Cultured Rabbit Lens Research Articles

Polyamine Oxidase Is Involved in Spermidine Reduction of Transglutaminase Type 2-Catalyzed βH-Crystallins Polymerization in Calcium-Induced Experimental Cataract.

In an in vitro Ca2+-induced cataract model, the progression of opacification is paralleled by a rapid decrease of the endogenous levels of spermidine (SPD) and an increase of transglutaminase type 2 (TG2, EC 2.3.2.13)-catalyzed lens crystallins cross-linking by protein-bound N1-N8-bis(γ-glutamyl) SPD. This pattern was reversed adding exogenous SPD to the incubation resulting in a delayed loss of transparency of the rabbit lens. The present report shows evidence on the main incorporation of SPD by the catalytic activity of TG2, toward βH-crystallins and in particular to the βB2- and mostly in βB3-crystallins. The increase of endogenous SPD in the cultured rabbit lens showed the activation of a flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAO EC 1.5.3.11). As it is known that FAD-PAO degrades the N8-terminal reactive portion of N1-mono(γ-glutamyl) SPD, the protein-bound N8-mono(γ-glutamyl) SPD was found the mainly available derivative for the potential formation of βB3-crystallins cross-links by protein-bound N1-N8-bis(γ-glutamyl)SPD. In conclusion, FAD-PAO degradation of the N8-terminal reactive residue of the crystallins bound N1-mono(γ-glutamyl)SPD together with the increased concentration of exogenous SPD, leading to saturation of glutamine residues on the substrate proteins, drastically reduces N1-N8-bis(γ-glutamyl)SPD crosslinks formation, preventing crystallins polymerization and avoiding rabbit lens opacification. The ability of SPD and MDL 72527 to modulate the activities of TG2 and FAD-PAO involved in the mechanism of lens opacification suggests a potential strategy for the prevention of senile cataract.

Mechanism of DADS in the bystander effect of HSV-tk/GCV suicide gene therapy system in lens epithelial cells

To investigate the mechanism and effect of diallyl disulfide (DADS) on the bystander effect of herpes simplex virus kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy system which was mediated by recombinant adeno-associated virus (rAAV) in lens epithelial cells. Immunohistochemistry method was used to determine the distribution of connexin 43 (Cx43) protein in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene. Cx43 protein was measured and analyzed in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene that was induced by various DADS. Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. DADS increased the Cx43 protein expression in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene, and especially in 50 μmol/L DADS. After combining with DADS, the bystander effect was significantly improved (P<0.05). DADS can elevate the expression of Cx43 protein and enhance the bystander effect of HSV-tk/GCV suicide gene therapy system.

Prevention of posterior capsule opacification by the induction of therapeutic apoptosis of residual lens cells

Posterior capsule opacification (PCO) is a common complication of cataract surgery. Using adenovirus(Ad)-mediated gene transfer, we overexpressed the proapoptotic molecules p53, procaspase 3, Bax, and TRAIL to induce therapeutic programmed cell death of residual lens cells to prevent PCO. Overexpressed TRAIL did not induce apoptosis in cultured rabbit lens cells or in human lens cells. Overexpressed p53 induced apoptosis of lens cells in vitro and ex vivo, but was unable to prevent PCO in vivo. Overexpressed procaspase 3 was associated with engagement of many components of the apoptotic pathway, including cleavage of intracellular caspase targets such as PARP and inter-nucleosome DNA fragmentation. Even when only slightly overexpressed, Bax caused apoptosis of transduced rabbit and human lens cells by engaging the mitochondrial pathway, including catalytic activation of the caspases. A single in vivo injection of Ad vectors expressing either Bax or procaspase 3 into the capsular bag at the end of phacoemulsification prevented PCO in rabbits. These experiments show that Ad-mediated Bax or procaspase 3 overexpression is capable of inducing therapeutic programmed cell death in vitro and in vivo in residual lens cells and preventing PCO in a rabbit model of PCO. Manipulation of proapoptotic molecule expression could be a novel gene therapy approach for prevention of PCO.

Low-intensity microwave blockes cell cycle and regulate cell cycle related gene expression in rabbit lens epithelial cells

To study the cell cycle arrest induced by different doses of microwave in cultured rabbit lens epithelial cells (RLECs) and its effects on the related gene expression of P21(WAF1), P27(Kip1) and c-myc. Cultured rabbit lens epithelial cells were exposed to microwave radiation with frequency of 2450 MHz and power density of 0.10 mW/cm(2) (A group), 0.25 mW/cm(2) (B group), 0.50 mW/cm(2) (C group), 1.00 mW/cm(2) (D group) and 2.00 mW/cm(2) (E group) for 8 hours. The morphological change of RLECs was observed under microscope, effects of microwave radiation on RLECs cell cycle were measured by flow cytometry. Expression of P21(WAF1), P27(Kip1) and c-myc were evaluated by western blot after 2.00 mW/cm(2) microwave radiation for 4 hours, 6 hours and 8 hours respectively. All the results were compared with F group, which was as controls without microwave radiation. 8 hours after radiation, RLECs of C, D and E groups showed RLECs cell swelling, congregation and detachment, the percentage of cells in G(0)/G(1) phase obviously was increased (P < 0.01). No obvious change could be detected in 0.10 mW/cm(2) and 0.25 mW/cm(2) microwave groups. The gene expression of P27(Kip1) was increased and c-myc was decreased after 2.00 mW/cm(2) radiation, it did not affect the gene expression of P21(WAF1). Microwave exceeding 0.50 mW/cm(2) can block RLECs cell cycle at G(0)/G(1) phase, which may be related to regulation of P27(Kip1) and c-myc expression, but not P21(WAF1).

Effects of Tranilast on cultured rabbit lens epithelial cells

目的 观察曲尼司特(Tranilast)对培养兔晶状体上皮细胞增殖的作用及其对细胞周期的影响.方法将传代的兔晶状体上皮细胞用含不同曲尼司特浓度的培养液培养,每天计数,绘制细胞生长曲线,收集细胞做流式细胞仪检查.空白培养液培养的细胞做阴性对照,含0.004%丝裂霉素C (MMC)培养液培养的细胞为阳性对照.结果用180μmol/L的培养液培养细胞时细胞增殖明显受到抑制,随药物浓度增加,细胞增殖受抑制越明显,细胞生长曲线越低平,细胞形态变化不大.经流式细胞术进行细胞周期分析,随药物浓度增加G0/G1期细胞数增多(67.47%～79%),而S期细胞数减少(21.19%～4.32%).阳性对照组细胞于48h全部死亡.结论曲尼司特具有抑制晶状体上皮细胞增殖的效果,且呈浓度依赖性.曲尼司特将细胞抑制在G1期的限制点内。

Identification of caveolae and their signature proteins caveolin 1 and 2 in the lens

This study shows that caveolae are present in lens epithelia of rabbit and guinea pig under normal conditions. Caveolae are unique lipid membrane microdomains observed in many cell types. They are believed to play crucial roles in a variety of basic physiological functions including signal transduction, lipid and transcellular transport. Using TEM, immunocytochemistry and immunoblotting we show for the first time the existence of caveolae and the co-localization of their signature marker integral proteins, caveolin-1 and caveolin-2, in the intact lens of rabbit and guinea pig. Thin-section TEM shows that among several species studied, lens epithelia of rabbit and guinea pig exhibited a large number of caveolae. The caveolae were pear shaped, approximately 70 nm in diameter, and were found frequently along the lateral membranes of epithelial cells in the intact lens. In the intact cortical fibers, only a small number of caveolae was seen in the superficial cells. In cultured lens epithelial cells, however, caveolae were observed along all membrane surfaces, but were more abundant at the apical membrane of the cells. Immunofluorescence and immunoblot analyses confirmed the presence of caveolin-1 and caveolin-2 in the lens epithelium. In addition, caveolin-1 and caveolin-2 co-exist in the lens epithelium of both rabbit and guinea pig. HRP tracer study demonstrated that caveolae could carry out endocytosis, suggesting their involvement in molecular transport. Cultured rabbit lens epithelial cells (line N/N1003A) were used to examine the response of caveolae to methyl-β-cyclodextrin (MBCD), a specific cholesterol-depleting drug. The lens epithelial cells were incubated in freshly prepared MEM medium plus 8% rabbit serum containing 10 m m MBCD for 0 (control), 15, 30 or 60 min. Controls for MBCD treatment were cultured in MEM plus 8% rabbit serum. MBCD treatment for 30 min revealed that depletion of cholesterol abolished the majority of caveolae in cultured lens epithelial cells. This result strongly suggests that caveolae are cholesterol-rich lipid rafts that are likely to play important roles in the lens.

The Effects of Hyperbaric Oxygen on the Crystallins of Cultured Rabbit Lenses: a Possible Catalytic Role for Copper

Oxidative effects on lens proteins have been linked with the formation of human age-related cataract, particularly nuclear cataract. This study investigated the effects of hyperbaric oxygen (HBO)-induced oxidative stress on nuclear and cortical α-, β- and γ-crystallins of cultured rabbit lenses, using high performance liquid chromatography (HPLC). The lenses were treated with 50atm of either 100% N2(control) or 100% O2(experimental) for 3, 6, 16 and 48hr. The levels of reduced glutathione (GSH) and water-soluble (WS) protein decreased more rapidly in the nucleus of the O2-treated lens than in the cortex. The first significant loss of WS protein in each of the two regions occurred when levels of GSH had decreased by at least 90% in either the nucleus (at 6hr) or the cortex (at 16hr). HPLC analysis of the nuclear WS proteins indicated that β-crystallins were the first proteins affected by the oxidative stress. Soon after HBO-treatment was initiated (at 6hr) and prior to insolubilization of protein, nuclear β- and γ-crystallins moved to the higher molecular weight α-crystallin fraction; 2-D gel electrophoresis and Western blotting indicated the presence of disulfide-crosslinked and non-crosslinked β- and γ-crystallins in this fraction. Significantly different HBO-induced effects were observed on lens cortical crystallins compared to those for the nucleus. For example, γ-crystallins in the cortex shifted very soon after HBO-treatment (at 3hr) to slightly higher molecular weights, possibly the result of protein/glutathione mixed disulfide formation; however, this phenomenon was not observed in the nucleus. Cortical β- and γ-crystallins remained in solution longer than nuclear proteins following HBO-treatment of the lenses, presumably the result of protection from the four-fold higher level of GSH (22 vs 6m M) present in the lens periphery. Surprisingly, there was no movement of β- and γ-crystallins to αH- and α-crystallin fractions in the cortex of the O2-treated lens, in contrast to that observed for the nucleus. Cortical crystallins appeared to go directly from being soluble to being insoluble with no high molecular weight intermediate stage. The data suggested a possible chaperone-like function for α-crystallin in the nucleus of the stressed lenses, but not in the cortex. HBO-induced effects on lens nuclear supernatants, which mimicked those observed for intact lenses, could be nearly completely prevented by the copper-chelator bathocuproine, but not by the iron-chelator deferoxamine. Overall, the results provide additional evidence demonstrating an increased susceptibility of the lens nucleus to oxidative stress; the greater protective ability of the cortex may be linked to a higher capacity for β- and γ-crystallin/glutathione mixed disulfide formation, inhibiting disulfide-crosslinked insolubilization. The data also implicate copper as a catalyst for the autoxidation of -SH groups in the lens, and suggest that α-crystallin chaperone-like activity may play a greater role in the lens nucleus than in the cortex in preventing oxidative insolubilization of crystallins.

Modulation of Lens Glycolytic Pathway by Thioltransferase

The observation that the level of S-thiolated proteins (protein–thiol mixed disulfides) was transiently increased in the lens epithelial cells correlation with the transient inactivation of glyceraldehyde-3-phosphate dehydrogenase (G-3PD), a key glycolytic enzyme, when the cells were treated with a bolus of hydrogen peroxide, prompted our speculation that G-3PD may have been transiently thiolated at the SH sensitive active center. In the meantime, thioltransferase (TTase), a thiol regulating enzyme, whose activity remained constant under the same condition, may be regulating G-3PD and other sulfhydryl-sensitive glycolytic enzymes through thiol–disulfide exchange reactions (11). To prove this hypothesis, several purified glycolytic enzymes from a commercial source, including hexokinase (HK), G-3PD, pyruvate kinase (PK) and fructose 1,6-bisphosphatase (FBPase), an enzyme in gluconeogenesis, were made into protein–thiol mixed disulfide and used for this study. Glycolytic enzymes in cultured rabbit lens epithelial cells pre-exposed to H2O2(0.5m M for 15min) were also studied for this purpose. Recombinant human lens thioltransferase (RHLT), which was isolated and purified previously in this laboratory, reactivated these pure glycolytic enzymes inactivated by forming protein-S-S-gluthathione (PSSG), protein-S-S-cysteine (PSSC) or, protein-S-S-cysteamine after thiolating with oxidized glutathione, cystine or cystamine respectively. RHLT also reactivated these enzymes in the cell extract of cultured rabbit lens epithelial cells after being briefly exposed to 0.5m M H2O2. The S-thiolation and dethiolation of FBPase however, showed an opposite effect to that of glycolytic enzymes. These results suggest that TTase may participate in the repair process of glycolytic enzymes during oxidative stress and restore their activities in situ.

Thiol Regulation in the Lens

The high content of glutathione (GSH) in the lens is believed to protect the thiols in structural proteins and enzymes for proper biological functions. The lens has both biosynthetic and regenerating systems for GSH to maintain its large pool size (4-6 mM). However, we have observed that, in aging lenses or lenses under oxidative stress, the size of GSH pool is diminished; and some protein thiols are being S-thiolated by oxidized nonprotein thiols to form protein-thiol mixed disulfides, either as protein-S-S-glutathione (PSSG) or protein-S-S-cysteine (PSSC). We have shown in an H2O2-induced cataract model that PSSG formation precedes a cascade of events starting with protein disulfide crosslinks, protein solubility loss, and eventual lens opacification. Recently, we discovered that this early oxidative damage in protein thiols could be spontaneously reversed in H2O2 pretreated lenses if the oxidant was removed in time. This dethiolation process is likely mediated through a redox regulating enzyme, thioltransferase (TTase), which has been discovered recently in the lens. To understand if the role of oxidative defense and repair is the physiological function of TTase in the lens, we cloned the TTase gene and purified the recombinant human lens TTase. Although TTase required GSH for its activity, TTase was far more efficient in dethiolating lens proteins than GSH alone. It favored PSSG over PSSC and dethiolated gamma-crystallin-S-S-G better than the alpha-crystallin counterparts. Furthermore, TTase showed a remarkable resistance to oxidation (H2O2) in cultured rabbit lens epithelial cells when GSH peroxidase, GSH reductase, and glyceraldehyde-3-phosphate dehydrogenase were severely inactivated. We further showed that activity loss in those SH sensitive enzymes could be attributed to S-thiolation, but reactivation via dethiolation could be attributed to TTase. We conclude that TTase can regulate and repair the thiols in lens proteins and enzymes through its dethiolase activity, thus contributing to the maintenance of the function of the lens.

Protection from Oxidative Insult in Glutathione Depleted Lens Epithelial Cells

It has previously been shown that TEMPOL, n-propyl gallate and deferoxamine, compounds that limit the availability of Fe+2and prevent the generation of hydroxyl radicals, protect cultured rabbit lens epithelial cells from H2O2-induced damage. In view of the importance of glutathione as an antioxidant and the decrease in GSH that is known to accompany most forms of cataract, we investigated whether these compounds protected cultured lens epithelial cells from H2O2when the cells were artificially depleted of glutathione. Treatment of lens epithelial cells with 1-chloro-2,4-dinitrobenzene (CDNB), a compound that irreversibly binds to glutathione, or buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis, reduced the glutathione content to an average of 15–20% of the control values without a concomitant increase in oxidized glutathione. Morphological changes were assessed by phase contrast and electron microscopy. In order to assess growth, cells in 5 ml serum-free MEM were exposed to an initial concentration of 0.05 mmH2O2(for 50,000 cells) or 2 doses of 0.5 mmH2O2(for 800,000 cells). After exposure to H2O2, medium was replaced with MEM plus 8% rabbit serum; cells were fed on days 3 and 6 and counted on day 7.When 50,000 or 800,000 cells with decreased glutathione were exposed to 0.05 or 0.5 mmH2O2the H2O2was cytotoxic, whereas cells treated with H2O2alone remained viable but showed inhibited proliferation. An unexpected finding was that cells continued to remove H2O2from the medium at normal rates even when the GSH level was reduced. Cells treated with CDNB or BSO alone exhibited morphological and growth properties comparable to untreated cells. Cells treated with CDNB or BSO and then with H2O2exhibited decreased cell-to-cell contact, nuclear shrinkage, and arborization when viewed with phase-contrast microscopy and showed extensive nuclear and cytoplasmic degeneration at the EM level. Cell death was determined by dye exclusion and confirmed by video microscopy. When cells were treated with CDNB or BSO and subsequently treated with TEMPOL, n-propyl gallate or deferoxamine and then challenged with H2O2cytotoxicity was prevented and the cells were capable of growth. The data show that H2O2was not lethal to glutathione-depleted lens epithelial cells when they were treated with compounds that prevented the generation of reactive oxygen species. In addition, the results indicate that GSH has an important protective role independent of its ability to decompose H2O2via glutathione peroxidase.

Nuclear cataract and light scattering in cultured lenses from guinea pig and rabbit

Purpose. To compare calcium ionophore-induced cataract formation and in vitro light scattering in cultured lenses from guinea pig and rabbit.Methods. Lenses from guinea pig and rabbit were cultured for 5 or 6 days with calcium ionophore A23187. To assess the involvement of calpain in cataract formation; SDS-PAGE, immunoblotting and calcium determinations were performed. For in vitro light scattering, lens soluble proteins from rabbit were hydrolyzed for 24 h by either endogenous lens calpain, or by addition of purified m-calpain and then further incubated for up to 10 days. Light scattering was measured daily at 405 nm.Results. Lenses from younger guinea pigs cultured in A23187 first developed outer cortical opacities followed by nuclear cataract. Total calcium was markedly increased by A23187 in lenses of all ages. Proteolysis of crystallins and a-spectrin were observed in nuclear cataract in younger guinea pigs. This was attenuated with age, in association with the attenuation of cataract formation with age. Calpain 80 kDa subunit in the lenses cultured with A23187 was also decreased. Co-culture with SJA6017 or E64d (reversible and irreversible inhibitors of calpain, respectively) reduced A23187-induced nuclear opacities, proteolysis of crystallins and a-spectrin, and loss of calpain without affecting increased total calcium. In contrast, rabbit lenses cultured in A23187 did not develop nuclear cataract, although biochemical changes in cultured rabbit lenses were similar to those in cultured guinea pig lenses. Furthermore, no appreciable in vitro light scattering occurred in soluble proteins from rabbit lenses after activation of endogenous m-calpain, or after addition of exogenous purified m-calpain, although crystallins were partially hydrolyzed by calpain.Conclusions. Both rabbit and guinea pig lenses undergo calpain-induced proteolysis upon elevation of lenticular calcium. However, factors in intact guinea pig lenses may promote light scattering and insolubilization after proteolysis by calpain, but these factors were not functional in rabbit lenses. Discovery of the factors promoting light scatter and insolubilization after proteolysis will help to explain the role of certain crystallin polypeptides in cataract formation. Curr. Eye Res. 17: 623–635, 1998.

Nuclear cataract and light scattering in cultured lenses from guinea pig and rabbit

To compare calcium ionophore-induced cataract formation and in vitro light scattering in cultured lenses from guinea pig and rabbit. Lenses from guinea pig and rabbit were cultured for 5 or 6 days with calcium ionophore A23187. To assess the involvement of calpain in cataract formation; SDS-PAGE, immunoblotting and calcium determinations were performed. For in vitro light scattering, lens soluble proteins from rabbit were hydrolyzed for 24 h by either endogenous lens calpain, or by addition of purified m-calpain and then further incubated for up to 10 days. Light scattering was measured daily at 405 nm. Lenses from younger guinea pigs cultured in A23187 first developed outer cortical opacities followed by nuclear cataract. Total calcium was markedly increased by A23187 in lenses of all ages. Proteolysis of crystallins and alpha-spectrin were observed in nuclear cataract in younger guinea pigs. This was attenuated with age, in association with the attenuation of cataract formation with age. Calpain 80 kDa subunit in the lenses cultured with A23187 was also decreased. Co-culture with SJA6017 or E64d (reversible and irreversible inhibitors of calpain, respectively) reduced A23187-induced nuclear opacities, proteolysis of crystallins and alpha-spectrin, and loss of calpain without affecting increased total calcium. In contrast, rabbit lenses cultured in A23187 did not develop nuclear cataract, although biochemical changes in cultured rabbit lenses were similar to those in cultured guinea pig lenses. Furthermore, no appreciable in vitro light scattering occurred in soluble proteins from rabbit lenses after activation of endogenous m-calpain, or after addition of exogenous purified m-calpain, although crystallins were partially hydrolyzed by calpain. Both rabbit and guinea pig lenses undergo calpain-induced proteolysis upon elevation of lenticular calcium. However, factors in intact guinea pig lenses may promote light scattering and insolubilization after proteolysis by calpain, but these factors were not functional in rabbit lenses. Discovery of the factors promoting light scatter and insolubilization after proteolysis will help to explain the role of certain crystallin polypeptides in cataract formation.

Heme Oxygenase Synthesis is Induced in Cultured Lens Epithelium by Hyperbaric Oxygen or Puromycin

We showed previously that treatment of cultured rabbit lens epithelial cells (LECs) with hyperbaric oxygen (HBO) produced DNA strand-breaks, caused reversible inhibition of protein synthesis and induced the synthesis of a 32 kD protein. In the present work, we employed immunostaining procedures to identify the 32 kD protein as heme oxygenase-1 (HO-1). Increased synthesis of the enzyme was observed as early as 12 hr after HBO-treatment, reached a maximum at 18 hr and was not detectable at 36 hr. Exposure of the cells to hemin also increased the synthesis of HO-1. An HBO-induced inhibition of protein synthesis and the subsequent induction of HO-1 was also observed in the capsule-epithelium of cultured rabbit lenses. For both LECs and the cultured lens, only HO-1 and not heme oxygenase-2 was HBO-inducible. Use of the antioxidant dimethylthiourea with HBO-treated lenses or LECs did not alter the observed effects on protein synthesis or the induction of HO-1. In contrast to results obtained with 50 atm O2, a pressure of 25 atm O2inhibited protein synthesis only slightly and failed to induce synthesis of the 32 kD protein (although, as shown previously, identical exposure of LECs to 25 atm O2significantly damaged DNA). Inhibition of protein synthesis in LECs and cultured lenses with the use of puromycin also induced synthesis of HO-1. Both hemin (10 μm), a source of iron, and 50 atm O2produced a three-fold increase in the concentration of ferritin, a natural iron chelator, in LECs two days after exposure; no effects on ferritin levels were observed after 1 or 3 days. The finding that the increase in ferritin concentration occurred in the cells significantly after hemin- or HBO-induced synthesis of heme oxygenase indicates that chelatable iron rather than the heme molecule itself may have been the primary agent responsible for inducing ferritin synthesis. The data suggest that HBO-induced synthesis of HO-1 in the lens epithelium may be the result of an inhibition of protein synthesis, possibly leading to an accumulation of heme, rather than a direct protective response against oxidative stress.

Oxidative Stress Increases Production of β-Amyloid Precursor Protein and β-Amyloid (Aβ) in Mammalian Lenses, and Aβ Has Toxic Effects on Lens Epithelial Cells

Many amyloid diseases are characterized by protein aggregations linked to oxidative stress. Such diseases including those of the brain, muscle, and blood vessels exhibit plaques containing beta-amyloid (Abeta). Here we demonstrate that Alzheimer's precursor protein (betaAPP) and A beta are present at low levels in normal lenses and increase in intact cultured monkey lenses treated with H2O2 or UV radiation (known cataractogenic agents), and with phorbol 12-myristate 13-acetate. AP-1 factor binding, shown by others to up-regulate betaAPP expression, increased in the monkey lenses treated with H2O2, UV radiation, or phorbol 12-myristate 13-acetate and paralleled the increase in betaAPP expression. Rat lenses exposed to oxidative stress showed increased betaAPP in the anterior epithelium and cortex. Incubation of cultured rabbit lens N/N1003A epithelial cells with Abeta induced inclusions and vacuoles and was cytotoxic. Abeta cross-reacting protein was readily detected in the cortex of a cataractous human lens. Our data show that betaAPP and Abeta increase in mammalian lenses as part of a response to H2O2 or UV radiation and suggest that they may contribute to the mechanism by which oxidative damage leads to lens opacification.

Lens Epithelium: a Primary Target of UVB Irradiation

Cultured rabbit lenses and cultured rabbit lens epithelial cells were irradiated with UV to correlate morphological changes in the epithelium with physiological changes in the whole lens during the development of UV-induced cataract. Two UV spectral ranges were utilized; one spanned 290 to 340 nm and was designated near-UV, the other was a narrower, pure UVB region: 303 to 313 nm, designated UVB. Irradiation with either spectrum of the anterior surface of whole lenses caused opacification and a dose-dependent loss of ion homeostasis as measured by Na+ and Ca2+ concentrations in whole lenses. It was determined that cation pump activity, assessed by 86 Rb uptake, continued to decline steadily during culture after UV irradiation. Whole mount preparations of the epithelial cell layer of UVB-irradiated lenses revealed morphological changes within 2 hr of irradiation and cell death after 20 hr. Following posterior irradiation of whole lenses, the epithelial cells remained viable and lenses remained transparent during 3 days of culture, presumably because UV photons did not reach the epithelium. Absorption of UV photons by posterior fiber cell membranes and proteins did not cause opacification. To learn more about the epithelial damage, cultured rabbit lens epithelial cells were irradiated. UVB treatment retarded growth over a 7-day period in cultured cells. The surviving cells at day 7 were abnormal in appearance and the potassium concentration was approximately 50% less than controls, a finding which may explain the previously reported reduction in protein synthesis by UVB irradiation. Collectively, the data suggest that UV cataract is initiated by damage to the epithelium, including a change in membrane permeability leading to loss of ion homeostasis in the lens.

The Superoxide Dismutase Mimic TEMPOL Protects Cultured Rabbit Lens Epithelial Cells from Hydrogen Peroxide Insult

The superoxide dismutase mimic, 4-hydroxy TEMPO (TEMPOL), was used to investigate the mechanism by which H 2O 2 damages cultured rabbit lens epithelial cells and to identify some of the targets of H 2O 2 insult. Most studies aimed at determining the mechanism by which H 2O 2 exerts its cytotoxic effect have used iron chelators to prevent the generation of the damaging hydroxyl radical. Since TEMPOL does not chelate transition metals, we were afforded an additional means of investigating the mechanism by which H 2O 2 exerts its cytotoxicity. Cells at low or high density were cultured in MEM containing 5 mM TEMPOL and exposed to a single sub-lethal dose of 0·05 or 0·5 mM H 2O 2, respectively. Analysis of EPR spectra indicated that TEMPOL was stable in MEM, did not destroy H 2O 2 and penetrated the intracellular fluid. TEMPOL prevented or curtailed the H 2O 2-induced inhibition of cell growth, blebbing of the cell membrane, the decrease in NAD +, the activation of poly ADP-ribose polymerase, an enzyme involved in DNA repair, and limited the induction of single strand breaks in DNA normally brought about by H 2O 2. TEMPOL did not prevent the H 2O 2-induced decrease in reduced glutathione, lactate production, and the activity of glyceraldehyde 3-phosphate dehydrogenase, or the H 2O 2-induced increases in oxidized glutathione and hexose monophosphate shunt activity. Addition of TEMPOL 1-15 min after exposure of cells to H 2O 2 offered partial protection from the inhibition of cell division. TEMPOL at 5 mM did not inhibit cell growth. These results, coupled with our other findings suggest that some of the H 2O 2-induced damage in cultured rabbit LECs is mediated by intracellular redox-active metals involved in the Haber-Weiss cycle. Cellular changes not protected by TEMPOL, including attack of H 2O 2 on the thiol groups of GSH (mediated through glutathione peroxidase) and G3PDH, are likely brought about by H 2O 2 itself and not by reactions of oxygen free-radicals generated from H 2O 2.

Hyperbaric Oxygen Inhibits the Growth of Cultured Rabbit Lens Epithelial Cells Without Affecting Glutathione Level

Studies on human patients and experimental animals indicate that hyperbaric O 2 can opacity the lens nucleus and damage the lens epithelium in vivo. Here we investigate the effects of hyperbaric O 2 on cultured rabbit lens epithelial cells (LECs). When the cells were exposed to 50 atm O 2 (99% O 2 + 1% CO 2) for 3 hr there were no immediate effects on morphology, viability and transport processes (uptake of 86 Rb and 14C-αAIB). In addition, the O 2 treatment did not lower the high level of reduced glutathione or increase the low level of oxidized glutathione. However, 50 atm O 2 did produce a near doubling in the glycolytic rate which maintained ATP at levels only slightly lower than normal. Although the 3-hr O 2 treatment was not lethal, it completely inhibited cell division for 2 days. After 2 days, growth was initiated and, at day 7 the rate of growth was faster than the controls (control cells were treated with ambient air or 50 atm N 2 for 3 hr). Cells treated with 8 atm O 2 for 3 hr exhibited a slowed rate of growth, relative to controls, while exposure to 2 atm O 2, did not inhibit mitosis. Changes in morphology (multilayering and elongation) of cells exposed to 50 atm O 2, but not the controls, were evident 7 days after the 3-hr exposure. The incorporation of [ 35 S]methionine into individual polypeptides and [ 3H]thymidine into DNA was significantly inhibited immediately following a 3-hr treatment with 50 atm O 2, but both parameters recovered within 2 days. DNA strand breaks were observed in LECs following hyperbaric O 2 treatment as low as 4 atm O 2 for 3 hr and increased with higher pressures of O 2, but not N 2. Treatment with 50 atm O 2 nearly doubled the activity of the DNA repair enzyme, poly-ADP-ribose polymerase, and decreased the level of its substrate NAD +; the latter effect was reduced by 3-aminobenzamide, an inhibitor of the enzyme. Thus, although LECs tolerated brief exposures to high pressures of O 2 without cell death, DNA damage occurred at relatively low pressures of O 2. All of the effects of hyperbaric O 2 on LECs occurred without any alteration of the normal levels of reduced and oxidized glutathione. It appears that GSH is important in maintaining cell viability during exposure to an elevated level of O 2, but that it is incapable of preventing O 2-induced effects on growth and DNA.

DNA damage and repair in rabbit lens epithelial cells following UVA radiation.

Since ultraviolet light may be a contributing factor to cataractogenesis, we investigated the response of the lens epithelium, a potential target for UV insult, to UVA radiation. Cell survival and the induction and repair of DNA single-strand breaks (SSBs) were measured in cultured rabbit lens epithelial cells following UVA exposure. The light was passed through a filter which eliminated wavelengths below 335 nm in order to ensure that the cells were exposed only to UVA. In order to study the effect of various fluences of UVA on cell survival, 2 x 10(6) cells suspended in Tyrode's buffer were exposed to UVA. During all irradiations the cells were maintained at 0.5 degrees C in order to minimize DNA repair. Following UVA treatment, 200 cells were cultured in minimal essential medium containing 10% rabbit serum, and a colony forming assay was used to quantify cell survival. UVA induced cell death in a dose-dependent manner. In additional experiments, confluent epithelial cells on glass slides immersed in Tyrode's buffer were irradiated and SSBs were quantified using the alkaline elution technique. A 30 min exposure to UVA (180 KJ/m2) induced measurable SSBs. An increase in UVA fluence brought about an increase in the number of DNA SSBs. Rejoining of SSBs was measured after the cells were irradiated in Tyrode's for 2 hrs and allowed to repair in the dark for 4 hrs at 36 degrees C in MEM containing 10% serum. Eighty percent of the DNA SSBs were repaired within 4 hrs as determined by analysis of the alkaline elution profile. The repair kinetics were biphasic with an initial fast and subsequently slower component. The results indicate that UVA can induce SSBs in lens epithelial cells, that the cells can repair most UVA-induced SSBs, and that UVA treatment can be toxic to the epithelium.

TEM-SEM investigations on the effect of LPC on cultured rabbit lens

TEM-SEM investigations on the effect of LPC on cultured rabbit lens

Growth regulation of the mammalian ocular lens by vitreous humor.

Experiments were performed in our laboratory to study the effects of a mammalian 8 kD vitreous humor (VH) factor on the DNA synthesis and mitosis of the epithelial cells of organ cultured rabbit lens. The 8 kD polypeptide factor was purified from mature rabbit vitreous humor by liquid chromatography. Proliferative activities of the epithelial cells of organ cultured lenses were stimulated by 3% rabbit serum. The data from our experiments depicted that the 8 kD VH factor effectively inhibits DNA synthesis and mitosis by the epithelial cells of the organ cultured lens. Our experiments also showed that this 8 kD VH factor can maintain its growth inhibitory activity even when heated for 3 min at 95 degrees C. The growth inhibitory effect of the 8 kD VH factor was dose dependent. Using iodinated vitreal proteins it was demonstrated that the VH proteins are able to enter or bind to lens epithelial cells. The growth inhibitory effect of the 8 kD VH factor was also tested on tissue cultured lens epithelial cells. These experiments showed that the 8 kD VH factor has no growth inhibitory effect on the tissue cultured lens epithelial cells. This experiment has been repeated many times using different concentrations of the factor. These observations suggest that the 8 kD VH factor may have receptors in the lens capsular material (extracellular matrix) and the factor-receptor binding is essential for the growth inhibitory effect.