ultrastructural aging after D-gal treatment. At 20 wk, control RPE–choroid appears unaffected (Upper Left); D-gal-treated RPE– deposits (Fig. 2). By 8 wk, D-gal RPE had severe dilation and loss choroid (Upper Right) shows basal laminar deposit (arrows) and prominent of basolateral infoldings, basal laminar deposits with long- outer collagenous layer deposit (*s). (Lower Left) A prominent basal laminar spacing collagen, and prominent outer collagenous layer depos- deposit (arrows) adjacent to an intercapillary region (*). (Upper Right) Lipid- its (Fig. 3). By 12 and 20 wk, control RPE had mildly dilated like membranous vacuoles within the RPE. (Scale bar, 1 ␮m.) basolateral infoldings and outer collagenous layer deposits, but D-gal-treated eyes showed more severe changes than controls (Fig. 4). lial changes were more frequent in D-gal-treated eyes than in The severity and frequency of ultrastructural aging was semi- control eyes (see the supporting information). quantified by using a modified, established grading scale (12, 14). Although aging severity increased with time in controls, more Differential of the RPE–Choroid by D-gal Treatment. severe changes were seen in D-gal-treated eyes at each time At 4 wk, 39 genes were up-regulated and 42 genes were down- point. The aging severity in D-gal-treated eyes peaked at 8 wk and regulated by D-gal-treated RPE–choroid. Clusters of nine genes was similar thereafter. The frequency of ultrastructural aging related to inflammation, eight genes related to matrix regulation, was identical between groups at 4 wk. From 8 to 20 wk, and six genes related to cell structure were differentially ex- ultrastructural aging was more frequent in D-gal-treated eyes pressed (Table 1). At 8 wk, 22 genes were up-regulated and 30 than in control eyes. In particular, severe basal laminar deposits, genes were down-regulated by D-gal. A cluster of 10 genes outer collagenous layer deposits, and choriocapillaris endothe- related to cell structure, 9 genes related to lipid metabolism͞

Table 1. Genes differentially expressed by the RPE–choroid after 4 wk of D-gal treatment Fold change, Gene UniGene cluster ID D-gal͞control Biological function

Myh3 Mm.2780 3.2 Cell structure Tpm3 Mm.17306 2.3 Cell structure Eva Mm.33240 2.0 Cell structure Myh2 Mm.34425 1.5 Cell structure Cldn5 Mm.22768 Ϫ2.9 Cell structure Mbp Mm.2992 Ϫ4.3 Cell structure PAI1 Mm.1263 1.9 ECM; increased in atherosclerosis Fmod Mm.41573 1.8 ECM; increased in atherosclerosis Fbln1 Mm.219663 1.7 ECM TGF-␤2 Mm.18213 1.8 ECM; matrix regulation Fbln3 EFEMP1 Mm.44176 1.5 ECM; Malattia Leventinese gene Cspg2 Mm.4575 1.5 ECM; increased in atherosclerosis Cst3 Mm.4263 Ϫ2.7 ECM; involved in atherosclerosis Pdgfr-␤ Mm.4146 Ϫ3.4 ECM; matrix regulation; inflammation; atherosclerosis Ifi202b Mm.89990 7.8 Inflammation; increased in atherosclerosis and apoptosis Il-6 Mm.1019 5.7 Inflammation; aging; increased in atherosclerosis Ptx3 Mm.4663 4.7 Inflammation; increased in atherosclerosis CXCL2 Mm.4979 4.1 Inflammation; increased in atherosclerosis CXCL1 Mm.21013 3.5 Inflammation; increased in atherosclerosis Lcn2 Mm.9537 2.7 Inflammation; increased in atherosclerosis Tnfrsf21 Mm.200792 1.6 Inflammation; apoptosis Oasl2 Mm.27162 Ϫ2.6 Inflammation; cell growth differentiation, and apoptosis CEBPD Mm.4639 1.6 Inflammation transcription factor; induces inflammation genes

11848 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504759102 Tian et al. Downloaded by guest on October 2, 2021 Table 2. Genes differentially expressed by the RPE–choroid after 8 wk of D-gal treatment Fold change, Gene UniGene cluster ID D-gal͞control Biological function

Myh3 Mm.2780 3.7 Cell structure Pvrl3 Mm.46724 2.2 Cell structure Cntn1 Mm.4911 1.7 Cell structure Tnnt1 Mm.711 1.5 Cell structure Tmsb10 Mm.3532 Ϫ1.9 Cell structure Bgpc Mm.14114 Ϫ2 Cell structure; immune response Ceacam1 Mm.14114 Ϫ2.5 Cell structure; immune response Sprr1a Mm.625 Ϫ3.5 Cell structure Mbp Mm.2992 Ϫ6.4 Cell structure Krt1–14 Mm.6974 Ϫ11.4 Cell structure Col18a1 Mm.196000 1.8 ECM Col4a5 Mm.155579 1.5 ECM Fbn1 Mm.735 1.7 ECM; Marfan’s syndrome Fmod Mm.41573 1.5 ECM Dpt Mm.28935 1.5 ECM ECM1 Mm.3433 Ϫ1.7 ECM Prpmp5 Mm.4491 Ϫ2.2 ECM Mmp3 Mm.4993 Ϫ2.9 ECM Prg4 Mm.212696 Ϫ3 ECM; cell proliferation Acrp30 Mm.3969 5.4 Inflammation DF Mm.4407 4.4 Inflammation͞lipid metabolism Fabp4 Mm.582 3.5 Lipid metabolism͞processing Scd1 Mm.140785 1.7 Lipid metabolism͞processing Lpl Mm.1514 1.5 Lipid metabolism͞processing Apod Mm.2082 Ϫ1.9 Lipid metabolism͞processing MEDICAL SCIENCES Cyp2f2 Mm.4515 Ϫ2 Lipid metabolism͞processing Angptl4 Mm.196189 Ϫ2.1 Lipid metabolism͞processing Fabp5 Mm.741 Ϫ2.7 Lipid metabolism͞processing S100a8 Mm.21567 Ϫ3 Lipid metabolism͞processing; FA–p34 complex S100a9 Mm.2128 Ϫ4 Lipid metabolism͞processing; FA–p34 complex

transport, and 9 genes related to matrix regulation were iden- similar to the chronological aging response by diverse cell types tified (Table 2). Distinct gene expression changes continued and include clusters of susceptibility for age-related disease. This after D-gal treatment. At 12 wk, 27 genes were up-regulated in transcriptional response underscores the potential relevance of D-gal treated eyes, of which 17 genes have function related to cell studying a single environmental factor in the context of aging. structure. At 20 wk, 18 genes were up-regulated and 29 genes Our results show a global transcriptional response after AGE were down-regulated by D-gal treatment. The expression of cell stimulus by the RPE–choroid that has significant similarity to the structure genes (n ϭ 18) was altered; six energy metabolism generalized aging response of brain, skeletal, and cardiac muscle, genes were down-regulated, and five crystallins were up- including increased inflammation, reduced metabolism and bio- regulated by D-gal (Table 3). Tables of all of the differentially synthesis, and increased stress response (1–3). Because these expressed genes for each time point appear in the supporting tissues are partially composed of postmitotic cells like the RPE, information. To validate the array expression, 19 genes were these overlapping transcriptional features may be common to evaluated by real-time RT-PCR, and all were found to be in cells that have limited regenerative capacity after insult. The agreement with the arrays (Table 4). similarity in response of the RPE–choroid, brain, and muscle suggests that AGEs contribute to aging more than previously Discussion recognized. Genetic and environmental factors influence physiological deg- Within each aging profile are unique changes related to the radation associated with aging. The transcriptional response of tissue demands of each microenvironment. For example, the the RPE–choroid in vivo to environmental factors is poorly aging cardiac myocyte shifts from a predominantly lipid to understood. Because we previously demonstrated an age- carbohydrate-dependent metabolism, which is the opposite of dependent increase of AGEs in human BM and because gly- aging skeletal muscle (1, 2). Likewise, in the RPE–choroid, a coxidation modifications have been identified in the RPE with cluster of energy metabolism genes were down-regulated and aging and AMD (10, 16, 17), we evaluated global gene expression four stress response crystalline genes were up-regulated by D-gal. changes after this single environmental stimulus on aging with The crystallins are increased as a general stress response in many the goal of identifying environmentally induced transcriptional cell types, but in the RPE–choroid, they may have particular susceptibility for aging and age-related disease. With progressive importance because they prevent oxidative stress-induced apo- ultrastructural changes after an AGE stimulus to the RPE–BM– ptosis (18, 19). The four crystallins (␤-A3, ␤-A4, ␤-B2, and ␤-S) choroid that mimic human aging, we associate these sequential up-regulated by D-gal have been identified as the most common transcriptional changes consisting of inflammation, matrix ex- identified in drusen from AMD samples (20). The pansion, and altered lipid metabolism͞processing and subse- crystallins are ideal defense molecules in an energy-deficient cell quent modifications to cell structure, reduced energy metabo- because they don’t require ATP while preventing stress-induced lism, and increased stress response. These alterations are both protein aggregation (21). We interpret this expression pattern as

Tian et al. PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 ͉ 11849 Downloaded by guest on October 2, 2021 Table 3. Genes differentially expressed by the RPE–choroid 20 Table 4. Differential expression of selected genes by wk after D-gal treatment real-time RT-PCR UniGene Fold change, Gene Time, wk Change array Change RT-PCR Gene cluster ID D-gal͞control Biological function Il6 4 5.7 7.2 Krt2–6b Mm.196352 6.4 Cell structure Ptx3 4 4.7 3.7 Eva Mm.33240 2 Cell structure CXCL1 4 3.5 2.4 Scel Mm.852 1.6 Cell structure Cldn5 4 Ϫ2.9 Ϫ3.7 Actn3 Mm.5316 Ϫ2.8 Cell structure Fbn3 4 1.5 4.3 Actc1 Mm.686 Ϫ2.9 Cell structure Acrp30 8 5.4 3.5 Mylpf Mm.14526 Ϫ3 Cell structure DF 8 5.3 9.1 Tncs Mm.1716 Ϫ3.2 Cell structure Angptl4 8 Ϫ2.1 Ϫ4.2 Tnni2 Mm.39469 Ϫ3.2 Cell structure FABP4 8 3.5 4.0 Acta1 Mm.214950 Ϫ3.2 Cell structure FABP5 8 Ϫ2.7 Ϫ2.0 Mylf Mm.1000 Ϫ3.3 Cell structure LPL 8 1.5 1.5 Smpx Mm.140340 Ϫ3.5 Cell structure MMP3 8 Ϫ2.9 Ϫ3.2 Ttid Mm.143804 Ϫ3.6 Cell structure S100A8 8 Ϫ3.0 Ϫ2.6 Myhc-IIB Mm.35531 Ϫ3.7 Cell structure S100A9 8 Ϫ4.0 Ϫ2.6 Myh2 Mm.34425 Ϫ4.1 Cell structure SCD1 8 1.7 1.9 Tpm2 Mm.121878 Ϫ4.5 Cell structure Acta1 12 2.6 4.3 Trdn Mm.55320 Ϫ4.6 Cell structure Crbaa 20 3.7 6.7 Ldb3 Mm.29733 Ϫ5.2 Cell structure Cryba3 20 4.1 6.7 Myh3 Mm.2780 Ϫ7.1 Cell structure Crbb2 20 3.3 3.9 Pdha1 Mm.34775 Ϫ2.4 Energy metabolism Fold change is expressed as D-gal͞control. Ckmm Mm.2375 Ϫ3.1 Energy metabolism Eno3 Mm.29994 Ϫ3.3 Energy metabolism Ϫ Adss1 Mm.3440 3.5 Energy metabolism brane component expansion (38–40). Further evidence of over- Ckmt2 Mm.20240 Ϫ4.8 Energy metabolism lap between D-gal-induced transcriptional changes in the RPE– Ϫ Pgam2 Mm.219627 14.5 Energy metabolism choroid and atherosclerosis is the altered expression of matrix Crygs Mm.6253 4.3 Crystallin; stress response genes. D-gal and atherosclerosis are both characterized by up- Cryba1 Mm.22830 4.1 Crystallin; stress response regulation of Cspg2, Fmod, plasminogen activator inhibitor-1, and Cryaa Mm.1228 3.7 Crystallin; stress response Fbln-1 and -3 and down-regulation of Cst-3 and matrix metal- Cryba4 Mm.40324 3.4 Crystallin; stress response loproteinase type 3 (41–44). Fbln-3 (EFEMP1) associates with Crybb2 Mm.1215 3.3 Crystallin; stress response drusen in AMD (45), and Fbln-3 mutations are linked to Malattia Leventinese, a macular dystrophy with a phenotype that is remarkably similar to AMD (46). Interestingly, an allelic a protective response to D-gal protein modifications in an variant of Cst-3 has also been associated with AMD (47). D-gal energy-deficient cell. also altered a cluster of matrix genes that differed from those in A subset of the transcriptional response by the RPE–choroid atherosclerosis, including the up-regulation of Col4a5, Col18a1, is reminiscent of the ‘‘response-to-retention hypothesis’’ of Fmod, Fbn-1, and Dpt, which highlights the complexity of matrix atherosclerosis, where lipoprotein cholesterol retention and expansion. oxidative modification in the vascular intima initiate both Cholesterol deposition in the inner collagenous layer is an chronic inflammation and matrix expansion (22). All of the important antecedent event of basal linear deposits, the most inflammatory genes that were up-regulated by D-gal in the specific basal deposit for AMD (48). Recent evidence supports RPE–choroid are up-regulated in atherosclerosis, potentially local lipoprotein secretion by the RPE into BM (49, 50). linking AGEs with inflammation, atherosclerosis, and BM aging D-gal-related transcriptional changes involving intracellular lipid associated with AMD. Mechanistically, it is possible that this transport (i.e., fatty acid-binding protein (FABP) 4 and 5 and the inflammatory cascade is induced by the up-regulation of FA–p34 complex), cholesterol esterification [stearoyl CoA de- CEBPD, which activates inflammation in atherosclerosis (23). saturase-1 (SCD-1)], and extracellular lipid processing [lipopro- IL-6 up-regulation induces oxidative stress, promotes dyslipide- tein lipase (LPL) and Angptl4) all play a critical role in athero- mia, and increases C-reactive protein (CRP), an established sclerosis and could promote lipoprotein secretion by the RPE cardiovascular disease marker (24, 25) that has recently been with subsequent retention in BM. For example, the cytoplasmic associated with AMD (26). The up-regulation of the long FABPs facilitate fatty acid uptake and intracellular fatty acid pentraxin PTX-3, which shares homology to CRP, activates transport, which would increase fatty acid accumulation and complement in atherosclerotic plaques (27). Complement accu- promote their delivery for lipoprotein assembly (51, 52). In mulates both in BM with AMD (28, 29). A role for complement monocytes, the accumulation of cholesterol esters is a critical in our model is suggested by up-regulation of DF (complement event in atherosclerosis that is regulated by FABP4, in part factor D), which also accumulates in atherosclerotic plaques through up-regulation of IL-6 (53, 54), whereas in adipocytes, (28). The up-regulation of the inflammatory genes Ifi202b, total FABP4 and FABP5 content controls the degree of fatty acid TNFRSF21, and Lcn2 all induce apoptosis in atherosclerosis and accumulation (53, 55). We also identified altered expression of could promote RPE apoptosis, a recognized mechanism of RPE the FA–p34 complex of S100A8 and S100A9, another intracel- cell loss during aging and AMD (30–37). We interpret this lular fatty acid transporter (56) that regulates cell structure, transcriptional response as a local inflammatory response that is proliferation, differentiation, and inflammation (57). SCD-1 is triggered, in part, by AGE formation. the rate-limiting microsomal enzyme that catalyzes the synthesis Analogous to the arterial wall in atherosclerosis, aging BM of monounsaturated fatty acids into phospholipids, , accumulates heterogeneous debris known as basal deposits. and cholesterol esters, thereby preventing the toxic accumula- Early deposit formation is the result of normal basement mem- tion of free cholesterol (58) and stimulating lipoprotein assembly

11850 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504759102 Tian et al. Downloaded by guest on October 2, 2021 and secretion (59). LPL promotes lipid entry into cells, which gests that AGEs trigger a cascade of events that accelerate the would be enhanced by the down-regulation of Angptl4 through its aging response, and identifies clusters of factors that predispose inhibition on LPL activity (60). LPL also retains lipoproteins in to vulnerability to early disease, such as AMD. An advantage of the vascular wall by binding lipoproteins with Cspg2 (see above this model is that it can be superimposed in other aging-related discussion), which enhances lipid entry into cells (61, 62). models. Given the complex multifactorial nature of aging, this Transgenic mice overexpressing LPL have increased lipid accu- combinatorial approach merits further investigation. Finally, the mulation in cardiomyocytes and a dilated cardiomyopathy (63). results of this study may have application to other complex The proatherogenic LPL activity could promote either lipid age-related diseases as specific environmental and genetic in- entry into the RPE or lipoprotein retention in BM. Due to the fluences are elucidated. For example, although genetic predis- high oxidative stress microenvironment of the fundus, oxidation position influences age-related diseases like AMD and athero- of accumulated lipids and subsequent RPE injury is a mechanism sclerosis, similar gene expression responses induced by a single worth exploring. environmental stimulus such as AGEs underscore the impor- The ultrastructural and transcriptional changes identified in tance of understanding generalizable aging pathways for age- this model simulate aging. Important age-related gene expres- related disease. sion changes may not have been recognized because of the small sample size or the bias introduced with the array strategy. This work was supported by National Institutes of Health Grants EY Further work characterizing the transcriptional response of the 14055 (to J.T.H.), EY134020-04 (to P.G.), and JDRFI 10-2001-412 (to specific cell types involved is scientifically indicated, although it P.G.); the Alexander and Margaret Stewart Trust (P.G.); the Michael remains technically difficult to reliably separate RPE from Panitch Macular Degeneration Research Fund (J.T.H.); gifts from choroidal cells, particularly in mice. Aging is associated with Aleda Wright (to J.T.H.) and Rick and Sandy Forsythe (to J.T.H.); and posttranscriptional modifications that are missed in a purely an unrestricted award from Research to Prevent Blindness (RPB) to the transcriptional analysis. This investigation, however, identifies Wilmer Eye Institute. J.T.H. is the recipient of a Lew R. Wasserman multiple changes induced by AGEs in the RPE–choroid, sug- Merit Award from RPB.

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