Shalesh Kaushal

Vitreous Substitutes: A Comprehensive Review

Vitreoretinal disorders constitute a significant portion of treatable ocular disease. Advances in vitreoretinal surgery have included the development and characterization of suitable substitutes for the vitreous. Air, balanced salt solutions, perfluorocarbons, expansile gases, and silicone oil serve integral roles in modern vitreoretinal surgery. Vitreous substitutes vary widely in their properties, serve different clinical functions, and present different shortcomings. Permanent vitreous replacement has been attempted with collagen, hyaluronic acid, hydroxypropylmethylcellulose, and natural hydrogel polymers. None, however, have proven to be clinically viable. A long-term vitreous substitute remains to be found, and recent research suggests promise in the area of synthetic polymers. Here we review the currently available vitreous substitutes, as well those in the experimental phase. We classify these compounds based on their functionality, composition, and properties. We also discuss the clinical use, advantages, and shortcomings of the various substitutes. In addition we define the ideal vitreous substitute and highlight the need for a permanent substitute with long-term viability and compatibility. Finally, we attempt to define the future role of biomaterials research and the various functions they may serve in the area of vitreous substitutes.

Rapamycin reduces intrahepatic alpha-1-antitrypsin mutant Z protein polymers and liver injury in a mouse model

Alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in one in 2000 Americans. The Z mutation confers an abnormal conformation on the a1AT mutant Z protein, resulting in accumulation within the endoplasmic reticulum of hepatocytes and chronic liver injury. Autophagy is one of several proteolytic mechanisms activated to cope with this hepatocellular protein burden, and is likely important in disposal of the unique polymerized conformation of the a1AT mutant Z protein, which is thought to be especially injurious to the cell. Recent data indicate that rapamycin may more efficiently upregulate autophagy when given in weekly dose pulses, as compared with a daily regimen. Therefore, we evaluated the effect of rapamycin on PiZ mice, a well-characterized model which recapitulates human a1AT liver disease. Daily dosing had no effect on autophagy, on accumulation of a1AT mutant Z protein or on liver injury. Weekly dosing of rapamycin did increase autophagic activity, as shown by increased numbers of autophagic vacuoles. This was associated with reduction in the intrahepatic accumulation of a1AT mutant Z protein in the polymerized conformation. Markers of hepatocellular injury, including cleavage of caspase 12 and hepatic fibrosis, were also decreased. In conclusion, this is the first report of a successful in vivo method for reduction of intrahepatic a1AT mutant Z polymerized protein. Application of this finding may be therapeutic in patients with a1AT deficiency by reducing the intracellular burden of the polymerized, mutant Z protein and by reducing the progression of liver injury.

Therapeutic potential of valproic acid for retinitis pigmentosa

Background/aim To examine the efficacy and safety of valproic acid (VPA) in patients with retinitis pigmentosa (RP). Methods Thirteen eyes were examined before and after brief treatment (average 4u2005months) with VPA. Visual fields (VF) for each eye were defined using digitised Goldmann Kinetic Perimetry tracings. VF areas were log-transformed and VF loss/gain relative to baseline was calculated. Visual acuity was measured using a Snellen chart at a distance of 20u2005feet (6.1u2005m). Values were converted to the logarithm of the minimum angle of resolution (logMAR) score. Results Nine eyes had improved VF with treatment, two eyes had decreased VF and two eyes experienced no change, with an overall average increase of 11%. Assuming typical loss in VF area without treatment, this increase in VF was statistically significant (p<0.02). An average decrease (0.172) in the logMAR scores was seen in these 13 eyes, which translates to a positive change in Snellen score of approximately 20/47 to 20/32, which was significant (p<0.02) assuming no loss in acuity without treatment. Side effects were mild and well tolerated. Conclusion Treatment with VPA is suggestive of a therapeutic benefit to patients with RP. A placebo-controlled clinical trial will be necessary to assess the efficacy and safety of VPA for RP rigorously.

Novel Hydrophobically Modified Asymmetric RNAi Compounds (sd-rxRNA) Demonstrate Robust Efficacy in the Eye

PURPOSEnThe major challenges of developing an RNAi therapeutic include efficient delivery to and entry into the cell type of interest. Conventional (naked and chemically stabilized) small interfering RNAs (siRNAs) have been used in the eye in the past but they demonstrated limited clinical efficacy. Here we investigated a recently developed class of small, hydrophobic, asymmetric RNAi compounds. These compounds, termed self-delivering rxRNAs (sd-rxRNA(®)), are extensively modified, have a small duplex region of <15 base pairs, contain a fully phosphorothioated single-stranded tail, and readily enter cells and tissues without the requirement for a delivery vehicle.nnnMETHODSnWe compared sd-rxRNA compounds with stabilized siRNAs in vitro (in ARPE-19 cells) and in vivo (intravitreal injection in mouse and rabbit eyes). Specifically, we investigated the retinal uptake, distribution, efficacy, and preliminary safety of sd-rxRNAs.nnnRESULTSnTreatment with sd-rxRNAs resulted in uniform cellular uptake and full retina penetration in both animal models while no detectable cellular uptake was observed with stabilized siRNAs either in vitro or in vivo. Further, both in vitro and in vivo delivery (without any transfection reagent or formulation) resulted in a significant reduction of the targeted mRNA levels, which lasted 14-21 days in vivo. Retinal morphology and function were unaltered following a single administration of sd-rxRNAs.nnnCONCLUSIONnThese data support the potential of developing sd-rxRNAs as a therapeutic for ocular disease.

Protein Misfolding and Retinal Degeneration

The retina is a highly complex and specialized organ that performs preliminary analysis of visual information. Composed of highly metabolically active tissue, the retina requires a precise and well-balanced means of maintaining its functional activity during extended periods of time. Maintenance and regulation of a vast array of different structural and functional proteins is required for normal function of the retina. This process is referred to as protein homeostasis and involves a variety of activities, including protein synthesis, folding, transport, degradation, elimination, and recycling. Deregulation of any of these activities can lead to malfunctioning of the retina, from subtle subclinical signs to severe retinal degenerative diseases leading to blindness. Examples of retinal degenerative diseases caused by disruption of protein homeostasis include retinitis pigmentosa and Stargardts disease. A detailed discussion of the role of disruption in protein homeostasis in these and other retinal diseases is presented, followed by examples of some existing and potential treatments.

Photoactivation-Induced Instability of Rhodopsin Mutants T4K and T17M in Rod Outer Segments Underlies Retinal Degeneration in X. laevis Transgenic Models of Retinitis Pigmentosa

Retinitis pigmentosa (RP) is an inherited neurodegenerative disease involving progressive vision loss, and is often linked to mutations in the rhodopsin gene. Mutations that abolish N-terminal glycosylation of rhodopsin (T4K and T17M) cause sector RP in which the inferior retina preferentially degenerates, possibly due to greater light exposure of this region. Transgenic animal models expressing rhodopsin glycosylation mutants also exhibit light exacerbated retinal degeneration (RD). In this study, we used transgenic Xenopus laevis to investigate the pathogenic mechanism connecting light exposure and RD in photoreceptors expressing T4K or T17M rhodopsin. We demonstrate that increasing the thermal stability of these rhodopsins via a novel disulfide bond resulted in significantly less RD. Furthermore, T4K or T17M rhodopsins that were constitutively inactive (due to lack of the chromophore-binding site or dietary deprivation of the chromophore precursor vitamin A) induced less toxicity. In contrast, variants in the active conformation accumulated in the ER and caused RD even in the absence of light. In vitro, T4K and T17M rhodopsins showed reduced ability to regenerate pigment after light exposure. Finally, although multiple amino acid substitutions of T4 abolished glycosylation at N2 but were not toxic, similar substitutions of T17 were not tolerated, suggesting that the carbohydrate moiety at N15 is critical for cell viability. Our results identify a novel pathogenic mechanism in which the glycosylation-deficient rhodopsins are destabilized by light activation. These results have important implications for proposed RP therapies, such as vitamin A supplementation, which may be ineffective or even detrimental for certain RP genotypes.

Compromised mutant EFEMP1 secretion associated with macular dystrophy remedied by proteostasis network alteration

R345W EFEMP1 is secreted poorly, causing the macular dystrophy malattia leventinese. A novel assay shows that other substitutions (F, Y, P) at residue 345 impair secretion, partly by reducing native disulfide bonds. EFEMP1 secretion is rescued by reduced growth temperature and translational attenuation—potential strategies to delay disease.

Formation of lipofuscin-like material in the RPE Cell by different components of rod outer segments.

The mechanisms that control the natural rate of lipofuscin accumulation in the retinal pigment epithelial (RPE) cell and its stability over time are not well understood. Similarly, the contributions of retinoids, phospholipids and oxidation to the rate of accumulation of lipofuscin are uncertain. The experiments in this study were conducted to explore the individual contribution of rod outer segments (ROS) components to lipofuscin formation and its accumulation and stability over time. During the period of 14 days incubation of ROS, lipofuscin-like autofluorescence (LLAF) determined at two wavelengths (530 and 585xa0nm) by fluorescence-activated cell sorting (FACS) was measured from RPE cells. The autofluorescence increased in an exponential manner with a strong linear component between days 1 and 7. The magnitude of the increase was larger in cells incubated with 4-hydroxynonenal (HNE-ROS) compared with cells incubated with either bleached or unbleached ROS, but with a different spectral profile. A small (10-15%) decrease in LLAF was observed after stopping the ROS feeding for 14 days. The phagocytosis rate of HNE-ROS was higher than that of either bleached or unbleached ROS during the first 24xa0hxa0of supplementation. Among the different ROS components, the increase of LLAF was highest in cells incubated with all-trans-retinal. Surprisingly, incubation with 11-cis-retinal and 9-cis-retinal also resulted in strong LLAF increase, comparable to the increase induced by all-trans-retinal. Supplementation with liposomes containing phosphatidylethanolamine (22: 6-PE) and phosphatidylcholine (18:1-PC) also increased LLAF, while incubation with opsin had little effect. Cells incubated with retinoids demonstrated strong dose-dependence in LLAF increase, and the magnitude of the increase was 2-3 times higher at 585xa0nm compared to 530xa0nm, while cells incubated with liposomes showed little dose-dependence and similar increase at both wavelengths. Very little difference in LLAF was noted between cells incubated with either unbleached or bleached ROS under any conditions. In summary, results from this study suggest that supplementation with various ROS components can lead to an increase in LLAF, although the autofluorescence generated by the different classes of components has distinct spectral profiles, where the autofluorescence induced by retinoids results in a spectral profile closest to the one observed from human lipofuscin. Future fluorescence characterization of LLAF inxa0vitro would benefit from an analysis of multiple wavelengths to better match the spectral characteristics of lipofuscin inxa0vivo.

Inhibition or Stimulation of Autophagy Affects Early Formation of Lipofuscin-Like Autofluorescence in the Retinal Pigment Epithelium Cell

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) is dependent on the effectiveness of photoreceptor outer segment material degradation. This study explored the role of autophagy in the fate of RPE lipofuscin degradation. After seven days of feeding with either native or modified rod outer segments, ARPE-19 cells were treated with enhancers or inhibitors of autophagy and the autofluorescence was detected by fluorescence-activated cell sorting. Supplementation with different types of rod outer segments increased lipofuscin-like autofluorescence (LLAF) after the inhibition of autophagy, while the induction of autophagy (e.g., application of rapamycin) decreased LLAF. The effects of autophagy induction were further confirmed by Western blotting, which showed the conversion of LC3-I to LC3-II, and by immunofluorescence microscopy, which detected the lysosomal activity of the autophagy inducers. We also monitored LLAF after the application of several autophagy inhibitors by RNA-interference and confocal microscopy. The results showed that, in general, the inhibition of the autophagy-related proteins resulted in an increase in LLAF when cells were fed with rod outer segments, which further confirms the effect of autophagy in the fate of RPE lipofuscin degradation. These results emphasize the complex role of autophagy in modulating RPE autofluorescence and confirm the possibility of the pharmacological clearance of RPE lipofuscin by small molecules.