Organ Cultured Rat Lenses Research Articles

Chaperone Peptides of α-Crystallin Inhibit Epithelial Cell Apoptosis, Protein Insolubilization, and Opacification in Experimental Cataracts

α-Crystallin is a member of the small heat-shock protein (sHSP) family and consists of two subunits, αA and αB. Both αA- and αB-crystallin act as chaperones and anti-apoptotic proteins. Previous studies have identified the peptide (70)KFVIFLDVKHFSPEDLTVK(88) in αA-crystallin and the peptide (73)DRFSVNLDVKHFSPEELKVK(92) in αB-crystallin as mini-chaperones. In the human lens, lysine 70 (Lys(70)) of αA and Lys(92) of αB (in the mini-chaperone sequences) are acetylated. In this study, we investigated the cellular effects of the unmodified and acetyl mini-chaperones. The αA- and αB-crystallin peptides inhibited stress-induced aggregation of four client proteins, and the αA-acetyl peptide was more effective than the native peptide against three of the client proteins. Both the acetyl and native crystallin peptides inhibited stress-induced apoptosis in two mammalian cell types, and this property was directly related to the inhibition of cytochrome c release from mitochondria and the activity of caspase-3 and -9. In organ-cultured rat lenses, the peptides inhibited calcimycin-induced epithelial cell apoptosis. Intraperitoneal injection of the peptides inhibited cataract development in selenite-treated rats, which was accompanied by inhibition of oxidative stress, protein insolubilization, and caspase activity in the lens. These inhibitory effects were more pronounced for acetyl peptides than native peptides. A scrambled αA-crystallin peptide produced no such effects. The results suggest that the α-crystallin chaperone peptides could be used as therapeutic agents to treat cataracts and diseases in which protein aggregation and apoptosis are contributing factors.

Bioactive Derivatives of Curcumin Attenuate Cataract Formation In Vitro

In this study, curcumin derivatives salicylidenecurcumin (CD1) and benzalidenecurcumin (CD2)] were prepared, and their biological activity was compared in in vitro selenite-induced cataract model. The antioxidant activity was studied using DPPH radical scavenging assay. Knoevenagel condensates of curcumin exhibited higher DPPH radical scavenging activity compared with curcumin. The anticataractogenic potential of curcumin derivatives was analyzed using lens organ culture method. The activity of antioxidant enzymes and calcium homeostasis was reversed to near normal levels following treatment in organ cultured rat lenses. These results indicated that curcumin and its derivatives--CD1 and CD2--are beneficial against selenite-induced cataract in vitro. Of these, CD1 is having higher bioactive potential compared with curcumin and CD2.

Protective Effects of l- and d-Carnosine on α-Crystallin Amyloid Fibril Formation: Implications for Cataract Disease

Mildly denaturing conditions induce bovine alpha-crystallin, the major structural lens protein, to self-assemble into fibrillar structures in vitro. The natural dipeptide l-carnosine has been shown to have potential protective and therapeutic significance in many diseases. Carnosine derivatives have been proposed as potent agents for ophthalmic therapies of senile cataracts and diabetic ocular complications. Here we report the inhibitory effect induced by the peptide (l- and d-enantiomeric form) on alpha-crystallin fibrillation and the almost complete restoration of the chaperone activity lost after denaturant and/or heat stress. Scanning force microscopy (SFM), thioflavin T, and a turbidimetry assay have been used to determine the morphology of alpha-crystallin aggregates in the presence and absence of carnosine. DSC and a near-UV CD assay evidenced that the structural precursors of amyloid fibrils are polypeptide chain segments that lack stable structural elements. Moreover, we have found a disassembling effect of carnosine on alpha-crystallin amyloid fibrils. Finally, we show the ability of carnosine to restore most of the lens transparency in organ-cultured rat lenses exposed to similar denaturing conditions that were used for the in vitro experiments.

Methylglyoxal inhibits glycation-mediated loss in chaperone function and synthesis of pentosidine in α-crystallin

α-Crystallin is a major protein in the eye lens and it functions as a molecular chaperone by preventing aggregation of mildly denatured proteins. Glycation, the reaction of sugars and ascorbate with proteins, causes covalent cross-linking and reduces the chaperone function of α-crystallin. We demonstrated that methylglyoxal (MGO), a metabolic α-dicarbonyl compound, modifies arginine residues in α-crystallin and enhances its chaperone function. We wanted to determine whether modification by MGO could protect α-crystallin from glycation-mediated cross-linking and loss of chaperone function. Our results show that MGO-modification of isolated bovine lens α-crystallin inhibits formation of pentosidine, a glycation-derived protein crosslink. Proteins in organ cultured rat lenses were similarly protected from pentosidine formation. Glycation by sugars and ascorbate resulted in almost complete loss of chaperone function of α-crystallin. Surprisingly, addition of MGO during or before glycation not only inhibited the loss of chaperone function, but it actually enhanced the chaperone function of α-crystallin. Together, these data suggest that in the aging lens, MGO inhibits glycation-mediated pentosidine synthesis and the loss of chaperone function of α-crystallin.

Roles for KCC Transporters in the Maintenance of Lens Transparency

To determine whether the potassium chloride cotransporter (KCC) family is expressed in the rat lens and to ascertain whether the transporters are involved in the regulation of lens volume and transparency. RT-PCR was performed on RNA extracted from fiber cells to identify members of the KCC family expressed in the lens. Western blot analysis and immunocytochemistry, using KCC isoform-specific antibodies, were used to verify expression at the protein level and to localize KCC isoform expression. Organ-cultured rat lenses were incubated in isotonic artificial aqueous humor (AAH) that contained either the KCC-specific inhibitor [(dihydronindenyl)oxy] alkanoic acid (DIOA), the KCC activator N-ethylmaleimide (NEM), or the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) for up to 18 hours. Lens wet weight was monitored, and lens transparency and tissue morphology were recorded with dark-field and confocal microscopy, respectively. Molecular experiments to characterize KCC isoform expression showed that KCC1, -3, and -4 were all expressed in the lens at both the transcript and protein levels and that KCC2 was not. Immunocytochemistry indicated that the three KCC isoforms exhibited distinct differentiation-dependent expression patterns, with KCC1 and -3 being restricted to the lens cortex, whereas KCC4 was found throughout the entire lens, including the lens core. In the lens cortex, most of the labeling for all KCC isoforms was cytoplasmic, whereas in the lens core, KCC4 labeling was associated with the membrane. Incubation of lenses in 100 microM DIOA for 18 hours caused lenses to increase their wet weight and induced a cortical opacity that was caused by extensive damage to peripheral fiber cells located up to 150 microm in from the lens capsule, whereas deeper fiber cells appeared unaffected by DIOA exposure. Lower concentrations of DIOA (10 microM) revealed that this damage was initiated primarily by the swelling of peripheral fiber cells. In contrast, NPPB-treated lenses exhibited a deeper zone (>100 microm) of cell damage that was initiated by the dilation of the extracellular space between fiber cells. Exposure of lenses to the KCC activator NEM caused cell shrinkage in peripheral fiber cells but extensive cell swelling in deeper fiber cells. Peripheral cell swelling caused a differential recruitment of KCC isoforms from a cytoplasmic pool to the plasma membrane. DIOA-induced cell swelling increased the association of KCC4 with membrane, whereas hypotonic cell swelling dramatically increased the association of KCC1 with the membrane. The rat lens expresses three KCC transporter isoforms (KCC1, -3, and -4) in a differentiation-dependent manner. Modulation of transporter activity and subcellular localization suggests that multiple KCC transporters mediate KCl efflux in peripheral fiber cells in a dynamic fashion. These results indicate that, in addition to Cl- channels, KCC transporters play a role in mediating a circulating flux of Cl- ions, which contributes to the maintenance of lens transparency through controlling the steady state volume of lens fiber cells.

Pyridoxamine Inhibits Maillard Reactions in Diabetic Rat Lenses

Purpose: Advanced glycation end products (AGEs) play an important role in protein modification during cataract formation. Along with sugars, α-dicarbonyl compounds, such as methylglyoxal (MGO), have been implicated in AGE formation. Here we report the effect of pyridoxamine (PM) on AGEs and AGE-precursor-metabolizing enzymes in diabetic rat lenses and organ-cultured rat lenses. Methods: Diabetes was induced in rats by injecting streptozotocin. Diabetic and nondiabetic control rats were treated with PM in drinking water for 20 weeks. Rat lenses were organ cultured with normal or high glucose. We measured lens glutathione (GSH), MGO, AGEs and activities of aldose reductase and glyoxalase I. Results: Treatment of diabetic rats with PM inhibited both argpyrimidine and pentosidine formation when compared to untreated diabetic animals and nondiabetic control animals. Incubation of lenses with 30 mMD-glucose caused an elevation of these AGEs. Addition of 250 µM PM along with glucose resulted in inhibition of AGE formation in organ-cultured lenses. The glyoxalase I activity was significantly reduced in diabetic rats; PM treatment inhibited such a reduction. The activity of aldose reductase was elevated in diabetic lenses; PM treatment further enhanced its activity. Conclusion: Our results suggest that PM can inhibit AGE formation in the diabetic lens by enhancing the activity of aldose reductase and reacting with precursors of AGEs.

Alterations in lens protein tyrosine phosphorylation and phosphatidylinositol 3-kinase signaling during selenite cataract formation

Purpose. Protein tyrosine phosphorylation is an important event in the cell signal transduction process. Phosphatidylinositol-3kinase (PI-3K) is an intracellular signal mediator and plays a key role in many cellular functions. In this study we have examined the changes in lens protein tyrosine phosphorylation and its impact on phosphatidylinositol 3-kinase (PI-3K) signaling during selenite cataract development. Methods. Cataract was induced in 10 days old rat pups by a single sub-cutaneous injection of sodium selenite (30µM/Kg body weight) and lenses were collected at different stages of cataract development. Immunoprecipitation and Western immunoblotting were employed to determine protein tyrosine phosphorylation, PI-3K activity and protein in lens cell extracts. Tyrosine kinase activity in lens membrane preparations was assayed in the presence of a synthetic substrate peptide and [ 32 P]ATP. Results. Protein tyrosine phosphorylation in the lens was disrupted before the onset of cataract. A decrease in tyrosine phosphorylation of lens proteins was observed within 2–3 days of selenite injection (pre-cataract stage). The effect was much more prominent with the progression of cataract. The decrease in protein tyrosine phosphorylation correlated with the decrease in tyrosine kinase activity associated with the lens membrane fraction. Stimulation of normal rat lenses in organ culture with insulin and IGF-1 caused an increase in the phosphorylation of proteins, whose tyrosine phosphorylation status appeared to be diminished during cataract development. Insulin and IGF-1 also stimulated rat lens PI-3K activity. While there was no change in total PI-3K activity during the onset of cataract, the activity of PI-3K associated with tyrosine phosphorylated proteins decreased markedly in pre-cataract lenses. Further, the ability of IGF-1 to stimulate PI-3K activity was significantly reduced in lens epithelial cells treated with selenium. Conclusions. These studies show that signaling events involving the protein tyrosine phosphorylation process and activation of PI-3K are altered during selenite cataract formation and implicate defects in signal transduction mechanisms as contributing factors in the development of cataract.

Effect of Perillic Acid, a Putative Isoprenylation Inhibitor, on the Cultured Rat Lens

Previous studies have demonstrated that agents affecting the cholesterol synthetic pathway can have cataractogenic effects. We have suggested that opacification of cultured lenses resulting from exposure to the cholesterol-lowering agent lovastatin is caused by inhibition of isoprenylation of small GTPases. To test that hypothesis we have investigated the effects of perillic acid, an agent reported to inhibit isoprenylation, on rat lenses in organ culture. Perillic acid caused dose and time dependent opacification of cultured lenses. While the opacities appeared grossly similar to those produced by lovastatin, they differed dramatically when analysed histologically. It also produced marked morphological changes to lens epithelial cells in culture. Analysis of small GTPases in the perillic acid treated cells failed to detect any accumulation in the water soluble fraction as would be expected if isoprenylation was inhibited. Further, studies on the isoprenylation of radiolabelled isoprenoids into proteins in cultured lenses showed no significant decrease following perillic acid exposure. It was concluded that perillic acid causes cataract in this system by a mechanism different from lovastatin and that inhibition of isoprenylation is unlikely to be a primary factor in the perillic acid cataract.

Maillard Reactions in Lens Proteins: Methylglyoxal-mediated Modifications in the Rat Lens

The nonenzymatic Maillard reaction is thought to contribute to aging and cataract formation in the lens. As levels of methylglyoxal (MG) and glutathione (GSH) affect the reaction, we examined the relationship of these factors and determined the effect of a glyoxalase I inhibitor on the Maillard reaction. Rat lens cultures were maintained for up to 3 days in TC-199 medium with or without 20 m m glyceraldehyde (GLD) and 250 μm S-[N-hydroxy-N-(4-chlorophenyl) carbamoyl] glutathione diethyl ester (HCCG diester). We measured GSH, MG, D-lactate, glyoxalase I activity, immunoreactive MG-derived advanced glycation endproducts (MG-AGEs) and imidazolysine in organ cultured rat lenses. In vitro experiments with isolated rat lens proteins revealed that HCCG alone inhibited glyoxalase I activity in a dose-dependent manner. In organ cultured rat lens protein, GLD increased MG levels 24-fold, and the addition of HCCG diester further increased it by about two-fold. GSH levels fell sharply in the presence of GLD and this was prevented to some extent by the presence of HCCG diester. D-lactate production in the lens was suppressed by HCCG diester treatment. Dialysed lens proteins retained glyoxalase I activity, indicating that the enzyme was unaltered during incubation. MG-AGEs and imidazolysine levels were significantly higher (P<0.05) in GLD-treated lenses, but a combination of HCCG diester and GLD lowered immunoreactive MG-AGEs and imidazolysine levels compared to GLD alone. HCCG had no significant effect on MG-AGE formation in lens proteins incubated with GLD or MG. We conclude that exogenous GLD enhances MG and MG-AGE levels in the rat lens and that this increase is accompanied by a loss in GSH. In addition, inhibition of glyoxalase I promotes MG accumulation.

A brief photochemically induced oxidative insult causes irreversible lens damage and cataract II. Mechanism of action

Using photochemically induced oxidative stress and rat lenses in organ culture with 4% O2 and 4 microM riboflavin, it has been found that the observed changes in lens parameters are, in most cases, irreversible. This has made possible the elucidation of the sequence of biological changes leading to cataract. The earliest detectable changes in lens cell biology are observed in the epithelial cell redox set point and at the DNA level in terms of DNA integrity and 3H-thymidine incorporation followed by decreased membrane transport and changes in gene expression. Significant modification in classical cataract parameters such as hydration, steady state non-protein thiol, glyceraldehyde-phosphate-dehydrogenase activity and transparency occur at later times. The data suggest a definitive pattern of lens breakdown resulting in opacity starting at the epithelial cell level and leading to subsequent fibre cell involvement.

Effect of smoke condensate on the physiological integrity and morphology of organ cultured rat lenses.

Smoke, either from cigarette smoking or from burning of organic fuels, has been proposed to be a major environmental risk factor for a variety of human diseases. Recently, smoke was implicated in cataract, an eye lens opacification which is a major cause of blindness. We have undertaken a study to investigate the effect of wood smoke condensate on the physiological integrity and morphology of organ cultured lenses. Lenses in organ culture are metabolically active and have functional defense systems, thus they provide an appropriate model for studying effects of smoke condensate. Our present study indicates that metabolites of wood smoke condensate accumulate in the lens. The ability of the lenses to accumulate rubidium-86 (mimic of potassium) and choline from the medium is compromised by exposure to smoke condensate. Rubidium efflux studies suggest that the damage is primarily at the uptake level and does not involve an overall increase in membrane permeability. Protein leakage experiments corroborate this suggestion. Histological data show distinct morphological changes such as hyperplasia, hypertrophy and multilayering of epithelial cells.

Heat shock response of the rat lens.

The sequence relationship between the small heat shock proteins and the eye lens protein alpha-crystallin (Ingolia, T. D., and E. E. Craig, 1982, Proc. Natl. Acad. Sci. USA, 79: 2360-2364) prompted us to subject rat lenses in organ culture to heat shock and other forms of stress. The effects on protein synthesis were followed by labeling with [35S]methionine and analysis by one- and two-dimensional gel electrophoresis and fluorography. Heat shock gave a pronounced induction of a protein that could be characterized as the stress protein SP71. This protein probably corresponds to the major mammalian heat shock protein hsp70. Also two minor proteins of 16 and 85 kD were induced, while the synthesis of a constitutive heat shock-related protein, P73, was considerably increased. The synthesis of SP71 started between 30 and 60 min after heat shock, reached its highest level after 3 h, and had stopped again after 8 h. In rat lenses that were preconditioned by an initial mild heat shock, a subsequent shock did not cause renewed synthesis of SP71. This effect resembles the thermotolerance phenomenon observed in cultured cells. The proline analogue azetidine-2-carboxylic acid, zinc chloride, ethanol, and calcium chloride did not, under the conditions used, induce stress proteins in the rat lens. Sodium arsenite, however, had very much the same effects as heat shock. Calcium ionophore A23187 specifically and effectively induced the synthesis of the glucose-regulated protein GRP78. No special response to stress on crystallin synthesis was noticed.

The effects of “oxygen radicals” generated in the medium on lenses in organ culture: Inhibition of damage by chelated iron

Rat lenses in organ culture were exposed to activated species of oxygen generated in the culture medium either by xanthine oxidase and hypoxanthine or by riboflavin and visible light, two systems which have been shown to produce superoxide and H 2O 2. In each case there was marked damage to carrier-mediated transport systems of the lens. Under standard culture conditions this damage was strongly inhibited by catalase, but not by superoxide dismutase (SOD). By the addition to the medium of chelated iron, hydroxyl radicals were produced in a Fenton reaction with a concomitant decrease in H 2O 2 levels. With both oxygen radical-generating systems, the addition of chelated iron strongly inhibited lens damage. This inhibitory effect could be reversed by the addition of SOD with the chelated iron. Under such conditions SOD converts superoxide anion to H 2O 2, thereby preventing reduction of the chelated iron and thus stopping the generation of hydroxyl radicals. Increased lens damage following addition of SOD to the iron-containing systems correlated with higher H 2O 2 concentrations, and was inhibited by catalase. These findings suggest that, when generated in the fluids surrounding the lens, H 2O 2 poses a much greater oxidative stress for the lens than do the superoxide or hydroxyl free radicals.

Effects of lipid peroxidation products on the rat lens in organ culture: A possible mechanism of cataract initiation in retinal degenerative disease

Rat lenses in organ culture which are exposed to bovine rod outer segments (ROS) or to the major fatty acid of ROS, docosahexaenoic acid, are impaired in their ability to accumulate radiolabeled compounds which lenses normally accumulate by active processes. The extent of lens damage correlates well with the extent of lipid peroxidation in the culture medium as assessed by the thiobarbituric acid assay. Addition of vitamin E to the medium inhibits the effect on the lens while addition of Fe-ADP complexes potentiates the effect. Thus, the lens damage appears to be attributable to toxic species generated by peroxidation of the polyunsaturated lipid added to the culture medium. Toxic aldehyde products appear to be major mediators of the lens damage, since semi-carbazide, which avidly reacts with aldehydes, can protect lenses in this system. These findings may have relevance to the cataracts clinically associated with retinal degenerative diseases such as retinitis pigmentosa. The highly membranous photoreceptor cells are extremely rich in polyunsaturated lipid. Degeneration of these cells, which is the primary pathology in such diseases, would likely lead to peroxidation with generation of toxic products within the eye. Such products could potentially produce secondary damage to other ocular structures including the lens.