Suppression of post-vitrectomy lens changes in the rabbit by novel benzopyranyl esters and amides.

This study reports for the first time a therapeutic modality for the suppression of posterior subcapsular cataract (PSC) formation in an animal model (rabbit) of vitrectomy. This therapeutic modality may also have the potential to attenuate/prevent the high incidence of loss of vision due to cataract formation in patients that undergo vitrectomy. Unilateral, partial vitrectomy was performed on 2.5 month old Dutch Belted rabbits with vitreous replaced by either commercially available BSS((R)) or BSS PLUS((R)) (n=16). Alternatively, vitreous was replaced with a proprietary, modified BSS PLUS((R)) irrigating solution containing 1.25 microM AL-8417 (n=12), 5.0 microM AL-12615 (n=5) or 5.0 microM AL-17052 (n=9). Age matched, non-operated rabbits were used as controls (n=16). Lenses were analysed by correlative structural (light, scanning electron microscopic and three-dimensional computer-assisted drawings) and optical (low power helium-neon laser scan) quality analysis 6 months following surgery. Results demonstrate that vitreous replacement with an irrigating solution that contains the ester-linked benzopyran, AL-8417, the amide-linked benzopyran pro-drug, AL-17052, or its active metabolite, AL-12615, prevented abnormal post-vitrectomy lens growth, or fiber formation. Focal length variability (FLV) assessments (sharpness of focus) confirmed the beneficial drug effects detected morphologically, with FLV being essentially equal to that of age-matched, non-surgical controls. In contrast, lenses of animals with vitreous replaced solely with BSS((R)) or BSS PLUS((R)) exhibited significantly higher FLV than both age-matched controls and animals that underwent vitrectomy with drug-containing irrigating solutions. The ability of AL-8417, AL-17052 and its active metabolite, AL-12615, to suppress vitrectomy-induced posterior lens fiber changes appears to reside in their unique pharmacological profile, acting as antioxidant, anti-inflammatory and cytostatic agents.

The effect of partial vitrectomy on blood-ocular barrier function in the rabbit.

PURPOSE: To compare ocular vascular permeability in the rabbit after vitrectomy as assessed by contrast-enhanced magnetic imaging (CE-MRI) and measurements of aqueous and vitreous humor protein concentration. METHODS: Partial vitrectomies were performed, irrigating with BSS or BSS PLUS. Post-operative vascular leakage was determined by CE-MRI following intravenous administration of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). Aqueous and vitreous protein concentrations were quantified by standard biochemical assay. ERG evaluations were performed on postoperative days 1, 3, and 7. RESULTS: Using BSS as irrigant, breakdown of the inner blood-retinal barrier (BRB) occurred in 4/7 eyes on post-operative day 1. The rate of Gd-DTPA leakage was significantly greater on postoperative day 1 than that in unoperated, control eyes, but declined approximately 50% by day 3. At both time points, outer BRB breakdown was restricted to the sclerotomy wounds. No BRB leakage was detectable in control eyes. Blood-aqueous barrier (BAB) leakage was bilateral on day 1. Significantly greater Gd-DTPA leakage occurred in the operated eye than in the nonsurgical contralateral eye. On day 3, approximately 40% bilateral reduction in leakage indicated resolution of BAB leakage. Notably, Gd-DTPA leakage of the BAB and BRB was significantly reduced in the BSS PLUS treated group. In contrast to MRI assessments, protein concentrations of the aqueous and vitreous in the surgical eye showed no detectable differences between BSS and BSS PLUS. Concurrent with the transient loss of ocular barrier function, ERG responses also declined. However, by day 7 greater than 90% recovery was noted in BSS PLUS treated animals but not in the BSS treatment group. CONCLUSIONS: CE-MRI is capable of detecting subtle changes in vascular permeability following ocular surgery. Advantages of using BSS PLUS compared to BSS as the irrigating solution can be detected using this technique. BSS PLUS's protection of barrier function is consistent with a rapid recovery in retinal function not observed in BSS treated eyes.

Menadione-induced oxidative stress accelerates onset of Emory mouse cataract in vivo.

PURPOSE: We evaluated the effect of the free-radical generator, menadione, on the time of occurrence of cataract in the Emory mouse, a model for human cataract. Concomitant experiments were done to compare production and level of reactive metabolites of oxygen in the Emory mouse and its cataract resistant (CR) genetic control. METHODS: Test and control mice were fed both a normal diet and a diet supplemented with menadione, and the lenses were evaluated for the time of occurrence of cataract and the level of membrane ATPases. Effects of menadione were determined on incubated lenses of Emory and CR mice, assaying reactive species of oxygen, levels of antioxidant enzymes, and formation of the lipid peroxidation product, malondialdehyde. RESULTS: Systemic administration of menadione markedly accelerated the onset of Emory mouse cataract, and decreased ATPase activities suggested oxidative damage to membrane proteins. Cumulative levels of O2.-, H2O2, .OH and malondialdehyde were significantly higher than controls in the lenses incubated in the presence of menadione, showing that it generates oxidative stress. However, [GSH] in lenses decreased equally in test and control mice. The observed increases in catalase and glutathione peroxidase activities in the test lenses indicated an early protective response to oxidative insult. CONCLUSIONS: Acceleration by menadione of the appearance of cataract in the Emory mouse demonstrates that oxidative stress is a risk factor in late-onset cataract. This quinone caused a greater increase in the production and levels of reactive metabolites of oxygen in Emory mice than in CR mice, indicative of a higher susceptibility of the former to oxidative insult.

Spectral characterization of lipid peroxidation in rabbit lens membranes induced by hydrogen peroxide in the presence of Fe2+/Fe3+ cations: a site-specific catalyzed oxidation.

The role of free-radical-induced lipid peroxidation (LPO) in relation to lens opacity is investigated using Fourier transform infrared spectroscopy. Phospholipids extracted from nuclear and cortical regions of the rabbit lens membranes are subjected to oxidative-damage induced by hydrogen peroxide and Fe2+/Fe3+ cations. Vibrational data suggest a homolytic decomposition of the unsaturated membrane hydrocarbon chains at cis-double bonds, as well as structural modifications at the carbonyl and phosphate-oxygen sites of the fiber cell membranes upon metal oxidation. This is also evident from a substantial induction of the carbonyl groups and a significant dephosphorylation of the phosphate groups in lens phospholipids. These covalent modifications and/or alterations of the carbonyl and phosphate groups, and specificity of certain vibrational modes only to iron oxidation, may serve as a diagnostic probe of the metal-catalyzed LPO in lens membranes. Despite covalent modifications of the hydrophilic part of the lens membranes, hydrocarbon chain region remains largely intact at physiological concentrations of hydrogen peroxide. However, at elevated concentrations of hydrogen peroxide, a substantial breakdown of the acyl chains occurs. Striking similarities observed between the spectral features of the oxidized rabbit lens phospholipids and those of the cataractous human lenses suggest that the mechanism and pathways of lipid oxidation in model animal membranes and in human lenses are similar. Differences in the nuclear or cortical regions are also evident upon metal oxidation. Nuclear lipids experience increased effects of the metal oxidation compared to cortical lipids. Both the nuclear or the cortical lipids indicate effective penetration of the bilayer water creating segregated membrane domains, possibly through breakdown of headgroup-specific lipid-water interactions. This could effectively alter the lens membrane permeability and fluidity, rendering it susceptible to a host of toxic oxidants present in the eye. These findings also demonstrate that LPO can lead to acyl chain degradation that may effectively derange the lens membrane function, which could be a contributing factor in cataractogenesis.

Infrared study of the structure and composition of rabbit lens membranes: a comparative analysis of the lipids of the nucleus, cortex and epithelium.

Infrared spectra of the phospholipids extracted from the nuclear, cortical and epithelial regions of the rabbit lens membrane bilayers are examined for the first time. Major structural, conformational and compositional differences in the lens membrane are correlated at physiological temperature. Vibrational data distinguish two classes of the phospholipids present in the rabbit lens membranes. Sphingolipids with a sphingosine backbone are largely concentrated in the nucleus whereas phospholipids with a glycerol backbone (glycero-lipids) such as phosphatidylcholine and phosphatidylethanolamine are abundant in the cortical region of the lens. Nuclear lipids are more saturated by a factor of four over the lipids of the cortical region. Nuclear lipids also exhibit increased headgroup and interface hydration and stronger hydrogen bonding over cortical lipids which provide them additional structural stability needed for the clarity of the lens. The lipid composition of the epithelial membranes is structurally similar to that of the cortical region but the hydrocarbon chains are more saturated. Epithelial membranes are largely shielded from bilayer water indicating hydrophilic lipid-water interactions are not important for the stability of these membranes. These membranes exhibit much stronger lipid-lipid interactions, vital for many physical properties like membrane fluidity and permeability. The fiber cell membranes are stabilized by hydrogen bonding of the carbonyls of the interface region and by headgroup-specific lipid-water interactions, while epithelial membranes are stabilized primarily through hydrophobic lipid-lipid interactions. Data also exhibit two phase transitions around 16 and 35 degrees C corresponding to the melting of the hydrocarbon chains from two pools of the phospholipids i.e. glycero- and sphingophospholipids, respectively. The first transition is caused by disruption of the carbonyl hydrogen bonding and disordering of the acyl chains of the glycero-lipids, while second transition is driven mainly from the hydrogen bonding effects of the carbonyls of the sphingolipids followed by acyl/sphingosine chain disordering. Upon transition to the liquid-crystalline phase, a sizable amount of the fiber cell membranes are disordered (approximately 33%) due to increased conformational motion of the acyl chains resulting from a loss of extended CH2 trans segments. These chains of the fully hydrated lipids pack in an hexagonal or triclinic unit cell in rabbit lens membranes.

The prevention of cataract caused by oxidative stress in cultured rat lenses. I. H2O2 and photochemically induced cataract.

H2O2 stress is shown to produce cataract in cultured rat lenses. The loss of transparency begins in the equatorial region within 24 hours and the entire superficial cortex is opaque by 96 hours. No involvement of the nuclear region is observed. However after an additional 48 hours, the nuclear region becomes opaque. The loss of transparency is accompanied by a large uptake of H2O which occurs gradually over the 96 hour period, complete loss of glyceraldehyde phosphate dehydrogenase (GPD) activity, almost complete loss of non-protein thiol and a slight decrease in protein thiol. Control lenses show no change other than the establishment of a new non-protein thiol base line approximately 60% lower than 0 time levels. The Alcon glutathione peroxidase type mimic, AL-3823A, completely eliminates almost all of the H2O2 induced effects and the lens remains transparent. Utilizing a more severe photochemical model than may be anticipated physiologically with 10 microM riboflavin and exposure to daylight fluorescent lamps, significant concentrations of superoxide and low levels of OH. are produced as well as extraordinarily high concentrations of H2O2 ranging from about 400 to 1000 microM. As with the H2O2 model, opacification begins at the equator but the cataract develops more rapidly, the lens being completely opaque by 68 hours. Hydration, GPD activity, non-protein and protein thiol all decrease more rapidly than in the H2O2 model. AL-3823A prevents loss of transparency until approximately 92 hours and markedly decreases changes in other parameters. At 92 hours, slight loss of transparency is observed. Catalase is somewhat less effective. AL-3823A is shown to also significantly decrease superoxide levels. The marked delay in the onset of changes in lens biochemistry and physiology in the severe photochemical stress model and the maintenance of normal parameters in the H2O2 model in the presence of AL-3823A suggests that such compounds may prevent cataract caused by oxidative stress under physiological conditions.

Nuclear magnetic resonance microscopic ocular imaging for the detection of early-stage cataract.

A nuclear magnetic resonance (NMR) microscopic ocular imaging was performed at 7.0 Tesla to investigate its usefulness in the detection of early-stage cataracts. For this study, galactose cataracts were generated in experimental rabbits through diet (35% galactose), and enucleated eyes were imaged at various times after initiation of the diet. In previous studies using a 0.6 Tesla conventional magnetic resonance imager (MRI), the contrast between normal and cataractous tissues in the lens was not well defined, mainly due to the partial volume effect coming from the limitation of resolution and signal-to-noise ratio (SNR). With resolution of 60 X 60 X 80 microns, early localized precataractous tissue changes were clearly observed after 5 days diet. Precataractous tissue changes were seen histologically but no visible evidence of lens change was detected by the conventional slit lamp biomicroscope at this time. Substantially elongated spin-spin relaxation times (T2) in localized cataractous tissues (72.4 +/- 8.8 msec) were consistently observed compared with those in normal lens region (16.1 +/- 3.2 msec); however, the changes of the spin-lattice relaxation time (T1) were not significant. Some ocular NMR microscopic images with corresponding histological photographs are demonstrated to show the potential of NMR microscopy.

Structure analysis of bovine lens calf gamma-II crystallin: residue assignments of the five histidine CE1 resonances observed by proton-nuclear magnetic resonance spectroscopy.

Bovine lens calf gamma-II crystallin contains five histidine residues at sequence positions 14, 53, 84, 117, and 122. The protein was examined by proton nuclear magnetic resonance spectroscopy at 300 MHz. Five histidine epsilon-1 carbon (CE1) proton resonances were observed titrating with pH between 8.9 and 7.4 ppm. The chemical shift values as a function of pH were fitted to a Henderson-Hasselbalch equation. The experimental pK values and end points of titration were then compared to theoretical electrostatic and ring-current calculations based on the 1.6 A resolution x-ray coordinate data of the protein. A sufficiently close correspondence between the experimental and calculated values allowed histidine residue assignments to be made.

Sodium-23 magnetic resonance imaging of the eye and lens.

In order to develop a better understanding of cataract and to evaluate the effectiveness of potential drugs, noninvasive techniques must be devised to detect early metabolic changes. As a prelude to these goals, sodium-23 imaging experiments operating at 29.8 MHz (2.7 teslas) were performed on the bovine eye and lens. A spatially localized transverse relaxation time (T2)-weighted spin-density map of the sodium-23 within the lens is presented, with a resolution better than 250 micron. Due to the presence of short-T2 (3 msec) components within the lens, only the use of the planar-integral projection reconstruction (PPR) imaging scheme allowed sufficiently short echo-times (1 msec) to permit sodium-23 signal detection. These noninvasive imaging results show differences in the apparent sodium concentration within the lens that are consistent with separate, invasive measurements of sodium concentration. Separate analysis (with no spatial localization) at 79.4 MHz (7.2 teslas), using a shift reagent (dysprosium) to distinguish extracellular from intracellular sodium, indicates that approximately 62% of the detected sodium-23 signal is intracellular. These results are consistent with observations based on invasive measurements and further support the existence of the pump-leak system and a sodium gradient within the lens.

H2O2-modification of Na,K-ATPase. Alterations in external Na+ and K+ stimulation of K+ influx.

Studies, at steady state, of the Na,K-ATPase dependent influx of K+ into bovine lenses in organ culture are used to characterize further the H2O2-modification of the Na+ pump. Control lenses display constants for interaction with external Na+ and K+ similar to those obtained for the erythrocyte. H2O2 treatment of the bovine lens leads to total loss of external Na+ stimulation and alteration of external K+ stimulation.

Kinetic cooperativity change after H2O2 modification of (Na,K)-ATPase.

The kinetics of hydrolysis of ATP and p-nitrophenylphosphate and the action of the allosteric effectors, Na+ and K+, upon the hydrolysis of these substrates were used to study the H2O2-modified, uncoupled (Na,K)-ATPase isolated from cultured bovine lenses ( Garner , W. H., Garner , M. H., and Spector , A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2044-2048). Pure bovine renal (Na,K)-ATPase was modified by H2O2 in 150 mM KCl and 20 mM MgCl2 to yield an enzyme with kinetic properties similar to the enzyme isolated from the H2O2-treated, cultured bovine lens. H2O2 modification changes the interaction of the ATP hydrolysis site from negative to positive kinetic cooperativity. H2O2 modification dramatically alters Na+ stimulation of ATP hydrolysis and Na+ inhibition of p-nitrophenylphosphate hydrolysis while having little effect upon K+ control of the hydrolysis of these two substrates.

Non-invasive techniques in the study of cataract development at the metabolic and protein molecular level.

Metabolic changes may precede changes in lens protein structure and cataract opacification. Since many of the effects associated with cataract are oxidative in nature, changes in the redox state may be caused by alterations in the level of various metabolic intermediates such as ATP and NAD(P)H. Abnormal levels of H2O2 have been found in the aqueous fluid of cataract patients. Lenses have been treated with 1 mM-H2O2 in organ culture as a cataract model. H2O2 in this system uncouples Na+, K+-ATPase. This metabolic stress has been further evaluated non-invasively by 31P NMR to show that H2O2 can reduce ATP levels without any immediate effects on visual transparency. However, further treatment by this oxidant leads to definitive visual changes in lens clarity. These changes may be due to further changes in structural lens proteins caused by denaturation and aggregation induced by H2O2. The effects of H2O2 on isolated lens proteins is being examined in molecular detail by NMR to ascertain how the lens proteins become denatured in solution. The relevance of the H2O2 model to cataract formation can only be evaluated by using several non-invasive techniques beyond NMR, and then critically comparing the model systems with human cataract tissue samples.

Determination of the solvent accessibility of specific aromatic residues in gamma-crystallin by photo-CIDNP NMR measurements.

The surface or solvent accessibility of certain individual aromatic residues of calf-gamma II crystallin in solution (1 mM) were measured by the dramatic intensity enhancements of NMR lines generated by the interactions of cyclic radical pair formation of the 3-N-carboxymethyl lumiflavin (flavin I) dye excited (488nm) by an argon laser (5 watts) with the protein. This effect is called photo-chemically induced dynamic nuclear polarization: photo-CIDNP. The "light" and "dark" NMR spectra were taken in alternating scans in the pulsed Fourier transform mode on a Bruker 360 MHz instrument. Subtraction results in the photo-CIDNP difference spectrum containing lines of the polarized residues. With flavin dyes only tyrosine, histidine, and tryptophan can be polarized. The respective theoretical static accessibility of these residues based upon van der Waal's contact radii have been calculated from the atomic coordinates and provide a basis for evaluating the dynamic NMR photo-CIDNP results and for assigning the resonances. These results suggest that while the four tryptophan residues are completely buried, His-113 and His-14 of the five histidines; and Tyr-165 and Tyr-62 of the fifteen tyrosines are sufficiently exposed to elicit a photo-CIDNP effect. These results confirm and extend the observations previously obtained with theoretical electrostatic programs and FT-NMR measurements.

H2O2-induced uncoupling of bovine lens Na+,K+-ATPase.

A 1-hr exposure of bovine lenses in organ culture to H2O2 concentrations in the range found in the aqueous fluid of patients with cataracts inhibits 86Rb+ influx. At 1 mM H2O2, complete inhibition was observed and further investigated. Membrane permeability is slightly decreased. Although lactate concentrations increase 2-fold, lens ATP concentrations decrease approximately equal to 10%, suggesting that glycolysis may be stimulated but ATP production is not able to keep up with the demand for energy. Examination of epithelial cell Mg2+-stimulated Na+,K+-ATPase isolated from the cultured lenses indicates H2O2-induced modification. At 5 mM MgATP, ATP hydrolysis is accelerated 30%; at 3 mM MgATP, hydrolysis is normal; and at 0.75 mM MgATP, it is inhibited 75%. p-Nitrophenyl phosphate hydrolysis and eosin maleimide binding indicate that K+ control of the enzyme is modified. Thus, a very early effect of H2O2 upon the lens, well before the formation of opacity, appears to be the uncoupling of Na+ and K+ transport from ATP hydrolysis.

Biochemical evidence for membrane disintegration in human cataracts.

Biochemical evidence is presented for the disintegration of the lens fiber plasma membrane in human cataracts. The intrinsic membrane proteins are found in both the water-soluble and water-insoluble nonmembrane fractions of the cataract lens but not in the normal tissue. Furthermore, in contrast to the normal lens, not all of the lipid found in the cataractous lens is isolated with the membrane fraction. In cataracts, both the membrane and membrane fragments are involved in covalent high molecular weight aggregates with an extrinsic membrane protein (43,000 daltons) and a cytoplasmic protein (gamma-crystallin).

Evolution of the amino acid substitution in the mammalian myoglobin gene.

Multivariate statistical analyses were applied to 16 physical and chemical properties of amino acids. Four of these properties; volume, polarity, isoelectric point (charge), and hydrophobicity were found to explain adequately 96% of the total variance of amino acid attributes. Using these four quantitative measures of amino acid properties, a structural discriminate function in the form of a weighted difference sum of squares equation was developed. The discriminate function is weighted by the location of each particular residue within a given tertiary structure and yields a numerical discriminate or difference value for the replacement of these residues by different amino acids. This resulting discriminate value represents an expression of the perturbation in the local positional environment of a protein when an amino acid substitution occurs. With the use of this structural discriminate function, a residue by residue comparison of the known mammalian myoglobin sequences was carried out in an attempt to elucidate the positions of possible deviations from the known tertiary structure of sperm whale myoglobin. Only 11 of the 153 residue positions in myoglobin demonstrated possible structural deviations. From this analysis, indices of difference were calculated for all amino acid exchanges between the various myoglobins. All comparisons yielded indices of difference that were considerably lower than would be expected if mutations had been fixed at random, even if the organization of the genetic code is taken into consideration. On the basis of these results, it is inferred that some form of selection has acted in the evolution of mammalian myoglobins to favor amino acid substitutions that are compatible with the retention of the original conformation of the protein.