scholarly journals Carrot solution culture bioproduction of uniformly labeled 13C-lutein and in vivo dosing in non-human primates

2016 ◽  
Vol 242 (3) ◽  
pp. 305-315 ◽  
Author(s):  
Joshua W Smith ◽  
Randy B Rogers ◽  
Sookyoung Jeon ◽  
Stanislav S Rubakhin ◽  
Lin Wang ◽  
...  
Keyword(s):  
Liquid Solution ◽  
Macaque Monkey ◽  
Solution Culture ◽  
Age Related Macular Degeneration ◽  
Tracer Studies ◽  
Human Diet ◽  
Age Related ◽  
High Enrichment ◽  
And Function

Lutein is a xanthophyll abundant in nature and most commonly present in the human diet through consumption of leafy green vegetables. With zeaxanthin and meso-zeaxanthin, lutein is a component of the macular pigment of the retina, where it protects against photooxidation and age-related macular degeneration. Recent studies have suggested that lutein may positively impact cognition throughout the lifespan, but outside of the retina, the deposition, metabolism, and function(s) of lutein are poorly understood. Using a novel botanical cell culture system ( Daucus carota), the present study aimed to produce a stable isotope lutein tracer for use in future investigations of dietary lutein distribution and metabolism. Carrot cultivars were initiated into liquid solution culture, lutein production conditions optimized, and uniformly labeled 13C-glucose was provided as the sole media carbon source for four serial growth cycles. Lutein yield was 2.58 ± 0.24 µg/g, and mass spectrometry confirmed high enrichment of 13C: 64.9% of lutein was uniformly labeled and 100% of lutein was labeled on at least 37 of 40 possible carbons. Purification of carrot extracts yielded a lutein dose of 1.92 mg with 96.0 ± 0.60% purity. 13C-lutein signals were detectable in hepatic extracts of an adult rhesus macaque monkey ( Macaca mulatta) dosed with 13C-lutein, but not in hepatic samples collected from control animals. This novel botanical biofactory approach can be used to produce sufficient quantities of highly enriched and pure 13C-lutein doses for use in tracer studies investigating lutein distribution, metabolism, and function.

scholarly journals The retinal toxicity profile towards assemblies of Amyloid-β indicate the predominant pathophysiological activity of oligomeric species

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Efrat Naaman ◽  
Sarah Ya’ari ◽  
Chen Itzkovich ◽  
Shadi Safuri ◽  
Flora Macsi ◽  
...  
Keyword(s):  
Toxic Effect ◽  
Self Assembly ◽  
Amyloid Β ◽  
Age Related Macular Degeneration ◽  
Retinal Tissue ◽  
Age Related ◽  
Aβ Aggregation ◽  
And Function ◽  
The Impact

AbstractAmyloid-β (Aβ), reported as a significant constituent of drusen, was implicated in the pathophysiology of age-related macular degeneration (AMD), yet the identity of the major pathogenic Aβ species in the retina has remained hitherto unclear. Here, we examined the in-vivo retinal impact of distinct supramolecular assemblies of Aβ. Fibrillar (Aβ40, Aβ42) and oligomeric (Aβ42) preparations showed clear biophysical hallmarks of amyloid assemblies. Measures of retinal structure and function were studied longitudinally following intravitreal administration of the various Aβ assemblies in rats. Electroretinography (ERG) delineated differential retinal neurotoxicity of Aβ species. Oligomeric Aβ42 inflicted the major toxic effect, exerting diminished ERG responses through 30 days post injection. A lesser degree of retinal dysfunction was noted following treatment with fibrillar Aβ42, whereas no retinal compromise was recorded in response to Aβ40 fibrils. The toxic effect of Aβ42 architectures was further reflected by retinal glial response. Fluorescence labelling of Aβ42 species was used to detect their accumulation into the retinal tissue. These results provide conceptual evidence of the differential toxicity of particular Aβ species in-vivo, and promote the mechanistic understanding of their retinal pathogenicity. Stratifying the impact of pathological Aβ aggregation in the retina may merit further investigation to decipher the pathophysiological relevance of processes of molecular self-assembly in retinal disorders.

scholarly journals Complement C5 is not critical for the formation of sub-RPE deposits in Efemp1 mutant mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Donita L. Garland ◽  
Eric A. Pierce ◽  
Rosario Fernandez-Godino
Keyword(s):  
Macular Degeneration ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Deposit Formation ◽  
Genetic Ablation ◽  
Age Related ◽  
Complement Dysregulation

AbstractThe complement system plays a role in the formation of sub-retinal pigment epithelial (RPE) deposits in early stages of age-related macular degeneration (AMD). But the specific mechanisms that connect complement activation and deposit formation in AMD patients are unknown, which limits the development of efficient therapies to reduce or stop disease progression. We have previously demonstrated that C3 blockage prevents the formation of sub-RPE deposits in a mouse model of EFEMP1-associated macular degeneration. In this study, we have used double mutant Efemp1R345W/R345W:C5-/- mice to investigate the role of C5 in the formation of sub-RPE deposits in vivo and in vitro. The data revealed that the genetic ablation of C5 does not eliminate the formation of sub-RPE deposits. Contrarily, the absence of C5 in RPE cultures promotes complement dysregulation that results in increased activation of C3, which likely contributes to deposit formation even in the absence of EFEMP1-R345W mutant protein. The results also suggest that genetic ablation of C5 alters the extracellular matrix turnover through an effect on matrix metalloproteinases in RPE cell cultures. These results confirm that C3 rather than C5 could be an effective therapeutic target to treat early AMD.

scholarly journals The anti-angiogenic isoforms of VEGF in health and disease

2009 ◽  
Vol 37 (6) ◽  
pp. 1207-1213 ◽  
Author(s):  
Yan Qiu ◽  
Coralie Hoareau-Aveilla ◽  
Sebastian Oltean ◽  
Steven J. Harper ◽  
David O. Bates
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Age Related Macular Degeneration ◽  
Splicing Factor ◽  
Splice Site Selection ◽  
Age Related ◽  
Normal Human ◽  
Radical Revision

Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys–Drash syndrome and pre-eclampsia). Administration of recombinant VEGF165b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGFxxxb isoforms to the pro-angiogenic VEGFxxx isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGFxxxb isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology.

scholarly journals Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells

Gene Therapy ◽  
2021 ◽  
Author(s):  
Anna K. Dreismann ◽  
Michelle E. McClements ◽  
Alun R. Barnard ◽  
Elise Orhan ◽  
Jane P. Hughes ◽  
...  
Keyword(s):  
Complement System ◽  
Functional Expression ◽  
Geographic Atrophy ◽  
Age Related Macular Degeneration ◽  
Complement Factor ◽  
Factor I ◽  
Complement Factor I ◽  
Age Related ◽  
The Complement System

AbstractDry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).

scholarly journals Hyperhomocysteinemia disrupts retinal pigment epithelial structure and function with features of age-related macular degeneration

Oncotarget ◽  
2016 ◽  
Vol 7 (8) ◽  
pp. 8532-8545 ◽  
Author(s):  
Ahmed S. Ibrahim ◽  
Suchreet Mander ◽  
Khaled A. Hussein ◽  
Nehal M. Elsherbiny ◽  
Sylvia B. Smith ◽  
...  
Keyword(s):  
Macular Degeneration ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Structure And Function ◽  
Age Related Macular Degeneration ◽  
Pigment Epithelial ◽  
Age Related ◽  
Epithelial Structure ◽  
And Function

scholarly journals Functional optical coherence tomography of retinal photoreceptors

2018 ◽  
Vol 243 (17-18) ◽  
pp. 1256-1264 ◽  
Author(s):  
Xincheng Yao ◽  
Taeyoon Son ◽  
Tae-Hoon Kim ◽  
Yiming Lu
Keyword(s):  
Vision Loss ◽  
Objective Assessment ◽  
Age Related Macular Degeneration ◽  
Retinal Stimulation ◽  
Age Related ◽  
Retinal Photoreceptors ◽  
Severe Vision Loss ◽  
Further Development

Age-related macular degeneration (AMD) is the leading cause of severe vision loss and legal blindness. It is known that retinal photoreceptors are the primary target of AMD. Therefore, a reliable method for objective assessment of photoreceptor function is needed for early detection and reliable treatment evaluation of AMD and other eye diseases such as retinitis pigmentosa that are known to cause photoreceptor dysfunctions. Stimulus-evoked intrinsic optical signal (IOS) changes promise a unique opportunity for objective assessment of physiological function of retinal photoreceptor and inner neurons. Instead of a comprehensive review, this mini-review is to provide a brief summary of our recent in vitro and in vivo optical coherence tomography (OCT) studies of stimulus-evoked IOS changes in animal retinas. By providing excellent axial resolution to differentiate individual retinal layers, depth-resolved OCT revealed rapid IOS response at the photoreceptor outer segment. The fast photoreceptor-IOS occurred almost right away (∼ 2 ms) after the onset of retinal stimulation, differentiating itself from slow IOS changes correlated with inner neural and hemodynamic changes. Further development of the functional IOS instruments and retinal stimulation protocols may provide a feasible solution to pursue clinical application of functional IOS imaging for objective assessment of human photoreceptors. Impact statement Retinal photoreceptors are the primary target of age-related macular degeneration (AMD) which is the leading cause of severe vision loss and legal blindness. An objective method for functional assessment of photoreceptor physiology can benefit early detection and better treatment evaluation of AMD and other eye diseases that are known to cause photoreceptor dysfunctions. This article summarizes in vitro study of IOS mechanisms and in vivo demonstration of IOS imaging of intact animals. Further development of the functional IOS imaging may provide a revolutionary solution to achieve objective assessment of human photoreceptors.

Cell-based therapy for ocular disorders: A promising frontier

2021 ◽  
Vol 16 ◽  
Author(s):  
Milad Ahani-Nahayati ◽  
Vahid Niazi ◽  
Alireza Moradi ◽  
Bahareh Pourjabbar ◽  
Reza Roozafzoon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wide Spectrum ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Ocular Tissue ◽  
Ocular Diseases ◽  
Cell Based Therapy ◽  
Age Related ◽  
Ocular Disorders

: As the ocular disorders causing long-term blindness or optical abnormalities of the ocular tissue affect the quality of life of patients to a large extent, awareness of their corresponding pathogenesis and the earlier detection and treatment need more consideration. Though current therapeutics result in desirable outcomes, they do not offer an inclusive solution for development of visual impairment to blindness. Accordingly, stem cells, because of their particular competencies, have gained extensive attention for application in regenerative medicine of ocular diseases. In the last decades, a wide spectrum of stem cells surrounding mesenchymal stem/stromal cells (MSC), neural stem cells (NSCs), and embryonic/induced pluripotent stem cells (ESCs/iPSCs) accompanied by Müller glia, ciliary epithelia-derived stem cells, and retinal pigment epithelial (RPE) stem cells have been widely investigated to report their safety and efficacy in preclinical models and also human subjects. In this regard, in the first interventions, RPE cell suspensions were successfully utilized to ameliorate visual defects of the patients suffering from age-related macular degeneration (AMD) after subretinal transplantation. Herein, we will explain the pathogenesis of ocular diseases and highlight the novel discoveries and recent findings in the context of stem cell-based therapies in these disorders, focusing on the in vivo reports published during the last decade.

Quantitative Action Spectroscopy Reveals ARPE-19 Sensitivity to Long-Wave Ultraviolet Radiation at 350 nm and 380 nm

2021 ◽  
Author(s):  
Graham Anderson ◽  
Andrew McLeod ◽  
Pierre Bagnaninchi ◽  
Baljean Dhillon
Keyword(s):  
Ultraviolet Radiation ◽  
Cell Viability ◽  
Age Related Macular Degeneration ◽  
Cell Dynamics ◽  
Cell Viability Assay ◽  
Cell Parameters ◽  
Viability Analysis ◽  
Age Related

The role of ultraviolet radiation (UVR) exposure in the pathology of age-related macular degeneration (AMD) has been debated for decades with epidemiological evidence failing to find a clear consensus for or against it playing a role. A key reason for this is a lack of foundational research into the response of living retinal tissue to UVR in regard to AMD-specific parameters of tissue function. We, therefore, explored the response of cultured retinal pigmented epithelium (RPE), the loss of which heralds advanced AMD, to specific wavelengths of UVR across the UV-B and UV-A bands found in natural sunlight. Using a bespoke in vitro UVR exposure apparatus coupled with bandpass filters we exposed the immortalised RPE cell line, ARPE-19, to 10nm bands of UVR between 290 and 405nm. Physical cell dynamics were assessed during exposure in cells cultured upon specialist electrode culture plates which allow for continuous, non-invasive electrostatic interrogation of key cell parameters during exposure such as monolayer coverage and tight-junction integrity. UVR exposures were also utilised to quantify wavelength-specific effects using a rapid cell viability assay and a phenotypic profiling assay which was leveraged to simultaneously quantify intracellular reactive oxygen species (ROS), nuclear morphology, mitochondrial stress, epithelial integrity and cell viability as part of a phenotypic profiling approach to quantifying the effects of UVR. Electrical impedance assessment revealed unforeseen detrimental effects of UV-A, beginning at 350nm, alongside previously demonstrated UV-B impacts. Cell viability analysis also highlighted increased effects at 350nm as well as 380nm. Effects at 350nm were further substantiated by high content image analysis which highlighted increased mitochondrial dysfunction and oxidative stress. We conclude that ARPE-19 cells exhibit a previously uncharacterised sensitivity to UV-A radiation, specifically at 350nm and somewhat less at 380nm. If upheld in vivo, such sensitivity will have impacts upon geoepidemiological risk scoring of AMD.

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