Advanced Glycation Endproduct Changes to Bruch's Membrane Promotes Lipoprotein Retention by Lipoprotein Lipase
2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.04.010
ISSN1525-2191
AutoresMarisol Cano, Natalia Fijalkowski, Naoshi Kondo, Sonny Dike, James T. Handa,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoLipoprotein particles accumulate in Bruch's membrane before the development of basal deposits and drusen, two histopathologic lesions that define age-related macular degeneration (AMD). We therefore, sought to determine which molecules could participate in lipoprotein retention. Wild-type or lipoprotein lipase–deficient mice were injected with low-dose d-galactose or PBS subcutaneously for 8 weeks to induce advanced glycation endproduct (AGE) formation. Some mice were also injected with the AGE breaker phenacylphiazolium bromide and d-galactose. Rhodamine-labeled low-density lipoproteins were injected into mice, and the fluorescence was measured up to 72 hours later. AGEs, proteoglycans, and other lipid-retaining molecules were evaluated by IHC. Lipoprotein lipase distribution was assessed in AMD samples by IHC. d-galactose–treated mice retained lipoproteins in the retinal pigment epithelial and Bruch's membrane to a greater extent than either PBS- or phenacylphiazolium bromide/d-galactose–treated mice at 24 and 72 hours after injection (P ≤ 0.04). Immunolabeling for carboxymethyllysine, biglycan, and lipoprotein lipase was found in d-galactose–treated mice only. Mice deficient for lipoprotein lipase treated with d-galactose did not retain lipoproteins to any measureable extent. Human AMD samples had lipoprotein lipase labeling within drusen, basal deposits, and the choroid. Mice treated with d-galactose to induce AGE formation in Bruch's membrane retain intravenously injected lipoproteins. Our results suggest that lipoprotein retention in Bruch's membrane is mediated by lipoprotein lipase. Lipoprotein particles accumulate in Bruch's membrane before the development of basal deposits and drusen, two histopathologic lesions that define age-related macular degeneration (AMD). We therefore, sought to determine which molecules could participate in lipoprotein retention. Wild-type or lipoprotein lipase–deficient mice were injected with low-dose d-galactose or PBS subcutaneously for 8 weeks to induce advanced glycation endproduct (AGE) formation. Some mice were also injected with the AGE breaker phenacylphiazolium bromide and d-galactose. Rhodamine-labeled low-density lipoproteins were injected into mice, and the fluorescence was measured up to 72 hours later. AGEs, proteoglycans, and other lipid-retaining molecules were evaluated by IHC. Lipoprotein lipase distribution was assessed in AMD samples by IHC. d-galactose–treated mice retained lipoproteins in the retinal pigment epithelial and Bruch's membrane to a greater extent than either PBS- or phenacylphiazolium bromide/d-galactose–treated mice at 24 and 72 hours after injection (P ≤ 0.04). Immunolabeling for carboxymethyllysine, biglycan, and lipoprotein lipase was found in d-galactose–treated mice only. Mice deficient for lipoprotein lipase treated with d-galactose did not retain lipoproteins to any measureable extent. Human AMD samples had lipoprotein lipase labeling within drusen, basal deposits, and the choroid. Mice treated with d-galactose to induce AGE formation in Bruch's membrane retain intravenously injected lipoproteins. Our results suggest that lipoprotein retention in Bruch's membrane is mediated by lipoprotein lipase. Age-related macular degeneration (AMD) is the most common cause of vision loss among the elderly in the United States. 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Complement factor H polymorphism and age-related macular degeneration.Science. 2005; 308: 421-424Crossref PubMed Scopus (2078) Google Scholar The influence of these genetic alterations on disease development remains unresolved at present. Bruch's membrane is a pentilaminar structure that is composed of the retinal pigment epithelial (RPE) basement membrane, inner collagenous layer, middle elastic layer, outer collagenous layer, and the choriocapillaris basement membrane. During aging, Bruch's membrane steadily accumulates neutral lipids contained within apolipoprotein B100 (apoB100) lipoprotein particles. These particles accumulate as a “wall” in the inner collagenous layer.12Ruberti J.W. Curcio C.A. Millican C.L. Menco B.P. Huang J.D. Johnson M. Quick-freeze/deep-etch visualization of age-related lipid accumulation in Bruch's membrane.Invest Ophthalmol Vis Sci. 2003; 44: 1753-1759Crossref PubMed Scopus (106) Google Scholar Lipoprotein particles also accumulate in basal laminar deposits, basal linear deposits, and drusen, the pathognomonic histopathologic Bruch's membrane lesions of AMD.12Ruberti J.W. Curcio C.A. Millican C.L. Menco B.P. Huang J.D. Johnson M. Quick-freeze/deep-etch visualization of age-related lipid accumulation in Bruch's membrane.Invest Ophthalmol Vis Sci. 2003; 44: 1753-1759Crossref PubMed Scopus (106) Google Scholar, 13Green W.R. McDonnell P.J. Yeo J.H. Pathologic features of senile macular degeneration.Ophthalmology. 1985; 92: 615-627Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 14Curcio C.A. Millican C.L. Bailey T. Kruth H.S. Accumulation of cholesterol with age in human Bruch's membrane.Invest Ophthalmol Vis Sci. 2001; 42: 265-274PubMed Google Scholar, 15Malek G. Li C.M. Guidry C. 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Esterified and unesterified cholesterol in drusen and basal deposits of eyes with age-related maculopathy.Exp Eye Res. 2005; 81: 731-741Crossref PubMed Scopus (192) Google Scholar The identical laminar location of lipoprotein deposition within Bruch's membrane before the development of these hallmark lesions suggests that lipoproteins, in addition to the genetic and environmental influences, play a critical role in the pathophysiology of AMD. The relative importance of lipoproteins in AMD pathogenesis is further suggested by studies that have identified polymorphisms in lipid-processing genes, including hepatic lipase and high-density lipoprotein c-associated alleles near lipoprotein lipase (LPL).19Chen W. Stambolian D. Edwards A.O. Branham K.E. Othman M. 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Gordiyenko N.V. Fariss R.N. Lee J.W. Fliesler S.J. Rodriguez I.R. Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.Mol Vis. 2006; 12: 1306-1318PubMed Google Scholar showed that LDLs are the preferred carrier of lipids into the retina and that they are internalized principally by the RPE through LDL receptors. In atherosclerosis, lipoproteins in excess and their subsequent retention within the vessel wall initiates a chronic, pathologic inflammatory response from the oxidation of retained lipoproteins.22Williams K.J. Tabas I. The response-to-retention hypothesis of early atherogenesis.Arterioscler Thromb Vasc Biol. 1995; 15: 551-561Crossref PubMed Google Scholar, 23Patel S. Celermajer D.S. Bao S. Atherosclerosis-underlying inflammatory mechanisms and clinical implications.Int J Biochem Cell Biol. 2008; 40: 576-580Crossref PubMed Scopus (47) Google Scholar Our laboratory has shown that oxidized lipoproteins accumulate in Bruch's membrane and basal deposits and drusen of patients with early AMD and that oxidized LDLs induce a pathologic phenotype of cultured RPE cells.24Yamada Y. Tian J. Yang Y. Cutler R.G. Wu T. Telljohann R.S. Mattson M.P. Handa J.T. Oxidized low density lipoproteins induce a pathologic response by retinal pigmented epithelial cells.J Neurochem. 2008; 105: 1187-1197Crossref PubMed Scopus (81) Google Scholar This work suggests that lipoproteins that accumulate within Bruch's membrane are retained for a sufficient period to become oxidized and can induce a phenotypic change to the RPE. Several changes to the subendothelial matrix promote lipoprotein retention during the development of atherosclerosis. These include an accumulation of certain matrix proteoglycans such as biglycan, decoran, and versican that retain lipoproteins due to their electronegative charge.22Williams K.J. Tabas I. The response-to-retention hypothesis of early atherogenesis.Arterioscler Thromb Vasc Biol. 1995; 15: 551-561Crossref PubMed Google Scholar, 25Nakashima Y. Wight T.N. Sueishi K. Early atherosclerosis in humans: role of diffuse intimal thickening and extracellular matrix proteoglycans.Cardiovasc Res. 2008; 79: 14-23Crossref PubMed Scopus (205) Google Scholar Secreted sphingomyelinases and LPL, which are distinct from hepatic lipase, endothelial lipase, and pancreatic lipase (PL), in the subendothelial matrix promote lipoprotein retention.26Olin K.L. Potter-Perigo S. Barrett P.H. Wight T.N. Chait A. 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Molecular interactions leading to lipoprotein retention and the initiation of atherosclerosis.Arterioscler Thromb Vasc Biol. 2004; 24: 2211-2218Crossref PubMed Scopus (80) Google Scholar, 30Pillarisetti S. Paka L. Obunike J.C. Berglund L. Goldberg I.J. Subendothelial retention of lipoprotein (a) Evidence that reduced heparan sulfate promotes lipoprotein binding to subendothelial matrix.J Clin Invest. 1997; 100: 867-874Crossref PubMed Scopus (98) Google Scholar Because of the cross-linking of AGEs, the supramolecular matrix architecture is modified so that the normal diffusion of metabolites or molecules such as lipoproteins might be impaired. We previously demonstrated that AGEs develop within Bruch's membrane, including basal deposits and drusen during aging and AMD.31Farboud B. Aotaki-Keen A. Miyata T. Hjelmeland L.M. Handa J.T. Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in Bruch's membrane of the aging eye.Mol Vis. 1999; 5: 11PubMed Google Scholar In mice, we have also shown that low-dose d-galactose (D-gal) treatment promotes AGE formation in Bruch's membrane and induces an accelerated aging phenotype with features of AMD in Bruch's membrane.32Ida H. Ishibashi K. Reiser K. Hjelmeland L.M. Handa J.T. Ultrastructural aging of the RPE-Bruch's membrane-choriocapillaris complex in the D-galactose-treated mouse.Invest Ophthalmol Vis Sci. 2004; 45: 2348-2354Crossref PubMed Scopus (44) Google Scholar, 33Tian J. Ishibashi K. Ishibashi K. Reiser K. Grebe R. Biswal S. Gehlbach P. Handa J.T. Advanced glycation endproduct-induced aging of the retinal pigment epithelium and choroid: a comprehensive transcriptional response.Proc Natl Acad Sci U S A. 2005; 102: 11846-11851Crossref PubMed Scopus (106) Google Scholar Given the accumulation of lipoproteins in Bruch's membrane, and their oxidation, we hypothesized that aging changes to Bruch's membrane encourages lipoprotein retention, which then leads to their oxidation. Oxidized lipoproteins are known to stimulate complement.34Miller Y.I. Chang M.K. Binder C.J. Shaw P.X. Witztum J.L. Oxidized low density lipoprotein and innate immune receptors.Curr Opin Lipidol. 2003; 14: 437-445Crossref PubMed Scopus (172) Google Scholar Accumulation of oxidized lipoproteins, therefore, could induce complement-mediated inflammation, leading to either RPE cell injury and/or drusen formation within Bruch's membrane. We, therefore, treated mice with low-dose D-gal to induce AGE formation as an experimental model of Bruch's membrane aging to determine whether lipoproteins are retained. An equal number of male and female C57Bl6 (The Jackson Laboratories, Bar Harbor, ME), B6.129 (The Jackson Laboratories) or LPL-deficient mice (B6.129S4-Lpltm1Ijg/J; The Jackson Laboratories) were fed standard rodent chow and water ad libitum and kept in a 12-hour light-dark cycle. All experiments were conducted according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the research was approved by the institutional research board at Johns Hopkins Medical Institutions. Five-month-old mice were divided into three groups. Group 1 mice were injected subcutaneously with D-gal (50 mg/kg; Sigma-Aldrich, St Louis, MO) daily for 8 weeks, following our previously published protocol.33Tian J. Ishibashi K. Ishibashi K. Reiser K. Grebe R. Biswal S. Gehlbach P. Handa J.T. Advanced glycation endproduct-induced aging of the retinal pigment epithelium and choroid: a comprehensive transcriptional response.Proc Natl Acad Sci U S A. 2005; 102: 11846-11851Crossref PubMed Scopus (106) Google Scholar Group 2 mice were injected with the AGE breaker phenacylphiazolium bromide (PTB; 10 mg/kg; Prime Organics, Woburn, MA) and D-gal (50 mg/kg) for 8 weeks, and group 3 mice were injected subcutaneously with PBS for 8 weeks. Human LDL or oxidized LDL was purchased from (Calbiochem, Gibbstown, NJ; Intracel, Frederick, MD, respectively). According to the manufacturer, the LDL preparations were purified by gradient-based ultracentrifugation. LDLs were verified by isoelectric focusing with the use of immunoelectrophoresis to obtain a single arc. LDL content was evaluated by staining for lipids by Fast Red 70 and proteins by Coomassie Blue staining. To avoid aggregation, LDL and oxidized LDL preparations were used within 1 week of purchase. LDLs were labeled with rhodamine with the use of the Fluororeporter Rhodamine Red Protein Labeling Kit (Invitrogen, Inc., Carlsbad, CA) according to the manufacturer's instructions. After the 8-week D-gal treatment, mice were injected intravenously via the tail vein with 500 μg of rhodamine-labeled LDL or oxidized LDL and sacrificed at 5 minutes, 24 hours, and 72 hours later to evaluate LDL retention in the fundus. Mouse eyes were enucleated at the appropriate time points and lightly fixed in 2% paraformaldehyde (Sigma-Aldrich), cryopreserved, and sectioned for histochemical evaluation. Human globes from 49- to 93-year-old donors were obtained from the National Disease Research Interchange (Philadelphia, PA). Eyes had been enucleated within 6 hours of death, were on life support for 8 μm), as defined by Sarks35Sarks S.H. Ageing and degeneration in the macular region: a clinico-pathological study.Br J Ophthalmol. 1976; 60: 324-341Crossref PubMed Scopus (723) Google Scholar with the use of our previously described protocol.36Yamada Y. Ishibashi K. Ishibashi K. Bhutto I.A. Tian J. Lutty G.A. Handa J.T. The expression of advanced glycation endproduct receptors in RPE cells associated with basal deposits in human maculas.Exp Eye Res. 2006; 82: 840-848Crossref PubMed Scopus (89) Google Scholar Macular calottes were fixed for 1 hour in 2% paraformaldehyde and then cryoprotected by progressive infiltration in 10% and 20% sucrose in PBS (w/v) before freezing in 2:1 sucrose 20% (w/v):OCT compound at −80°C.Table 1Donor CharacteristicsPatientAMDAge (years)SexEnucleation (hours)Cause of death1No49F5:10Lung cancer2No91F5:25Lung cancer3No91F5:25Lung cancer4No91M2:20Subdural hematoma5No90F3:53Respiratory arrest6No90M2:45Stroke7Yes70F6:10Lung cancer8Yes72F6:11Cardiac arrest9Yes85F2:28Multiorgan failure10Yes90F3:53Respiratory arrest11Yes93F5:46Renal failureAMD, age-related macular degeneration; F, female; M, male. Open table in a new tab AMD, age-related macular degeneration; F, female; M, male. For fluorescence immunohistochemistry (IHC), mouse cryosections (7 μm) were first blocked with 2% goat or donkey serum (Jackson ImmunoResearch, West Grove, PA) followed by blocking with avidin/biotin blocking reagent (Vector Labs, Burlingame, CA), and then treated with a mouse on mouse blocking reagent (anti-mouse IgG blocking reagent; Vector Labs) if necessary for 1 hour at room temperature. Sections were then incubated with the primary antibody: a monoclonal anti-carboxymethyl lysine (CML) monoclonal antibody (1:50; Wako, Richmond, VA), a rabbit polyclonal antibody to LPL (1:50; Santa Cruz Biotechnologies, Santa Cruz, CA), a rabbit polyclonal antibody to versican (1:50; Santa Cruz Biotechnologies), a goat polyclonal antibody to biglycan (1:50; Santa Cruz Biotechnologies), a rabbit polyclonal antibody to acid and neutral sphingomyelinase (1:50; Santa Cruz Biotechnologies), or the appropriate isotype IgG. The anti-LPL antibody was raised against amino acids 28 to 80, mapping near the N-terminus of LPL of human origin (Santa Cruz Biotechnologies). This antibody was Protein A purified and then glutathione S-transferase subtracted. It does not cross-react with other lipases and is suitable for use in human and mouse tissues.37Fernandez-Veledo S. Nieto-Vazquez I. de Castro J. Ramos M.P. Bruderlein S. Moller P. Lorenzo M. Hyperinsulinemia induces insulin resistance on glucose and lipid metabolism in a human adipocytic cell line: paracrine interaction with myocytes.J Clin Endocrinol Metab. 2008; 93: 2866-2876Crossref PubMed Scopus (36) Google Scholar, 38Li Y. Sugiyama E. Yokoyama S. Jiang L. Tanaka N. Aoyama T. Molecular mechanism of age-specific hepatic lipid accumulation in PPARalpha (+/−): LDLR (+/−) mice, an obese mouse model.Lipids. 2008; 43: 301-312Crossref PubMed Scopus (9) Google Scholar, 39Kirpich IA, Gobejishvili LN, Bon Homme M, Waigel S, Cave M, Arteel G, Barve SS, McClain CJ, Deaciuc IV: Integrated hepatic transcriptome and proteome analysis of mice with high-fat diet-induced nonalcoholic fatty liver disease. J Nutr Biochem 22:38–45Google Scholar Sections were incubated with primary antibody overnight at 4°C, washed with PBS, and incubated with goat anti-rabbit and donkey anti-goat secondary antibodies, respectively (1:400; Santa Cruz Biotechnologies). Appropriate mouse, rabbit, and goat IgGs (Santa Cruz Biotechnologies) were used as isotype controls. Sections were imaged with a confocal microscope. Human macular cryosections (7 μm) were blocked with avidin/biotin blocking reagent (Vector Labs) and then incubated with rabbit anti-LPL antibody (1:1000; Santa Cruz Biotechnologies). Sections were then incubated with biotinylated anti-rabbit IgG (Vector Labs) for 30 minutes at room temperature and then with alkaline phosphatase–conjugated avidin streptavidin (Vectastain ABC Kit; Vector Labs) and the appropriate substrate (Alkaline Phosphatase Substrate III; Vector Labs). Sections were counterstained with Nuclear Fast Red (Vector Labs). To permit interpretation of immunoreaction product in the RPE and choroid, some sections were fixed in 4% paraformaldehyde overnight at 4°C immediately after streptavidin APase IHC. Slides were washed in distilled water at room temperature, immersed in 0.05% potassium permanganate solution (Aldrich Chemical Co., Milwaukee, WI) for 25 minutes, and then rinsed in distilled water for 5 minutes. Sections were treated with 35% paracetic acid (Sigma-Aldrich) in a humidified chamber for 60 to 90 minutes at room temperature followed by washing in distilled water for 10 minutes and imaged in a axiovert 200M microscope (Carl Zeiss Micro Imaging, Inc., Thornwood, NY). Confocal microscopy was performed on a laser scanning confocal microscope (Zeiss 510 META confocal microscope; Carl Zeiss Micro Imaging, Inc.). Rhodamine fluorescence was visualized with the use of an excitation wavelength of 543 nm and an emission wavelength between 560 and 585 nm. Green fluorescence staining was visualized by exciting with a 488-nm laser beam and collecting emissions between 500 and 552 nm. The fluorescence intensity was quantified with Zeiss software (AxioVision). Results were compared with animals that received an i.v. injection of PBS. Specifically, the measured fluorescence of the PBS-injected mice was subtracted from the fluorescence obtained from eyes in each of the experimental groups (D-gal, PTB/D-gal, and PBS). Separate intensities were calculated for the RPE, Bruch's membrane, and choroid. The fluorescence values were standardized by cross-sectional area of each tissue (n = 3 animals per time point per group). Statistical significance was determined with analysis of variance and Bonferroni procedure for post hoc analysis. Our laboratory has previously shown that AGEs form in Bruch's membrane of mice treated with low-dose D-gal, coincident with the development of ultrastructural features of early AMD.32Ida H. Ishibashi K. Reiser K. Hjelmeland L.M. Handa J.T. Ultrastructural aging of the RPE-Bruch's membrane-choriocapillaris complex in the D-galactose-treated mouse.Invest Ophthalmol Vis Sci. 2004; 45: 2348-2354Crossref PubMed Scopus (44) Google Scholar, 33Tian J. Ishibashi K. Ishibashi K. Reiser K. Grebe R. Biswal S. Gehlbach P. Handa J.T. Advanced glycation endproduct-induced aging of the retinal pigment epithelium and choroid: a comprehensive transcriptional response.Proc Natl Acad Sci U S A. 2005; 102: 11846-11851Crossref PubMed Scopus (106) Google Scholar To verify that this treatment induced AGE formation in Bruch's membrane, sections were evaluated for CML, a well-established AGE found to accumulate in basal deposits and drusen in AMD.31Farboud B. Aotaki-Keen A. Miyata T. Hjelmeland L.M. Handa J.T. Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in Bruch's membrane of the aging eye.Mol Vis. 1999; 5: 11PubMed Google Scholar Figure 1 (A and B) shows that mice treated with D-gal develop CML in Bruch's membrane, whereas PBS-treated mice did not develop CML (Figure 1, E and F). Mice treated with PTB, the AGE cross-link breaker, had reduced but detectable staining for CML (Figure 1, C and D). The RPE also showed CML labeling and an altered cell structure, as we have previously reported32Ida H. Ishibashi K. Reiser K. Hjelmeland L.M. Handa J.T. Ultrastructural aging of the RPE-Bruch's membrane-choriocapillaris complex in the D-galactose-treated mouse.Invest Ophthalmol Vis Sci. 2004; 45: 2348-2354Crossref PubMed Scopus (44) Google Scholar (Figure 1A). To show that AGE formation in Bruch's membrane is acutely associated with LDL retention, C57Bl6 mice treated with D-gal for 8 weeks were then injected intravenously with rhodamine-labeled LDLs. At 5 minutes, rhodamine-labeled LDL was found in the RPE, Bruch's membrane, and choroid of all three groups without a difference in fluorescence (data not shown). Although minimal labeling was observed in PTB/D-gal– and PBS-treated mice at 24 and 72 hours after injection, rhodamine-labeled LDL was more prominently retained in the RPE and Bruch's membrane, as shown in Figure 2A. As graphically quantified in Figure 2B, the RPE of D-gal mice retained LDLs 24 hours after injection significantly more than the PTB/D-gal– and PBS-injected mice (P < 0.05). LDLs were similarly retained in the RPE 72 hours after injection in the D-gal group relative to the PTB/D-gal and PBS controls (P < 0.01). In Bruch's membrane of the D-gal–treated mice, LDLs were increased at 24 and 72 hours after injection compared with the PTB/D-gal and PBS controls. At 24 hours, they were significantly higher in the D-gal group than in the PTB/D-gal and PBS controls (P ≤ 0.01). At 72 hours, the relative fluorescence remained higher in the D-gal group than in the PTB/D-gal and PBS groups (P < 0.01). A similar trend was seen with LDLs in the choroid, but the magnitude of retention was lower than the other tissues. It is possible that oxidized LDLs from the systemic circulation can get retained in Bruch's membrane instead of LDLs being oxidized after being retained. To evaluate this possibility, rhodamine-labeled oxidized LDLs were injected with the use of the same protocol. Although labeling was seen at 5 minutes, confirming that oxidized LDLs transit to the RPE/choroidal com
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