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Advanced Glycation as a Basis for Understanding Retinal Aging and Noninvasive Risk Prediction
Abstract
The retina is exquisitely sensitive to age-related processes, and, in many cases, these can precipitate progressive and profound loss of vision. Many asymptomatic abnormalities that accrue in the outer retina as we get older can serve as a sinister preamble to age-related macular degeneration (AMD). This condition remains the leading cause of irreversible blindness in industrialized countries, but its precise pathogenesis has yet to be completely elucidated. Over recent years, increasing evidence has suggested that the accumulation of advanced glycation end products (AGEs) and activation of the receptor for AGEs in the outer retina could play a significant role in the initiation and progression of AMD. The current review outlines this evidence and indicates how products of Maillard chemistry could be used as robust markers for outer retinal aging and susceptibility to AMD. The utility of Raman spectroscopy to measure AGE adducts in human tissues is presented. The methodology reinforces the association between AGE formation and retinal aging and provides exciting possibilities for assessing these pathogenic agents in the living eye and, perhaps, for providing a valuable index for AMD susceptibility.
... (Hammes et al. 1996;Li & Dickson 1997;Schutt et al. 2003;Tabaton et al. 1997) Because accumulation of AGEs plays an important role in AMD pathogenesis, recently studies have tried to use the fluorescent property of AGE adducts to develop non-invasive predictors for AMD. (Mulder et al. 2010;Pawlak et al. 2008) AGEs influence the physiological functions of RPE. For example, in vitro AGEs induce RPE to up-regulate the expression of VEGF and platelet-derived growth factor-B (PDGF-B), both of which are important regulators in angiogenesis. ...
... 08) atherosclerosis, (Ehlermann et al. 2006), dysfunction of cardiomyocytes (Gao et al. 2008), and retinal diseases . The RAGE-ligand signals activate the wide range of patho-physiological responses linked to downstream transcriptional activity of NF-κB (Fig. 5), which induces pro-inflammatory cytokines and oxidative stress. (Bierhaus et al. 2005.;Pawlak et al. 2008) In the context of the outer retina, studies have showed that RAGE is expressed on RPE and that RAGE levels are significantly increased in AMD (in postmortem tissue), especially on cells adjacent to drusen. (Howes et al. 2004;Pawlak et al. 2008;Yamada et al. 2006) Activation of the RAGE axis in RPE cells up-regulates the expression and s ...
... et al. 2008) In the context of the outer retina, studies have showed that RAGE is expressed on RPE and that RAGE levels are significantly increased in AMD (in postmortem tissue), especially on cells adjacent to drusen. (Howes et al. 2004;Pawlak et al. 2008;Yamada et al. 2006) Activation of the RAGE axis in RPE cells up-regulates the expression and secretion of VEGF. Yamada et al. 2006) This can elicit or propagate neovascularization. ...
The glycemic index (GI) indicates how fast blood glucose is raised after consuming a carbohydrate-containing food. Human metabolic studies indicate that GI is related to patho-physiological responses after meals. Compared with a low-GI meal, a high-GI meal is characterized with hyperglycemia during the early postprandial stage (0–2 h) and a compensatory hyperlipidemia associated with counter-regulatory hormone responses during late postprandial stage (4–6 h). Over the past three decades, several human health disorders have been related to GI. The strongest relationship suggests that consuming low-GI foods prevents diabetic complications. Diabetic retinopathy (DR) is a complication of diabetes. In this aspect, GI appears to be useful as a practical guideline to help diabetic people choose foods. Abundant epidemiological evidence also indicates positive associations between GI and risk for type 2 diabetes, cardiovascular disease, and more recently, age-related macular degeneration (AMD) in people without diabetes. Although data from randomized controlled intervention trials are scanty, these observations are strongly supported by evolving molecular mechanisms which explain the pathogenesis of hyperglycemia. This wide range of evidence implies that dietary hyperglycemia is etiologically related to human aging and diseases, including DR and AMD. In this context, these diseases can be considered as metabolic retinal diseases.
... Composition of ligament [26][27][28] Spectra of proteins [29][30][31][32] , amino acids [33][34][35][36][37] Non-enzymatic modification of collagen/elastin: molecular composition [38][39][40][41][42][43] and spectroscopy [44][45][46] Young's modulus [47][48][49][50][51][52][53][54] To use less certain sources as part of the input (to reflect situations were information may not be readily accessible or may be incomplete) diabetes was modelled on publicly available information [55] . ...
... The R 2 against Young's modulus is predicted to be poor (close to 0) for young populations with high glucose intake. It is best for middle aged populations (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49) with moderate glucose intake (83-125 g/day), both biased below the outpatient population means (49 years old and 125 g glucose/day). Performance is moderately impacted for an older population with low glucose intake. ...
Increasing use of real‐world experiments embeds scientific study in settings relevant to everyday life but brings a number of complications. Traditional study design has evaluated experimental risk based on a limited number of factors that are tightly controlled while others are standardised. In real‐world data, many unstandardised factors risk confounding the study in unanticipated ways. Some simulation tools and processes are evaluated for use in capturing current knowledge of complex scenarios to evaluate validation of multivariate models in real‐world settings. Further, it examines the value of probing the range of uncertainty of the assumptions used to build the model. Copulas simulate correlation of multiple underlying factors where the web of causal interactions cannot be untangled. These factors influence the biochemical status of an individual, determining what pathways are activated in that individual. The final biochemical status of the individuals is then transformed into Raman spectroscopic, reference and pathological measurements. Evaluating different validation scenarios (same population, subset of that population and a new population) allows evaluation of the risks of the model being deployed for different intended uses. Technical factors are stress tested to determine the analytical bottlenecks. Individual socio‐economic factors are stress tested to determine the leverage that current uncertainty has on the risks to the validation study. Use of simulation was able to identify the limitations of the current model for new scenarios and also to assess the specific changes in expected sensitivity versus specificity in the new populations, which would be relevant for benefit risk analysis.
... All spectra were pre-processed to remove background fluorescence using an iterative polynomial fitting algorithm [24], then cropped to the region 800-1800 cm −1 ; this range was selected in order to match the spectral region of the advanced glycation end products (AGEs) reported in the literature [25,26]. This polynomial fitting algorithm was proposed by Zhao et al. [24] and is also known as the Vancouver Raman algorithm (VRA); it is widely used for fluorescence background removing in biomedical applications due to effectiveness and simplicity. ...
... In order to explore the relationship between the acquired spectra and the molecules related to diabetic complications, such as AGEs [33], Raman spectra from several AGEs (glyoxallysine dimer GOLD, methylglyoxal-derived hydroimidazolone MG-H2, and pentosidine) and AGEs precursors (3-deoxyglucosone, glyoxal, and methylglyoxal) were digitized from the literature [25,26]. GOLD and MG-H2 have been found to accumulate in tissue in diabetes, through oxidation of polyunsaturated fatty acids [34], while pentosidine has been identified with Raman microscopy in the progression of DM2 [35]. ...
Type 2 diabetes mellitus (DM2) is one of the most widely prevalent diseases worldwide and is currently screened by invasive techniques based on enzymatic assays that measure plasma glucose concentration in a laboratory setting. A promising plan of action for screening DM2 is to identify molecular signatures in a non-invasive fashion. This work describes the application of portable Raman spectroscopy coupled with several supervised machine-learning techniques, to discern between diabetic patients and healthy controls (Ctrl), with a high degree of accuracy. Using artificial neural networks (ANN), we accurately discriminated between DM2 and Ctrl groups with 88.9-90.9% accuracy, depending on the sampling site. In order to compare the ANN performance to more traditional methods used in spectroscopy, principal component analysis (PCA) was carried out. A subset of features from PCA was used to generate a support vector machine (SVM) model, albeit with decreased accuracy (76.0-82.5%). The 10-fold cross-validation model was performed to validate both classifiers. This technique is relatively low-cost, harmless, simple and comfortable for the patient, yielding rapid diagnosis. Furthermore, the performance of the ANN-based method was better than the typical performance of the invasive measurement of capillary blood glucose. These characteristics make our method a promising screening tool for identifying DM2 in a non-invasive and automated fashion. spectroscopy and PCA-SVM as a non-invasive diagnostic tool to identify and classify qualitatively glycated hemoglobin levels in vivo," J. spectroscopy provides a powerful diagnostic tool for accurate determination of albumin glycation," PLoS One 7(2), e32406 (2012). 9. A. fasting Blood glucose levels by invasive and indigenously developed noninvasive technology and its correlation with the glycated hemoglobin (HbA1c) biomarker in healthy and diabetic subjects," Res.
... Some studies that have looked into the processes involved in AMD pathogenesis, have suggested that AGEs can be strongly associated with AMD pathogenesis [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. AGEs are protein or lipid products of nonenzymatic glycation that is one of the most important post-translational modifications in cells [23,24]. ...
... To our knowledge, no studies analyzing the impact of genes' polymorphisms on the development of early or exudative AMD have been carried out. Previous studies on the morphogenesis of AMD drew attention to the role of advanced glycation end products (AGEs) formation in Bruch's membrane overlying deposits [8][9][10][11][12][13][14][15][16][17][18][19][20][21] as well as in retinal pigment epithelium (RPE) [22] but not genetic predisposition. ...
BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in people aged 65 years and older in developed countries. The pathogenesis of AMD has been linked to mechanisms involving inflammation, oxidative stress, and basal laminar deposit formation between retinal pigment epithelium (RPE) cells and the basal membrane, caused by advanced glycation end products (AGEs). AGEs are implicated in the pathogenesis of AMD through the AGE-and receptor for AGE (RAGE) interaction, which can be altered by polymorphisms of the RAGE gene. We examined RAGE rs1800624 and rs1800625 gene polymorphisms contributing to AMD development.
MATERIAL AND METHODS: The study enrolled 300 patients with early AMD, 300 patients with exudative AMD, and 800 healthy controls. The genotyping was carried out using the RT-PCR method.
RESULTS: The analysis of two single nucleotide polymorphisms (SNPs) in the RAGE gene showed that rs1800624 was associated with a 1.6-fold decreased risk for exudative AMD under the dominant model after adjustment for age (OR=0.616; 95% CI: 0.394–0.963; p=0.034) and each copy of allele T at rs1800624 was associated with a 1.4-fold decreased risk for exudative AMD development under the additive model after adjustment for age (OR=0.701; 95% CI: 0.510–0.962; p=0.028). Analysis revealed that the rs1800625 allele G at rs1800625 was associated with a 1.5-fold increased risk for exudative AMD after adjustment for age (OR=1.545; 95% CI: 1.003–2.379; p=0.048). These results suggested that the allele G at rs1800625 was a risk-allele for exudative AMD development. In haplotype analysis, A-G haplotype was significantly more frequently observed in exudative AMD patients compared to healthy controls (3.3% versus 1.4%, p=0.035).
CONCLUSIONS: We revealed a significant association between RAGE gene rs1800624 and rs1800625 polymorphisms and AMD risk. We considered T allele at rs1800624 to be protective against AMD development, while allele G at rs1800625 was considered to be a marker of poor prognosis in AMD development.
... Age-related cellular dysfunction includes increased chemical protein damage, accumulation of intracellular and extracellular injuries, and decreased antioxidant defense (Laimer et al., 2010;Shin et al., 2014). The aging process is best studied through long-lived, post-mitotic cells such as those of the retinal pigment epithelium (RPE) (Pawlak et al., 2008). ...
... The representative disease associated with RPE dysfunction is early age-related maculopathy. This maculopathy sometimes progresses to age-related macular degeneration (Pawlak et al., 2008). ...
PurposeThis study used five clinical assessments and Raman spectroscopy to investigate the age- and hyperglycemia-related properties of the murine retina over an eight-week experimental period.Method
Acute hyperglycemia and chronic hyperglycemia were assessed with blood glucose (BG) and glycated hemoglobin (HbA1c) levels, respectively. Changes in the retinal thickness and neovascularization were evaluated with optical coherence tomography and fluorescein angiography (FAG). Histological changes in the retina were studied after periodic acid-Schiff (PAS) staining. Raman spectroscopy was used to examine the molecular structures and chemical compositions of the retinal tissues.ResultsThe young hyperglycemic group had acute hyperglycemia with a BG level of 576 ± 22 mg/dL and HbA1c of 5.9 ± 0.6%, while the aged hyperglycemic group displayed chronic hyperglycemia with a BG level of 607 ± 28 mg/dL and HbA1c of 11.2 ± 1.5%. Aged hyperglycemic retinas showed an insignificant (5%) decrease in thickness and no presence of vascular leaky lesions with FAG. There was no histological evidence of the retinal neovascularization with PAS staining of these aged hyperglycemic retinas. In the aged group, Raman intensities assigned to the C–C symmetric breathing of the aromatic ring in phenylalanine (1003 cm−1), the NH2 amide III α-helix deformation in the protein structure (1265 cm−1), and the C=O stretching vibration of amide I α-helix structure in collagen (1657 cm−1) were all decreased.Conclusion
These decreased Raman intensities indicate elevated reactive oxygen species (ROS) and oxidative damage to proteins such as those involved in cell apoptosis. A decrease in these ROS-related Raman peaks indicates an aging effect on the retina. Microsc. Res. Tech., 2014. © 2014 Wiley Periodicals, Inc.
... We also compared modes intensities ratio between and for amide I (1,670 cm -1 ) and AGE products (1,550-1,600 cm -1 ) [12,13]. An increase of this ratio indicates altered collagen quality induced by Maillard reaction. ...
... Sebag et al. used a non-microscopy-based approach to show Raman spectral changes in the human vitreous that were attributed to AGEs [22]. Confocal Raman microscopy was used to quantify AGEs adducts in Bruch membrane [12,13]. Table 1 gathers the Raman intensities of amide I and AGE's related product bands as well as their ratio. ...
Objectives
Our aim was to determine the origin of the red fluorescence of carious dentine observed with the Soprolife® camera.
Methods
We conducted in vitro studies to evaluate the origin of the red fluorescence using acids and matrix metalloproteinase (MMP) to mimic caries and methylglycoxal (MGO) to evaluate the effect of glycation reactions on the red fluorescence. In every step of these models, we detected the changes of dentin photonic response with Soprolife® in daylight mode and in treatment mode. A Raman spectroscopy analysis was performed to determine the variations of the dentin organic during the in vitro caries processes. Raman microscopy was performed to identify change in the collagen matrix of dentine.
Results
The red fluorescence observed in carious dentine using a Soprolife® camera corresponds to the brownish color observed using daylight. Demineralization using nitric acid induces a loss of the green fluorescence of dentine. The red fluorescence of carious dentine is resistant to acid treatment. Immersion of demineralized dentine in MGO induces a change of color from white to orange-red. This indicates that the Maillard reaction contributes to lesion coloration. Immersion of demineralized dentine in an MMP-1 solution followed by MGO treatment results in a similar red fluorescence. Raman microspectroscopy analysis reveals accumulation of AGE's product in red-colored dentine.
Conclusions
Our results provide important information on the origin of the fluorescence variation of dentine observed with the Soprolife® camera. We demonstrate that the red fluorescence of carious dentine is linked to the accumulation of Advanced Glycation End products (AGE).
Clinical relevance
The study provides a new biological basis for the red fluorescence of carious dentine and reinforces the importance of the Soprolife® camera in caries diagnostics.
... AGEs accumulate in various tissues during aging, including skin, neural, vascular, renal and cardiac tissues, collagens and crystalline lens. In the skin, glycation is involved in many metabolic processes and, along with aging, affects the functionality of certain cells, such as the synthesis of fibroblasts, enzyme activation of matrix degradation (metalloproteinases) and the organization of the matrix (Hartog et al., 2007;Pageon, 2010;Pawlak et al., 2008). It is proposed that the accumulation of the advanced glycation end products (AGEs) and the activation of the receptor for AGEs in the retina could play a significant role in the initiation and progression of age-related macular degeneration and cataracts (Pawlak et al., 2008). ...
... In the skin, glycation is involved in many metabolic processes and, along with aging, affects the functionality of certain cells, such as the synthesis of fibroblasts, enzyme activation of matrix degradation (metalloproteinases) and the organization of the matrix (Hartog et al., 2007;Pageon, 2010;Pawlak et al., 2008). It is proposed that the accumulation of the advanced glycation end products (AGEs) and the activation of the receptor for AGEs in the retina could play a significant role in the initiation and progression of age-related macular degeneration and cataracts (Pawlak et al., 2008). Kalousová et al. (2002) and most recently, Mostafa et al. (2007) showed that AGEs level in plasma proteins are elevated in patients with diabetes. ...
... All of this indicates that reactive carbonyls formed by photooxidation of lipofuscin bisretinoids can modify molecular structures in RPE cells by forming adducts with proteins and phospholipids. Glycative stress, accompanied by the deposition and accumulation of damaged proteins, as well as the activation of inflammatory and para-inflammatory processes, significantly increases the risk of developing many age-related eye diseases-in particular, age-related macular degeneration [33][34][35][36]. ...
Aging of the retina is accompanied by a sharp increase in the content of lipofuscin granules and bisretinoid A2E in the cells of the retinal pigment epithelium (RPE) of the human eye. It is known that A2E can have a toxic effect on RPE cells. However, the specific mechanisms of the toxic effect of A2E are poorly understood. We investigated the effect of the products of photooxidative destruction of A2E on the modification of bovine serum albumin (BSA) and hemoglobin from bovine erythrocytes. A2E was irradiated with a blue light-emitting diode (LED) source (450 nm) or full visible light (400–700 nm) of a halogen lamp, and the resulting water-soluble products of photooxidative destruction were investigated for the content of carbonyl compounds by mass spectrometry and reaction with thiobarbituric acid. It has been shown that water-soluble products formed during A2E photooxidation and containing carbonyl compounds cause modification of serum albumin and hemoglobin, measured by an increase in fluorescence intensity at 440–455 nm. The antiglycation agent aminoguanidine inhibited the process of modification of proteins. It is assumed that water-soluble carbonyl products formed as a result of A2E photodestruction led to the formation of modified proteins, activation of the inflammation process, and, as a consequence, to the progression of various senile eye pathologies.
... Thereby, a higher specificity and a greater diagnostic value would be achieved. Although it has been already demonstrated that nonresonant Raman spectroscopy is a promising method for studying the biochemical composition and disease related changes of ex vivo eye samples, [31][32][33] in vivo studies of the human retina have not yet been performed. The transition of Raman spectroscopy into the clinics is challenging especially for ophthalmology. ...
Retinal diseases, such as age-related macular degeneration, are leading causes of vision impairment, increasing in incidence worldwide due to an aging society. If diagnosed early, most cases could be prevented. In contrast to standard ophthalmic diagnostic tools, Raman spectroscopy can provide a comprehensive overview of the biochemical composition of the retina in a label-free manner. A proof of concept study of the applicability of nonresonant Raman spectroscopy for retinal investigations is presented. Raman imaging provides valuable insights into the molecular composition of an isolated ex vivo human retina sample by probing the entire molecular fingerprint, i.e., the lipid, protein, carotenoid, and nucleic acid content. The results are compared to morphological information obtained by optical coherence tomography of the sample. The challenges of in vivo Raman studies due to laser safety limitations and predefined optical parameters given by the eye itself are explored. An in-house built setup simulating the optical pathway in the human eye was developed and used to demonstrate that even under laser safety regulations and the above-mentioned optical restrictions, Raman spectra of isolated ex vivo human retinas can be recorded. The results strongly support that in vivo studies using nonresonant Raman spectroscopy are feasible and that these studies provide comprehensive molecular information of the human retina.
... In dental caries autofluorescence properties are most likely elicited from denatured collagen structure. Pentosidine, a glycation end-product after Maillard reaction, is also forming fluorescent cross-link between arginine and lysine residues (Pawlak et al., 2008;Levallois et al., 2012). Other fluorophores in caries are porphyrines, presumably synthesized by oral bacteria that penetrate dentinal tubules (König et al., 1999). ...
Isotope analysis of collagen from high-resolution sequential samples of dentine has become a popular tool to provide diachronic insights into individual life histories including childhood diet, stress episodes, and mobility during the tooth formation period. New microsampling approaches improve temporal resolution enabling tracking of diet shifts within relatively short time-spans. These methods, however, necessarily deliver small samples and depend upon good collagen preservation at similar high-resolution scales. Yet present methods indicate state of collagen preservation only after sampling and isotope analysis. Here we present a method for prior determination of collagen preservation, based on differential autofluorescence imaging of tooth longitudinal thin sections to map regions of intact and degraded collagen in the corresponding sampling areas. We find that even in dentine areas that were apparently intact based on histology, decay was detected in fluorescence images and that these could be correlated to lower collagen amounts and higher C/N ratios. Areas identified as ‘severely decayed’ dissolved after demineralization. The presented dual-colour autofluorescence imaging approach allows determination of areas of degraded collagen at high resolution. This can be used to detect diagenetic changes in collagen ahead of sampling and facilitates selection of well-preserved samples.
... However, Streptococcus mutans and lactobacilli [42][43][44][45] seemed to have a weaker or no porphyrin fluorescence in the red wavelength region. The red signal certainly involved the Maillard product [46][47][48] and in part the bacteria species, which formed a highly complex ecosystem, depending on the activity and depth of decay, with variable excitation wavelengths. ...
The goals of this trial were, first, to produce a Raman mapping of decay and sound dentin samples, through accurate analysis of the Raman band spectra variations of mineral and organic components. The second goal was to confirm the correlation between the Raman signal and the signal of a fluorescent camera, by assaying the concentration of pentosidine and natural collagen fluorescent crosslink using reverse phase high-pressure liquid chromatography. The first correlation assumed a possible relationship between the signal observed with the camera and Raman spectroscopy. The second correlation assumed an association with the Maillard reaction. Absence of a correlation for this trial was that no association could be found between Raman spectra characteristics, fluorescence variation and the HPLC assay. Our results void this absence (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
... The AGEs accumulate gradually in tissues with aging and in patients with specific diseases, such as diabetes, Alzheimer's disease, and retinal dysfunction (7)(8)(9)(10)(11). These AGEs include fructolysine, N-(carboxymethyl)lysine, N-(carboxymethyl)hydrolysine, and pentosidine (12,13), a fluorescent cross-link formed between arginine and lysine residues. The Maillard reaction can be studied using methylglycoxal (MGO) as an intermediate glycating agent. ...
In clinical situations carious dentine tissues can be discriminated by most caries fluorescence detection tools, including a new fluorescence intra-oral camera. The objectives of this study were: (i) to analyze the Raman spectra of sound, carious, and demineralized dentine, (ii) to compare this spectral analysis with the fluorescence variation observed when using a fluorescence camera, and (iii) to evaluate the involvement of the Maillard reaction in the fluorescence variations. The first positive hypothesis tested was that the fluorescence of carious dentine obtained using a fluorescence camera and the Raman spectra variation were closely related. The second was that the variation of fluorescence could be linked with the Maillard reaction. Sound dentine, sound dentine demineralized in aqueous nitric acid solution, carious soft dentine, sound dentine demineralized in lactic acid solution, sound dentine demineralized in aqueous nitric acid solution and immersed in methylglycoxal solution, and sound dentine demineralized in aqueous nitric acid solution and immersed in methylglycoxal and glucose solutions, were studied using micro-Raman spectroscopy. Modifications in the band ratio of amide, phosphate, and carbonate were observed in the decayed and demineralized groups compared with the sound dentine group. The results indicate that a close relationship exists between the Maillard reaction and fluorescence variation.
... Reactive oxygen intermediates are involved in the formation of early glycation adducts such as CML [55,59]. Increasing published evidence suggests that the accumulation of AGEs in the retina could play a significant role in initiation and pathogenesis of AMD [60,61]. Cumulative prolonged blue light damage at the RPE level may contribute to the development of specific changes characteristic for early AMD, including RPE pigmentary abnormalities and formation of sub-RPE extracellular deposits [62,63]. ...
To evaluate the in vitro response of retinal pigment epithelial (RPE) cells to a nonlethal dose of blue light.
The human RPE cell line ARPE-19 was irradiated with blue light (405 nm) at an output power of 1 mW/cm(2) or 0.3 mW/cm(2). The following parameters were studied: metabolic activity; apoptosis; reactive oxygen species (ROS) production; mitochondrial membrane potential (MMP); ultrastructural changes of mitochondria; production of advanced glycation endproducts (AGEs); and stress-related cellular proteins.
Nonlethal doses of blue light irradiation significantly reduced ARPE-19 metabolic activity and MMP while increasing intracellular ROS levels and expression of stress-related proteins heme oxygenase-1 (HO-1), osteopontin, heat shock protein 27 (Hsp-27), manganese superoxide dismutase (SOD-Mn), and cathepsin D. Blue light irradiation also induced ultrastructural conformation changes in mitochondria, resulting in the appearance of giant mitochondria after 72 h. We further found enhanced formation of AGEs, particularly N(epsilon)-(carboxymethyl) lysine (CML) modifications, and a delay in the cell cycle.
ARPE-19 cells avoid cell death and recover from blue light irradiation by activating a host of defense mechanisms while simultaneously triggering cellular stress responses that may be involved in RPE disease development. Continuous light exposure can therefore detrimentally affect metabolically stressed RPE cells. This may have implications for pathogenesis of age-related macular degeneration.
Establishing a non‐destructive method for spatially assessing advanced glycation end‐products (AGEs) is a potentially useful step toward investigating the mechanistic role of AGEs in bone quality. To test the hypothesis that the shape of the Amide I in the Raman Spectroscopy (RS) analysis of bone matrix changes upon AGE accumulation, we incubated paired cadaveric cortical bone in ribose or glucose solutions and in control solutions for 4 and 16 weeks, respectively, at 37 °C. Acquiring 10 spectra per bone with a 20X objective and a 830 nm laser, RS was sensitive to AGE accumulation (confirmed by biochemical measurements of pentosidine and fluorescent AGEs). Hyp/Pro ratio increased upon glycation using either 0.1 M ribose, 0.5 M ribose, or 0.5 M glucose. Glycation also decreased the Amide I sub‐peak ratios (cm‐1) 1668/1638 and 1668/1610 when directly calculated using either second derivative spectrum or local maxima of difference spectrum, though the processing method (e.g., averaged spectrum vs. individual spectra) to minimize noise influenced detection of differences for the ribose‐incubated bones. Glycation however did not affect these sub‐peak ratios including the matrix maturity ratio (1668/1690) when calculated using indirect sub‐band fitting. The Amide I sub‐peak ratios likely reflected changes in the collagen I structure.
To determine the effects of chromosome 1 genotype and cigarette smoking on levels of complement activation and inflammation in the human macula.
Donor macular tissue was stratified into three groups by diplotype at the AMD-associated CFH-to-F13B locus: homozygous "risk" (n = 9, 56-78 years), homozygous neutral (n = 2, 64-79 years), and homozygous "protective" (n = 6, 61-78 years) diplotype. Importantly, all donors were homozygous nonrisk at the ARMS2/HTRA1 locus, so that purely chromosome 1-directed pathways were examined. Immunohistochemistry was performed by using 14 antibodies, mostly against markers of complement and inflammation, followed by confocal microscopy and immunofluorescence quantification (all masked to donor status).
Donors homozygous risk at CFH-to-F13B exhibited significantly higher levels of terminal complement complex (TCC) in macular Bruch's membrane (BM; P = 0.03), choriocapillaris (CC; P = 0.04), and choriocapillaris intercapillary septa (CC IS; P = 0.03), compared to homozygous protected donors. Smoking was associated with increased TCC in BM (P = 0.05), CC IS (P = 0.03), and choroidal stroma (CS; P = 0.01), and with substantially elevated C-reactive protein (CRP) levels in RPE (P = 0.04), BM (P = 0.01), CC (P = 0.05), and CS (P = 0.05). Smoking was associated with higher levels of oxidative stress in macular RPE (P = 0.04) and CS (P = 0.01).
Genetic risk at the CFH-to-F13B locus was associated with higher levels of complement activation at the human macular RPE-choroid interface, as was cigarette smoking. Levels of CRP were substantially elevated in risk donors with smoking history. Examination of human macular tissue from donors with "pure" diplotypes allows assessment of AMD-associated pathways driven solely by CFH-to-F13B. These findings have important implications for identifying chromosome 1-directed pathways and therapeutic targets.
Advanced glycation end products (AGEs) have been implicated in vision loss associated with macula degeneration, cataract formation, diabetic retinopathy and glaucoma. This pathogenic potential is mainly attributed to their accumulation in ocular tissues where they mediate aberrant crosslinking of extracellular matrix proteins and disruption of endothelial junctional complexes that affects cell permeability, mediates angiogenesis and breakdown of the inner blood-retinal barrier. Furthermore, AGEs severely affect cellular metabolism by disrupting ATP production, enhancing oxidative stress and modulating gene expression of anti-angiogenic and anti-inflammatory genes. Elucidation of AGE-induced mechanisms of action in different eye compartments will help in the understanding of the complex cellular and molecular processes associated with eye diseases. Several pharmaceutical agents with anti-glycating and anti-oxidant properties as well as AGE crosslink 'breakers' have been currently applied to eye diseases. The role of diet and the beneficial effects of certain nutriceuticals provide an alternative way to manage chronic visual disorders that affect the quality of life of millions of people.
Ocular calcification is a biological mineralization. It is also a well-known human ophthalmic disease but is a complex process because the pathophysiology of calcium deposition in ocular tissues depends on site-specific factors. Calcified deposits in the eyes are asymptomatic lesions; a serious consequence of ocular plaques is the potential partial or total loss of vision in the affected eye. Therefore, a better understanding of the exact compositional components and origin of calcified plaques in different eye tissues is necessary and merits exploration. The present review article includes information on different diagnostic or analytical techniques to examine human ocular calcification collected and compiled from 2005 to 2013, together with data on the application of vibrational spectroscopy in ophthalmology studies from 1990 onwards. This overview of ocular calcification is mainly focused on two aspects: the pathogenesis and mechanism of calcification in different ocular tissues and biomedical applications of vibrational spectroscopy to spectral biodiagnosis of eye tissues. A summary of the in vitro, in situ, and in vivo spectral information that is expected to assist physicians in the diagnosis and treatment of patients or satisfy patients of the clinical competence of the diagnosis is provided.
Raman microscopy is used to investigate the spectral features of selected compounds known to be involved in the development of the eye disease age-related macular degeneration (AMD). Diagnostic features were identified in synthetic samples of these compounds and in a biological matrix. The study demonstrates the potential of Raman microscopy for the development of diagnostic markers of the onset of AMD. Copyright © 2008 John Wiley & Sons, Ltd.
Raman spectroscopy has been revolutionised in recent decades by major technological advances such as lasers, charge-coupled detectors (CCD) and notch/edge filters. In contrast the development of signal processing algorithms has progressed at a slower pace. Spectroscopic applications increasingly focus on ‘real-world’ applications that are not under highly controlled conditions and with more stringent limitations placed on acquisition conditions (e.g. low power for in vivo and explosives analysis). Often it is necessary to work with signals of a quality traditionally considered poor. In this study an alternative paradigm for signal processing poor quality signals is presented and rigorously assessed. Instead of estimating the background on the individual signals it is estimated on the results of a multivariate analysis. Under this paradigm prediction reproducibility is unaffected by the signal processing, unlike the traditional paradigm of correcting individual signals which induces errors that propagate through to the prediction. The paradigms were tested on a ‘real-world’ dataset to predict the concentration of a pathologically relevant protein modification, carboxymethyl lysine (CML). Use of the new paradigm allowed signals with a signal to noise ratio (SNR) of 2.4 to give a prediction with variance just 8.7% of the mean, with the traditional paradigm giving a variance of over 140% of the mean. Significant improvement in reproducibility could even be observed with signals as good as SNR 85. The ability to obtain reproducible predictions from low quality signals allows shorter acquisition (e.g. mapping or on-line analysis), use of low powers (in vivo diagnostics, hazardous materials analysis (HAZMAT)) or use of cheaper equipment. Copyright © 2011 John Wiley & Sons, Ltd.
Raman microscopy, a rapid nondestructive technique that profiles the composition of biological samples, was used to characterize retinal biochemistry in the retinal dysplasia and degeneration (rdd) and wild-type (wt) chick retina during retinogenesis and at hatching.
Embryonic day (E)13 and posthatch day (P)1 rdd and wt retinal cross-sections (n = 3 of each line at each age) were profiled using 633 helium-neon laser excitation. The biochemical composition was determined using computational analysis of the Raman spectra. In parallel histology, TUNEL and glial fibrillary acidic protein (GFAP) immunostaining were used to visualize retinal dysfunction.
Principal component (PC) analysis of the Raman spectra identified 50 major biochemical profiles, but only PCs that made significant contributions to variation within rdd and wt retina were mapped. These significant PCs were shown to arise from DNA, various fatty acids, melanin, and a number of proteins. Distinct patterns of GFAP immunostaining and a larger population of TUNEL-positive nuclei were observed in the rdd versus wt retina.
This study has demonstrated that Raman microscopy can discriminate between major retinal biomolecules, thus providing an unbiased account of how their composition varies due to the impact of the MPDZ null mutation in the rdd chick relative to expression in the normal wt retina.
Raman spectroscopy is an effective probe of advanced glycation end products (AGEs) in Bruch's membrane. However, because it is the outermost layer of the retina, this extracellular matrix is difficult to analyze in vivo with current technology. The sclera shares many compositional characteristics with Bruch's membrane, but it is much easier to access for in vivo Raman analysis. This study investigated whether sclera could act as a surrogate tissue for Raman-based investigation of pathogenic AGEs in Bruch's membrane.
Human sclera and Bruch's membrane were dissected from postmortem eyes (n = 67) across a wide age range (33-92 years) and were probed by Raman spectroscopy. The biochemical composition, AGEs, and their age-related trends were determined from data reduction of the Raman spectra and compared for the two tissues.
Raman microscopy demonstrated that Bruch's membrane and sclera are composed of a similar range of biomolecules but with distinct relative quantities, such as in the heme/collagen and the elastin/collagen ratios. Both tissues accumulated AGEs, and these correlated with chronological age (R(2) = 0.824 and R(2) = 0.717 for sclera and Bruch's membrane, respectively). The sclera accumulated AGE adducts at a lower rate than Bruch's membrane, and the models of overall age-related changes exhibited a lower rate (one-fourth that of Bruch's membrane) but a significant increase with age (P < 0.05).
The results suggest that the sclera is a viable surrogate marker for estimating AGE accumulation in Bruch's membrane and for reliably predicting chronological age. These findings also suggest that sclera could be a useful target tissue for future patient-based, Raman spectroscopy studies.
Aging of the human retina is characterized by progressive pathology, which can lead to vision loss. This progression is believed to involve reactive metabolic intermediates reacting with constituents of Bruch's membrane, significantly altering its physiochemical nature and function. We aimed to replace a myriad of techniques following these changes with one, Raman spectroscopy. We used multiplexed Raman spectroscopy to analyze the age-related changes in 7 proteins, 3 lipids, and 8 advanced glycation/lipoxidation endproducts (AGEs/ALEs) in 63 postmortem human donors. We provided an important database for Raman spectra from a broad range of AGEs and ALEs, each with a characteristic fingerprint. Many of these adducts were shown for the first time in human Bruch's membrane and are significantly associated with aging. The study also introduced the previously unreported up-regulation of heme during aging of Bruch's membrane, which is associated with AGE/ALE formation. Selection of donors ranged from ages 32 to 92 yr. We demonstrated that Raman spectroscopy can identify and quantify age-related changes in a single nondestructive measurement, with potential to measure age-related changes in vivo. We present the first directly recorded evidence of the key role of heme in AGE/ALE formation.
The evolution of light sensitive cells probably began with a primitive functional unit composed of a photoreceptor cell and a pigmented cell. Even during embryonic development this functional unit is formed in a differentiation process in which the two interacting partners depend on each other. For some of the most important forms of retinal degeneration this knowledge on the functional cooperation between retinal pigment epithelium and photoreceptors is of great importance for analysis and development of therapeutic approaches. In this way mutations of genes which are expressed in photoreceptors can lead to diseases which start in the retinal pigment epithelium and vice versa. This article summarizes the variety of different functions of the retinal pigment epithelium and describes the failure of those functions which are of most clinical importance.
The accelerated formation of advanced glycation end products (AGEs) and the overexpression of transforming growth factor beta (TGF-beta) have both been implicated in the pathogenesis of diabetic microvascular and macrovascular complications. Previous studies in our laboratory have demonstrated that the vascular changes in diabetes include hypertrophy of the mesenteric vasculature. To examine the role of AGEs in this process, streptozotocin-induced diabetic rats and control animals were randomized to receive aminoguanidine, an inhibitor of AGE formation, or no treatment. Animals were studied at 7 d, 3 wk, and 8 mo after induction of diabetes. When compared with control animals, diabetes was associated with an increase in mesenteric vascular weight and an increase in media wall/lumen area. By Northern analysis, TGF-beta1 gene expression was increased 100-150% (P < 0.01) and alpha1 (IV) collagen gene expression was similarly elevated to 30-110% compared to controls (P < 0.05). AGEs and extracellular matrix were present in abundance in diabetic but not in control vessels. Treatment of diabetic rats with aminoguanidine resulted in significant amelioration of the described pathological changes including overexpression of TGF-beta1 and alpha1 (IV) collagen. These data implicate the formation of AGEs in TGF-beta overexpression and tissue changes which accompany the diabetic state.
The free radical theory of aging, conceived in 1956, has turned 40 and is rapidly attracting the interest of the mainstream of biological research. From its origins in radiation biology, through a decade or so of dormancy and two decades of steady phenomenological research, it has attracted an increasing number of scientists from an expanding circle of fields. During the past decade, several lines of evidence have convinced a number of scientists that oxidants play an important role in aging. (For the sake of simplicity, we use the term oxidant to refer to all "reactive oxygen species," including O2-., H2O2, and .OH, even though the former often acts as a reductant and produces oxidants indirectly.) The pace and scope of research in the last few years have been particularly impressive and diverse. The only disadvantage of the current intellectual ferment is the difficulty in digesting the literature. Therefore, we have systematically reviewed the status of the free radical theory, by categorizing the literature in terms of the various types of experiments that have been performed. These include phenomenological measurements of age-associated oxidative stress, interspecies comparisons, dietary restriction, the manipulation of metabolic activity and oxygen tension, treatment with dietary and pharmacological antioxidants, in vitro senescence, classical and population genetics, molecular genetics, transgenic organisms, the study of human diseases of aging, epidemiological studies, and the ongoing elucidation of the role of active oxygen in biology.
To study the correlations between age, Bruch's membrane (BM) thickness, retinal pigment epithelial (RPE) autofluorescence, and RPE residual body content.
Eight-millimeter-diameter macular discs from 88 unpaired human eye bank eyes were obtained within 72 hours of death, fixed in 10% neutral buffered formalin, and hemisected horizontally. One portion of the macular disc was embedded in paraffin and stained with periodic acid-Schiff for the measurement of BM thickness. RPE autofluorescence measurements were performed on unstained, deparaffinized sections. A second portion of the macular disc was prepared for electron microscopy to evaluate RPE residual body content. Linear and polynomial regression techniques were used to investigate the correlations between age, BM thickness, RPE autofluorescence, and RPE residual body content.
Bruch's membrane thickness increased with age according to the linear model. RPE autofluorescence and RPE residual body content also increased with age, but the correlations were best approximated by a quadratic model. The correlations between RPE autofluorescence and residual body content and between BM thickness and RPE autofluorescence were best approximated by a linear regression model. There was considerable variation in these correlations between specimens and within the same age group.
Although the changes in RPE and Bruch's membrane increased with age and there was a direct correlation between changes in the two tissues, there was considerable variation within each age group and between specimens. This probably reflects the multifactorial nature of the process.
To determine whether there is an age-related increase of pentosidine in human Bruch's membranes and to localize pentosidine and carboxymethyllysine (CML), two well-characterized, advanced glycation end products (AGEs) in aged human Bruch's membranes and choroid in vivo.
Human Bruch's membrane samples were isolated from the retinal pigment epithelium (RPE) and choroid and subjected to reversed-phase high-performance liquid chromatography to determine pentosidine content. A polyclonal anti-pentosidine antibody and a monoclonal antibody specific for carboxymethyllysine were used to localize AGEs in 20-month-old nondiabetic, 82-year-old nondiabetic, and 82-year-old diabetic globes.
Human Bruch's membranes (n = 20) showed a linear age-dependent increase in pentosidine that reached approximately 0.17 millimoles pentosidine per mole hydroxyproline in late life (r = 0.896; P < 0.001). Immunohistochemical evaluation showed evidence of pentosidine in Bruch's membrane, choroidal extracellular matrix, and vessel walls in the 82-year-old nondiabetic and diabetic globes. A similar staining pattern was found with the anti-CML antibody. Basal laminar deposits and drusen stained with both antibodies in the elderly nondiabetic eye. In contrast, neither antibody stained the 20-month-old tissue.
We provide biochemical and immunohistochemical evidence for the formation of pentosidine and CML structures in human Bruch's membrane and choroid with age. These changes could promote aging of the RPE-Bruch's membrane-choroid complex.
The formation of advanced glycation end products (AGEs) is a key pathophysiological event with links to a range of important human diseases. It is now clear that AGEs may act as mediators, not only of diabetic complications(1 2) but also of widespread age related pathology such as Alzheimer's disease,(3) decreased skin elasticity,(4) (5) male erectile dysfunction,(6) (7) pulmonary fibrosis,(8) and atherosclerosis.(9 10) Since many cells and tissues of the eye are profoundly influenced by both diabetes and ageing, it is fitting that advanced glycation is now receiving considerable attention as a possible modulator in important visual disorders. An increasing number of reports confirm widespread AGE accumulation at sites of known ocular pathology and demonstrate how these products mediate crosslinking of long lived molecules in the eye. Such studies also underscore the putative pathophysiological role of advanced glycation in ocular cell dysfunction in vitro and in vivo. This article reviews some of the important effects that advanced glycation has on ocular tissues and the role that AGEs, and their specific receptors, have in the initiation and progression of sight threatening disorders such as diabetic retinopathy, glaucoma, cataract formation, and age related macular degeneration (AMD). This review also considers pharmacological strategies to prevent or neutralise the effects of AGEs and the recent development of potential therapies for AGE induced disease processes.
The advanced stage of the Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it has been proposed that the intermediates contributing to AGE formation include dicarbonyl intermediates such as glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). In the present study, we developed a novel, non-carboxymethyllysine (CML) anti-AGE antibody that recognizes serum proteins and peptides modified by 3-DG in vivo.
AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with 3-DG or D-glucose. After immunization of rabbits, anti-AGE antisera were subjected to affinity chromatography on a Sepharose 4B column coupled with CML-BSA, or AGE-BSA created by incubation with 3-DG (AGE-6) or D-glucose (AGE-1). The AGE-Ab-6 and AGE-Ab-1 thus obtained was used to investigate AGEs in serum from diabetic patients on hemodialysis.
Characterization of the novel AGE-Ab-6 obtained by immunoaffinity chromatography was performed with a competitive ELISA and immunoblot analysis. This antibody specifically cross-reacted with proteins modified by 3-DG. AGE-6 was detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD, while AGE-1 was detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD.
This study provides new data on the pathways of AGE formation from 3-DG and methods for the immunochemical detection of AGEs. We also provide immunochemical evidence for the existence of six distinct AGEs in vivo among the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis.
In adults, advanced glycation end products (AGEs) rise slowly in tissues and circulation during aging, and accumulate at an accelerated rate both in diabetes and chronic renal insufficiency (CRI). We aimed to investigate the pattern of AGE accumulation in children/adolescents with CRI and on renal replacement therapy by dialysis and transplantation. Concentrations of fluorescent AGEs, carboxymethyllysine (CML) and lipofuscin-like substance (LFLS, a marker of lipid peroxidation) were followed. Data were obtained from 11 CRI patients on conservative treatment (age 12.6+/-1.7 years, serum creatinine: 205.7+/-17.5 micromol/l), ten patients on renal replacement therapy with dialysis (13.6+/-1.7 years, 698.2+/-48.9 micromol/l) and nine patients after kidney transplantation (15.9+/-1.1 years, 115.9+/-12.0 micromol/l) and comparison made with the data from 28 healthy controls (11.8+/-8.2 years, 44.1+/-8.2 micromol/l). In controls, an age-dependent rise of fluorescent AGE and CML levels was observed. In the CRI group, fluorescent AGEs [0.38+/-0.03x105 arbitrary units (AU)] and CML (369+/-26 ng/ml) concentrations were doubled compared with controls (0.16+/-0.03x105 AU and 189+/-42 ng/ml, respectively) and even higher levels were revealed in dialyzed patients (0.80+/-0.05x105 AU; 650+/-94 ng/ml). Successful kidney transplantation significantly reduced but did not normalize fluorescent AGE levels (0.39+/-0.03 x105 AU), while the decline in CML levels (550+/-47 ng/ml) was insignificant. Plasma LFLS was elevated in CRI (19.6+/- 1.7 AU) and was even higher in dialyzed children (32.0+/-5.3 AU) compared with healthy controls (7.1+/- 1.4 AU). Kidney transplantation did not normalize LFLS levels (20.3+/-5.3 AU), pointing to persistently enhanced lipid peroxidation. Our study provides the first data on enhanced fluorescent AGEs and CML levels in children/adolescents with CRI and on dialysis. Successful renal transplantation decreased but did not normalize AGE levels, probably because of still-impaired renal function with enhanced oxidative stress, as well as the influence of immunosuppressive therapy.
Raman spectroscopy using a safe laser energy of 95 μJ was used to determine noninvasive the presence of silicone oil in the anterior chamber of a patient with a central opaque cornea.
Hyperglycaemia is the major risk factor for the development of complications in both Type I and Type II diabetes; however, there is growing evidence from several clinical trials that dyslipidaemia, including hypertriglyceridaemia, is a significant and independent risk factor for diabetic complications. In this paper, we propose that chemical modification of proteins by lipids may be a underlying pathogenic mechanism linking dyslipidaemia to diabetic complications. Thus the major AGEs (advanced glycation end-products) in tissues, such as carboxymethyl-lysine, carboxyethyl-lysine and hydroimidazolones, may, in fact, be ALEs (advanced lipoxidation end-products), derived from lipids. Increased lipid peroxidation and accelerated ALE formation, possibly catalysed by hyperglycaemia and oxidative stress, may be the mechanistic link between dyslipidaemia and diabetic complications. If correct, this proposal would suggest that inhibition or reversal of glycation, which is a central theme of this symposium, may not be sufficient for protection against diabetic complications.
PURPOSE. To determine whether there is an age-related increase of pentosidine in human Bruch's membranes and to localize pentosidine and carboxymethyllysine (CML), two well-characterized, advanced glycation end products (AGEs) in aged human Bruch's membranes and choroid in vivo. METHODS. Human Bruch's membrane samples were isolated from the retinal pigment epithelium (RPE) and choroid and subjected to reversed-phase high-performance liquid chromatography to determine pentosidine content. A polyclonal anti-pentosidine antibody and a monoclonal antibody specific for carboxymethyllysine were used to localize AGEs in 20-month-old nondiabetic, 82-year-old nondiabetic, and 82-year-old diabetic globes. RESULTS. Human Bruch's membranes (n = 20) showed a linear age-dependent increase in pentosidine that reached approximately 0.17 millimoles pentosidine per mole hp droxyproline in late life (r = 0.896; P < 0.001). Immunohistochemical evaluation showed evidence of pentosidine in Bruch's membrane, choroidal extracellular matrix, and vessel walls in the 82-year-old nondiabetic and diabetic globes. A similar staining pattern was found with the anti-CML antibody. Basal laminar deposits and drusen stained with both antibodies in the elderly nondiabetic eye. In contrast, neither antibody stained the 20-month-old tissue. CONCLUSIONS. We provide biochemical and immunohistochemical evidence for the formation of pentosidine and CML structures in human Bruch's membrane and choroid with age. These changes could promote aging of the RPE-Bruch's membrane-choroid complex.
PURPOSE. Lipofuscin (LF) accumulation in the retinal pigment epithelium (RPE) is associated with age and various retinal diseases. Toxic LF compounds may interfere with normal RPE function. Oxidative modification of proteins was determined in LF granules from human eyes. METHODS. LF was isolated from the RPE-choroid complex of 10 pairs of donor eyes by gradient ultracentrifugation. Protein compounds were separated by two-dimensional (2-D) gel electrophoresis and screened by Western blot analysis for lipid peroxidation- or glucoxidation-induced damage-in particular, by malondialdehyde (MDA), 4-hydroxynonenal (HNE), and advanced glycation end products (AGEs). Identity of the immunostained proteins was revealed using 2-D software for comparison of the spot position with Coomassie-stained 2-D gels of the same samples. RESULTS. By comparing the results taken from the authors' previous proteome analysis of RPE LF with an immunoblot analysis of the same samples, this study shows that a variety of LF-associated proteins were damaged by aberrant covalent modifications of MDA, 4-HNE, and AGEs. Several proteins were altered by two or three different modification types. Modified mitochondrial proteins indicated that autophagy of altered proteins also contributed to lipofuscin formation. CONCLUSIONS. The identification of lipid peroxidation and glucoxidation products in proteinaceous LF components in human RPE supports the hypothesis that these compounds are involved in lipofuscinogenesis and may contribute to the cytotoxic effects of LF in retinal diseases such as age-related macular degeneration and Stargardt disease. Their identification may help to identify potential future treatment targets.
Objective To investigate the localization of Nϵ -(carboxymethyl)lysine (CML), a component and major immunologic epitope of advanced glycation end products, in aged eyes and choroidal neovascular membranes (CNVMs) surgically excised from eyes with age-related macular degeneration. Methods Immunohistochemistry for CML was performed using 8 snap-frozen, surgically excised CNVMs. Twelve eyes from patients aged 69 to 82 years and 2 donor eyes, 1 each from a 23-week-old fetus and 21-year-old patient, without age-related macular degeneration or diabetic retinopathy were also examined. To determine if retinal pigment epithelial cells in CNVMs accumulate advanced glycation end products, cytokeratin and CML were stained in paired serial sections. Results Soft, macular drusen and/or basal laminar and basal linear deposits were observed in 8 of 12 aged eyes. Each case showed CML accumulation, while overlying retinal pigment epithelial cells showed no accumulation in all 12 eyes. In CNVMs, however, retinal pigment epithelial cells showed CML accumulation in their cytoplasm. Conclusion The additional accumulation of advanced glycation end products in soft, macular drusen and/or retinal pigment epithelial cells may play a role in the pathogenesis of CNVM formation in age-related macular degeneration. Clinical Relevance Recently, advanced glycation end products have been found to play a role both in aging changes and neovascularization. Localization of advanced glycation end products in the above-mentioned tissue may lead to a better understanding of the pathogenesis of age-related macular degeneration.
In this study, we show the feasibility of a new type of cell based biosensor which uses spectroscopic in situ real time detection of biochemical changes in living cells exposed to toxic chemical agents. We used a high power 785 nm laser to measure the time dependent changes in the Raman spectrum of individual living human lung cells (A549 cell line) treated with a toxic agent (Triton X-100, 250 μM solution).Individual cells were monitored by Raman spectroscopy over a total time span of 420 min, with 30 min sampling intervals. During this period of time, the A549 cells were maintained in a purpose designed temperature controlled cell chamber, which allowed the cells to be maintained in physiological conditions.The time dependent changes in the Raman spectra were correlated with the sequences of events that occur during cell death. The molecular mechanisms involved in cell death are indicated by the decrease in the magnitude of Raman peaks corresponding to proteins (1322, 1342 and 1005 cm−1) and DNA (decrease by 80–90% in the 786 cm−1 phosphodiester bonds C5′–O–P–O–C3′). To support these conclusions, viability tests and Western blotting analysis of PARP protein were carried out.This technique could overcome the limitations of other detection systems available, since the specific time dependent biochemical changes in the living cells can be used for the identification and quantification of a large range of toxic agents. This technique could also be used with cellular microarrays for high throughput in vitro toxicological testing of pharmaceuticals and in situ monitoring of the growth of engineered tissues.
The retina represents a paradox, in that, while light and oxygen are essential for vision, these conditions also favour the formation of reactive oxygen species leading to photochemical damage to the retina. Such light damage seems to be multi-factorial and is dependent on the photoreactivity of a variety of chromophores (e.g., vitamin A metabolites, lipofuscin, melanin, flavins, porphyrins, carotenoids) endogenous to the retina. The aim of this article is to provide a detailed review of our current understanding of the photochemistry and photobiology of these chromophores and to consider how they may contribute to retinal ageing and pathology.
A common detection and classification system is needed for epidemiologic studies of age-related maculopathy (ARM). Such a grading scheme for ARM is described in this paper. ARM is defined as a degenerative disorder in persons ≥50 years of age characterized on grading of color fundus transparencies by the presence of the following abnormalities in the macular area: soft drusen ≥63μm, hyperpigmentation and/or hypopigmentation of the retinal pigment epithelium (RPE), RPE and associated neurosensory detachment, (peri)retinal hemorrhages, geographic atrophy of the RPE, or (peri)retinal fibrous scarring in the absence of other retinal (vascular) disorders. Visual acuity is not used to define the presence of ARM. Early ARM is defined as the presence of drusen and RPE pigmentary abnormalities described above; late ARM is similar to age-related macular degeneration (AMD) and includes dry AMD (geographic atrophy of the RPE in the absence of neovascular AMD) or neovascular AMD (RPE detachment, hemorrhages, and/or scars as described above). Methods to take and grade fundus transparencies are described.
N-epsilon-(Carboxymethyl)lysine (CML) adduct, a major structure of advanced glycation end product, facilitated production of immature microvessels from choroidal explant cultured in fibrin gel. The present study was investigated an action of endogenous CML adduct on neovascularization of cultured choroidal explants of aged Wistar rats with 9 months of age. The number of microvessels budded from explants was counted under optical microscope and used as an index of in vitro neovascularization. Aged choroidal explants increased the neovascularization in an age-dependent manner. Anti-CML antibody decreased age-facilitated neovascularization as well as CML-human serum albumin (HSA)-facilitated neovascularization. Both the aged explant and CML-HSA-treated explant significantly released vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) alpha and platelet-derived growth factor (PDGF)-B during the culture period. The release of TNF alpha and PDGF-B was earlier than that of VEGF from the aged explants. The antibodies against these factors decreased the CML-facilitated and age-facilitated neovascularization in the choroidal explants. The inhibitory capacity of anti-TNF alpha antibody was greater than those of anti-VEGF and anti-PDGF-B antibodies. In conclusion, endogenous CML adduct overproduced the neovascularization of the aged choroidal explant. The CML adduct releases TNF alpha which might induce the production and release of VEGF for the abnormal choroidal neovascularization in the patients of age-related macular degeneration.
PurposeThe aim of the study was to describe the 10-year incidence and progression of retinal drusen, retinal pigmentary abnormalities, and signs of late age-related maculopathy.
The glycation of proteins by glucose has been linked to the development of diabetic complications and other diseases. Early glycation is thought to involve the reaction of glucose with N-terminal and lysyl side chain amino groups to form Schiffs base and fructosamine adducts. The formation of the alpha-oxoaldehydes, glyoxal, methylglyoxal and 3-deoxyglucosone, in early glycation was investigated. Glucose (50 mM) degraded slowly at pH 7.4 and 37 degrees C to form glyoxal, methylglyoxal and 3-deoxyglucosone throughout a 3-week incubation period. Addition of t-BOC-lysine and human serum albumin increased the rate of formation of alpha-oxoaldehydes - except glyoxal and methylglyoxal concentrations were low with albumin, as expected from the high reactivity of glyoxal and methylglyoxal with arginine residues. The degradation of fructosyl-lysine also formed glyoxal, methyl-glyoxal and 3-deoxyglucosone. alpha-Oxoaldehyde formation was dependent on the concentration of phosphate buffer and availability of trace metal ions. This suggests that alpha-oxoaldehydes were formed in early glycation from the degradation of glucose and Schiffs base adduct. Since alpha-oxoaldehydes are important precursors of advanced glycation adducts, these adducts may be formed from early and advanced glycation processes. Short periods of hyperglycaemia, as occur in impaired glucose tolerance, may be sufficient to increase the concentrations of alpha-oxoaldehydes in vivo.
Receptor for advanced glycation end products (RAGE) is a multiligand member of the immunoglobulin superfamily of cell surface molecules whose repertoire of ligands includes advanced glycation end products (AGEs), amyloid fibrils, amphoterins and S100/calgranulins. The overlapping distribution of these ligands and cells overexpressing RAGE results in sustained receptor expression which is magnified via the apparent capacity of ligands to upregulate the receptor. We hypothesize that RAGE-ligand interaction is a propagation factor in a range of chronic disorders, based on the enhanced accumulation of the ligands in diseased tissues. For example, increased levels of AGEs in diabetes and renal insufficiency, amyloid fibrils in Alzheimer’s disease brain, amphoterin in tumors and S100/calgranulins at sites of inflammation have been identified. The engagement of RAGE by its ligands can be considered the ‘first hit’ in a two-stage model, in which the second phase of cellular perturbation is mediated by superimposed accumulation of modified lipoproteins (in atherosclerosis), invading bacterial pathogens, ischemic stress and other factors. Taken together, these ‘two hits’ eventuate in a cellular response with a propensity towards tissue destruction rather than resolution of the offending pathogenic stimulus. Experimental data are cited regarding this hypothesis, though further studies will be required, especially with selective low molecular weight inhibitors of RAGE and RAGE knockout mice, to obtain additional proof in support of our concept.
Recent progress in structure elucidation of products of the advanced Maillard reaction now allows probing specifically for the role of this reaction in the pathogenesis of age- and diabetes-related complications. Pyrraline is a glucose-derived advanced glycation end product against which polyclonal and monoclonal antibodies have been raised. Immunohistochemical localization studies revealed that pyrraline is found predominantly in the sclerosed extracellular matrix of glomerular and arteriolar renal tissues from both diabetic and aged nondiabetic individuals. Pentosidine and carboxymethyllysine are Maillard end products derived from both glucose and ascorbate. In addition, pentosidine can be formed from several other sugars under oxidative conditions, and in vitro studies suggest that a common intermediate involving a pentose is a necessary precursor molecule. The highest levels of these advanced Maillard products are generally found in the extracellular matrix, but these products are also present in lens proteins and in proteins with a fast turnover such as plasma proteins. Diabetes, and especially uremia, greatly catalyzes pentosidine formation. Both conditions are characterized by accelerated cataractogenesis, atherosclerosis, and neuropathy, suggesting that molecular damage by advanced Maillard reaction products may be a common mechanism in their development.
Pentosidine is a fluorescent advanced Maillard/glycosylation product and protein cross-link present in elevated amounts in skin from diabetic and uremic subjects. A high-performance liquid chromatographic (HPLC) assay was developed to quantitate pentosidine in plasma and erythrocytes and other tissue proteins with low levels of pentosidine. High protein content and presence of basic amino acids and O2 during acid hydrolysis led to the formation of fluorescent artifacts that could be separated from true pentosidine through combined reverse-phase ion-exchange HPLC. No true pentosidine was formed during acid hydrolysis of ribated protein, suggesting that Amadori products do not generate artifactual pentosidine during hydrolysis. With the combined reverse-phase ion-exchange chromatographic assay, we found a 2.5-fold (P less than 0.001) and a 23-fold (P less than 0.001) elevation of mean +/- SD plasma protein pentosidine in diabetic (2.4 +/- 1.2 pmol/mg) and uremic (21.5 +/- 10.8 pmol/mg) subjects compared with healthy (0.95 +/- 0.33 pmol/mg) subjects. Pentosidine in hemolysate was normal in diabetes but dramatically elevated in uremia (0.6 +/- 0.4 pmol/mg hemoglobin, P less than 0.001). Although the precise nature of the pentosidine precursor sugar is unknown, plasma pentosidine may be a useful marker for monitoring the biochemical efficacy of trials with aminoguanidine or other treatment modalities. Furthermore, pentosidine in plasma proteins may act as a signal for advanced glycosylation end product-mediated receptor uptake by macrophages and other cells and contribute to accelerated atherosclerosis in diabetes and uremia.
Phagosomes are converted to phagolysosomes and then to residual bodies (also known as lipofuscin granules or age pigment). Lipofuscin granules of retinal pigment epithelial (RPE) cells of single human eyes were isolated and analysed for enzyme content and fluorescence spectra. The granules are low in lysosomal enzymes and they fluoresce yellow-gold. Thin layer chromatography reveals several separable constituents. Lipofuscin granules accumulate with age, the largest increase occurring after the first decade of life in humans. Macular RPE cells accumulate granules more than non-macular cells.
A common detection and classification system is needed for epidemiologic studies of age-related maculopathy (ARM). Such a grading scheme for ARM is described in this paper. ARM is defined as a degenerative disorder in persons > or = 50 years of age characterized on grading of color fundus transparencies by the presence of the following abnormalities in the macular area: soft drusen > or = 63 microns, hyperpigmentation and/or hypopigmentation of the retinal pigment epithelium (RPE), RPE and associated neurosensory detachment, (peri)retinal hemorrhages, geographic atrophy of the RPE, or (peri)retinal fibrous scarring in the absence of other retinal (vascular) disorders. Visual acuity is not used to define the presence of ARM. Early ARM is defined as the presence of drusen and RPE pigmentary abnormalities described above; late ARM is similar to age-related macular degeneration (AMD) and includes dry AMD (geographic atrophy of the RPE in the absence of neovascular AMD) or neovascular AMD (RPE detachment, hemorrhages, and/or scars as described above). Methods to take and grade fundus transparencies are described.
Pyrraline (epsilon-2-(formyl-5-hydroxymethyl-pyrrol-1-yl)-L-norleucine) is an advanced Maillard reaction product formed from 3-deoxyglucosone in the non-enzymatic reaction between glucose and the epsilon-amino group of lysine residues on proteins. Although its presence in vivo as well as in in vitro incubations of proteins with sugars has been documented by immunochemical methods using polyclonal and monoclonal antibodies, its formation in proteins has recently been questioned by similar methodology. To clarify this issue, we investigated pyrraline formation in proteins following alkaline hydrolysis and quantitation by high-performance liquid chromatography on a C18 reverse-phase column. Time- and sugar concentration-dependent increase in pyrraline formation was noted in serum albumin incubated with either 100 mM glucose or 50 mM 3-deoxyglucosone. Formation of pyrraline from 3-deoxyglucosone was rapid at slightly acidic pH, confirming its synthetic pathway through this Maillard reaction intermediate. Low levels of pyrraline (< 10 pmol/mg protein) were also detected in a pool of human skin collagen by this method, but no age effect was apparent. Using a slightly different approach, pyrraline-like material was detected in human plasma proteins following enzyme digestion and analysis by high performance liquid chromatography. Plasma from diabetic patients showed a significant increase in pyrraline-like material compared to controls. The levels in diabetic and normal individuals were 21.6 +/- 9.56 and 12.8 +/- 5.6 pmol per mg protein, respectively (P = 0.005), reflecting thereby the elevated levels of the immediate precursor of pyrraline, 3-deoxyglucosone, in diabetic plasma.
The hydrodynamic properties of isolated human Bruch's membrane and choroid were investigated as a function of age and retinal location. Macular and peripheral regions of the fundus showed an exponential decline of hydraulic conductivity with half-lives of 15 and 22 years respectively. Comparison of age profiles for hydraulic conductivity and lipid deposits suggests the involvement of two discrete processes for reduction in transport capability. The first appears to involve 'membrane remodelling' with a programmed decay rate leading to a major reduction in hydraulic conductivity by the fifth decade of life. The second commences in the fourth decade and is apparently dependent on the lipid content of Bruch's membrane.
Advanced glycation endproducts have been implicated in a number of diabetic and aging changes. Some of these effects occur in part through induction of cytokines such as platelet-derived growth factor (PDGF), which is expressed by the retinal pigment epithelium (RPE). In this study, cultures of RPE were evaluated for PDGF expression after treatment with pentosidine, a well characterized advanced glycation endproduct. Northern analysis provided evidence for the increased expression of a 3.7 kb PDGF-B transcript over unstimulated controls in the established ARPE-19 cell line. Western analysis demonstrated increased PDGF-BB protein in conditioned medium compared to controls of ARPE-19 cells. In addition, two different early passage cultures of RPE showed increased PDGF-BB protein after pentosidine treatment compared to unstimulated controls. The enhanced production of PDGF-BB could play a role in the maintenance of the RPE-Bruch's membrane complex and influence changes associated with diabetes and aging.
To investigate the localization of N epsilon-(carboxymethyl)lysine (CML), a component and major immunologic epitope of advanced glycation end products, in aged eyes and choroidal neovascular membranes (CNVMs) surgically excised from eyes with age-related macular degeneration.
Immunohistochemistry for CML was performed using 8 snap-frozen, surgically excised CNVMs. Twelve eyes from patients aged 69 to 82 years and 2 donor eyes, 1 each from a 23-week-old fetus and 21-year-old patient, without age-related macular degeneration or diabetic retinopathy were also examined. To determine if retinal pigment epithelial cells in CNVMs accumulate advanced glycation end products, cytokeratin and CML were stained in paired serial sections.
Soft, macular drusen and/or basal laminar and basal linear deposits were observed in 8 of 12 aged eyes. Each case showed CML accumulation, while overlying retinal pigment epithelial cells showed no accumulation in all 12 eyes. In CNVMs, however, retinal pigment epithelial cells showed CML accumulation in their cytoplasm.
The additional accumulation of advanced glycation end products in soft, macular drusen and/or retinal pigment epithelial cells may play a role in the pathogenesis of CNVM formation in age-related macular degeneration.
Recently, advanced glycation end products have been found to play a role both in aging changes and neovascularization. Localization of advanced glycation end products in the above-mentioned tissue may lead to a better understanding of the pathogenesis of age-related macular degeneration.
Age-related macular disease is a major and growing public health burden in developed Caucasian societies, accounting for about 50% of blind registration. Evidence exists that this is an emerging problem in Eastern Asia, although the phenotype appears to differ from that seen in Western society. It is likely that several genes are involved, and that the genes or allelic variants conferring are common. Environment plays a major role in its pathogenesis, and it is believed that genetic susceptibility becomes apparent only if there are sufficient environmental pressures. There is no therapy currently available that will have an impact on the prevalence of blindness from age-related macular disease. It has been shown that visual loss occurs as a reaction to ageing changes in Bruch's membrane, which is interposed between the choriocapillaris and the retinal pigment epithelium. The age changes in all three structures have been partly characterised, and as a consequence, multiple putative pathogenic mechanisms have been proposed. Cross-sectional studies of populations with different genetic background and life styles would serve to prove the importance of inheritance and environment. Molecular genetic analysis of blood from affected sibling pairs from these sources may indicate the relevant genes, the prevalence of which may differ in different communities. Enquiries as to life styles may determine important environmental influences. Examination of donor eyes from these communities may reveal distinctive features that may reflect the variation in genetic predisposition and environmental pressures. It is hoped that the findings from such studies will lead to novel and potentially successful management strategies.
The glycation of proteins by glucose has been linked to the development of diabetic complications and other diseases. Early glycation is thought to involve the reaction of glucose with N-terminal and lysyl side chain amino groups to form Schiff's base and fructosamine adducts. The formation of the alpha-oxoaldehydes, glyoxal, methylglyoxal and 3-deoxyglucosone, in early glycation was investigated. Glucose (50 mM) degraded slowly at pH 7.4 and 37 degrees C to form glyoxal, methylglyoxal and 3-deoxyglucosone throughout a 3-week incubation period. Addition of t-BOC-lysine and human serum albumin increased the rate of formation of alpha-oxoaldehydes - except glyoxal and methylglyoxal concentrations were low with albumin, as expected from the high reactivity of glyoxal and methylglyoxal with arginine residues. The degradation of fructosyl-lysine also formed glyoxal, methylglyoxal and 3-deoxyglucosone. alpha-Oxoaldehyde formation was dependent on the concentration of phosphate buffer and availability of trace metal ions. This suggests that alpha-oxoaldehydes were formed in early glycation from the degradation of glucose and Schiff's base adduct. Since alpha-oxoaldehydes are important precursors of advanced glycation adducts, these adducts may be formed from early and advanced glycation processes. Short periods of hyperglycaemia, as occur in impaired glucose tolerance, may be sufficient to increase the concentrations of alpha-oxoaldehydes in vivo.
Senescence of human cells has largely been studied as an in vitro phenomenon resulting from replicative exhaustion. The literature contains many studies of retinal pigment epithelium (RPE) cells which document replicative senescence. Several studies by Burke and others illustrate the relationship between donor age and replicative lifespan, the relationship between geographical location of RPE in the posterior pole and replicative lifespan, and the phenomena of altered cellular morphology and decreased culture saturation density for senescent RPE cells. Other studies have focused on the alterations of the expression of specific genes or the alteration of enzymatic activities during the senescence of RPE cells in vitro. Recently, a technique utilizing a histochemical staining procedure for beta galactosidase has been developed which identifies senescent cells. Normal beta galactosidase histochemistry which identifies the lysosomal form of the enzyme is performed at pH 4.0, while senescence-associated beta galactosidase activity is observed at pH 6.0 and is observed in the cytoplasm. We have studied the replicative senescence of human RPE cells in vitro using this procedure and have also measured the length of chromosomal telomeres to identify the aging of cultures in vitro. Our results show that RPE cultures accumulate beta galactosidase positive cells as a function of the number of population doublings and that these data correlate with the shortening of chromosomal telomeres to a functional limit observed for many human cell types at senescence. We have also recently extended this work to the development of a senescence-associated beta galactosidase procedure for observing senescent RPE cells in vivo. Basically, the same histochemical procedure is used with a post-staining bleaching step to clearly visualize staining in the RPE. Our first studies were performed on globes from Rhesus monkeys at a variety of ages from 1 year to 29 years of age. The results show the accumulation of beta galactosidase positive cells in the older monkey eyes. We have also examined several human eyes in an attempt to observe whether any relationship exists between beta galactosidase staining and age, pathology (diabetes, basal laminar deposits), and geographical location (macula vrs. periphery). These studies represent a first effort to determine if senescent RPE are present in vivo. It will be important to extend these studies so that these data might be expressed on a quantitative bases.
Collagen molecules in articular cartilage have an exceptionally long lifetime, which makes them susceptible to the accumulation
of advanced glycation end products (AGEs). In fact, in comparison to other collagen-rich tissues, articular cartilage contains
relatively high amounts of the AGE pentosidine. To test the hypothesis that this higher AGE accumulation is primarily the
result of the slow turnover of cartilage collagen, AGE levels in cartilage and skin collagen were compared with the degree
of racemization of aspartic acid (% d-Asp, a measure of the residence time of a protein). AGE (N
ε-(carboxymethyl)lysine,N
ε-(carboxyethyl)lysine, and pentosidine) and % d-Asp concentrations increased linearly with age in both cartilage and skin collagen (p < 0.0001). The rate of increase in AGEs was greater in cartilage collagen than in skin collagen (p < 0.0001). % d-Asp was also higher in cartilage collagen than in skin collagen (p< 0.0001), indicating that cartilage collagen has a longer residence time in the tissue, and thus a slower turnover, than
skin collagen. In both types of collagen, AGE concentrations increased linearly with % d-Asp (p < 0.0005). Interestingly, the slopes of the curves of AGEs versus% d-Asp, i.e. the rates of accumulation of AGEs corrected for turnover, were identical for cartilage and skin collagen. The present study
thus provides the first experimental evidence that protein turnover is a major determinant in AGE accumulation in different
collagen types. From the age-related increases in % d-Asp the half-life of cartilage collagen was calculated to be 117 years and that of skin collagen 15 years, thereby providing
the first reasonable estimates of the half-lives of these collagens.
Age-related macular degeneration (AMD) is the leading cause of blind registration in the developed world, and yet its pathogenesis remains poorly understood. Oxidative stress, which refers to cellular damage caused by reactive oxygen intermediates (ROI), has been implicated in many disease processes, especially age-related disorders. ROIs include free radicals, hydrogen peroxide, and singlet oxygen, and they are often the byproducts of oxygen metabolism. The retina is particularly susceptible to oxidative stress because of its high consumption of oxygen, its high proportion of polyunsaturated fatty acids, and its exposure to visible light. In vitro studies have consistently shown that photochemical retinal injury is attributable to oxidative stress and that the antioxidant vitamins A, C, and E protect against this type of injury. Furthermore, there is strong evidence suggesting that lipofuscin is derived, at least in part, from oxidatively damaged photoreceptor outer segments and that it is itself a photoreactive substance. However, the relationships between dietary and serum levels of the antioxidant vitamins and age-related macular disease are less clear, although a protective effect of high plasma concentrations of alpha-tocopherol has been convincingly demonstrated. Macular pigment is also believed to limit retinal oxidative damage by absorbing incoming blue light and/or quenching ROIs. Many putative risk-factors for AMD have been linked to a lack of macular pigment, including female gender, lens density, tobacco use, light iris color, and reduced visual sensitivity. Moreover, the Eye Disease Case-Control Study found that high plasma levels of lutein and zeaxanthin were associated with reduced risk of neovascular AMD. The concept that AMD can be attributed to cumulative oxidative stress is enticing, but remains unproven. With a view to reducing oxidative damage, the effect of nutritional antioxidant supplements on the onset and natural course of age-related macular disease is currently being evaluated.
Aetiological and immunological aspects of AMD, a leading cause of blindness in Western countries, have been reviewed. Developmental studies suggest that anatomical features unique to the fovea result in a critical relationship between metabolic demand and blood supply at the macula, which is maintained throughout life. Recent studies show a sufficient degree of consistency in the link between smoking and both dry and wet AMD to regard it as causative. Dry AMD is considered to be the natural endstage of the disease; epidemiological and morphological studies point to choroidal vascular atrophy as the causative event and it is suggested that signals associated with acute vascular compromise lead to the development of subretinal neovascularisation. The relationship between sub-pigment epithelial deposits, including basal laminar deposit, and the pathogenesis of AMD is examined. Much of the literature is consistent with a choroidal origin for the constituents of drusen. The blood-retinal barrier preserves the physiological environment of the neural retina and limits inflammatory responses. The factors, including cytokines, adhesion molecules and the presence of resident immunocompetent cells (microglia), which determine the immune status of the retina are considered. Historical descriptions of the involvement of inflammatory cells are provided, evidence implicating inflammation in the pathogenesis of AMD involving macrophages, giant cells and microglia has been derived from observations of human and animal subretinal neovascular lesions. The role of humoral factors such as anti-retinal autoantibodies and acute phase proteins together with clinical observations has been surveyed. Taken together these data demonstrate the involvement of both cellular and humoral immunity in the pathogenesis of AMD. It remains to be determined to what degree the influence of immunity is causative or contributory in both wet and dry AMD, however, the use of anti-inflammatory agents to ameliorate the condition further indicates the existence of an inflammatory component.
We have previously shown that Raman spectroscopy can be used for chemical analysis of intact human coronary artery atherosclerotic lesions ex vivo without tissue homogenization or extraction. Here, we report the chemical analysis of individual cellular and extracellular components of atherosclerotic lesions in different stages of disease progression in situ using Raman microspectroscopy.
Thirty-five coronary artery samples were taken from 16 explanted transplant recipient hearts, and thin sections were prepared. Using a high-resolution confocal Raman microspectrometer system with an 830-nm laser light, high signal-to-noise Raman spectra were obtained from the following morphologic structures: internal and external elastic lamina, collagen fibers, fat, foam cells, smooth muscle cells, necrotic core, beta-carotene, cholesterol crystals, and calcium mineralizations. Their Raman spectra were modeled by using a linear combination of basis Raman spectra from the major biochemicals present in arterial tissue, including collagen, elastin, actin, myosin, tropomyosin, cholesterol monohydrate, cholesterol linoleate, phosphatidyl choline, triolein, calcium hydroxyapatite, calcium carbonate, and beta-carotene.
The results show that the various morphologic structures have characteristic Raman spectra, which vary little from structure to structure and from artery to artery. The biochemical model described the spectrum of each morphologic structure quite well, indicating that the most essential biochemical components were included in the model. Furthermore, the biochemical composition of each structure, indicated by the fit contributions of the biochemical basis spectra of the morphologic structure spectrum, was very consistent.
The Raman spectra of various morphologic structures in normal and atherosclerotic coronary artery may be used as basis spectra in a linear combination model to analyze the morphologic composition of atherosclerotic coronary artery lesions.
The retinal pigment epithelium (RPE) is a single layer of post-mitotic cells, which functions both as a selective barrier to and a vegetative regulator of the overlying photoreceptor layer, thereby playing a key role in its maintenance. Through the expression and activity of specific proteins, it regulates the transport of nutrients and waste products to and from the retina, it contributes to outer segment renewal by ingesting and degrading the spent tips of photoreceptor outer segments, it protects the outer retina from excessive high-energy light and light-generated oxygen reactive species and maintains retinal homeostasis through the release of diffusible factors. The ageing characteristics of the RPE suggest that in addition to cell loss, pleomorphic changes and loss of intact melanin granules, significant metabolic changes occur resulting, at least in part, from the intracellular accumulation of lipofuscin. This pigment has been shown to be highly phototoxic and has been linked to several oxidative changes, some leading to cell death. While the aetiology of age-related macular degeneration is complex and is as yet unresolved, it is likely that accelerated ageing-like changes in the RPE play a fundamental role in the development of this condition.
Over a dozen advanced glycation end-products (AGEs) have been identified in tissue proteins by chemical or immunological methods. Of these, about half are known to accumulate with age in collagen at a rate that correlates with the half-life of the collagen. AGEs may be formed by oxidative and non-oxidative reactions and are in some cases identical to advanced lipoxidation end-products (ALEs) formed in protein during lipid peroxidation reactions. AGEs affect the biochemical and physical properties of proteins and the extracellular matrix (ECM), including the charge, hydrophobicity, turnover and elasticity of collagen, and the cell adhesion, permeability and pro-inflammatory properties of the ECM. A number of scavenger and AGE-specific receptors have been identified that may mediate the turnover of AGE-proteins, catalyze the local production of reactive oxygen species and attract and activate tissue macrophages. Although AGEs in proteins are probably correlative, rather than causative, with respect to aging, they accumulate to high levels in tissues in age-related chronic diseases, such as atherosclerosis, diabetes, arthritis and neurodegenerative disease. Inhibition of AGE formation in these diseases may limit oxidative and inflammatory damage in tissues, retarding the progression of pathophysiology and improve the quality of life during aging.
The human eye is constantly exposed to sunlight and artificial lighting. Therefore the eye is exposed to UV-B (295-320 nm), UV-A (320-400 nm), and visible light (400-700 nm). Light is transmitted through the eye and then signals the brain directing both sight and circadian rhythm. Therefore light absorbed by the eye must be benign. Damage to the young and adult eye by intense ambient light is avoided because the eye is protected by a very efficient antioxidant system. In addition, there are protective pigments such as the kynurenines, located in the human lens, and melanin, in the uvea and retina, which absorb ambient radiation and dissipate its energy without causing damage. After middle age there is a decrease in the production of antioxidants and antioxidant enzymes. At the same time, the protective pigments are chemically modified (lenticular 3-hydroxy kynurenine pigment is enzymatically converted into the phototoxic chromophore xanthurenic acid; melanin is altered from an antioxidant to pro-oxidant) and fluorescent chromophores (lipofuscin) accumulate to concentrations high enough to produce reactive oxygen species. We have known for some time that exposure to intense artificial light and sunlight either causes or exacerbates age-related ocular diseases. We now know many of the reasons for these effects, and with this knowledge methods are being developed to interfere with these damaging processes.
A considerable amount of new information on putative pathogenetic mechanisms in age-related maculopathy and degeneration has emerged in recent years. This comprises experimental studies on retinal pigment epithelium (RPE) and rod photoreceptor ageing, lipofuscin accumulation, the roles of oxidative stress and free radical formation, as well as antioxidants and other defensive mechanisms operating against environmental factors and ageing.
The current application of photodynamic therapy (PDT) using verteporfin marks a new era in the treatment of subfoveal classical choroidal neovascularization. Several new treatment modalities, such as transpupillary thermotherapy (TTT) and anti-vascular endothelial growth factor (VEGF) agents for inhibition of neovascularization, have emerged and are undergoing multicentre clinical trials. A period of dynamic development in this field has commenced.
The aim of the study was to describe the 10-year incidence and progression of retinal drusen, retinal pigmentary abnormalities, and signs of late age-related maculopathy.
Population-based cohort study.
The study included 4926 persons, 43 to 86 years of age at the time of a baseline examination from 1988 through 1990, living in Beaver Dam, Wisconsin, of whom 3684 participated in a 5-year follow-up examination and 2764 participated in a 10-year follow-up.
Characteristics of drusen and other lesions typical of age-related maculopathy were determined by grading stereoscopic color fundus photographs using the Wisconsin Age-Related Maculopathy Grading System.
Incidence of drusen type and size, pigmentary abnormalities, geographic atrophy, and exudative degeneration.
The 10-year incidence of early age-related maculopathy was 12.1% and of late age-related maculopathy it was 2.1%. There was a statistically significant increased incidence of age-related maculopathy lesions with age (P < 0.05). Individuals 75 years of age or older at baseline had significantly (P < 0.01) higher 10-year incidences of the following characteristics than people 43 to 54 years of age: larger sized drusen (125 micro m-249 micro m, 26.3% vs. 3.3%; > or =250 micro m, 16.2% vs. 1.0%), soft indistinct drusen (22.2% vs. 2.2%), retinal pigment abnormalities (19.5% vs. 0.8%), exudative macular degeneration (4.1% vs. 0%), and pure geographic atrophy (3.1% vs. 0%). Compared with those with small numbers of only small, hard drusen (1-2), those with large numbers of only hard drusen (8 or more) had an increased 10-year incidence of both soft drusen (12.3% vs. 6.7%) and pigmentary abnormalities (4.9% vs. 1.7%). Eyes with soft indistinct drusen or retinal pigmentary abnormalities at baseline, were more likely to develop late age-related macular degeneration at follow-up than eyes without these lesions (15.1% vs. 0.4% and 20.0% vs. 0.8%, respectively).
These population-based estimates document the high incidence of signs of age-related maculopathy in people 75 years of age or older. Our findings demonstrate that large numbers of hard drusen predict the incidence of soft drusen and pigmentary abnormalities and that the presence of the latter lesions significantly increases the risk for the development of geographic atrophy and exudative macular degeneration.
The proteasomal pathway is responsible for processes essential for cell viability, including the selective degradation of oxidized proteins. An age-dependent loss in proteasome function has been reported in many tissues, but has not been examined in the retina. In this study, we evaluated proteasome function and protein oxidation in retinal homogenates from young adult and old F344BN rats. For retinal proteasome from old rats, we observed an 80% decrease in the rate of casein degradation and a 75% loss in chymotrypsin-like activity. This loss in activity could be partially accounted for by a 50% reduction in expression of the 20S proteasome. The regulatory complex PA700 and the inducible beta-subunit, LMP7, which is associated with the chymotrypsin-like activity, were expressed in equivalent concentrations relative to the 20S catalytic core in both young and old rats. Immunochemical analysis using antibodies that recognize the protein oxidative modifications, nitrotyrosine and 4-hydroxy-2-nonenal, showed that retinal proteins from old rats exhibited the greatest immunoreactivity. These results suggest that the age-related loss in proteasome function contributes to the accumulation of oxidized retinal proteins. Thus, the combined effect of an increase in oxidized proteins and inactivation of the protease responsible for ridding the cell of oxidized proteins places the aged retina at greater risk for irreversible damage caused by oxidative stress.
Earlier studies have revealed the association of advanced glycation end products (AGE) with the pathogenesis of various micro and macro vascular complications. The purpose of the present study is to localize AGEs, namely carboxy methyl lysine (CML-AGE) and methyl glyoxal-derived AGEs (MG-AGE), in retinal neovascular membranes and to quantify them in serum samples.
Surgically excised retinal neovascular membranes and serum samples obtained from patients with diabetic retinopathy, Eales' disease and nondiabetics were studied. Immunolocalization of AGEs namely CML-AGE and MG-derived AGEs was done using avidin biotin complex method and quantification was done by enzyme linked immunosorbent assay (ELISA).
CML-AGE immunoreactivity was detected in all cases of Eales' disease and 61% cases of diabetic retinopathy and none in idiopathic epiretinal membrane (ERM). MG-AGE immunoreactivity was observed in approximately 15% of diabetic retinopathy and none in Eales' disease and and idiopathic ERM. Quantification of AGEs in serum samples revealed statistically significant increased levels of MG-AGE in diabetes, in relation to nondiabetics with idiopathic ERM and CML-AGE in Eales' disease, in relation to diabetics and nondiabetics with idiopathic ERM.
Results from this study suggest that AGEs formed through glycation and glycoxidation may play an important role in the development of retinal neovascularization. The immunoreactivity of CML-AGEs in neovascular membrane and its increased levels in serum suggest that inspite of the normoglycemic status, glycoxidation and lipid peroxidation due to oxidative stress may trigger retinal neovascularization in Eales' disease, while MG-AGEs in diabetic membrane and serum suggest the role of glycation. Thus the mechanism of neovascularization in different pathological conditions could be different.
Age-related macular degeneration is the principal cause of registered legal blindness among those aged over 65 in the United States, western Europe, Australia, and Japan. Despite intensive research, the precise etiology of molecular events that underlie age-related macular degeneration is poorly understood. However, investigations on parallel fronts are addressing this prevalent public health problem. Sophisticated biochemical and biophysical techniques have refined our understanding of the pathobiology of drusen, geographic atrophy, and retinal pigment epithelial detachments. Epidemiological identification of risk factors has facilitated an intelligent search for underlying mechanisms and fueled clinical investigation of behavior modification. Gene searches have not only brought us to the cusp of identifying the culpable gene loci in age-related macular degeneration, but also localized genes responsible for other macular dystrophies. Recent and ongoing investigations, often cued by tumor biology, have revealed an important role for various growth factors, particularly in the neovascular form of the condition. Transgenic and knockout studies have provided important mechanistic insights into the development of choroidal neovascularization, the principal cause of vision loss in age-related macular degeneration. This in turn has culminated in preclinical and clinical trials of directed molecular interventions.
Lipofuscin (LF) accumulation in the retinal pigment epithelium (RPE) is associated with age and various retinal diseases. Toxic LF compounds may interfere with normal RPE function. Oxidative modification of proteins was determined in LF granules from human eyes.
LF was isolated from the RPE-choroid complex of 10 pairs of donor eyes by gradient ultracentrifugation. Protein compounds were separated by two-dimensional (2-D) gel electrophoresis and screened by Western blot analysis for lipid peroxidation- or glucoxidation-induced damage-in particular, by malondialdehyde (MDA), 4-hydroxynonenal (HNE), and advanced glycation end products (AGEs). Identity of the immunostained proteins was revealed using 2-D software for comparison of the spot position with Coomassie-stained 2-D gels of the same samples.
By comparing the results taken from the authors' previous proteome analysis of RPE LF with an immunoblot analysis of the same samples, this study shows that a variety of LF-associated proteins were damaged by aberrant covalent modifications of MDA, 4-HNE, and AGEs. Several proteins were altered by two or three different modification types. Modified mitochondrial proteins indicated that autophagy of altered proteins also contributed to lipofuscin formation.
The identification of lipid peroxidation and glucoxidation products in proteinaceous LF components in human RPE supports the hypothesis that these compounds are involved in lipofuscinogenesis and may contribute to the cytotoxic effects of LF in retinal diseases such as age-related macular degeneration and Stargardt disease. Their identification may help to identify potential future treatment targets.
In Alzheimer's disease, neurons in affected regions re-enter the cell cycle, leave the G0 state and appear to be arrested at both the G1/S and G2/M phase with resulting cell death, predominantly by apoptosis. Further hallmarks of AD are crosslinked protein deposits (amyloid plaques and neurofibrillary tangles), which time-dependently become modified by "advanced glycation endproducts (AGEs)". Since AGEs activate both mitogenic and redox-sensitive pathways, they might be involved both in cell cycle re-entry and arrest.
Alterations in a wide array of physiological functions are a normal consequence of aging. Importantly, aged individuals exhibit an enhanced susceptibility to various degenerative diseases and appear less able than their young and adult counterparts to withstand (patho)physiological stress. Elucidation of mechanisms at play in the aging process would benefit the development of effective strategies for enhancing the quality of life for the elderly. It is likely that decrements in cellular and physiological function that occur during aging are the net result of numerous interacting factors. The current review focuses on the potential contribution(s) of free radical-mediated modifications to protein structure/function and alterations in the activities of two major proteolytic systems within cells, lysosomes and the proteasome, to the age-dependent accumulation of fluorescent intracellular granules, termed lipofuscin. Specifically, aging appears to influence the interplay between the occurrences of free radical-derived modifications to protein and the ability of cells to carry out critical proteolytic functions. We present immunochemical and ultrastructural evidence demonstrating the occurrence of a fluorescent protein cross-link derived from free radical-mediated reaction(s) within lipofuscin granules of rat cerebral cortex neurons. In addition, we provide evidence that a fluorophore-modified protein present in lipofuscin granules is the alpha subunit of F1F0-ATP synthase, a mitochondrial protein. It has previously been shown that protein(s) bearing this particular fluorescent cross-link are resistant to proteolysis and can inhibit the proteasome in a non-competitive fashion (J. Biol. Chem. 269 (1994a) 21639; FEBS Lett. 405 (1997) 21). Therefore, the current findings demonstrate that free radical-mediated modifications to protein(s) that lead to the production of inhibitor(s) of cellular proteolytic systems are present on specific protein components of lipofuscin. In addition, the mitochondrial origin of one of these proteins indicates specific intracellular pathways likely to be influenced by free radical events and participate in the formation of lipofuscin. The results of these studies are related to previous in vitro and in vivo observations in the field, thus shedding light on potential consequences to cellular function. In addition, future research directions suggested by the available evidence are discussed.
The chemical modification of protein by nonenzymatic browning or Maillard reactions increases with age and in disease. Maillard products are formed by reactions of both carbohydrate- and lipid-derived intermediates with proteins, leading to formation of advanced glycation and lipoxidation end-products (AGE/ALEs). These modifications and other oxidative modifications of amino acids increase together in proteins and are indicators of tissue aging and pathology. In this review, we describe the major pathways and characteristic products of chemical modification of proteins by carbohydrates and lipids during the Maillard reactions and identify major intersections between these pathways. We also describe a new class of intracellular sulfhydryl modifications, Cys-AGE/ALEs, that may play an important role in regulatory biology and represent a primitive link between nonenzymatic and enzymatic chemistry in biological systems.
This review will discuss some of the implications for using cells from aged donors for retinal pigment epithelium (RPE) transplantation. It will consider age-related changes in the structure and function of RPE cells and the accumulation of potentially damaging photoreactive constituents. The review will focus on the role of the ocular pigments lipofuscin and melanin in respect to age-related changes in composition, photoreactivity and potential role in retinal ageing and age-related macular degeneration. The article concludes by considering the suitability of senescent RPE cells for transplantation and whether such cells can be rejuvenated.