ABSTRACT
Hyperbaric oxygen (HBO) treatment of animals or ocular lenses in culture recapitulates many molecular changes observed in human age-related nuclear cataract. The guinea pig HBO model has been one of the best examples of such treatment leading to dose-dependent development of lens nuclear opacities. In this study, complimentary mass spectrometry methods were employed to examine protein truncation after HBO treatment of aged guinea pigs. Quantitative liquid chromatography-mass spectrometry (LC-MS) analysis of the membrane fraction of guinea pig lenses showed statistically significant increases in aquaporin-0 (AQP0) C-terminal truncation, consistent with previous reports of accelerated loss of membrane and cytoskeletal proteins. In addition, imaging mass spectrometry (IMS) analysis spatially mapped the acceleration of age-related αA-crystallin truncation in the lens nucleus. The truncation sites in αA-crystallin closely match those observed in human lenses with age. Taken together, our results suggest that HBO accelerates the normal lens aging process and leads to nuclear cataract.
Subject(s)
Aging/physiology , Cataract/etiology , Crystallins/metabolism , Hyperbaric Oxygenation/adverse effects , Lens Nucleus, Crystalline/metabolism , Proteolysis/drug effects , Animals , Aquaporins/metabolism , Cataract/metabolism , Cataract/pathology , Chromatography, Liquid , Cytoskeletal Proteins/metabolism , Disease Models, Animal , Eye Proteins/metabolism , Guinea Pigs , Lens Nucleus, Crystalline/pathology , Tandem Mass Spectrometry , alpha-Crystallin A Chain/metabolismABSTRACT
This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The senior author contacted the journal in a forthright manner, in an effort to preserve the scientific integrity of the literature, after discovering a significant error in the results reported in the article. The authors were recently made aware of a paper by Kim et al. (Nature Commun. 2019) which shows a spirosome structure (the enzyme aldehyde-alcohol dehydrogenase) present in E. coli (Fig. 5a) that is very similar to the structure the authors thought formed when synthetic alpha A crystallin (66-80) peptide was incubated for 24 h with recombinant guinea pig alpha A insert crystallin (see Kumarasamy et al. Figs. 7C and F, and Fig. 9). Subsequent to publication of their report, the authors later found a number of images that showed what appeared to be the same structure present in samples of their presumably purified recombinant guinea pig alpha A insert crystallin which had been incubated without peptide for 24 h. Hence, the authors now conclude that the structures shown in Figs. 7C and F, and Fig. 9 of their article published in this journal are actually due to E. coli contaminant aldehyde-alcohol dehydrogenase. The authors deeply regret this error and any inconvenience it may have caused.
ABSTRACT
Blood-retinal barrier (BRB) dysfunction represents one of the most significant changes occurring during diabetic retinopathy. We set up a high-reproducible human-based in vitro BRB model using retinal pericytes, retinal astrocytes, and retinal endothelial cells in order to replicate the human in vivo environment with the same numerical ratio and layer order. Our findings showed that high glucose exposure elicited BRB breakdown, enhanced permeability, and reduced the levels of junction proteins such as ZO-1 and VE-cadherin. Furthermore, an increased expression of pro-inflammatory mediators (IL-1ß, IL-6) and oxidative stress-related enzymes (iNOS, Nox2) along with an increased production of reactive oxygen species were observed in our triple co-culture paradigm. Finally, we found an activation of immune response-regulating signaling pathways (Nrf2 and HO-1). In conclusion, the present model mimics the closest human in vivo milieu, providing a valuable tool to study the impact of high glucose in the retina and to develop novel molecules with potential effect on diabetic retinopathy.
Subject(s)
Astrocytes/metabolism , Blood-Retinal Barrier/metabolism , Endothelial Cells/metabolism , Glucose/metabolism , Pericytes/metabolism , Retina/metabolism , Antigens, CD/metabolism , Biomarkers/metabolism , Blood-Retinal Barrier/enzymology , Cadherins/metabolism , Coculture Techniques , Glucose/pharmacology , Heme Oxygenase-1/metabolism , Humans , In Vitro Techniques , Inflammation/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Models, Biological , NADPH Oxidase 2/genetics , NADPH Oxidase 2/metabolism , NF-E2-Related Factor 2/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Oxidative Stress/genetics , Protein Serine-Threonine Kinases/metabolism , Reactive Oxygen Species/metabolism , Zonula Occludens-1 Protein/metabolism , NF-kappaB-Inducing KinaseABSTRACT
Deletion of dystrobrevin binding protein 1 has been linked to Hermansky-Pudlak syndrome type 7 (HPS-7), a rare disease characterized by oculocutaneous albinism and retinal dysfunction. We studied dysbindin-1 null mutant mice (Dys-/-) to shed light on retinal neurodevelopment defects in HPS-7. We analyzed the expression of a focused set of miRNAs in retina of wild type (WT), Dys+/- and Dys-/- mice. We also investigated the retinal function of these mice through electroretinography (ERG). We found that miR-101-3p, miR-137, miR-186-5p, miR-326, miR-382-5p and miR-876-5p were up-regulated in Dys-/-mice retina. Dys-/- mice showed significant increased b-wave in ERG, compared to WT mice. Bioinformatic analysis highlighted that dysregulated miRNAs target synaptic plasticity and dopaminergic signaling pathways, affecting retinal functions of Dys-/- mice. Overall, the data indicate potential mechanisms in retinal neurodevelopment of Dys-/- mice, which may have translational significance in HSP-7 patients, both in terms of diagnostic/prognostic biomarkers and novel pharmacological targets.
Subject(s)
Hermanski-Pudlak Syndrome/drug therapy , Hermanski-Pudlak Syndrome/metabolism , MicroRNAs/metabolism , Molecular Targeted Therapy , Retina/drug effects , Retina/metabolism , Animals , Computational Biology , Gene Expression Regulation/drug effects , Hermanski-Pudlak Syndrome/diagnosis , Hermanski-Pudlak Syndrome/genetics , Mice , Mice, Inbred C57BL , MicroRNAs/blood , MicroRNAs/genetics , PrognosisABSTRACT
This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The senior author contacted the journal in a forthright manner, in an effort to preserve the scientific integrity of the literature, after discovering a significant error in the results reported in the article. The authors were recently made aware of a paper by Kim et al. (Nature Commun. 2019) which shows a spirosome structure (the enzyme aldehyde-alcohol dehydrogenase) present in E. coli (Fig. 5a) that is very similar to the structure the authors thought formed when synthetic alpha A crystallin (66-80) peptide was incubated for 24 h with recombinant guinea pig alpha A insert crystallin (see Kumarasamy et al., Figs. 7C and F, and Fig. 9). Subsequent to publication of their report, the authors later found a number of images that showed what appeared to be the same structure present in samples of their presumably purified recombinant guinea pig alpha A insert crystallin which had been incubated without peptide for 24 h. Hence, the authors now conclude that the structures shown in Figs. 7C and F, and Fig. 9 of their article published in this journal are actually due to E. coli contaminant aldehyde-alcohol dehydrogenase. The authors deeply regret this error and any inconvenience it may have caused.
Subject(s)
Amyloid beta-Peptides/metabolism , Amyloid/metabolism , Lens, Crystalline/drug effects , Peptide Fragments/pharmacology , Protein Aggregates , Temperature , alpha-Crystallin A Chain/metabolism , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Guinea Pigs , Humans , Hydrogen-Ion Concentration , Lens, Crystalline/metabolism , Lens, Crystalline/ultrastructure , Microscopy, Electron, Transmission , Molecular Sequence Data , Recombinant ProteinsABSTRACT
This study investigated poly(ADP-ribose) polymerase-1 (PARP-1) activation in cultured human lens epithelial cells exposed to two levels of UVB light (312 nm peak wavelength), 0.014 and 0.14 J cm-2 ("low" and "high" dose, respectively). At the low dose, PARP-1 and poly(ADP-ribose) (PAR) polymers acted to repair DNA strand breaks rapidly with no subsequent major effects on either cell morphology or viability. However, following the high UVB dose, there was a dramatic second phase of PARP-1 activation, 90 min later, which included a sudden reappearance of DNA strand breaks, bursts of reactive oxygen species (ROS) formation within both the mitochondria and nucleus, a translocation of PAR from the nucleus to the mitochondria and an ultimate 70% loss of cell viability occurring after 24 h. The results provide evidence for an important role for PARP-1 in protecting the human lens epithelium against low levels of UVB light, and possibly participating in the triggering of cell death following exposure to toxic levels of radiation.
Subject(s)
Lens, Crystalline/enzymology , Lens, Crystalline/radiation effects , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolism , Ultraviolet Rays/adverse effects , Cell Death , Cell Line , Cell Nucleus/metabolism , Cell Survival , DNA Damage , Epithelial Cells/cytology , Epithelial Cells/enzymology , Epithelial Cells/radiation effects , Humans , Lens, Crystalline/cytology , Membrane Proteins/genetics , Mitochondria/metabolism , Neoplasm Proteins/genetics , Poly (ADP-Ribose) Polymerase-1/genetics , Reactive Oxygen Species/metabolismABSTRACT
Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide-mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways.
Subject(s)
Cataract/metabolism , Hyperbaric Oxygenation , Lens, Crystalline/metabolism , Peptide Fragments/physiology , alpha-Crystallins/physiology , Animals , Disease Models, Animal , Guinea Pigs , Lens, Crystalline/chemistry , Mass Spectrometry , Molecular Chaperones/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolismABSTRACT
This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2 , 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm(-2) of UVA radiation (338-400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage.
Subject(s)
Epithelial Cells/radiation effects , Glutathione/metabolism , Lens, Crystalline/radiation effects , Thioredoxin Reductase 1/metabolism , Catalase/metabolism , Cell Count , Cell Line, Transformed , Cell Survival/drug effects , Cell Survival/radiation effects , Dinitrochlorobenzene/pharmacology , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Glutathione/antagonists & inhibitors , Humans , Lens, Crystalline/cytology , Lens, Crystalline/drug effects , Lens, Crystalline/metabolism , Methionine Sulfoximine/analogs & derivatives , Methionine Sulfoximine/pharmacology , Oxidative Stress , Oxygen/pharmacology , Thioredoxins/metabolism , Ultraviolet RaysABSTRACT
Structural analysis of a novel UV filter present in the lens of the thirteen-lined ground squirrel has shown that it is related in structure to N-acetyl-3-hydroxykynurenine. This finding is consistent with the fact that the squirrel lenses also contain high levels of this tryptophan metabolite. Analysis of both NMR and mass spectrometric data suggested that the novel UV filter compound forms by condensation of proline with N-acetyl-3-hydroxykynurenine. Its absorption maximum at 340 nm is more than 20 nm lower than that of the kynurenines and it may therefore assist in filtering the more damaging shorter wavelengths of UVA.
Subject(s)
Kynurenine/analogs & derivatives , Lens, Crystalline/chemistry , Radiation-Protective Agents/chemistry , Sciuridae/physiology , Ultraviolet Rays , Animals , Chromatography, High Pressure Liquid , Kynurenine/chemistry , Kynurenine/isolation & purification , Magnetic Resonance Spectroscopy , Pigments, Biological/analysis , Radiation-Protective Agents/isolation & purification , Tandem Mass SpectrometryABSTRACT
PURPOSE: To compare levels of S-glutathiolation and S-cysteinylation occurring at more than 60 cysteine residues of 12 different guinea pig lens water-soluble nuclear crystallins following treatment of the animals with hyperbaric oxygen (HBO). METHODS: Guinea pigs (initially 18 months old) were treated 30X (3X per week for 10 weeks) with HBO (2.5 atm 100% O(2) for 2.5 h) as a model to study the formation of nuclear cataract. This treatment produces a moderate increase in lens nuclear light scatter (compared to denser scatter occurring after 80 HBO treatments), with five- to sixfold increases in levels of protein-bound glutathione (PSSG) and protein-bound cysteine (PSSC). Trypsin digests of lens nuclear water-soluble proteins were analyzed with two-dimensional liquid chromatography and mass spectrometry to identify specific cysteine residues binding either glutathione or cysteine. Lens nuclei of age-matched untreated animals were used as controls. RESULTS: All major crystallins, except αB, were modified to some extent by either S-glutathiolation or S-cysteinylation. Overall, 72% of the cysteine residues of guinea pig lens nuclear crystallins were shown to be capable of binding glutathione, cysteine, or both molecules. The crystallin with the highest level of modification was ßA1/A3 (six of eight -SH groups), and that with the lowest (two of five -SH groups) was ßA2. O(2)-induced increases in PSSG levels were 2.8, 2.4, and 4.1 times control for γA-, γB-, and γC-crystallins, respectively. Comparable increases in PSSC levels for the three γ-crystallins were 2.3, 2.7, and 2.4 times control, respectively. ßB2-crystallin showed the highest amount of O(2)-induced PSSG formation of any of the crystallins, as well as a substantial level of control PSSG, and nearly all of this was due to a single residue, C67, a site also present in human ßB2-crystallin. Overall, 32 of the 44 modified cysteine residues were homologous with the human. CONCLUSIONS: This large-scale study successfully identified lens crystallin cysteine residues that bound glutathione and/or cysteine under normal or oxidative stress conditions. The high percentage of protein -SH groups that are modified by S-thiolation in the guinea pig lens nucleus demonstrates the substantial protein sulfhydryl redox buffer capability present in the center of the lens. The results suggest that PSSG and PSSC formation may act to delay O(2)-induced insolubilization of γA-, γB-, and γC-crystallins, and ß-crystallins, but with a greater effect on the γ-crystallins at an early stage of oxidative stress. The study has shown that technological approaches are now available to investigate in considerable detail the role of specific lens -SH groups in nuclear cataractogenesis.
Subject(s)
Crystallins/chemistry , Crystallins/metabolism , Lens Nucleus, Crystalline/metabolism , Proteomics/methods , Animals , Binding Sites , Cataract/etiology , Cataract/metabolism , Cysteine/chemistry , Disease Models, Animal , Glutathione/chemistry , Guinea Pigs , Hyperoxia/metabolism , Oxidative Stress , Protein Processing, Post-Translational , Solubility , Sulfhydryl Compounds/chemistry , Tandem Mass SpectrometryABSTRACT
It is known that fluorescence, much of it caused by UVA light excitation, increases in the aging human lens, resulting in loss of sharp vision. This study used an in vivo animal model to investigate UVA-excited fluorescence in the rabbit lens, which contains a high level of the UVA chromophore NADH, existing both free and bound to λ-crystallin. Also, the ability of a Class I (senofilcon A) soft contact lens to protect against UVA-induced effects on the rabbit lens was tested. Rabbit eyes were irradiated with UVA light in vivo (100 mW/cm(2) on the cornea) for 1 h using monochromatic 365 nm light. Irradiation was conducted in the presence of either a senofilcon A contact lens, a minimally UV-absorbing lotrafilcon A contact lens, or no contact lens at all. Eyes irradiated without a contact lens showed blue 365 nm-excited fluorescence initially, but this changed to intense yellow fluorescence after 1 h. Isolated, previously irradiated lenses exhibited yellow fluorescence originating from the lens nucleus when viewed under 365 nm light, but showed normal blue fluorescence arising from the cortex. Previously irradiated lenses also exhibited a faint yellow color when observed under visible light. The senofilcon A contact lens protected completely against the UVA-induced effects on fluorescence and lens yellowing, whereas the lotrafilcon A lens showed no protection. The UVA-exposure also produced a 53% loss of total NADH (free plus bound) in the lens nucleus, with only a 13% drop in the anterior cortex. NADH loss in the nucleus was completely prevented with use of a senofilcon A contact lens, but no significant protection was observed with a lotrafilcon A lens. Overall, the senofilcon A lens provided an average of 67% protection against UVA-induced loss of four pyridine nucleotides in four different regions of the lens. HPLC analysis with fluorescence detection indicated a nearly six-fold increase in 365 nm-excited yellow fluorescence arising from lens nuclear λ-crystallin after the in vivo UVA exposure. It is concluded that UVA-induced loss of free NADH (which fluoresces blue) may have allowed the natural yellow fluorescence of λ-crystallin and other proteins in the lens nucleus to become visible. Increased fluorescence exhibited by UVA-exposed λ-crystallin may have been the result of a UVA-induced change in the conformation of the protein occurring during the initial UVA-exposure in vivo. The results demonstrate the greater susceptibility of the lens nucleus to UVA-induced stress, and may relate to the formation of human nuclear cataract. The senofilcon A contact lens was shown to be beneficial in protecting the rabbit lens against effects of UVA light, including changes in fluorescence, increased yellowing and loss of pyridine nucleotides.
Subject(s)
Contact Lenses, Hydrophilic , Fluorescence , Hydrogels , Lens Nucleus, Crystalline/radiation effects , NAD/metabolism , Radiation Injuries, Experimental/prevention & control , Silicones , Ultraviolet Rays/adverse effects , Animals , Cataract/enzymology , Cataract/prevention & control , Chromatography, High Pressure Liquid , Crystallins/metabolism , Electrophoresis, Polyacrylamide Gel , Eye/radiation effects , Lens Nucleus, Crystalline/pathology , Malate Dehydrogenase/metabolism , Rabbits , Radiation Injuries, Experimental/enzymology , Radiation Injuries, Experimental/pathology , Radiation Protection/instrumentationSubject(s)
Fibrinolysin/pharmacokinetics , Fibrinolytic Agents/pharmacokinetics , Intravitreal Injections/instrumentation , Needles/classification , Vitreous Body/metabolism , Fibrinolysin/administration & dosage , Fibrinolytic Agents/administration & dosage , Humans , Intravitreal Injections/methodsABSTRACT
Glutaredoxin belongs to the oxidoreductase family, with cytosolic glutaredoxin 1 (Grx1) and mitochondrial glutaredoxin 2 (Grx2) isoforms. Of the two isozymes, the function of Grx2 is not well understood. This paper describes the effects of Grx2 deletion on cellular function using primary lens epithelial cell cultures isolated from Grx2 gene knockout (KO) and wild-type (WT) mice. We found that both cell types showed similar growth patterns and morphology and comparable mitochondrial glutathione pool and complex I activity. Cells with deleted Grx2 did not show affected Grx1 or thioredoxin expression but exhibited high sensitivity to oxidative stress. Under treatment with H(2)O(2), the KO cells showed less viability, higher membrane leakage, enhanced ATP loss and complex I inactivation, and weakened ability to detoxify H(2)O(2) in comparison with the WT cells. The KO cells had higher glutathionylation in the mitochondrial proteins, particularly the 75-kDa subunit of complex I. Recombinant Grx2 deglutathionylated complex I and restored most of its activity. We conclude that Grx2 has a function that protects cells against H(2)O(2)-induced injury via its peroxidase and dethiolase activities; particularly, Grx2 prevents complex I inactivation and preserves mitochondrial function.
Subject(s)
Epithelial Cells/metabolism , Glutaredoxins/metabolism , Lens, Crystalline/cytology , Oxidative Stress , Animals , Cell Survival/drug effects , Epithelial Cells/drug effects , Glutaredoxins/deficiency , Hydrogen Peroxide/pharmacology , Lens, Crystalline/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Oxidative Stress/drug effectsABSTRACT
PURPOSE: UVB radiation from sunlight is known to be a risk factor for human cataract. The purpose in this study was to investigate the ability of a class I UV-blocking soft contact lens to protect against UVB-induced effects on the ocular tissues of the rabbit in vivo. METHODS: Eyes of rabbits were exposed to UVB light for 30 minutes (270-360 nm, peak at 310 nm, 1.7 mW/cm(2) on the cornea). Eyes were irradiated in the presence of either a UV-blocking senofilcon A contact lens, a minimally UV-blocking lotrafilcon A contact lens, or no contact lens at all. Effects on the cornea and lens were evaluated at various times after exposure. RESULTS: Eyes irradiated with no contact lens protection showed corneal epithelial cell loss plus lens epithelial cell swelling, vacuole formation, and DNA single-strand breaks, as well as lens anterior subcapsular opacification. The senofilcon A lens protected nearly completely against the UVB-induced effects, whereas the lotrafilcon A lens showed no protection. CONCLUSIONS: The results indicate that use of a senofilcon A contact lens is beneficial in protecting ocular tissues of the rabbit against the harmful effects of UVB light, including photokeratitis and cataract.
Subject(s)
Cataract/prevention & control , Contact Lenses, Hydrophilic , Eye/radiation effects , Hydrogels , Radiation Injuries, Experimental/prevention & control , Radiation Protection/instrumentation , Silicones , Ultraviolet Rays/adverse effects , Animals , Cataract/etiology , Cataract/pathology , Cell Death , DNA Damage/radiation effects , Epithelial Cells/radiation effects , Epithelial Cells/ultrastructure , Epithelium, Corneal/pathology , Epithelium, Corneal/radiation effects , Keratitis/etiology , Keratitis/pathology , Keratitis/prevention & control , Lens, Crystalline/radiation effects , Lens, Crystalline/ultrastructure , Rabbits , Radiation Injuries, Experimental/etiology , Radiation Injuries, Experimental/pathologyABSTRACT
PURPOSE: Intravitreal bevacizumab (BV) (Avastin, Genentech Inc., South San Francisco, CA) is frequently used for the treatment of age-related macular degeneration. Previous studies have demonstrated full-thickness retinal penetration. Intravitreal recombinant microplasmin (MP) has been shown to successfully induce a posterior vitreous detachment (PVD) and vitreous liquefaction in animals. It has been suggested that a PVD may alter the retinal penetration of molecules in the vitreous cavity. The aim of this study was to compare BV retinal penetration in rabbit eyes with and without an MP-induced PVD. METHODS: Twelve adult rabbits were injected with 0.1 mL (0.4 mg) of MP into the vitreous cavity of 1 eye. One week later, the rabbits were injected with 0.05 mL (1.25 mg) of BV into both eyes. Both eyes of 3 rabbits were harvested at 6 hours, 12 hours, 24 hours, and 72 hours after the BV injection. Frozen retinal cross sections were prepared, and BV retinal penetration was evaluated with immunohistochemistry using a fluorescence-labeled antibody against BV. Two eyes from one rabbit were not injected with either agent and used as controls to compare the background autofluorescence. Peripapillary retinal sections were recorded with a digital camera, and intraretinal BV fluorescence-labeled antibody was measured by qualitative photographic interpretation. Two additional rabbits received an intravitreal injection of 0.1 mL of MP in 1 eye. One week later, both eyes from each rabbit were enucleated, and frozen retinal sections were prepared and analyzed with light microscopy to evaluate histologic damage. RESULTS: Full-thickness BV retinal penetration was observed throughout the retina in both eyes of each rabbit. All the MP-injected eyes exhibited increased antibody labeling in retinas evaluated at 6 hours, 12 hours, and 24 hours after BV injection when compared with the contralateral non-MP-injected eyes. By 3 days after BV injection, all eyes demonstrated decreased antibody labeling compared with earlier periods. At 3 days, 1 rabbit showed increased antibody labeling in the retina of the non-MP-injected eye compared with the contralateral MP-injected eye, and 2 rabbits exhibited similar antibody labeling in both eyes. When compared with control eyes, light microscopy demonstrated normal retinal histologic findings in eyes injected only with MP. CONCLUSION: Increased BV retinal penetration is observed initially in eyes with an MP-induced PVD, and the mechanism is likely multifactorial. By 3 days, retinal penetration is similar in eyes with and without a PVD. Although it is difficult to directly extrapolate to humans, our study suggests that a PVD may alter the retinal penetration of BV.
Subject(s)
Angiogenesis Inhibitors/pharmacokinetics , Antibodies, Monoclonal/pharmacokinetics , Fibrinolysin/toxicity , Peptide Fragments/toxicity , Retina/metabolism , Vitreous Body/drug effects , Vitreous Detachment/metabolism , Animals , Antibodies, Monoclonal, Humanized , Bevacizumab , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique, Indirect , Intravitreal Injections , Rabbits , Recombinant Proteins/toxicity , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vitreous Body/metabolism , Vitreous Detachment/etiologySubject(s)
Angiogenesis Inhibitors/adverse effects , Endophthalmitis/etiology , Eye Infections/etiology , Intravitreal Injections/adverse effects , Staphylococcal Infections/etiology , Anti-Bacterial Agents/therapeutic use , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal, Humanized , Aptamers, Nucleotide/adverse effects , Bevacizumab , Ceftazidime/therapeutic use , Drug Therapy, Combination , Endophthalmitis/drug therapy , Eye Infections/drug therapy , Humans , Ranibizumab , Risk Factors , Staphylococcal Infections/drug therapy , Vancomycin/therapeutic use , Vitreous Body/microbiologyABSTRACT
Zeta-crystallin is an NADPH-binding protein consisting of four identical 35kD subunits. The protein possesses quinone oxidoreductase activity, and is present in large amounts in the lenses of camelids, certain hystricomorphic rodents, and the Japanese tree frog, and in lower catalytic amounts in certain tissues of various species. In this study, recombinant methods were used to produce substantial quantities of his-tagged recombinant mouse zeta-crystallin, which was then purified to homogeneity. The yield of pure recombinant mouse zeta-crystallin was five times that obtained previously for purification of recombinant guinea pig zeta-crystallin. The quinone oxidoreductase activity of purified his-tagged recombinant mouse zeta-crystallin was comparable to that of purified native guinea pig lens zeta-crystallin, and to that previously reported for recombinant guinea pig zeta-crystallin. The method permits production of substantial amounts of recombinant zeta-crystallin for conducting studies on the biological role of this interesting protein, which exists in such high concentration in the lenses of certain species.
Subject(s)
Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , zeta-Crystallins/isolation & purification , zeta-Crystallins/metabolism , Animals , Guinea Pigs , Lens, Crystalline/chemistry , Lens, Crystalline/metabolism , Mice , NADP/metabolism , Quinone Reductases/metabolism , Recombinant Proteins/genetics , zeta-Crystallins/geneticsABSTRACT
Oxidative mechanisms during nuclear sclerosis of the lens are poorly understood, in particular metal-catalyzed oxidation. The lysyl oxidation product adipic semialdehyde (allysine, ALL) and its oxidized end-product 2-aminoadipic acid (2-AAA) were determined as a function of age and presence of diabetes. Surprisingly, whereas both ALL and 2-AAA increased with age and strongly correlated with cataract grade and protein absorbance at 350 nm, only ALL formation but not 2-AAA was increased by diabetes. To clarify the mechanism of oxidation, rabbit lenses were treated with hyperbaric oxygen (HBO) for 48 h, and proteins were analyzed by gas and liquid chromatography mass spectrometry for ALL, 2-AAA, and multiple glycation products. Upon exposure to HBO, rabbit lenses were swollen, and nuclei were yellow. Protein-bound ALL increased 8-fold in the nuclear protein fractions versus controls. A dramatic increase in methyl-glyoxal hydroimidazolone and carboxyethyl-lysine but no increase of 2-AAA occurred, suggesting more drastic conditions are needed to oxidize ALL into 2-AAA. Indeed the latter formed only upon depletion of glutathione and was catalyzed by H(2)O(2). Neither carboxymethyl-lysine nor glyoxal hydroimidazolone, two markers of glyco-/lipoxidation, nor markers of lenticular glycemia (fructose-lysine, glucospane) were elevated by HBO, excluding significant lipid peroxidation and glucose involvement. The findings strongly implicate dicarbonyl/metal catalyzed oxidation of lysine to allysine, whereby low GSH combined with ascorbate-derived H(2)O(2) likely contributes toward 2-AAA formation, since virtually no 2-AAA formed in the presence of methylglyoxal instead of ascorbate. An important translational conclusion is that chelating agents might help delay nuclear sclerosis.
Subject(s)
Aging/physiology , Crystallins/metabolism , Diabetes Mellitus/metabolism , Lens, Crystalline/metabolism , Lysine/metabolism , 2-Aminoadipic Acid/analogs & derivatives , 2-Aminoadipic Acid/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Cattle , Child , Crystallins/chemistry , Crystallins/genetics , Diabetes Mellitus/pathology , Humans , Hydrogen Peroxide/metabolism , Hyperbaric Oxygenation , Lens, Crystalline/chemistry , Lens, Crystalline/pathology , Leucine/chemistry , Lysine/chemistry , Metals/chemistry , Mice , Mice, Inbred C57BL , Middle Aged , Molecular Structure , Oxidants/metabolism , Oxidation-Reduction , Rabbits , Regression Analysis , Young AdultABSTRACT
BACKGROUND: Lens cataract is associated with protein oxidation and aggregation. Two proteins that cause cataract when deleted from the lens are methionine sulfoxide reductase A (MsrA) that repairs protein methionine sulfoxide (PMSO) oxidized proteins and alpha-crystallin which is a two-subunit (alphaA and alphaB) chaperone. Here, we tested whether PMSO formation damages alpha-crystallin chaperone function and whether MsrA could repair PMSO-alpha-crystallin. METHODS: Total alpha-crystallin was oxidized to PMSO and evaluated by CNBr-cleavage and mass spectrometry. Chaperone activity was measured by light scattering using lysozyme as target. PMSO-alpha-crystallin was treated with MsrA, and repair was assessed by CNBr cleavage, mass spectrometry and recovery of chaperone function. The levels of alpha-crystallin-PMSO in the lenses of MsrA-knockout relative to wild-type mice were determined. RESULTS: PMSO oxidation of total alpha-crystallin (met 138 of alphaA and met 68 of alphaB) resulted in loss of alpha-crystallin chaperone activity. MsrA treatment of PMSO-alpha-crystallin repaired its chaperone activity through reduction of PMSO. Deletion of MsrA in mice resulted in increased levels of PMSO-alpha-crystallin. CONCLUSIONS: Methionine oxidation damages alpha-crystallin chaperone function and MsrA can repair PMSO-alpha-crystallin restoring its chaperone function. MsrA is required for maintaining the reduced state of alpha-crystallin methionines in the lens. SIGNIFICANCE: Methionine oxidation of alpha-crystallin in combination with loss of MsrA repair causes loss of alpha-crystallin chaperone function. Since increased PMSO levels and loss of alpha-crystallin function are hallmarks of cataract, these results provide insight into the mechanisms of cataract development and likely those of other age-related diseases.