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1.  Nucleophilic compounds decrease advanced glycation end products (AGEs) from ascorbic acid in the hSVCT2 transgenic mouse model of lenticular aging 
Purpose
Senile cataracts are associated with oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. This process is partially explained by advanced glycation endproducts (AGEs) from ascorbic acid (ASA), as unequivocally demonstrated in our hSVCT2 transgenic mouse(PNAS 103:16912, 2006). We now present the first pharmacological intervention study against ascorbylation in these mice.
Methods
Five groups of mice (10 mice/group) were fed from two to nine months a diet containing 0.1% (wt/wt) aminoguanidine (AG), pyridoxamine (PM), penicillamine (PA), and nucleophilic compounds NC-I and NC-II. AGEs were determined in crystallin digests using HPLC, LC-MS or GC-MS. In vitro incubations of lens protein extract with ASA or dehydroascorbic aicd (DHA) were also performed.
Results
ASA level increased ~10 fold in all groups and was unaffected by treatment. AGEs were several fold increased in transgenic compared to control lenses. Body weight, food intake, lenticular glutathione and glycated lysine level were unaltered. In vitro, all compounds inhibited AGE formation. In vivo, NC-I and NC-II significantly decreased protein fluorescence at λex335/em385 (p=0.045, 0.017, respectively) and λex370/em440 (p=0.029, 0.007, respectively). Other inhibitors had no effect. After 7 months, only NC-1 and NC-2 induced a 50 % reduction in pentosidine (n.s, p=0.035 respectively). NC-1 also decreased carboxymethyllysine (CML) (p=0.032) and carboxyethyllysine (CEL) (p= n.s). Fluorescent crosslink K2P was decreased by NC-1, NC-2, AG and PM (p= n.s).
Conclusions
Pharmacologically blocking protein ascorbylation with absorbable guanidino compounds is feasible and may represent a new strategy for the delay of age-related nuclear sclerosis of the lens.
doi:10.1167/iovs.08-1813
PMCID: PMC2576497  PMID: 18421088
2.  Glycation by Ascorbic Acid Oxidation Products Leads to the Aggregation of Lens Proteins 
Biochimica et biophysica acta  2007;1782(1):22-34.
Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 × 106 up to 3.0 × 108 Da. This compared to proteins ranging from 1.8 × 106 up to 3.6 × 108 Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-14C] ascorbate was incorporated into the aggregate fraction and because CNBH3, which reduces the initial Schiff base, prevented any protein aggregation.
Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of α-crystallin, significant aggregation of βH-crystallin, but rapid precipitation of purified βL- and γ-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, α-crystallin prevented the precipitation of the purified βL- and γ-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with βL-, or γ-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of α- and βL-, or γ-crystallins showed similar molar masses (up to 108 Da) and hydrodynamic diameter (up to 80 nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.
doi:10.1016/j.bbadis.2007.10.003
PMCID: PMC2274899  PMID: 18023423
ascorbic acid; glycation; lens proteins; protein aggregation; light scattering
3.  ANAEROBIC VS. AEROBIC PATHWAYS OF CARBONYL AND OXIDANT STRESS IN HUMAN LENS AND SKIN DURING AGING AND IN DIABETES: A COMPARATIVE ANALYSIS 
Free radical biology & medicine  2010;49(5):847-856.
The effects of anaerobic (lens) vs aerobic (skin) environment on carbonyl and oxidant stress are compared using de novo and existing data on advanced glycation and oxidation products in human crystallins and collagen. Almost all modifications increase with age. Methylglyoxal hydroimidazolones (MG-H1), carboxymethyl-lysine (CML), and carboxyethyl-lysine (CEL) are several folds higher in lens than skin, and markedly increase upon incubation of lens crystallins with 5 mM ascorbic acid. Vice-versa, fructose-lysine, glucosepane crosslinks, glyoxal hydroimidazolones (G-H1), metal catalyzed oxidation (allysine) and H2O2 dependent modifications (2-aminoapidic acid and methionine sulfoxide) are markedly elevated in skin, but relatively suppressed in the aging lens. In both tissues ornithine is the dominant modification, implicating arginine residues as the principal target of the Maillard reaction in vivo. Diabetes (here mostly type 2 studied) increases significantly fructose-lysine and glucosepane in both tissues (P<0.001) but has surprisingly little effect on the absolute level of most other advanced glycation end products (AGEs) . However, diabetes strengthens the Spearman correlation coefficients for age-related accumulation of hydrogen peroxide mediated modifications in the lens. Overall, the data suggest oxoaldehyde stress involving methylglyoxal from either glucose or ascorbate is predominant in the aging non-cataractous lens, while aging skin collagen undergoes combined attack by non-oxidative glucose mediated modifications, as well as those from metal catalyzed oxidation and H2O2.
doi:10.1016/j.freeradbiomed.2010.06.003
PMCID: PMC2910832  PMID: 20541005
crystallins; collagen; glycation; oxidative stress; methylglyoxal; metals
4.  A Class I UV-Blocking (senofilcon A) Soft Contact Lens Prevents UVA-induced Yellow Fluorescence and NADH loss in the Rabbit Lens Nucleus in vivo 
Experimental eye research  2012;102C:17-27.
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/cm2 on the cornea) for 1 hour 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 hour. 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.
doi:10.1016/j.exer.2012.06.007
PMCID: PMC3432665  PMID: 22766154
UVA light; rabbit; in vivo; lens; yellowing; fluorescence; pyridine nucleotides; nuclear cataract
5.  N-Acetylcarnosine sustained drug delivery eye drops to control the signs of ageless vision: Glare sensitivity, cataract amelioration and quality of vision currently available treatment for the challenging 50,000-patient population 
Background:
Innovative Vision Products, Inc. (IVP)’s scientists developed the lubricant eye drops (Can-C™) designed as 1% N-acetylcarnosine (NAC) prodrug of l-carnosine containing a mucoadhesive cellulose-based compound combined with corneal absorption promoters in a sustained drug delivery system. Only the natural l-isomeric form of NAC raw material was specifically synthesized at the cGMP facility and employed for the manufacturing of Can-C™ eye drops.
Objective and study design:
In the present clinical study the authors assessed vision before and after 9 month term of topical ocular administration of NAC lubricant eye drops or placebo in 75 symptomatic patients with age-related uncomplicated cataracts in one or both eyes, with acuity in one eye of 20/40 or worse (best-corrected distance), and no previous cataract surgery in either eye and no other ocular abnormality and 72 noncataract subjects ranged in age from 54 to 78 years.
Setting:
Subjects in these subsample groups have reported complaints of glare and wanted to administer eye drops to get quick eye relief and quality of vision for their daily activities including driving and computer works. Following 9 months of treatment with NAC lubricant eye drops, most patients’ glare scores were improved or returned to normal in disability glare tests with Halometer DG. Improvement in disability glare was accompanied with independent improvement in acuity. Furthermore, patients with the poorest pretreatment vision were as likely to regain certain better visual function after 9 months of treatment with N-acetylcarnosine lubricant eye drops as those with the worth pretreatment vision.
Patients or other participants:
The authors made a reference to electronic records of the product sales to patients who have been made the repurchase of the Can-C™ eye drops since December 2001.
Intervention:
Based on this analysis of recorded adjustments to inventory, various parameters were analyzed during the continued repurchase behavior program, including testimonials from buyers. With these figures, researchers judged on the patients’ compliance rate to self-administer NAC eye-drops.
Main outcome measure and results:
The ophthalmic drug showed potential for the non-surgical treatment of age-related cataracts for participants after controlling for age, gender and daily activities and on a combined basis of repurchases behavior reports in more than 50,000 various cohort survivors, has been demonstrated to have a high efficacy and good tolerability for prevention and treatment of visual impairment determined for the older population with relative stable pattern of causes for blindness and visual impairment. The mechanisms of prevention and reversal of cataracts with NAC ophthalmic drug are considered which include prevention by the intraocular released carnosine of free-radical-induced inactivation of proprietary lens antioxidant enzymes (superoxide dismutase); prevention of carbohydrate and metal-catalyzed autooxidation of ascorbic acid-induced cross-linking glycation reactions to the lens proteins; transglycation properties of carnosine, allowing it to compete for the glycating agent, protecting proteins (lens crystallins) against modification; universal antioxidant and scavenging activity towards lipid hydroperoxides, aldehydes and oxygen radicals; activation with l-carnosine ingredient of proteasome activity in the lens; chaperone-like disaggregating to lens crystallins activity of NAC and of its bioactivated principal carnosine. Blindness incidence increased with advancing age, such as cataract and glaucoma, which are by far the commonest causes of blindness in our sample and in all age groups, glaucomatous neurodegeneration can be treated with developed NAC autoinduction prodrug eye drops equipped with corneal absorption promoters. The common blinding affections presenting in developed countries such as, senile macular degeneration, hereditary chorioretinal dystrophies, diabetic retinopathy are poorly represented in our current summary of vital-statistics and will be reported inherent in next N-acetylcarnosine ophthalmic drug studies.
Conclusion:
The authors present evidence, about why only a certain kind of NAC is safe, and why only certain formulas designed by IVP for drug discovery are efficacious in the prevention and treatment of senile cataract for long-term use. Overall cumulated studies demonstrate that the designed by IVP new vision-saving drug NAC eye drops help the aging eye to recover by improving its clarity, glare sensitivity, color perception and overall vision.
PMCID: PMC2685223  PMID: 19503764
age-related ophthalmic diseases; cataract; disability-glare; halos; Halometer; visual-acuity; N-acetylcarnosine lubricant eye drops; repurchase behavior analysis; 50,000-patients’ compliance to self-administer eye drops
6.  Lens Aging: Effects of Crystallins 
Biochimica et biophysica acta  2009;1790(10):1095-1108.
The primary function of the eye lens is to focus light on the retina. The major proteins in the lens—a, b, and g-crystallins—are constantly subjected to age-related changes such as oxidation, deamidation, truncation, glycation, and methylation. Such age-related modifications are cumulative and affect crystallin structure and function. With time, the modified crystallins aggregate, causing the lens to increasingly scatter light on the retina instead of focusing light on it and causing the lens to lose its transparency gradually and become opaque. Age-related lens opacity, or cataract, is the major cause of blindness worldwide. We review deamidation, and glycation that occur in the lenses during aging keeping in mind the structural and functional changes that these modifications bring about in the proteins. In addition, we review proteolysis and discuss recent observations on how crystallin fragments generated in vivo, through their anti-chaperone activity may cause crystallin aggregation in aging lenses. We also review hyperbaric oxygen treatment induced guinea pig and ‘humanized’ ascorbate transporting mouse models as suitable options for studies on age-related changes in lens proteins.
doi:10.1016/j.bbagen.2009.05.008
PMCID: PMC2743770  PMID: 19463898
lens crystallins; aging; lens opacity; chaperones; deamidation; glycation; oxidation; peptides
7.  Isolation, Purification and Characterization of Histidino-Threosidine, a Novel Maillard Reaction Protein Crosslink from Threose, Lysine and Histidine 
We isolated a novel acid-labile yellow chromophore from the incubation of lysine, histidine and D-threose and identified its chemical structure by one and two-dimensional 1H and DEPT NMR spectroscopy combined with LC-tandem mass spectrometry. This new cross-link exhibits a UV absorbance maximum at 305 nm and a molecular mass of 451 Da. The proposed structure is 2-amino-5-(3-((4-(2-amino-2-carboxyethyl)-1H-imidazol-1-yl)methyl)-4-(1,2-dihydroxyethyl)-2-formyl-1H-pyrrol-1-yl)pentatonic acid, a cross-link between lysine and histidine with addition of two threose molecules. It was in part deduced and confirmed through synthesis of the analogous compound from n-butylamine, imidazole and D-threose. We assigned the compound the trivial name histidino-threosidine. Systemic incubation revealed that histidino-threosidine can be formed in low amounts from fructose, glyceraldehyde, methylglyoxal, glycolaldehyde, ascorbic acid, and dehydroascorbic acid, but at a much higher yield with degradation products of ascorbic acid, i.e. threose, erythrose, and erythrulose. Bovine lens protein incubated with 10 and 50 mM threose for two weeks yielded 560 and 2840 pmol/mg histidino-threosidine. Histidino-threosidine is to our knowledge the first Maillard reaction product known to involve histidine in a crosslink.
doi:10.1016/j.abb.2007.03.007
PMCID: PMC1978223  PMID: 17466255
advanced glycation end-product; yellow chromophore; fluorophore; ascorbic acid; lens; glycation; aging; erythrulose
8.  Role of lipid peroxidation in the pathogenesis of myopic and senile cataract. 
AIMS/BACKGROUND: Increased production of free radicals, consumption of antioxidant, and oxidation of unsaturated lipids have been observed recently in cataractous lenses and active participation of the retina in human cataractogenesis has been proposed. To verify this hypothesis, the total (GSH) and oxidised (GSSG) glutathione concentrations were assayed in the lens and the malondialdehyde (MDA) levels assayed in the vitreous and in the lens of normal controls and patients with senile or myopic cataract. METHODS: The study was conducted on 34 lenses (nucleus and epinucleus) (nine clear lenses, 14 lenses with idiopathic senile cataract, and 11 lenses affected by severe myopic cataract) and vitreous of 19 (seven non-myopic, seven myopic, and five control) subjects. Glutathione determination was performed following the method of Reed, while malondialdehyde was assayed using a modification of the method of Dahle. RESULTS: Cataractous lenses showed a decreased content of GSH and increased concentration of GSSG compared with clear lenses. A higher oxidative consumption of GSH was found in myopic cataracts compared with senile ones. Also, increased levels of MDA were observed both in cataractous lenses and in the vitreous of myopic patients compared with the control and the senile ones. CONCLUSION: The observed alterations strongly suggest that retinal lipid peroxidation might play a key role in human cataractogenesis, especially in the myopic type.
PMCID: PMC505624  PMID: 8942384
9.  Immunochemical detection of glycated lens crystallins and their circulating autoantibodies in human serum during aging 
Molecular Vision  2008;14:2056-2066.
Purpose
The aim of this investigation was to exploit lens-specific glycated crystallins as an immunogen to detect human glycated crystallins and their circulating autoantibodies in human serum during aging in relation to the development of cataract.
Methods
Polyclonal antibodies were produced against human total lens proteins (40–80 years) in rabbits. The specificity of the antibodies produced were determined by antibody capture assay using purified human lens crystallins (high molecular weight fraction [HMW]+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat β-, β-glycated, γ-, and γ-glycated lens crystallins was also analyzed. A non-competitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+α, HMW+α-glycated, β-, and β-glycated crystallins from humans and γ- and γ-glycated crystallins from rats as immobilized antigens. Circulating autoantibodies were also detected in human sera by antibody capture assay. The methodology was validated by evaluating 60 human serum samples collected from cataract patients and 30 human serum samples from apparently normal subjects belonging to the same age group.
Results
The polyclonal antibodies raised against human total lens proteins showed 90% and 65% cross-reactivity with rat γ- and β-crystallins, respectively, by ELISA. Further, these polyclonal antibodies were capable of detecting both native and in vitro synthesized glycated crystallins. Their IC50 values were observed to be (i) human total lens proteins (55 ng), (ii) human HMW+α (16.45 ng), (iii) human HMW+α-glycated (273 ng), (iv) human β- (37.82 ng), (v) human β-glycated (260 ng), (vi) rat γ- (105.34 ng), and (vii) rat γ-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating β-glycated and γ-glycated crystallins in the age group of 40–80 years with respect to their control groups. However, there was no statistically significant change in the levels of HMW+α-glycated crystallins in the age group of 40–80 years as compared to their age-matched controls. Notably, the levels of serum γ-glycated crystallins were found to be threefold higher than that of HMW+α-glycated and β-glycated crystallins in the age group of 70–80 years. Circulating autoantibodies to HMW+α-glycated, β-glycated, and γ-glycated crystallins were detected in the serum of both apparently normal and cataract patients in the age group of 40–80 years by antibody capture assay. The levels of these autoantibodies were significantly higher at every time point compared to their respective controls. Autoantibodies to γ-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to β-glycated and HMW+α-glycated crystallins, respectively. Western blot and immunohistochemical analysis substantiated the observations made in non-competitive ELISA.
Conclusions
During the course of aging, leakage of lens crystallins (HMW+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) elicit an immune response resulting in the formation of autoantibodies in cataract patients (40–80 years) as compared to age matched controls. This is the first experimental report where polyclonal antibodies raised against lens-specific glycated crystallins were capable of detecting the early leakage of glycated crystallins in human subjects. This immunochemical approach has implications in the early detection of senile cataract.
PMCID: PMC2584771  PMID: 19023447
10.  Elevated insulin signaling disrupts the growth and differentiation pattern of the mouse lens 
Molecular Vision  2007;13:397-407.
Purpose
Insulin and insulin-like growth factors (IGFs) are putative regulators of cell proliferation and differentiation during lens development. Transgenic mice that overexpress IGF-1 in the lens have been previously described. To further understand the ocular functions of this growth factor family, the in vivo effects of insulin expression on lens development were investigated using transgenic mice.
Methods
Expression of insulin receptor (IR) and IGF-1 receptor (IGF-1R) in mouse lens was examined by reverse-transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. Transgenic mice that overexpress insulin in the lens were generated using two different promoters: a fiber-cell specific αA-crystallin (αA) promoter and a modified αA-promoter linked to the chicken δ1-crystallin enhancer (called the δenαA promoter). The δenαA promoter is active in both lens epithelial and fiber cells. The lens phenotypes were analyzed by histology and immunohistochemistry. Protein expression was examined by western blotting.
Results
Normal mouse lenses express both the insulin receptor (IR) and the IGF-1 receptor (IGF-1R), and their expression is highest at the lens periphery where the germinative and transitional zones are located. In transgenic mice, insulin expression in the lens induced cataract formation. The severity of the cataracts reflected the level of transgene expression, independent of the type of promoter used. In severely affected families, the spherical shape of the lens was altered and the lenses were smaller than normal. Histological analysis showed no evidence of premature differentiation of the anterior epithelial cells. In contrast to the IGF-1 mice, insulin transgenic mice exhibited an anterior shift in the location of the germinative and transitional zones, leading to a reduction of the lens epithelial compartment. Additional alterations included expansion of the lens transitional zone, variable nuclear positioning in the lens bow region, and inhibition of fiber cell denucleation and terminal differentiation.
Conclusions
Elevated intraocular insulin does not enhance proliferation nor induce differentiation of mouse lens epithelial cells. Since an increase in IGF-1 causes a posterior shift of the lens geminative and transitional zones, while an increase in insulin causes an anterior shift of these zones, our results suggest that these two growth factors may work together to control the location of this structural domain during normal lens development. Our data also suggest that increased insulin-signaling activity in the lens can antagonize the endogenous signals that are responsible for fiber cell maturation and terminal differentiation.
PMCID: PMC2642934  PMID: 17417601
11.  Spectroscopic and biochemical correlations during the course of human lens aging 
BMC Ophthalmology  2006;6:10.
Background
With age, the human lens accumulates variety of substances that absorbs and fluorescence, which explains the color of yellow, brunescent and nigrescent cataract in terms of aging. The aim of this study was to assess lens fluorophores with properties comparable to those of advanced glycated end products (AGEs) in relation to age in human lenses. These fluorescent compounds are believed to be involved in the development of cataract.
Methods
Spectroscopic (UV-Vis-NIR) and fluorescence photography (CCD-Digital based image analysis) studies were carried out in randomly selected intact human lenses (2–85 years). AGE-like fluorophores were also measured in water soluble and insoluble (alkali soluble) fractions of human lenses (20–80 years).
Results
Our experimental findings suggest that there was a progressive shift in the absorbance characteristic of intact lens in the range of λ210 nm-λ470 nm. A relative increase in the absorptivity at λ(511–520 nm), with age, was also observed. In addition, the ratio of absorptivity at λ(511–520 nm) versus the maximum absorbance recorded at blue-end cut-off (210–470 nm) was also found to increase, with age. The fluorescent intensity in the intact lens at both UV-B (λEx312 nm) and UV-A (λEx365 nm) were found to be positively correlated (r2 = 0.91 & 0.94, respectively; Confidence interval 95%) upto 50 years of age. In addition, a concomitant changes in AGE- like fluorophores were also observed in the processed lens samples (soluble and insoluble fractions) along the age. A significant increase in the concentration of AGE- like fluorophores, both in intact and processed lens was observed during the period of 40 – 50 years.
Conclusion
Based on the present investigation, it was concluded that significant changes do occur in the AGE-like fluorophores of human lenses during the period of 40–50 years.
doi:10.1186/1471-2415-6-10
PMCID: PMC1450316  PMID: 16519820
12.  Antioxidant Capacity of Lenses with Age-Related Cataract 
The immediate cause of the occurrence of cataract is unknown, but oxidative damage and effects of reactive oxygen species are considered important in its etiopathogenesis. Our research was aimed at testing the nonenzyme antioxidant power of corticonuclear lens blocks, with different types and different maturity of age-related cataract. Clinical and biochemical researches were carried out in 101 patients with age-related cataract. In corticonuclear lens blocks of the patient, the concentration of nonprotein and total-SH groups and the concentration of total vitamin C and dehydroascorbic acid (DHA) were determined; the current redox balance of dehydroascorbate/ascorbate and total antioxidant power measured by ferric-reducing ability were examined. In corticonuclear lens blocks with incipient cataract a significantly higher concentration of GSH, total SH groups, concentration of total vitamin C and ascorbic acid (AA), and ferric-reducing ability were measured. The measured concentration of DHA is higher than the concentration of AA in the lenses with the incipient and mature cataract. The concentration ratio of redox couple DHA/AA is higher in lenses with mature cataract, where the measured concentration of AA was lower than in the incipient cataract. Timely removal of DHA from the lens is important because of its potential toxicity as an oxidant. An increase of the current concentration of DHA/AA redox balance can be an indicator of oxidative stress.
doi:10.1155/2012/467130
PMCID: PMC3272861  PMID: 22363833
13.  Drevogenin D prevents selenite-induced oxidative stress and calpain activation in cultured rat lens 
Molecular Vision  2007;13:1121-1129.
Purpose
Selenite-induced cataractogenesis is mediated by oxidative stress, accumulation of calcium and activation of lenticular calpains. Calpains are a super family of Ca2+ dependent proteases, which are involved in lens protein proteolysis and insolubilization. Many inhibitors could prevent calpain-induced proteolysis of α- and β-crystallins in rodent cataracts. Evaluating natural sources with antioxidant property and subsequent prevention of calpain activation may lead to the development of safer and more effective agents against cataractogenesis. There are no reports on the protective role of bioactive components against calpain-mediated proteolysis and subsequent cataractogenesis. The purpose of the study was to evaluate the role of Drevogenin D, a triterpenoid aglycone, isolated from Dregea volubilis in preventing selenite-induced, calcium-activated, calpain-mediated proteolysis in cultured rat lenses.
Methods
Lenses were extracted from Sprague-Dawley strain rats at the age of one month and were organ cultured in M-199 medium with HEPES buffer. The lenses were divided into three groups with eight lenses in each group as follows: lenses cultured in a normal medium (GI), lenses cultured in a sodium selenite supplemented medium (GII), and lenses cultured in a medium supplemented with sodium selenite and Drevogenin D-treated (GIII). Changes to transparency and opacity formation of lenses were monitored under microscopic observation. At the end of the experiment, biochemical parameters such as activity of lens superoxide dismutase (SOD), lens Ca2+ ATPase, concentration of Ca2+, levels of sulfhydryl content, and thiobarbituric acid reacting substances (TBARS) were determined. Changes to casein zymography for calpains, immunoblot for Lp82, and SDS-PAGE of lens water soluble protein fraction (WSF) were also done.
Results
Microscopic evaluation of lens morphology showed that Drevogenin D prevented the opacification in G-III. Drevogenin D inhibited the accumulation of calcium, the activation of calpain system, and lipid peroxidation. Activity of Ca2+ATPase, SOD, and SDS-PAGE profile of water soluble proteins was normalized following treatment with Drevogenin D.
Conclusions
Selenite-induced cataractogenesis is mediated by oxidative stress leading to a decrease in the activity of Ca2+ ATPase, resulting in the accumulation of calcium and the subsequent activation of lenticular calpains. The results obtained indicated that Drevogenin D treatment was effective in protecting the lens proteins by controlling stress-induced protein oxidation, maintenance of Ca2+ ATPase activity, calcium accumulation, lipid peroxidation, and prevention of calpain activation. Hence, Drevogenin D can be used as a potential therapeutic agent against oxidative stress-induced cataract.
PMCID: PMC2779145  PMID: 17653057
14.  Distribution of ferritin chains in canine lenses with and without age-related nuclear cataracts 
Molecular Vision  2009;15:2404-2410.
Purpose
It was determined in an earlier study that ferritin-heavy (H) and -light (L) chains in lens fiber cells are modified in comparison to those in lens epithelial cells. The purpose of the present study was to determine whether changes in ferritin chain characteristics are developmental, age-related, or associated with cataractogenesis, by analyzing the distribution of modified chains throughout the lens fiber mass.
Methods
After removing the capsule, noncataractous and cataractous lenses were separated into six layers of fiber cells. The content of ferritin H and L chains in each layer was determined by western blotting with chain-specific antibodies. The level of ferritin complex (450 kDa protein made up of assembled L and H chains) was determined using the enzyme-linked immunosorbent assay. The ability of ferritin complex to bind iron was assessed by in vitro labeling with 59Fe.
Results
Fiber cell ferritin L chains were 30 kDa (modified from the normal 19 kDa), and were present at the highest level in the outermost layers of both cataractous and non-cataractous lenses. The amount of modified L chains decreased gradually in the inner layers of the fiber mass, and was undetectable in the inner two layers of cataractous lenses. The ferritin H chains were also modified to 12 kDa (perhaps truncated from the normal 21 kDa size) in both cataractous and non-cataractous lenses. Similar levels of this modified H chain were found throughout the normal lens. Interestingly, in cataractous lenses, the modified H chains were found in decreasing amounts towards the interior of the lens, and were undetectable in the nucleus. However, in these cataractous lenses, the normal-sized ferritin H chains (21 kDA) appear in small quantities in the outer fiber layers, and increase in quantity and size (to 29 kDa) in the inner layers. This observation was best seen and demonstrated in advanced cataracts. Ferritin, which can bind iron, was found mainly in the outer layers of the lens fiber mass of normal lenses, but was more evenly distributed in fiber layers from cataractous lenses.
Conclusions
Both ferritin H and L chains were modified in lens fiber cells from normal and cataractous canine lenses. These modifications were not age-related, and most likely occur during the differentiation of epithelial cells to fiber cells, since only normal-sized chains have been found in lens epithelial cells. In addition, there was a specific and distinct distribution of these modified chains throughout the lens fiber mass. The most striking differences between normal and cataractous lenses fiber cells were the appearance of normal-sized ferritin H chains and the relatively even distribution of iron binding capacity throughout the fiber mass of the cataractous lenses. These differences may reflect a response of the lens to increased oxidative stress during cataractogenesis.
PMCID: PMC2785719  PMID: 19956561
15.  Effects of topically applied tocotrienol on cataractogenesis and lens redox status in galactosemic rats 
Molecular Vision  2014;20:822-835.
Purpose
Oxidative and nitrosative stress underlies cataractogenesis, and therefore, various antioxidants have been investigated for anticataract properties. Several vitamin E analogs have also been studied for anticataract effects due to their antioxidant properties; however, the anticataract properties of tocotrienols have not been investigated. In this study, we investigated the effects of topically applied tocotrienol on the onset and progression of cataract and lenticular oxidative and nitrosative stress in galactosemic rats.
Methods
In the first part of this study, we investigated the effects of topically applied microemulsion formulation of tocotrienol (TTE) using six concentrations ranging from 0.01% to 0.2%. Eight groups of Sprague-Dawley rats (n = 9) received distilled water, vehicle, or one of the six TTE concentrations as pretreatment topically twice daily for 3 weeks while on a normal diet. After pretreatment, animals in groups 2–8 received a 25% galactose diet whereas group 1 continued on the normal diet for 4 weeks. During this 4-week period, topical treatment continued as for pretreatment. Weekly slit-lamp examination was conducted to assess cataract progression. At the end of the experimental period, the animals were euthanized, and the proteins and oxidative stress parameters were estimated in the lenses. In the second part of the study, we compared the anticataract efficacy of the TTE with the liposomal formulation of tocotrienol (TTL) using five groups of Sprague-Dawley rats (n = 15) that received distilled water, TTE, TTL, or corresponding vehicle. The mode of administration and dosing schedule were the same as in study 1. Weekly ophthalmic examination and lens protein and oxidative stress estimates were performed as in study 1. Lens nitrosative stress was also estimated.
Results
During the 4-week treatment period, the groups treated with 0.03% and 0.02% tocotrienol showed slower progression of cataract compared to the vehicle-treated group (p<0.05), whereas the group treated with 0.2% tocotrienol showed faster progression of cataract compared to the vehicle-treated group (p<0.05). The lenticular protein content, malondialdehyde, superoxide dismutase, and catalase levels were normalized in the groups that received 0.03% and 0.02% tocotrienol. The lenticular reduced glutathione also showed a trend toward normalization in these groups. In contrast, the group treated with 0.2% tocotrienol showed increased lenticular oxidative stress. When the microemulsion and liposomal formulations were compared, the effects on cataract progression, lens oxidative and nitrosative stress, and lens protein content did not show significant differences.
Conclusions
Topically applied tocotrienol within the concentration range of less than 0.05% and more than 0.01% tends to delay the onset and progression of cataract in galactose-fed rats by reducing lenticular oxidative and nitrosative stress. However, topical tocotrienol at a concentration of 0.2% and higher aggravates cataractogenesis in galactose-fed rats by increasing lens oxidative stress. The anticataract efficacy of 0.03% microemulsion of tocotrienol did not differ from its liposomal formulations at the same concentration.
PMCID: PMC4057512  PMID: 24940038
16.  Nonenzymatic glycation of human lens crystallin. Effect of aging and diabetes mellitus. 
Journal of Clinical Investigation  1984;74(5):1742-1749.
We have examined the nonenzymatic glycation of human lens crystallin, an extremely long-lived protein, from 16 normal human ocular lenses 0.2-99 yr of age, and from 11 diabetic lenses 52-82-yr-old. The glucitol-lysine (Glc-Lys) content of soluble and insoluble crystallin was determined after reduction with H-borohydride followed by acid hydrolysis, boronic acid affinity chromatography, and high pressure cation exchange chromatography. Normal lens crystallin, soluble and insoluble, had 0.028 +/- 0.011 nanomoles Glc-Lys per nanomole crystallin monomer. Soluble and insoluble crystallins had equivalent levels of glycation. The content of Glc-Lys in normal lens crystallin increased with age in a linear fashion. Thus, the nonenzymatic glycation of nondiabetic lens crystallin may be regarded as a biological clock. The diabetic lens crystallin samples (n = 11) had a higher content of Glc-Lys (0.070 +/- 0.034 nmol/nmol monomer). Over an age range comparable to that of the control samples, the diabetic crystallin samples contained about twice as much Glc-Lys. The Glc-Lys content of the diabetic lens crystallin samples did not increase with lens age.
PMCID: PMC425353  PMID: 6438156
17.  Ubiquitin Proteasome Pathway–Mediated Degradation of Proteins: Effects Due to Site-Specific Substrate Deamidation 
Lens clarity requires maintenance of a perfect proteome. Deamidated crystallins are observed in cataractous lenses. The authors demonstrate for the first time that deamidated proteins are selective substrates for the ubiquitin proteolytic pathway. As such, they could be more efficiently cleared from lens systems, but they are not. These data raise the possibility that ubiquitination is not productive for degradation or that proteasome activity in lens cells and tissues is insufficient, resulting in the accumulation rather than the timely degradation of the deamidated proteins.
Purpose.
The accumulation, aggregation, and precipitation of proteins is etiologic for age-related diseases, particularly cataract, because the precipitates cloud the lens. Deamidation of crystallins is associated with protein precipitation, aging, and cataract. Among the roles of the ubiquitin proteasome pathway (UPP) is protein surveillance and maintenance of protein quality. The purpose of this study was to determine whether deamidation can alter clearance of crystallins by the UPP.
Methods.
Wild-type (WT) and deamidated crystallins were expressed and 125I-radiolabeled. Ubiquitination and degradation were monitored separately.
Results.
For βB2 crystallins, rates of ubiquitination and adenosine triphosphate–dependent degradation, both indicators of active UPP, occurred in the order Q70E/Q162E>Q162E> Q70E=WT βB2 using reticulocyte lysate as the source of degradation machinery. Human lens epithelial cell lysates and lens fiber cell lysates also catalyzed ubiquitination but only limited degradation. Supplementation with proteasome failed to enhance degradation. Rates of ubiquitination and degradation of WT and deamidated βB1 crystallins were rapid and showed little relationship to the site of deamidation using N157D and Q204E mutants. γD-Crystallins were not degraded by the UPP. Deamidation altered amine reactivity, circular dichroism spectra, surface hydrophobicity, and thermal stability.
Conclusions.
These data demonstrate for the first time that, like mild oxidative stress, deamidation of some proteins makes them preferred substrates for ubiquitination and, in some cells, for UPP-dependent degradation. Failure to properly execute ubiquitination and degrade the ubiquitin-conjugates may explain their accumulation on aging and in cataractogenesis.
doi:10.1167/iovs.09-4087
PMCID: PMC2910644  PMID: 20592226
18.  Phototransformations of Advanced Glycation End Products in the Human Eye Lens due to Ultraviolet A Light Irradiation 
Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP-lysine and argpyrimidine are two UVA-absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation. The changes of OP-lysine, OP-phenethylamine (a phenethylamine analogue of OP-lysine), and argpyrimidine due to irradiation with UVA light in the presence or absence of air and ascorbic acid were followed by different spectral methods. Aged human lenses were similarly irradiated in artificial aqueous humor. The amounts of OP-lysine in the irradiated lenses and in the corresponding dark controls were determined by HPLC. Both OP-lysine and argpyrimidine decreased 20% when irradiated with UVA light in the absence of ascorbic acid. Under the same conditions, OP-lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UVA light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP-phenethylamine after UVA irradiation in the presence of ascorbic acid was phenethylamine, which indicates that the entire heterocycle of this AGE was cleaved and the initial amino group was restored. Some AGEs in the human eye lens can be transformed by UVA light.
doi:10.1196/annals.1333.021
PMCID: PMC1564128  PMID: 16037236
ascorbic acid; OP-lysine; UVA light; eye lens; glycation
19.  Methionine Sulfoxide Reductases B1, B2, and B3 Are Present in the Human Lens and Confer Oxidative Stress Resistance to Lens Cells 
Purpose
Methionine-sulfoxide reductases are unique, in that their ability to repair oxidized proteins and MsrA, which reduces S-methionine sulfoxide, can protect lens cells against oxidative stress damage. To date, the roles of MsrB1, -B2 and -B3 which reduce R-methionine sulfoxide have not been established for any mammalian system. The present study was undertaken to identify those MsrBs expressed by the lens and to evaluate the enzyme activities, expression patterns, and abilities of the identified genes to defend lens cells against oxidative stress damage.
Methods
Enzyme activities were determined with bovine lens extracts. The identities and spatial expression patterns of MsrB1, -B2, and -B3 transcripts were examined by RT-PCR in human lens and 21 other tissues. Oxidative stress resistance was measured using short interfering (si)RNA–mediated gene-silencing in conjunction with exposure to tert-butyl hydroperoxide (tBHP) and MTS viability measurements in SRA04/01 human lens epithelial cells.
Results.
Forty percent of the Msr enzyme activity present in the lens was MsrB, whereas the remaining enzyme activity was MsrA. MsrB1 (selenoprotein R, localized in the cytosol and nucleus), MsrB2 (CBS-1, localized in the mitochondria), and MsrB3 (localized in the endoplasmic reticulum and mitochondria) were all expressed by the lens. These genes exhibit asymmetric expression patterns between different human tissues and different lens sublocations, including lens fibers. All three genes are required for lens cell viability, and their silencing in lens cells results in increased oxidative-stress–induced cell death.
Conclusions.
The present data suggest important roles for both MsrA and -Bs in lens cell viability and oxidative stress protection. The differential tissue distribution and lens expression patterns of these genes, coupled with increased oxidative-stress–induced cell death on their deletion provides evidence that they are important for lens cell function, resistance to oxidative stress, and, potentially, cataractogenesis.
doi:10.1167/iovs.05-0018
PMCID: PMC1351357  PMID: 15914630
20.  Advanced glycation end products in diabetic and non-diabetic human subjects suffering from cataract 
Age  2010;33(3):377-384.
Advanced glycation end products (AGEs) play a pivotal role in loss of lens transparency, i.e., cataract. AGEs formation occurs as a result of sequential glycation and oxidation reaction between reducing sugars and protein. AGEs production takes place throughout the normal aging process but its accumulation is found to be more rapid in diabetic patients. In this study, we quantified AGEs and N-(carboxyethyl) lysine (CEL) in human cataractous lenses from non-diabetic (n = 50) and diabetic patients (n = 50) using ELISA. We observed significantly higher (p < 0.001) levels of lens AGEs and CEL in diabetic patients with cataract as compared with their respective controls. The presence of AGEs and CEL was also determined by western blotting and immuno-histochemical analysis. Furthermore, isolated β-crystallin from cataractous lenses of non-diabetic and diabetic patients was incubated with different sugars to evaluate the extent of glycation in a time dependent manner. Our data indicated more pronounced glycation in patients suffering from diabetes as compared to non-diabetics subjects demonstrating the need to focus on developing normoglycemic approaches. Such studies may provide an insight in developing therapeutic strategies and may have clinical implications.
doi:10.1007/s11357-010-9177-1
PMCID: PMC3168597  PMID: 20842534
AGEs; Cataract; Diabetes; CEL; Non-enzymatic glycation
21.  Analysis of nuclear fiber cell cytoplasmic texture in advanced cataractous lenses from Indian subjects using Debye-Bueche theory 
Experimental eye research  2007;86(2):434-444.
Alterations in ultrastructural features of the lens fiber cells lead to scattering and opacity typical of cataracts. The organelle-free cytoplasm of the lens nuclear fiber cell is one such component that contains vital information about the packing and organization of crystallins critical to lens transparency. The current work has extended analysis of the cytoplasmic texture to transparent and advanced cataractous lenses from India and related the extent of texturing to the nuclear scattering observed using the Debye-Bueche theory for inhomogeneous materials. Advanced age-related nuclear cataracts (age-range 38–75 years) and transparent lenses (age-range 48–78 years) were obtained following extracapsular cataract removal or from the eye bank, at the L. V. Prasad Eye Institute. Lens nuclei were Vibratome-sectioned, fixed and prepared for transmission electron microscopy using established techniques. Electron micrographs of the unstained thin sections of the cytoplasm were acquired at 6500X and percent scattering for wavelengths 400–700 nm was calculated using the Debye-Bueche theory. Electron micrographs from comparable areas in an oxidative-damage sensitive (OXYS) rat model and normal rat lenses preserved from an earlier study were used, as they have extremely textured and smooth cytoplasms, respectively. The Debye-Bueche theoretical approach produces plots that vary smoothly with wavelength and are sensitive to spatial fluctuations in density. The central lens fiber cells from advanced cataractous lenses from India and the OXYS rat, representing opaque lens nuclei, produced the greatest texture and scattering. The transparent human lenses from India had a smoother texture and less predicted scattering, similar to early cataracts from previous studies. The normal rat lens had a homogeneous cytoplasm and little scattering. The data indicate that this method allowed easy comparison of small variations in cytoplasmic texture and robustly detected differences between transparent and advanced cataractous human lenses. This may relate directly to the proportion of opacification contributed by the packing of crystallins. The percent scattering calculated using this method may thus be used to generate a range of curves with which to compare and quantify the relative contribution of the packing of crystallins to the loss of transparency and scattering observed.
doi:10.1016/j.exer.2007.11.018
PMCID: PMC2366113  PMID: 18191834
aged; lens nucleus; cataract; cytoplasm; humans; scattering; electron microscopy; Fourier analysis
22.  Perinuclear lens retrodots: a role for ascorbate in cataractogenesis. 
Lens retrodots are round, oblong, or oval features in the perinuclear zone of the adult lens after the fifth decade of life and associated with cataract. Retrodots were found in 47 out of 121 eyes with cataract (39%) in the present series. They show birefringence in vivo and in vitro, and chemical studies suggest that they contain calcium oxalate. It is proposed that ascorbic acid, which is abundant in the normal human lens, is the most likely source for this oxalate. Ascorbic acid is thought to have a protective role against oxidative stress in the lens and other parts of the eye, and its level is known to be reduced in senile cataract. The presence of the retrodots may identify lenses which have been exposed to oxidative stress and are less capable of resisting oxidative damage.
Images
PMCID: PMC1041096  PMID: 3828268
23.  High Affinity Nuclear and Nongenomic Estradiol Binding Sites in the Human and Mouse Lens 
Estrogen is reported to be protective against cataracts in women and animal models. Immunodection methods have identified the classic estrogen receptors (ER), ERα and ERβ, in human lens epithelial cells and their RNAs have been detected in the rat and human lens. To verify that estrogen binding occurs in the lens, sensitive [125I]-17β-estradiol binding analyses were performed on subcellular lens fractions from women (ages 39-78 years). The presence of high affinity estradiol binding sites in the nuclear, cytoplasmic, and membrane fractions indicate the lens is able to respond to estrogens, even up to age 78, although fewer binding sites were detected in the postmenopausal women. Additionally, due to the importance of mouse models in estrogen action and lens research, lenses from intact female mice were also analyzed. Both the C57BL/6 and FVB/N mouse strains also possessed high affinity binding sites in all three lens fractions. Furthermore, transcripts for ERα, ERβ, and G protein-coupled estrogen receptor (GPER; previously called GPR30) that bind estradiol with high affinity were expressed in the human and mouse lenses. These data provide the first evidence of GPER expression in the lens. Its role, functions, and subcellular location are currently unknown, but a G-shift assay in the membrane fractions of human and mouse lenses did not show evidence that estradiol induced classic G protein-coupled receptor activation. All three receptor transcripts were also detected in the lens capsule region isolated from female C57BL/6 mice, which is mainly comprised of epithelial cells. In contrast, only ERα and GPER were expressed in the cortex/nuclear region, which is primarily composed of differentiating and organelle-free fiber cells. No significant differences in specific estradiol binding and receptor RNA expression was observed in the lenses between male and female C57BL/6 mice. These findings indicate that the lens is an estrogen target tissue in both sexes. The identification of GPER, in addition to ERα and ERβ, in the lens also adds to the complexity of possible estrogen responses in the lens. Accordingly, the protective effects of estrogen in women and animals may be mediated by all three estrogen receptors in the lens. In addition, the similarities in binding and receptor RNA expression in the lenses of both species suggest that mice can be used to model estrogen action in the human lens.
doi:10.1016/j.exer.2013.04.002
PMCID: PMC3786782  PMID: 23597597
age; binding analysis; estradiol; estrogen receptor; female; GPER; lens; male
24.  Clinical and experimental advances in congenital and paediatric cataracts 
Cataracts (opacities of the lens) are frequent in the elderly, but rare in paediatric practice. Congenital cataracts (in industrialized countries) are mainly caused by mutations affecting lens development. Much of our knowledge about the underlying mechanisms of cataractogenesis has come from the genetic analysis of affected families: there are contributions from genes coding for transcription factors (such as FoxE3, Maf, Pitx3) and structural proteins such as crystallins or connexins. In addition, there are contributions from enzymes affecting sugar pathways (particularly the galactose pathway) and from a quite unexpected area: axon guidance molecules like ephrins and their receptors. Cataractous mouse lenses can be identified easily by visual inspection, and a remarkable number of mutant lines have now been characterized. Generally, most of the mouse mutants show a similar phenotype to their human counterparts; however, there are some remarkable differences. It should be noted that many mutations affect genes that are expressed not only in the lens, but also in tissues and organs outside the eye. There is increasing evidence for pleiotropic effects of these genes, and increasing consideration that cataracts may act as early and readily detectable biomarkers for a number of systemic syndromes.
doi:10.1098/rstb.2010.0227
PMCID: PMC3061104  PMID: 21402583
paediatric; cataracts; human; mouse; genetics
25.  Connections Between Connexins, Calcium, and Cataracts in the Lens 
The Journal of General Physiology  2004;124(4):289-300.
There is a good deal of evidence that the lens generates an internal micro circulatory system, which brings metabolites, like glucose, and antioxidants, like ascorbate, into the lens along the extracellular spaces between cells. Calcium also ought to be carried into the lens by this system. If so, the only path for Ca2+ to get out of the lens is to move down its electrochemical gradient into fiber cells, and then move by electrodiffusion from cell to cell through gap junctions to surface cells, where Ca-ATPase activity and Na/Ca exchange can transport it back into the aqueous or vitreous humors. The purpose of the present study was to test this calcium circulation hypothesis by studying calcium homeostasis in connexin (Cx46) knockout and (Cx46 for Cx50) knockin mouse lenses, which have different degrees of gap junction coupling. To measure intracellular calcium, FURA2 was injected into fiber cells, and the gradient in calcium concentration from center to surface was mapped in each type of lens. In wild-type lenses the coupling conductance of the mature fibers was ∼0.5 S/cm2 of cell to cell contact, and the best fit to the calcium concentration data varied from 700 nM in the center to 300 nM at the surface. In the knockin lenses, the coupling conductance was ∼1.0 S/cm2 and calcium varied from ∼500 nM at the center to 300 nM at the surface. Thus, when the coupling conductance doubled, the concentration gradient halved, as predicted by the model. In knockout lenses, the coupling conductance was zero, hence the efflux path was knocked out and calcium accumulated to ∼2 μM in central fibers. Knockout lenses also had a dense central cataract that extended from the center to about half the radius. Others have previously shown that this cataract involves activation of a calcium-dependent protease, Lp82. We can now expand on this finding to provide a hypothesis on each step that leads to cataract formation: knockout of Cx46 causes loss of coupling of mature fiber cells; the efflux path for calcium is therefore blocked; calcium accumulates in the central cells; at concentrations above ∼1 μM (from the center to about half way out of a 3-wk-old lens) Lp82 is activated; Lp82 cleaves cytoplasmic proteins (crystallins) in central cells; and the cleaved proteins aggregate and scatter light.
doi:10.1085/jgp.200409121
PMCID: PMC2233908  PMID: 15452195
connexin knockout; connexin knockin; intracellular calcium; gap junctions; coupling conductance

Results 1-25 (974553)