Reduction of Glut1 in the Neural Retina But Not the RPE Alleviates Polyol Accumulation and Normalizes Early Characteristics of Diabetic Retinopathy.

Hyperglycemia is a key determinant for development of diabetic retinopathy (DR). Inadequate glycemic control exacerbates retinopathy, while normalization of glucose levels delays its progression. In hyperglycemia, hexokinase is saturated and excess glucose is metabolized to sorbitol by aldose reductase via the polyol pathway. Therapies to reduce retinal polyol accumulation for the prevention of DR have been elusive because of low sorbitol dehydrogenase levels in the retina and inadequate inhibition of aldose reductase. Using systemic and conditional genetic inactivation, we targeted the primary facilitative glucose transporter in the retina, Glut1, as a preventative therapeutic in diabetic male and female mice. Unlike WT diabetics, diabetic Glut1+/- mice did not display elevated Glut1 levels in the retina. Furthermore, diabetic Glut1+/- mice exhibited ameliorated ERG defects, inflammation, and oxidative stress, which was correlated with a significant reduction in retinal sorbitol accumulation. Retinal pigment epithelium-specific reduction of Glut1 did not prevent an increase in retinal sorbitol content or early hallmarks of DR. However, like diabetic Glut1+/- mice, reduction of Glut1 specifically in the retina mitigated polyol accumulation and diminished retinal dysfunction and the elevation of markers for oxidative stress and inflammation associated with diabetes. These results suggest that modulation of retinal polyol accumulation via Glut1 in photoreceptors can circumvent the difficulties in regulating systemic glucose metabolism and be exploited to prevent DR.SIGNIFICANCE STATEMENT Diabetic retinopathy affects one-third of diabetic patients and is the primary cause of vision loss in adults 20-74 years of age. While anti-VEGF and photocoagulation treatments for the late-stage vision threatening complications can prevent vision loss, a significant proportion of patients do not respond to anti-VEGF therapies, and mechanisms to stop progression of early-stage symptoms remain elusive. Glut1 is the primary facilitative glucose transporter for the retina. We determined that a moderate reduction in Glut1 levels, specifically in the retina, but not the retinal pigment epithelium, was sufficient to prevent retinal polyol accumulation and the earliest functional defects to be identified in the diabetic retina. Our study defines modulation of Glut1 in retinal neurons as a targetable molecule for prevention of diabetic retinopathy.

A novel, small peptide with activity against human pancreatic cancer.

KTH-222 is a novel, 8-amino acid length peptide. It is derived from a motif identified in a group of peptides that are related to atrial natriuretic peptide and that are able to inhibit cancer cell growth. We report here that KTH-222 inhibits the attachment, proliferation, and development of an invasive morphology in cultured human pancreatic tumor cells (MIA PaCa-2 and HPAC). At a biochemical level, it inhibits tubulin polymerization which may underlie these cellular effects. We further report that KTH-222 reduces the rate of tumor growth and prolongs survival in mice implanted with MIA PaCa-2 cells. In this model system, KTH-222 is more effective than gemcitabine, a drug commonly used in the treatment of pancreatic cancer. Furthermore, KTH-222 does not decrease the rate of weight gain in the treated mice, suggesting the absence of gross toxicity. These activities of KTH-222 suggest that it may be useful in the treatment of pancreatic cancer.

Endogenous insulin signaling in the RPE contributes to the maintenance of rod photoreceptor function in diabetes.

In diabetes, there are two major physiological aberrations: (i) Loss of insulin signaling due to absence of insulin (type 1 diabetes) or insulin resistance (type 2 diabetes) and (ii) increased blood glucose levels. The retina has a high proclivity to damage following diabetes, and much of the pathology seen in diabetic retinopathy has been ascribed to hyperglycemia and downstream cascades activated by increased blood glucose. However, less attention has been focused on the direct role of insulin on retinal physiology, likely due to the fact that uptake of glucose in retinal cells is not insulin-dependent. The retinal pigment epithelium (RPE) is instrumental in maintaining the structural and functional integrity of the retina. Recent studies have suggested that RPE dysfunction is a precursor of, and contributes to, the development of diabetic retinopathy. To evaluate the role of insulin on RPE cell function directly, we generated a RPE specific insulin receptor (IR) knockout (RPEIRKO) mouse using the Cre-loxP system. Using this mouse, we sought to determine the impact of insulin-mediated signaling in the RPE on retinal function under physiological control conditions as well as in streptozotocin (STZ)-induced diabetes. We demonstrate that loss of RPE-specific IR expression resulted in lower a- and b-wave electroretinogram amplitudes in diabetic mice as compared to diabetic mice that expressed IR on the RPE. Interestingly, RPEIRKO mice did not exhibit significant differences in the amplitude of the RPE-dependent electroretinogram c-wave as compared to diabetic controls. However, loss of IR-mediated signaling in the RPE reduced levels of reactive oxygen species and the expression of pro-inflammatory cytokines in the retina of diabetic mice. These results imply that IR-mediated signaling in the RPE regulates photoreceptor function and may play a role in the generation of oxidative stress and inflammation in the retina in diabetes.

Senescent retinal pigment epithelial cells are more sensitive to vascular endothelial growth factor: implications for "wet" age-related macular degeneration.

PURPOSE: Senescence of the retinal pigment epithelial (RPE) cell layer has been implicated in the occurrence of age-related macular degeneration (AMD). The present study examines whether the ability of vascular endothelial growth factor (VEGF) to decrease the barrier function of RPE cells is enhanced in senescent RPE cells, which could contribute to the pathology of "wet" AMD. METHODS: Low or high population doubling level (PDL) range ARPE-19 human RPE cells were cultured in 6-well plates on membrane-containing inserts. After 2 weeks, the cells were treated with either VEGF or its vehicle and their transepithelial electrical resistance (TEER) was measured. One week later, the cells were stained for senescence-associated ß-galactosidase (SABG) activity. RESULTS: VEGF was significantly more effective in reducing the TEER of the high PDL ARPE-19 cell layers than the low PDL layers (25% decrease vs. 6% decrease; t-test, P=0.0013). The low PDL cell layers had a modest uniform level of SABG staining. In contrast, the high PDL layers displayed darker and more mottled SABG staining indicative of the presence of senescent cells. CONCLUSIONS: The present results show that the ability of VEGF to reduce the barrier function of RPE cell layers is greater in high PDL layers, which display signs of senescence, than in low PDL layers. Senescence-induced changes in the responsiveness of RPE cell layers to VEGF could contribute to the pathology of AMD. Agents that strengthen the barrier properties of RPE cells or reduce their responsiveness to VEGF could be effective in treating "wet" AMD.

The ARPE-19 cell line: mortality status and utility in macular degeneration research.

PURPOSE: The present report examines several subcultures of a single sample of ARPE-19 cells to determine their status with respect to cell mortality. If a transformation from mortal to immortal has occurred in these cells, it may impact their characteristics and, thereby, their utility for modeling natural retinal pigment epithelial (RPE) cells. METHODS: Five separate subcultures of ARPE-19 cells were grown as recommended by the supplier. During the course of culture, they were periodically monitored for signs of mortality including erosion of telomeres, increased senescence-associated beta-galactosidase (SABG) staining, altered morphology and reduced viability with an increased population doubling level (PDL). There were also observed for signs of immortality including continuous growth to very high population doubling levels and maintenance of short telomere lengths. RESULTS: Each of the subcultures showed both mortal and immortal characteristics. Telomere erosion, increased SABG staining, changes in cell morphology and a modest drop in cell viability took place within a range of population doublings (59-77) in which cell senescence would be expected to occur. The cultures, however, continued to proliferate even after signs of senescence had appeared, with one subculture propagating to 257 population doublings. In addition, little further telomere erosion occurred at high PDL. CONCLUSION: These results suggest that the ARPE-19 subcultures contained both mortal and immortal cells. Since no transformation event was witnessed during the study, it appears likely that both types of cells were present in the original sample. Based on the proportion of cells demonstrating senescence-related changes, the mortal cells were estimated to comprise approximately 27% of the total culture. Because of the differences that can exist between normal and immortalized cells, and given the large proportion of ARPE-19 cells that are immortalized, discretion should be exercised when using ARPE-19 cells to model native RPE cells for the study of retinal diseases such as AMD.

RPE cell senescence: a key contributor to age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in industrialized countries. Although much progress has been made recently in the management of later stages of the disease, no agents have yet been developed for the early stages or for prophylactic use. Furthermore, even the treatments for the later stages have limited effectiveness. The process of developing improved treatments for AMD is complicated by the existence of several theories concerning the cause of the disorder, each suggesting a different strategy for finding effective therapeutics. One of the potential contributors to AMD pathology is retinal pigment epithelial (RPE) cell senescence. The present paper hypothesizes that RPE senescence plays a central role in the etiology of AMD. This hypothesis is supported by the ability of RPE cell senescence to account for the signs, risk factors, and successful treatment modalities of the disorder. This hypothesis also points to several new prophylactic and treatment strategies for AMD.

DNA synthesis and neuronal apoptosis caused by familial Alzheimer disease mutants of the amyloid precursor protein are mediated by the p21 activated kinase PAK3.

Apoptotic pathways and DNA synthesis are activated in neurons in the brains of individuals with Alzheimer disease (AD). However, the signaling mechanisms that mediate these events have not been defined. We show that expression of familial AD (FAD) mutants of the amyloid precursor protein (APP) in primary neurons in culture causes apoptosis and DNA synthesis. Both the apoptosis and the DNA synthesis are mediated by the p21 activated kinase PAK3, a serine-threonine kinase that interacts with APP. A dominant-negative kinase mutant of PAK3 inhibits the neuronal apoptosis and DNA synthesis; this effect is abolished by deletion of the PAK3 APP-binding domain or by coexpression of a peptide representing this binding domain. The involvement of PAK3 specifically in FAD APP-mediated apoptosis rather than in general apoptotic pathways is suggested by the facts that a dominant-positive mutant of PAK3 does not alone cause neuronal apoptosis and that the dominant-negative mutant of PAK3 does not inhibit chemically induced apoptosis. Pertussis toxin, which inactivates the heterotrimeric G-proteins Go and Gi, inhibits the apoptosis and DNA synthesis caused by FAD APP mutants; the apoptosis and DNA synthesis are rescued by coexpression of a pertussis toxin-insensitive Go. FAD APP-mediated DNA synthesis precedes FAD APP-mediated apoptosis in neurons, and inhibition of neuronal entry into the cell cycle inhibits the apoptosis. These data suggest that a normal signaling pathway mediated by the interaction of APP, PAK3, and Go is constitutively activated in neurons by FAD mutations in APP and that this activation causes cell cycle entry and consequent apoptosis.