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SanGiovanni J.P.,U.S. National Institutes of Health | Rosen R.,New York Eye and Ear Infirmary | Kaushal S.,Retina Specialty Institute
Current Molecular Medicine | Year: 2014

Genome-wide association (GWA) studies apply broad DNA scans on hundreds-of-thousands of common sequence variants in thousands of people for the purpose of mapping trait- or disease-related loci. We provide examples of ligand- and target-based studies from the field of age-related macular degeneration (AMD) to demonstrate the value of the GWA approach in confirmatory and exploratory pharmacogenomics research. Complementing this genomic analysis, we used a simple biochemical retinal pigment epithelium (RPE) oxidative, apoptotic high throughput screening (HTS) assay to identify compounds. This ligand-to-target-to DNA sequence variant-to disease approach provided guidance on rational design of preclinical studies and identified associations between: 1) valproic acid and advanced AMD-associated genes with the capacity to alter GABA-succinate signaling (ALDH5A1, CACNA1C, SUCLA2, and GABBR2) and chromatin remodeling (HDAC9); and 2) Ropinirole and a geographic atrophy-associated gene (DRD3) with the capacity to alter systems involved in cAMP-PKA signaling. In both applications of our method, the breadth of GWA findings allowed efficient expansion of results to identify enriched pathways and additional ligands capable of targeting pathway constituents. A disease associated SNP-to gene-to target-to ligand approach provided guidance to inform preventive and therapeutic preclinical studies investigating roles of targets in: 1) PPAR-RXR transcription complex constituents for neovascular AMD; and 2) the stress activated MAPK signaling cascade constituents for advanced AMD. Our conclusion is that publically available data from GWA studies can be used successfully with open-access genomics, proteomics, structural chemistry, and pharmacogenomics databases in an efficient, rational approach to streamline the processes of planning and implementation for confirmatory and exploratory pre-clinical studies of preventive or therapeutic pharmacologic treatments for complex diseases. © 2014 Bentham Science Publishers. Source


Tam B.M.,University of British Columbia | Noorwez S.M.,University of Massachusetts Medical School | Kaushal S.,University of Massachusetts Medical School | Kaushal S.,Retina Specialty Institute | And 2 more authors.
Journal of Neuroscience | Year: 2014

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. © 2014 the authors. Source


Lei L.,Sun Yat Sen University | Lei L.,University of Massachusetts Medical School | Tzekov R.,University of Massachusetts Medical School | Tzekov R.,Roskamp Institute | And 5 more authors.
Experimental Eye Research | Year: 2013

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 585nm) 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 24hof 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 585nm compared to 530nm, 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 invitro would benefit from an analysis of multiple wavelengths to better match the spectral characteristics of lipofuscin invivo. © 2013 Elsevier Ltd. Source


Zimmer-Galler I.E.,Wilmer Eye Institute | Kimura A.E.,Aurora University | Gupta S.,Retina Specialty Institute
Current Opinion in Ophthalmology | Year: 2015

Purpose of review Evidence-based practice guidelines and treatments are highly effective in reducing vision loss from diabetic retinopathy. However, less than half of the total number of patients with diabetes mellitus receive recommended annual retinal evaluations, and vision loss due to diabetic retinopathy remains the leading cause of blindness in adults. Poor adherence to screening recommendations stems from a number of challenges which telemedicine technology may address to increase the evaluation rates and ultimately reduce vision loss. The aim of this review was to provide an update on the recent advances in tele-ophthalmology and how it may expand our current concept of eye care delivery for diabetic eye disease. Recent findings The benefits of telemedicine diabetic retinopathy are proven for large population-based systems. Outcomes information from community-based programs is now also beginning to emerge. Improved screening rates and less vision loss from diabetic retinopathy are being reported after implementation of telemedicine programs. New imaging platforms for telemedicine programs may enhance the ability to detect and grade diabetic retinopathy. However, financial factors remain a barrier to widespread implementation. Summary Telemedicine diabetic retinopathy screening programs may have a significant impact on reducing the vision complications and healthcare burden from the growing diabetes epidemic. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Source


Kaushal S.,University of Maryland Baltimore County | Sharma S.,University of Maryland Baltimore County | Mishra R.,University of Maryland Baltimore County | Simpson D.,University of Maryland Baltimore County | And 9 more authors.
Stem Cells | Year: 2015

We have demonstrated that human neonatal cardiosphere-derived cells (CDCs) derived from the young are more regenerative due to their robust secretome. However, it is unclear how the decompensated pediatric heart impacts the functional activity of their CDCs. Our aim was to characterize the potency of pediatric CDCs derived from normal functioning myocardium of control heart disease (CHD) patients to those generated from age-matched end stage heart failure (ESHF) patients and to determine the mechanisms involved. ESHF-derived CDCs contained a higher number of c-kit+, Islet-1+, and Sca-1+ cells. When transplanted into an infarcted rodent model, ESHF-derived CDCs significantly demonstrated higher restoration of ventricular function, prevented adverse remodeling, and enhanced angiogenesis when compared with CHD patients. The superior functional recovery of the ESHF-derived CDCs was mediated in part by increased SDF-1α and VEGF-A secretion resulting in augmented recruitment of endogenous stem cells and proliferation of cardiomyocytes. We determined the mechanism is due to the secretome directed by the heat shock response (HSR), which is supported by three lines of evidence. First, gain of function studies demonstrated that increased HSR induced the lower functioning CHD-derived CDCs to significantly restore myocardial function. Second, loss-of function studies targeting the HSR impaired the ability of the ESHF-derived CDCs to functionally recover the injured myocardium. Finally, the native ESHF myocardium had an increased number of c-kit+ cardiac stem cells. These findings suggest that the HSR enhances the functional activity of ESHF-derived CDCs by increasing their secretome activity, notably SDF-1α and VEGF-A. © 2015 Alpha Med Press. Source

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