Antiviral activity and ocular kinetics of antisense oligonucleotides designed to inhibit CMV replication.

PURPOSE: To compare the antiviral activity and ocular distribution of first- and second-generation antisense oligonucleotides intended for the treatment of cytomegalovirus (CMV) retinitis. METHODS: The antiviral activity of ISIS 13312 and ISIS 2922 (Isis Pharmaceuticals, Inc., Carlsbad, CA) against 10 clinical CMV isolates was compared with a plaque-reduction assay. The ocular pharmacokinetics were compared after intravitreal injection in rabbits (36-90 microg) and monkeys (125-500 microg). Vitreous and/or retina were collected after single and multiple injections to characterize ocular distribution, clearance, and accumulation. Oligonucleotide concentrations were measured by capillary gel electrophoresis and immunohistochemical techniques. RESULTS: ISIS 13312 and ISIS 2922 demonstrated comparable antiviral activity that was consistent among the 10 clinical isolates examined (50% inhibitory concentration [IC(50)], <1 microM). Activity was independent of the resistance of CMV isolates to DNA polymerase inhibitors. After intravitreal injection, the kinetics of ISIS 2922 and ISIS 13312 were characterized by clearance from vitreous and distribution to the retina; however, ISIS 2922 was cleared more quickly from the retina than ISIS 13312. The half-life of ISIS 13312 in the monkey retina was approximately 2 months. Retinal concentrations of ISIS 13312 were dose dependent, with approximately a twofold increase in concentration after once-monthly doses compared with single-dose concentrations. Immunohistochemical analysis indicated that both oligonucleotides were efficiently distributed to numerous ocular tissues, including retina, ciliary body, and optic nerve. CONCLUSIONS: ISIS 13312 possesses antiviral activity and pharmacokinetic properties that favor its use as a therapeutic agent in treatment of CMV retinitis. The half-life of ISIS 13312 in retina is longer than that of ISIS 2922, potentially allowing for less frequent administration.

Lutein and zeaxanthin concentrations in rod outer segment membranes from perifoveal and peripheral human retina.

PURPOSE: In addition to acting as an optical filter, macular (carotenoid) pigment has been hypothesized to function as an antioxidant in the human retina by inhibiting the peroxidation of long-chain polyunsaturated fatty acids. However, at its location of highest density in the inner (prereceptoral) layers of the foveal retina, a specific requirement for antioxidant protection would not be predicted. The purpose of this study was to determine whether lutein and zeaxanthin, the major carotenoids comprising the macular pigment, are present in rod outer segment (ROS) membranes where the concentration of long-chain polyunsaturated fatty acids, and susceptibility to oxidation, is highest. METHODS: Retinas from human donor eyes were dissected to obtain two regions: an annular ring of 1.5- to 4-mm eccentricity representing the area centralis excluding the fovea (perifoveal retina) and the remaining retina outside this region (peripheral retina). ROS and residual (ROS-depleted) retinal membranes were isolated from these regions by differential centrifugation and their purity checked by polyacrylamide gel electrophoresis and fatty acid analysis. Lutein and zeaxanthin were analyzed by high-performance liquid chromatography and their concentrations expressed relative to membrane protein. Preparation of membranes and analysis of carotenoids were performed in parallel on bovine retinas for comparison to a nonprimate species. Carotenoid concentrations were also determined for retinal pigment epithelium harvested from human eyes. RESULTS: ROS membranes prepared from perifoveal and peripheral regions of human retina were found to be of high purity as indicated by the presence of a dense opsin band on protein gels. Fatty acid analysis of human ROS membranes showed a characteristic enrichment of docosahexaenoic acid relative to residual membranes. Membranes prepared from bovine retinas had protein profiles and fatty acid composition similar to those from human retinas. Carotenoid analysis showed that lutein and zeaxanthin were present in ROS and residual human retinal membranes. The combined concentration of lutein plus zeaxanthin was 70% higher in human ROS than in residual membranes. Lutein plus zeaxanthin in human ROS membranes was 2.7 times more concentrated in the perifoveal than the peripheral retinal region. Lutein and zeaxanthin were consistently detected in human retinal pigment epithelium at relatively low concentrations. CONCLUSIONS: The presence of lutein and zeaxanthin in human ROS membranes raises the possibility that they function as antioxidants in this cell compartment. The finding of a higher concentration of these carotenoids in ROS of the perifoveal retina lends support to their proposed protective role in age-related macular degeneration.

Influence of UVA light stress on photoreceptor cell metabolism: decreased rates of rhodopsin regeneration and opsin synthesis.

There is considerable evidence indicating that rhodopsin is the chromophore mediating light damage to the rat retina caused by exposure to mid-visible wavelengths. Retinal damage is, however, more effectively produced by short-wavelength light, and little is known about the initiating events for this damage class. The present study sought to determine the involvement of rhodopsin bleaching in short-wavelength damage by examining rhodopsin levels and opsin synthesis at early time points following acute ultraviolet-A (UVA) exposures of the pigmented rat eye. A gradual decline in rhodopsin to 8% of the level in non-exposed control eyes occurred over a 1 hr exposure to 1500 microW cm-2of UVA light. When animals were placed in darkness following this exposure, rhodopsin had recovered to only 27% of control levels by 2 hr post-exposure indicating a very slow rate of regeneration. For later time points, animals were returned to dim cyclic light and by 2 days following exposure, rhodopsin levels had risen to 57% of control. In contrast, opsin levels at this same time point were unaffected by UVA exposure. Other observations indicating the UVA exposure affected photoreceptor cell metabolism included a 27% decrease in the rate of opsin synthesis between 1 and 2 days following exposure, and a 69% reduction in the rate of rod outer segment disk renewal during the initial 3 days following exposure. These data show that UVA light stress in the retina causes a gradual bleaching of rhodopsin followed by a slow rate of recovery and altered photoreceptor cell metabolism. These results are consistent with the concept that rhodopsin mediates UVA-induced retinal damage and the possible mechanisms by which this might occur are discussed in relation to alternative hypotheses currently in the literature.

Evaluation of retinal susceptibility to light damage in pigmented rats supplemented with beta-carotene.

The present study evaluated the influence of beta-carotene supplementation on the susceptibility of the retina to light damage. Long-Evans pigmented rats were supplemented with beta-carotene by either dietary or intraperitoneal administration, and beta-carotene levels in plasma, liver and retina were determined by high performance liquid chromatography. Other animals from each group were exposed to ultraviolet-A light at a dose of 8.1 J/cm2 in their right eye only, and photoreceptor cell losses determined by light microscopic morphometry. In supplemented animals, beta-carotene levels increased markedly in the liver, and were elevated from non-detectable to detectable in the plasma and retina, relative to nonsupplemented controls. In each tissue, beta-carotene levels were found to be higher in animals receiving intraperitoneal supplementation as compared to dietary. Beta-carotene supplementation by either route did not protect the retina against photoreceptor cell loss measured at two weeks following UVA exposure. Preliminary observations indicated that beta-carotene supplementation decreased the incidence of light-induced retinal pigment epithelium destruction.

In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure.

Recent studies have demonstrated that inhibition of mevalonate synthesis in cultured cells leads to altered cell morphology due to inhibition of protein prenylation. To investigate the effects in vivo of mevalonate deprivation in nondividing, terminally differentiated neural cells, we have analyzed the effects on retinal tissue of intravitreal injection of lovastatin, a potent inhibitor of the mevalonate-producing enzyme, HMG-CoA reductase. A single injection of lovastatin (0.25 mumol) produced profound dysplastic-like changes in adult rat retinas primarily involving the photoreceptor layer. Within 2 d after injection, photoreceptor nuclei migrated in a circular pattern resulting in the formation of rosette-like structures by 4 d. Also during this period, photoreceptor inner and outer segment degeneration was evident. By 21 d, intact photoreceptor nuclei with remnants of inner and outer segments were dispersed throughout all retinal layers. To investigate the biochemical specificity of the lovastatin-induced alterations, and to distinguish the relative importance of the various branches of the mevalonate pathway, the incorporation of [3H]acetate into retinal lipids was examined in the presence and absence of metabolic inhibitors. HPLC analysis of lovastatin-treated retinas revealed a dramatic reduction in the incorporation of intravitreally injected [3H]acetate into nonsaponifiable lipids, compared with controls. In contrast, intravitreal injection of NB-598, a specific inhibitor of squalene epoxidase, eliminated the conversion of newly synthesized squalene to sterols without obvious pathology. Hence, involvement to the sterol branch of isoprenoid metabolism in the lovastatin-induced morphologic disruption was obviated. Intravitreal injection of 0.27 mumol of N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), an inhibitor of carboxyl methyltransferase activity and prenylated protein function, produced morphologic changes that were virtually indistinguishable from those induced with lovastatin. These results implicate a defect in protein prenylation in the lovastatin-induced retinal degeneration, and suggest the presence of a dynamic pathway in the retina that requires isoprenylated proteins to maintain retinal cytoarchitecture.

Effect of dietary fat on the response of the rat retina to chronic and acute light stress.

We designed an experiment to study the role of light history and polyunsaturated fatty acids (PUFA) on the susceptibility of the albino rat retina to light damage. Albino rats were born to dams that had been kept in either 1 lx or 250 lx cyclic light for 0-4 days prior to delivery and fed one of three diets containing either 10% (by weight) hydrogenated coconut oil (COC, no n-3 nor n-6 fatty acids), safflower oil (SAF, high n-6, < 0.1% n-3 fatty acids), or linseed oil (LIN, high n-3, low n-6 fatty acids). After weaning, the rats were maintained in the same light environment and fed the same diets for 9 weeks, at which time some were killed and their retinas processed for morphometric analysis. Animals raised in bright cyclic light had shorter ROS lengths and thinner outer nuclear layers (ONL) than rats raised in dim cyclic light. The LIN animals had a thinner ONL than animals of the SAF or COC groups. Rats from each diet and light rearing groups were exposed to constant illumination of 2000 lx for 24 hr, after which they and non-exposed controls were placed in 1 lx cyclic light for 10 days and analysed for changes in ONL thickness. In the 250 lx (bright; B) groups, there was no effect of acute light stress on ONL thickness, although both control and stressed LIN animals had a thinner ONL than the corresponding COC and SAF groups. However, in the rats raised in 1 lx cyclic light, acute stress resulted in significant retinal damage (i.e. decrease in ONL thickness) in the three diet groups combined. The superior region was damaged the most and the severity was dependent on diet, as evidenced by the LIN group having a greater reduction in ONL thickness than the SAF group after light stress. From these results we conclude that rats on diets high in n-3 fatty acids are more susceptible to photoreceptor cell loss than animals fed n-6 or no polyunsaturated fatty acids when raised in dim cyclic light. These results show that both diet and light history play a role in the susceptibility of the retina to acute and chronic effects of light and suggest a role for lipid peroxidation in retinal light damage.

Reduced rate of rod outer segment disk synthesis in photoreceptor cells recovering from UVA light damage.

PURPOSE: Recovery of photoreceptor cells after light damage is thought to involve the physiologic process of disk renewal. However, only indirect evidence is available to support this hypothesis. The present study sought to examine more directly the mechanisms of photoreceptor cell recovery by quantitatively assessing the rate of rod outer segment (ROS) disk synthesis in retinas damaged by ultraviolet-A (UVA) light. METHODS: Pigmented rats were anesthetized, and their right eyes were exposed for 1 hour to a uniform field of UVA light at a dose of 6 J/cm2. Animals were returned to their dim cyclic light environment and were allowed to recover for various time points up to 42 days, at which time their eyes were enucleated for histologic examination and quantification of outer nuclear layer (ONL) thickness. Seven days before the 6- and 21-day time points, some animals were intravitreally injected with 3H-leucine in both eyes, and these eyes were used to analyze autoradiographically the rate of ROS disk synthesis. RESULTS: ROS disk synthesis in UVA-exposed eyes was 43% slower relative to nonexposed controls in the more severely damaged superior retina at postexposure day 6. Ultrastructural observations revealed a sharp demarcation between damaged and recovered ROS disks at this time. At postexposure day 21, there was a marked recovery in outer segment structure despite a further decrease in ONL thickness and a continued slow rate of disk synthesis. In the less severely damaged inferior region of the retina, the rate of disk synthesis was not altered by UVA exposure, although mild ROS disruption was evident at the earlier time points. CONCLUSIONS: These findings indicate that the rate of ROS disk synthesis is slowed in moderately damaged photoreceptor cells even though they eventually fully recover in structure. A slow-down of this rate after UVA exposure may be an adaptive change geared toward recovery mechanisms, or it may simply be a manifestation of cellular damage.

Impact of lipofuscin on the retinal pigment epithelium: electroretinographic evaluation of a protease inhibition model.

With aging, the retinal pigment epithelium (RPE) becomes increasingly congested with residual debris called lipofuscin. Little is known about the impact of lipofuscin on retinal function, and this was addressed in the present study by examining the influence of RPE debris on electroretinographic (ERG) parameters utilizing an experimental model of lipofuscin accumulation. Pigmented rats were injected intravitreally with the protease inhibitor leupeptin, and examined 1 week later by electroretinogram (ERG) recording and light and electron microscopy. Relative to vehicle-injected controls, leupeptin-treated retinas showed abundant accumulation throughout the RPE cytoplasm of inclusions that resembled lipofuscin. RPE cells filled with this debris showed a marked increase in height and a displacement of melanin from their apical border. Morphological changes in the RPE had no influence on retinal function since ERG threshold, a- and b-wave maximum amplitude, latency and implicit time were not significantly different between leupeptin-treated eyes and controls. Furthermore, leupeptin-induced RPE inclusions did not alter either the rate or extent of ERG dark adaptation. These findings suggest that filling of the RPE cytoplasm with residual debris is not in itself likely to be the cause of functional alterations in the aging eye.

Splice site selection in polyomavirus late pre-mRNA processing.

Polyomavirus late pre-mRNAs contain one 5' splice site and two message body 3' splice sites, which are not used at equal frequencies. As a result of alternative splicing, the total late mRNA population consists of about 5% mVP2 (no message body splice chosen), about 15% mVP3 (promoter-proximal 3' splice site chosen), and about 80% mVP1 (promoter-distal 3' splice site chosen). To determine whether it is splice site strength that determines the ratio of spliced products, constructs containing duplicated or rearranged 3' splice sites were created. In construct VP1,1, 160 bp surrounding the VP3 3' splice site was substituted with the corresponding region of the VP1 3' splice site. This construct resulted in the duplication of the VP1 3' splicing signal. VP3,3 (two identical VP3 3' splice sites) and VP1,3 (VP1 and VP3 3' splice sites reversed) were similarly created. Each construct maintained wild-type spacing between the 3' splice sites. Analysis of RNAs from transfections showed that in each construct, the 3' splice closest to the polyadenylation site was used preferentially. Analysis of a number of additional constructs indicated that there are no strong cis-acting positive or negative regulators of polyomavirus late splicing; rather, splicing choices appear to be determined largely by relative position of splice sites.

Free radicals and ocular disease.

Ames, Shigenaga, and Hagen recently published a thorough review of the relationship between oxidants, antioxidants, and degenerative diseases of ageing. They point out that only 9% of Americans daily consume the two fruits and three vegetables recommended by the National Cancer Institute and the National Research Council/National Academy of Science. In addition to antioxidants, these foodstuffs contain many essential micronutrients. To date, specific recommendations for antioxidant supplementation have not been made by any governmental agency or professional association. A number of clinical, basic, and epidemiological studies have implicated free radical induced lipid peroxidation in various ocular disorders. It would seem prudent that those persons at greatest risk for these disorders take some precautions, which could include sunglasses that filter ultraviolet light; hats that shield the eyes from direct sunlight; and the ingestion of fruits, vegetables, and antioxidants.

In vivo biosynthesis of cholesterol in the rat retina.

Previous reports have suggested that the rate of de novo cholesterol synthesis in the adult vertebrate retina is extremely slow. We investigated cholesterol biosynthesis in the adult rat retina in vivo, following intravitreal injection of [3H]acetate. HPLC analysis of retinal non-saponifiable lipid extracts revealed co-elution of radioactivity with endogenous cholesterol mass within 4.5 h post-injection. Incorporation of [3H]acetate into cholesterol was markedly reduced by co-injection of known inhibitors of the cholesterol pathway. In contrast to previous results with retinas from other species, no radiolabel or mass corresponded to squalene, except in lipid extracts from retinas treated with NB-598, a squalene epoxidase inhibitor. These results demonstrate, for the first time, the capacity of the adult vertebrate retina to rapidly synthesize cholesterol de novo.

Herpes simplex virus 1 alkaline nuclease is required for efficient egress of capsids from the nucleus.

We previously described the isolation of AN-1, a null mutant of the HSV-1 alkaline nuclease gene, which is able to synthesize near wild-type levels of viral DNA and late viral proteins under nonpermissive growth conditions; these results lead us to conclude that the alkaline nuclease is not essential for viral DNA synthesis (Weller, S. K., Seghatoleslami, R. M., Shao, L., Rowse, D., and Carmichael, E. P., 1990, J. Gen. Virol. 71, 2941-2952). AN-1 was found to be deficient in the production of infectious virions suggesting that the nuclease may play a role in processing or packaging of viral DNA into infectious virions. In this report we demonstrate that the defect is distinct from that observed in other late HSV-1 mutants which make but fail to process viral DNA under nonpermissive growth conditions. Following restriction enzyme digestion, specific terminal fragments are observed in DNA from AN-1-infected Vero cells, indicating that specific cleavage has occurred; moreover, the efficiency of cleavage is at near wild-type levels. Also in contrast to cells infected with previously described late mutants, DNase I or staphylococcal nuclease resistant DNA is observed in these cells further indicating that encapsidation has occurred. Three lines of evidence suggest, however, that maturation of DNA-containing AN-1 capsids is defective in some ways. First, in contrast to wild-type, very small amounts of protected DNA is detected in cytoplasmic extracts from AN-1-infected cells. Second, very few if any mature, DNA-containing C capsids are observed in the cytoplasm when analyzed by electron microscopy or sucrose gradient sedimentation. Finally, analysis of nuclei by sucrose gradient sedimentation indicates an elevated ratio of A to B capsids. These data indicate that AN-1 may be defective for the production of capsids competent to mature into the cytoplasm. Possible models for the nature of the defect in AN-1 will be discussed.

Morphologic comparisons between rhodopsin-mediated and short-wavelength classes of retinal light damage.

The histologic manifestations of rhodopsin-mediated versus short-wavelength classes of retinal phototoxicity were compared after spectral exposures of the albino rat retina. Animals were exposed to wave-bands of light centered at the peak of rhodopsin absorbance (green, 500 nm) or in the ultraviolet A (UVA; 360 nm). Intensity-damage curves generated for each wave-band indicated that UVA light was 50-80 times more effective than green light at causing photoreceptor cell losses. Examination of early ultrastructural changes in rod inner segments, outer segments, and retinal pigment epithelium revealed a remarkable degree of similarity between UVA and green light-induced damage. Furthermore, the two classes of damage were indistinguishable in terms of post-exposure recovery from threshold damage and regional distribution of photoreceptor cell loss along the vertical meridian. The finding of essentially identical histologic manifestations for the two classes of damage raises the possibility that they share a common biochemical etiology or pathway of cell destruction.

Evidence against melanin as the mediator of retinal phototoxicity by short-wavelength light.

Albino and pigmented (black-hooded) rats of the Sprague-Dawley and Long Evans strains, respectively, were compared in terms of their susceptibility to retinal damage by ultraviolet-A light. Anesthetized animals were exposed to ultraviolet-A light (lambda max = 360 nm) for 4 hr and retinal damage was assessed 1 week later by electroretinographic analysis and measurement of outer nuclear layer thickness. Albino and pigmented animals showed approximately the same severity of ultraviolet-A retinal damage as a function of exposure irradiance. Furthermore, both pigmentation strains showed swelling and vesiculation of rod inner segment mitochondria as an early manifestation of damage. An abbreviated study on a congenic rat strain (F344-c/+) of albino and pigmented littermates again demonstrated an equal susceptibility to ultraviolet-A phototoxicity for both pigmentation phenotypes. These findings provide evidence that melanin is not the mediator of short-wavelength phototoxicity to the retina, since damage readily occurred in albino animals completely lacking this chromophore.

Predisposing factors to light-induced photoreceptor cell damage: retinal changes in maturing rats.

Retinal changes occurring during the period of growth and maturation of Long Evans pigmented rats were examined to obtain a better understanding of the basis for the age-dependency of light-induced photoreceptor cell damage. Susceptibility to light damage increased markedly between 30 and 60 days of age and to a lesser extent between 60 and 90 days. Although the retinal antioxidant vitamins E and C, and taurine showed a significant increase during the age-period studied, retinal lipid phosphorus and total protein increased by similar amounts indicating that the concentration of these nutrients was not changing. In contrast, rhodopsin content of the retina increased progressively by 44% between 30 and 90 days of age. While ROS length showed no appreciable change with age, rhodopsin per ROS length increased by 31% between 30 and 60 days of age and by 48% between 30 and 90 days. Determinations of ROS phospholipid to rhodopsin ratio and disks per ROS length indicated that rhodopsin did not become more concentrated in photoreceptor cells between 30 and 90 days. However, the 12% increase in ROS diameter between 30 and 90 days of age may partially account for the rhodopsin difference. These findings demonstrate an age-dependent association between greater rhodopsin per ROS length and increased susceptibility to retinal light damage. An increased metabolic demand on photoreceptor cells with greater rhodopsin may be an important factor influencing their destruction by light.

Separate mechanisms for retinal damage by ultraviolet-A and mid-visible light.

Retinal damage by light has two distinct action spectra, one peaking in the ultraviolet-A (UVA) and the other in the mid-visible wavelengths (green light). Here we show in a single animal species, the Long Evans rat, that UVA and green light can produce histologically dissimilar types of damage. UVA light in particular produces severe retinal damage at low irradiation levels. Furthermore, the mechanism of damage by UVA light is different from that of green light as determined by their relative rhodopsin bleaching efficacies. These results provide convincing evidence that different chromophores mediate damage by UVA and green light. By producing both classes of damage in a single species, a sound model is provided for further investigation into the different forms of photic retinopathy.

Synergism between environmental lighting and taurine depletion in causing photoreceptor cell degeneration.

Experiments were conducted to examine the possible interaction between retinal taurine depletion and environmental lighting in causing photoreceptor cell degeneration. Albino rats were raised from birth in either dim (2 lx) or relatively bright (300 lx) cyclic light. Beginning at weaning, half the animals from both light environments were taurine-depleted by treating them for 10 weeks with guanidinoethyl sulfonate as a 1% solution in their drinking water. The remaining animals were given ordinary tap-water and served as controls. In the 2 lx light environment, taurine depletion caused a decrease in electroretinogram (ERG) a- and b-wave amplitude of 36 and 46%, respectively; however, no photoreceptor cells were lost in this group. Tap-water controls kept in the 300 lx light environment had a 59- and 43% decrease in ERG a- and b-wave amplitude, respectively, and a 21% reduction in the number of photoreceptor cells. In contrast to the other groups, animals that were taurine-depleted in the 300 lx environment showed a marked retinal degeneration. ERG a- and b-wave amplitude was decreased by 94- and 89% respectively and there was a 62% loss of photoreceptor cells. The greatest cell loss occurred in the central superior region of the retina, in which the outer nuclear layer was typically reduced to one to two rows of nuclei. The results of a two-way analysis of variance applied to the data indicated that the effects of taurine depletion on the retina were greater in the 300 lx as compared with the 2 lx environment in terms of loss of photoreceptor cells and reduction in log ERG a- and b-wave amplitude. These findings demonstrate a synergism between environmental lighting and taurine depletion in causing photoreceptor cell degeneration.