Microsphere antioxidant and sustained erythropoietin-R76E release functions cooperate to reduce traumatic optic neuropathy.

Wild-type erythropoietin (EPO) is promising for neuroprotection, but its therapeutic use is limited because it causes a systemic rise in hematocrit. We have developed an EPO-R76E derivative that maintains neuroprotective function without effects on hematocrit, but this protein has a short half-life in vivo. Here, we compare the efficacy and carrier-induced inflammatory response of two polymeric microparticle (MP) EPO-R76E sustained release formulations based on conventional hydrolytically degradable poly(lactic-co-glycolic acid) (PLGA) and reactive oxygen species (ROS)-degradable poly(propylene sulfide) (PPS). Both MP types effectively loaded EPO-R76E and achieved sustained release, providing detectable levels of EPO-R76E at the injection site in the eye in vivo for at least 28 days. Testing in an in vitro oxidative stress assay and a mouse model of blast-induced indirect traumatic optic neuropathy (bITON) showed that PPS and PLGA MP-mediated delivery of EPO-R76E provided therapeutic protection. While unloaded PLGA MPs inherently increase levels of pro-inflammatory cytokines in the bITON model, drug-free PPS MPs have innate antioxidant properties that provide therapeutic benefit both in vitro and in vivo. Both PLGA and PPS MPs enabled sustained release of EPO-R76E, providing therapeutic benefits including reduction in inflammation and axon degeneration, and preservation of visual function as measured by electroretinogram. The PPS-based MP platform is especially promising for further development, as the delivery system provides inherent antioxidant benefits that can be harnessed to work in complement with EPO-R76E or other drugs for neuroprotection in the setting of traumatic eye injury.

Safety and angiogenic effects of systemic gene delivery of a modified erythropoietin.

Erythropoietin (EPO) is critical for red blood cell production and is also an effective neuroprotective agent. However, it may contribute to pathological angiogenesis. Here we investigate the angiogenic potential of EPO and a mutant form with attenuated erythropoietic activity, EPO-R76E, on primary human retinal microvascular endothelial cells (HRMECs) and in the adult retina. Assays of death, proliferation and tube formation were performed on HRMECs exposed to EPO, EPO-R76E or media alone. Postnatal day-9 wild-type mice were injected intramuscularly with adeno-associated virus vectors expressing either enhanced green fluorescent protein or EpoR76E. At 3 months, levels of EPO-R76E in the eye were quantified, and the health of the retinal vasculature was assessed by fluorescein angiography and isolectin immunolabeling. Immunohistochemistry, histology and electroretinogram (ERG) assessments were performed as measures of retinal health. Neither EPO nor EPO-R76E induced proliferation or tube formation in HRMECs under the conditions used. EPO-R76E decreased HRMEC death in a dose-dependent manner. Long-term systemic gene delivery of EPO-R76E was safe in terms of retinal vasculature, histology and the ERG in vivo. Our results show that EPO-R76E can block HRMEC death, consistent with its role in erythropoiesis and neuroprotection. In addition, long-term gene delivery of EPO-R76E is safe in the adult retina.

A single intramuscular injection of rAAV-mediated mutant erythropoietin protects against MPTP-induced parkinsonism.

Erythropoietin (Epo) is neuroprotective in a number of preparations, but can lead to unacceptably high and even lethal hematocrit levels. Recent reports show that modified Epo variants confer neuroprotection in models of glaucoma and retinal degeneration without raising hematocrit. In this study, neuroprotective effects of two Epo variants (EpoR76E and EpoS71E) were assessed in a model of Parkinson's disease. The constructs were packaged in recombinant adeno-associated viral (rAAV) vectors and injected intramuscularly. After 3 weeks, mice received five daily injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and were killed 5 weeks later. The MPTP-lesioned mice pretreated with rAAV.eGFP (negative control) exhibited a 7- to 9-Hz tremor and slower latencies to move on a grid test (akinesia). Both of these symptomatic features were absent in mice pretreated with either modified Epo construct. The rAAV.eGFP-treated mice lesioned with MPTP exhibited a 41% reduction in tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. The rAAV.EpoS71E construct did not protect nigral neurons, but neuronal loss in mice pretreated with rAAV.EpoR76E was only half that of rAAV.eGFP controls. Although dopamine levels were normal in all groups, 3,4-dihydroxyphenylacetic acid (DOPAC) was significantly reduced only in MPTP-lesioned mice pretreated with rAAV.eGFP, indicating reduced dopamine turnover. Analysis of TH-positive fibers in the striatum showed normalized density in MPTP-lesioned mice pretreated with rAAV.EpoS71E, suggesting that enhanced sprouting induced by EpoS71E may have been responsible for normal behavior and dopaminergic tone in these mice. These results show that systemically administered rAAV-generated non-erythropoietic Epo may protect against MPTP-induced parkinsonism by a combination of neuroprotection and enhanced axonal sprouting.

Adenovirus-mediated delivery of catalase to retinal pigment epithelial cells protects neighboring photoreceptors from photo-oxidative stress.

Oxidative stress is involved in the pathogenesis of many diseases. Overexpression of antioxidant enzymes by gene therapy may protect tissues from oxidative damage. Because the reactive oxygen species hydrogen peroxide can diffuse across cell membranes, we hypothesized that overexpression of the antioxidant catalase within certain cells might protect neighboring cells. To test this hypothesis, we transduced retinal pigment epithelial (RPE) cells in vitro and in vivo with adenovirus carrying the catalase gene (Ad.CMV.catalase). After transduction of only a subset of RPE cells in vitro, all cells in the culture were protected from exogenous hydrogen peroxide. Similarly, in vivo, eyes injected with Ad. CMV. catalase had high catalase levels in the RPE, which protected the adjacent photoreceptors from light damage and reduced photoreceptor oxidative stress as measured by the markers 4-hydroxynonenal and nitrotyrosine. Both in vitro and in vivo, gene therapy with Ad. CMV. catalase protected neighboring cells from oxidative stress-induced terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) positivity. The data provide a paradigm for antioxidant gene therapy with catalase, designed to protect not only transduced cells, but also neighboring cells.

Distribution of S- and M-cones in normal and experimentally detached cat retina.

The lectin peanut agglutinin (PNA) and antibodies to short (S)- and medium to long wavelength (M/L)-sensitive cones were utilized in order to define the relative distributions of the two spectral types of cone across the domestic cat's retina. These values, in turn, were compared to those from retinas that had been experimentally detached from the retinal pigment epithelium. The pattern of cone distribution in the normal cat's retina is established by the preponderance of M-cones that constitute between 80% and 90% of all cones. Their peak density of over 26,000 cells/mm(2) resides at the area centralis. Though M-cone density decreases smoothly to the ora serrata where they have densities as low as 2,200 cells/mm(2), the density decrease along the nasotemporal axis is slower,creating a horizontal region of higher cone density. S-cones constitute between 10% and 20% of all cones, the number being quite variable even between individual animals of similar age. The highest S-cone densities are found in three distinct locations: at the superior far periphery near the ora serrata, immediately at the area centralis itself, and in a broad zone comprising the central and lower half of the inferior hemiretina. S-cones in the cat retina do not form a regular geometrical array at any eccentricity. As for the detached cat retina, the density of labeled S-cone outer segments (OS) decreases rapidly as early as 1 day postdetachment and continues decreasing to day 28 when the density of cones labeling with anti-S opsin has dropped to less than 10% of normal. This response points to a profound difference between rods and cones; essentially all rods, including those without OS, continue to express their opsin even in long-term detachments. The implications of these results for visual recovery after retinal reattachment are discussed.

The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective.

This article provides a comprehensive analysis of the redox reaction between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid. It includes an historical perspective of the progression of the experiments, first begun more than 60 years ago and continuing today with heightened importance. Indeed, the antioxidant capacity of glutathione and ascorbic acid, whether singly or in combination, linked via the redox couple, is a subject of intense interest for studies by bench scientists and clinicians, particularly because a growing body of evidence suggests that free radicals may be involved in a variety of diseases. The authors begin with a detailed summary of "test tube" experiments (the chemical perspective) that have revealed the conditions that regulate the rate of the redox coupling between glutathione and dehydroascorbic acid and that promote or inhibit the decomposition of dehydroascorbic acid in ordinary, buffered aqueous media; results obtained in the authors' laboratory are used for illustration purposes and uniformity of presentation. The authors then proceed to a critical examination of the extent to which the redox couple between glutathione and ascorbic acid operates in a cell, using the often published antioxidant cascade (See Fig. 1) as the model for the analysis (the physiological perspective). The evidence for and the evidence against the presence of the enzyme dehydroascorbate reductase in animal cells is outlined in a balanced way in an attempt to make sense of this continuing controversy. Next, the authors carefully document the many studies showing that exogenous dehydroascorbic acid is transported into cells where it is reduced to ascorbic acid by glutathione. Finally, they probe the functional significance and efficiency of the redox couple in monolayer cultures of human retinal pigment epithelial (RPE) cells, as a prototypical cellular model. The authors include the results of new experiments showing that incubation of RPE cells with a nitroxide, TEMPOL, leads to the selective oxidation of intracellular ascorbic acid. This approach is desirable because it dissects the cascade at a specific site and permits measurements of the levels of ascorbic acid and glutathione in the cells before, during, and after oxidation. The results show that only partial regeneration of ascorbic acid is obtained when control conditions are restored. However, if either ascorbic acid or dehydroascorbic acid is added to the media during the recovery period following treatment of cells with TEMPOL, then full recovery of ascorbic acid is observed.(ABSTRACT TRUNCATED AT 400 WORDS)