Publications by authors named "James W B Bainbridge"

43 Publications

Validation of a Vision-Guided Mobility Assessment for -Associated Retinal Dystrophy.

Transl Vis Sci Technol 2020 09 3;9(10). Epub 2020 Sep 3.

UCL Institute of Ophthalmology, University College London, London, UK.

Purpose: To validate a vision-guided mobility assessment for individuals affected by -associated retinal dystrophy (-RD).

Methods: In this comparative cross-sectional study, 29 subjects, comprising 19 subjects with -RD and 10 normally-sighted subjects undertook three assessments of mobility: following a straight line, navigating a simple maze, and stepping over a sidewalk "kerb." Performance was quantified as the time taken to complete each assessment, number of errors made, walking speed, and percent preferred walking speed, for each assessment. Subjects also undertook assessments of visual acuity, contrast sensitivity, full-field static perimetry, and age-appropriate quality of life questionnaires. To identify the most relevant metric to quantify vision-guided mobility, we investigated repeatability, as well as convergent, discriminant, and criterion validity. We also measured the effect of illumination on mobility.

Results: Walking speed through the maze assessment best discriminated between -RD and normally-sighted subjects, with both convergent and discriminant validity. Walking speed also approached statistical significance when assessed for criterion validity ( = 0.052). Subjects with -RD had quantifiably poorer mobility at lower illumination levels. A relatively small mean difference (-0.09 m/s) was identified in comparison to a relatively large repeatability coefficient (1.10 m/s).

Conclusions: We describe a novel, quantifiable, repeatable, and valid assessment of mobility designed specifically for subjects with -RD. The assessment is sensitive to the visual impairment of individuals with -RD in low illumination, identifies the known phenotypic heterogeneity and will furthermore provide an important outcome measure for -RD.

Translational Relevance: This assessment of vision-guided mobility, validated in a dedicated cohort of subjects with -RD, is a relevant and quantifiable outcome measure for -RD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/tvst.9.10.5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476654PMC
September 2020

Advancing Clinical Trials for Inherited Retinal Diseases: Recommendations from the Second Monaciano Symposium.

Transl Vis Sci Technol 2020 06 3;9(7). Epub 2020 Jun 3.

Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan Medical School, Ann Arbor, MI, USA.

Major advances in the study of inherited retinal diseases (IRDs) have placed efforts to develop treatments for these blinding conditions at the forefront of the emerging field of precision medicine. As a result, the growth of clinical trials for IRDs has increased rapidly over the past decade and is expected to further accelerate as more therapeutic possibilities emerge and qualified participants are identified. Although guided by established principles, these specialized trials, requiring analysis of novel outcome measures and endpoints in small patient populations, present multiple challenges relative to study design and ethical considerations. This position paper reviews recent accomplishments and existing challenges in clinical trials for IRDs and presents a set of recommendations aimed at rapidly advancing future progress. The goal is to stimulate discussions among researchers, funding agencies, industry, and policy makers that will further the design, conduct, and analysis of clinical trials needed to accelerate the approval of effective treatments for IRDs, while promoting advocacy and ensuring patient safety.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/tvst.9.7.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414644PMC
June 2020

Intravitreal Pharmacokinetic Study of the Antiangiogenic Glycoprotein Opticin.

Mol Pharm 2020 07 9;17(7):2390-2397. Epub 2020 Jun 9.

Division of Cardiovascular Sciences, School of Medical Sciences, FBMH, University of Manchester, Manchester M13 9PT, United Kingdom.

Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supranormal concentrations suppress preretinal neovascularization. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 μg/mL. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94% of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 μg) into both eyes of rabbits was followed by enucleation at 5, 24, and 72 h and 7, 14, and 28 days postinjection ( = 6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 mL, and the vitreous half-life was 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 μg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available antiangiogenic therapeutics. While opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the nonpigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate, and the turnover in vitreous is approximately 15% per day.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.0c00151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341526PMC
July 2020

Facedown Positioning Following Surgery for Large Full-Thickness Macular Hole: A Multicenter Randomized Clinical Trial.

JAMA Ophthalmol 2020 07;138(7):725-730

National Institute of Health Research Biomedical Research Centre (BRC), Moorfields Eye Hospital National Health Service (NHS) Foundation Trust, UCL Institute of Ophthalmology, London, England.

Importance: The value of facedown positioning following surgery for large full-thickness macular holes is unknown.

Objective: To determine whether advice to position facedown postoperatively improves the outcome for large macular holes.

Design, Setting, And Participants: This randomized, parallel group superiority trial with 1:1 randomization stratified by site with 3 months' follow-up was conducted at 9 sites across the United Kingdom and included participants with an idiopathic full-thickness macular hole of at least 400 μm minimum linear diameter and a duration of fewer than 12 months. All participants had vitrectomy surgery with peeling of the internal limiting membrane and injection of perfluoropropane (14%) gas, with or without simultaneous surgery for cataract.

Interventions: Following surgery, participants were randomly advised to position either facedown or face forward for 8 hours daily for 5 days.

Main Outcomes And Measures: The primary outcome was closure of the macular hole determined 3 months following surgery by masked optical coherence tomography evaluation. Secondary outcome measures at 3 months were visual acuity, participant-reported experience of positioning, and quality of life measured by the National Eye Institute Visual Function Questionnaire 25.

Results: A total of 185 participants (45 men [24.3%]; 156 white [84.3%]; 9 black [4.9%]; 10 Asian [5.4%]; median age, 69 years [interquartile range, 64-73 years]) were randomized. Macular hole closure was observed in 90 (85.6%) who were advised to position face forward and 88 (95.5%) advised to position facedown (adjusted odds ratio, 3.15; 95% CI, 0.87-11.41; P = .08). The mean (SD) improvement in best-corrected visual acuity at 3 months was 0.34 (0.69) logMAR (equivalent to 1 Snellen line) in the face-forward group and 0.57 (0.42) logMAR (equivalent to 3 Snellen lines) in the facedown group (adjusted mean difference, 0.22 [95 % CI, 0.05-0.38]; equivalent to 2 Snellen lines); 95% CI, 0.05-0.38; P = .01). The median National Eye Institute Visual Function Questionnaire 25 score was 89 (interquartile range, 76-94) in the facedown group and 87 (interquartile range, 73-93) in the face-forward group (mean [SD] change on a logistic scale, 0.08 [0.26] face forward and 0.11 [0.25] facedown; adjusted mean [SD] difference on a logistic scale, 0.02; 95% CI, -0.03 to 0.07; P = .41).

Conclusions And Relevance: The results do not prove that facedown positioning following surgery is more likely to close large macular holes compared with facing forward but do support the possibility that visual acuity outcomes may be superior.

Trial Registration: Isrctn.org Identifier: 12410596.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1001/jamaophthalmol.2020.0987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206530PMC
July 2020

Retinal Structure in RPE65-Associated Retinal Dystrophy.

Invest Ophthalmol Vis Sci 2020 04;61(4):47

,.

Purpose: RPE65-associated retinal dystrophy (RPE65-RD) is an early onset, progressive, severe retinal dystrophy. We sought to characterize the natural history of retinal degeneration in affected individuals.

Methods: We performed cross-sectional and longitudinal quantitative and qualitative assessments of retinal architecture in RPE65-RD using spectral domain optical coherence tomography (SD-OCT) and fundus autofluorescence (FAF) imaging. Twenty-six subjects (mean age, 14.8 years, range, 5-24 years) with RPE65-RD underwent SD-OCT and FAF imaging, of whom 14 subjects were followed up over time. Foveal thickness (FT), outer nuclear layer thickness (ONLT), ellipsoid zone width (EZW), and ellipsoid zone area (EZA) were calculated where possible. These were correlated with age, best corrected visual acuity (BCVA), and central 30° retinal sensitivity (V30). Intra-observer agreement, test-retest repeatability, and interocular symmetry were also investigated.

Results: We identified structural interocular symmetry, the presence of autofluorescence in 46% (12/26) of subjects, and the presence of foveal hypoplasia (associated with significantly worse BCVA) in 50% of subjects. EZW and EZA were measurable in 67% (35/52) and 37% (19/52) of eyes, respectively, with both demonstrating good agreement on repeated measurement. The annual rate of progression using EZW was -300.63 µm/year, and -1.17 mm2/year in EZA. EZW was found to have a statistically significant correlation with BCVA and V30.

Conclusions: We identified the presence of autofluorescence in half of our subjects, with foveal hypoplasia also noted in half of our cohort. EZW, and to a lesser extent EZA, were robust measures of retinal degeneration and represent valuable metrics to determine the impact of intervention. (ClinicalTrials.gov number NCT02714816.).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.61.4.47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401957PMC
April 2020

Gene therapy for neovascular age-related macular degeneration: rationale, clinical trials and future directions.

Br J Ophthalmol 2021 Feb 8;105(2):151-157. Epub 2020 Apr 8.

UCL Institute of Ophthalmology, University College London, London, UK

Age-related macular degeneration (AMD) is one of the leading causes of irreversible blindness in the developed world. Antivascular endothelial growth factor therapy has transformed the management and outcome of neovascular AMD (nAMD), although the need for repeated intravitreal injections-even lifelong-and the related complications, high drug costs, frequent clinic visits and repeated imaging have resulted in an enormous burden both to healthcare systems and patients. The application of gene therapy approaches for sustained delivery of a range of antiangiogenic proteins has the promise of helping to address these aforementioned challenges. A number of early phase clinical trials of gene therapy in nAMD have provided encouraging results, with many more ongoing or anticipated. There remain significant areas of controversy, including regarding the optimal treatment targets, routes of administration and potential safety concerns. In this review we aim to provide an update of the current status of gene therapy for nAMD and briefly discuss future prospects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/bjophthalmol-2020-316195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848059PMC
February 2021

Stabilization of myeloid-derived HIFs promotes vascular regeneration in retinal ischemia.

Angiogenesis 2020 05 3;23(2):83-90. Epub 2019 Oct 3.

Division of Genetics, UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK.

The retinal vasculature is tightly organized in a structure that provides for the high metabolic demand of neurons while minimizing interference with incident light. The adverse impact of retinal vascular insufficiency is mitigated by adaptive vascular regeneration but exacerbated by pathological neovascularization. Aberrant growth of neovessels in the retina is responsible for impairment of sight in common blinding disorders including retinopathy of prematurity, proliferative diabetic retinopathy, and age-related macular degeneration. Myeloid cells are key players in this process, with diverse roles that can either promote or protect against ocular neovascularization. We have previously demonstrated that myeloid-derived VEGF, HIF1, and HIF2 are not essential for pathological retinal neovascularization. Here, however, we show by cell-specific depletion of Vhl in a mouse model of retinal ischemia (oxygen-induced retinopathy, OIR) that myeloid-derived HIFs promote VEGF and bFGF expression and enhance vascular regeneration in association with improved density and organization of the astrocytic network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10456-019-09681-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160070PMC
May 2020

A Cross-Sectional and Longitudinal Study of Retinal Sensitivity in RPE65-Associated Leber Congenital Amaurosis.

Invest Ophthalmol Vis Sci 2018 07;59(8):3330-3339

UCL Institute of Ophthalmology, University College London, London, United Kingdom.

Purpose: RPE65-associated Leber congenital amaurosis (RPE65-LCA) is an early-onset severe retinal dystrophy associated with progressive visual field loss. Phase I/II and III gene therapy trials have identified improved retinal sensitivity but little is known about the natural history of retinal sensitivity in RPE65-LCA.

Methods: A total of 19 subjects (aged 9 to 23 years) undertook monocular full-field static perimetry of which 13 subjects were monitored longitudinally. Retinal sensitivity was measured as mean sensitivity (MS) and volumetrically quantified (in decibel-steradian) using visual field modeling and analysis software for the total (VTOT), central 30° (V30) and central 15° (V15) visual field. Correlation was evaluated between retinal sensitivity and age, best-corrected visual acuity (BCVA), contrast sensitivity, vision-related quality of life, and genotype. Test-retest reliability was also investigated.

Results: V30 was identified to have a strong, weak, and moderate correlation with age, BCVA and contrast sensitivity respectively. Furthermore, V30 was identified as having a weak linear relationship with the mobility and independence domains of the vision-related quality of life questionnaire. Longitudinal analysis demonstrated a slow loss of retinal sensitivity in this cohort. Subjects with at least one RPE65 nonsense variant appeared to show greater progressive loss of retinal sensitivity in the second decade of life than those without.

Conclusions: Volumetric assessment of central 30° visual field sensitivity, V30, is a useful independent measure of retinal function and, in our data, represented the best metric to monitor deterioration of retinal sensitivity in RPE65-LCA. Furthermore, functional correlation with genotype may enable more informed prognostic counseling. (ClinicalTrials.gov number, NCT02714816.)
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.18-23873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040235PMC
July 2018

Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in Macular Degeneration.

Ophthalmology 2018 11 5;125(11):1765-1775. Epub 2018 Jun 5.

NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital and University College London, London, United Kingdom; Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom; Institute of Ophthalmology, University College London, London, United Kingdom. Electronic address:

Purpose: Transplantation of human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells offers the potential for benefit in macular degeneration. Previous trials have reported improved visual acuity (VA), but lacked detailed analysis of retinal structure and function in the treated area.

Design: Phase 1/2 open-label dose-escalation trial to evaluate safety and potential efficacy (clinicaltrials.gov identifier, NCT01469832).

Participants: Twelve participants with advanced Stargardt disease (STGD1), the most common cause of macular degeneration in children and young adults.

Methods: Subretinal transplantation of up to 200 000 hESC-derived RPE cells with systemic immunosuppressive therapy for 13 weeks.

Main Outcome Measures: The primary end points were the safety and tolerability of hESC-derived RPE cell administration. We also investigated evidence of the survival of transplanted cells and measured retinal structure and function using microperimetry and spectral-domain OCT.

Results: Focal areas of subretinal hyperpigmentation developed in all participants in a dose-dependent manner in the recipient retina and persisted after withdrawal of systemic immunosuppression. We found no evidence of uncontrolled proliferation or inflammatory responses. Borderline improvements in best-corrected VA in 4 participants either were unsustained or were matched by a similar improvement in the untreated contralateral eye. Microperimetry demonstrated no evidence of benefit at 12 months in the 12 participants. In one instance at the highest dose, localized retinal thinning and reduced sensitivity in the area of hyperpigmentation suggested the potential for harm. Participant-reported quality of life using the 25-item National Eye Institute Visual Function Questionnaire indicated no significant change.

Conclusions: Subretinal hyperpigmentation is consistent with the survival of viable transplanted hESC-derived RPE cells, but may reflect released pigment in their absence. The findings demonstrate the value of detailed analysis of spatial correlation of retinal structure and function in determining with appropriate sensitivity the impact of cell transplantation and suggest that intervention in early stage of disease should be approached with caution. Given the slow rate of progressive degeneration at this advanced stage of disease, any protection against further deterioration may be evident only after a more extended period of observation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ophtha.2018.04.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195794PMC
November 2018

Late neuroprogenitors contribute to normal retinal vascular development in a Hif2a-dependent manner.

Development 2018 04 25;145(8). Epub 2018 Apr 25.

UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK

In the adult central nervous system, endothelial and neuronal cells engage in tight cross-talk as key components of the so-called neurovascular unit. Impairment of this important relationship adversely affects tissue homeostasis, as observed in neurodegenerative conditions including Alzheimer's and Parkinson's disease. In development, the influence of neuroprogenitor cells on angiogenesis is poorly understood. Here, we show in mouse that these cells interact intimately with the growing retinal vascular network, and we identify a novel regulatory mechanism of vasculature development mediated by hypoxia-inducible factor 2a (Hif2a). By gene excision, we show that Hif2a in retinal neuroprogenitor cells upregulates the expression of the pro-angiogenic mediators vascular endothelial growth factor and erythropoietin, whereas it locally downregulates the angiogenesis inhibitor endostatin. Importantly, absence of in retinal neuroprogenitor cells causes a marked reduction of proliferating endothelial cells at the angiogenic front. This results in delayed retinal vascular development, fewer major retinal vessels and reduced density of the peripheral deep retinal vascular plexus. Our findings demonstrate that retinal neuroprogenitor cells are a crucial component of the developing neurovascular unit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.157511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964651PMC
April 2018

Retinal gene therapy.

Br Med Bull 2018 06;126(1):13-25

NIHR Biomedical Research Centre for Ophthalmology at Moofields Eye Hospital and UCL, 162 City Road, London, UK.

Introduction: Inherited retinal diseases are the leading cause of sight impairment in people of working age in England and Wales, and the second commonest in childhood. Gene therapy offers the potential for benefit.

Sources Of Data: Pubmed and clinicaltrials.gov.

Areas Of Agreement: Gene therapy can improve vision in RPE65-associated Leber Congenital Amaurosis (RPE65-LCA). Potential benefit depends on efficient gene transfer and is limited by the extent of retinal degeneration.

Areas Of Controversy: The magnitude of vision improvement from RPE65-LCA gene therapy is suboptimal, and its durability may be limited by progressive retinal degeneration.

Growing Points: The safety and potential benefit of gene therapy for inherited and acquired retinal diseases is being explored in a rapidly expanding number of trials.

Areas Timely For Developing Research: Developments in vector design and delivery will enable greater efficiency and safety of gene transfer. Optimization of trial design will accelerate reliable assessment of outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bmb/ldy005DOI Listing
June 2018

Severe Loss of Tritan Color Discrimination in RPE65 Associated Leber Congenital Amaurosis.

Invest Ophthalmol Vis Sci 2018 01;59(1):85-93

UCL Institute of Ophthalmology, University College London, London, United Kingdom.

Purpose: RPE65-associated Leber congenital amaurosis (RPE65-LCA) is a progressive severe retinal dystrophy with early profound dysfunction of rod photoreceptors followed by progressive cone photoreceptor degeneration. We aim to provide detailed information about how cone dysfunction affects color discrimination.

Methods: Seven adults (aged 16-21) with RPE65-LCA underwent monocular color discrimination assessment using the Trivector and Ellipse versions of three computerized tests: Cambridge Colour Test (CCT), low vision version of the Cambridge Colour Test (lvvCCT), and the Universal Colour Discrimination Test (UCDT). For comparison, subjects were also tested using the American Optical Hardy Rand Rittler (AO-HRR) plates. Each assessment was repeated three times.

Results: The Trivector version of the tests demonstrated that color discrimination along the tritan axis was undetectable in four subjects, and severely reduced in three subjects. These findings were confirmed by the Ellipse version of the tests. Color discrimination along the protan and deutan axes was evident but reduced in six of seven subjects. Four of seven subjects were unable to read any of the HRR plates.

Conclusions: The computerized color vision tests adopted in this study provide detailed information about color discrimination in adult RPE65-LCA patients. The condition is associated with severe impairment of color discrimination, particularly along the tritan axis indicating possible early involvement of S-cones, with additional protan and deutan loss to a lesser extent. This psychophysical assessment strategy is likely to be valuable in measuring the impact of therapeutic intervention on cone function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.17-22905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769497PMC
January 2018

Isolation of Human Photoreceptor Precursors via a Cell Surface Marker Panel from Stem Cell-Derived Retinal Organoids and Fetal Retinae.

Stem Cells 2018 05 1;36(5):709-722. Epub 2018 Feb 1.

Stem Cells and Regenerative Medicine Section, University College London, London, United Kingdom.

Loss of photoreceptor cells due to retinal degeneration is one of the main causes of blindness in the developed world. Although there is currently no effective treatment, cell replacement therapy using stem-cell-derived photoreceptor cells may be a feasible future treatment option. In order to ensure safety and efficacy of this approach, robust cell isolation and purification protocols must be developed. To this end, we previously developed a biomarker panel for the isolation of mouse photoreceptor precursors from the developing mouse retina and mouse embryonic stem cell cultures. In the current study we applied this approach to the human pluripotent stem cell (hPSC) system, and identified novel biomarker combinations that can be leveraged for the isolation of human photoreceptors. Human retinal samples and hPSC-derived retinal organoid cultures were screened against 242 human monoclonal antibodies using a high through-put flow cytometry approach. We identified 46 biomarkers with significant expression levels in the human retina and hPSC differentiation cultures. Human retinal cell samples, either from fetal tissue or derived from embryonic and induced pluripotent stem cell cultures, were fluorescence-activated cell sorted (FACS) using selected candidate biomarkers that showed expression in discrete cell populations. Enrichment for photoreceptors and exclusion of mitotically active cells was demonstrated by immunocytochemical analysis with photoreceptor-specific antibodies and Ki-67. We established a biomarker combination, which enables the robust purification of viable human photoreceptors from both human retinae and hPSC-derived organoid cultures. Stem Cells 2018;36:709-722.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.2775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947711PMC
May 2018

Transplanted Donor- or Stem Cell-Derived Cone Photoreceptors Can Both Integrate and Undergo Material Transfer in an Environment-Dependent Manner.

Stem Cell Reports 2018 02 4;10(2):406-421. Epub 2018 Jan 4.

UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK. Electronic address:

Human vision relies heavily upon cone photoreceptors, and their loss results in permanent visual impairment. Transplantation of healthy photoreceptors can restore visual function in models of inherited blindness, a process previously understood to arise by donor cell integration within the host retina. However, we and others recently demonstrated that donor rod photoreceptors engage in material transfer with host photoreceptors, leading to the host cells acquiring proteins otherwise expressed only by donor cells. We sought to determine whether stem cell- and donor-derived cones undergo integration and/or material transfer. We find that material transfer accounts for a significant proportion of rescued cells following cone transplantation into non-degenerative hosts. Strikingly, however, substantial numbers of cones integrated into the Nrl and Prph2, but not Nrl;RPE65, murine models of retinal degeneration. This confirms the occurrence of photoreceptor integration in certain models of retinal degeneration and demonstrates the importance of the host environment in determining transplantation outcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2017.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830910PMC
February 2018

Recapitulation of Human Retinal Development from Human Pluripotent Stem Cells Generates Transplantable Populations of Cone Photoreceptors.

Stem Cell Reports 2017 09 24;9(3):820-837. Epub 2017 Aug 24.

Department of Genetics, University College London Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK; NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, City Road, London EC1V 2PD, UK. Electronic address:

Transplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium cones survive and become incorporated within the adult mouse retina, supporting the potential of photoreceptor transplantation for treating retinal degeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2017.07.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599247PMC
September 2017

Accelerated oxygen-induced retinopathy is a reliable model of ischemia-induced retinal neovascularization.

PLoS One 2017 26;12(6):e0179759. Epub 2017 Jun 26.

Division of Genetics, UCL Institute of Ophthalmology, University College London, London, United Kingdom.

Retinal ischemia and pathological angiogenesis cause severe impairment of sight. Oxygen-induced retinopathy (OIR) in young mice is widely used as a model to investigate the underlying pathological mechanisms and develop therapeutic interventions. We compared directly the conventional OIR model (exposure to 75% O2 from postnatal day (P) 7 to P12) with an alternative, accelerated version (85% O2 from P8 to P11). We found that accelerated OIR induces similar pre-retinal neovascularization but greater retinal vascular regression that recovers more rapidly. The extent of retinal gliosis is similar but neuroretinal function, as measured by electroretinography, is better maintained in the accelerated model. We found no systemic or maternal morbidity in either model. Accelerated OIR offers a safe, reliable and more rapid alternative model in which pre-retinal neovascularization is similar but retinal vascular regression is greater.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179759PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484470PMC
September 2017

A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye.

Dis Model Mech 2015 Nov 20;8(11):1479-87. Epub 2015 Aug 20.

University College London, Institute of Ophthalmology, London EC1V 9EL, UK

We have developed a method to label and image myeloid cells infiltrating the mouse retina and choroid in vivo, using a single depot injection of indocyanine green dye (ICG). This was demonstrated using the following ocular models of inflammation and angiogenesis: endotoxin-induced uveitis, experimental autoimmune uveoretinitis and laser-induced choroidal neovascularization model. A near-infrared scanning ophthalmoscope was used for in vivo imaging of the eye, and flow cytometry was used on blood and spleen to assess the number and phenotype of labelled cells. ICG was administered 72 h before the induction of inflammation to ensure clearance from the systemic circulation. We found that in vivo intravenous administration failed to label any leukocytes, whereas depot injection, either intraperitoneal or subcutaneous, was successful in labelling leukocytes infiltrating into the retina. Progression of inflammation in the retina could be traced over a period of 14 days following a single depot injection of ICG. Additionally, bright-field microscopy, spectrophotometry and flow cytometric analysis suggest that the predominant population of cells stained by ICG are circulating myeloid cells. The translation of this approach into clinical practice would enable visualization of immune cells in situ. This will not only provide a greater understanding of pathogenesis, monitoring and assessment of therapy in many human ocular diseases but might also open the ability to image immunity live for neurodegenerative disorders, cardiovascular disease and systemic immune-mediated disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dmm.019018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631782PMC
November 2015

Cd59a deficiency in mice leads to preferential innate immune activation in the retinal pigment epithelium-choroid with age.

Neurobiol Aging 2015 Sep 9;36(9):2637-48. Epub 2015 Jun 9.

Department of Genetics, UCL Institute of Ophthalmology, London, UK; NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital, London, UK.

Dysregulation of the complement system has been implicated in the pathogenesis of age-related macular degeneration. To investigate consequences of altered complement regulation in the eye with age, we examined Cd59a complement regulator deficient (Cd59a(-/-)) mice between 4 and 15 months. In vivo imaging revealed an increased age-related accumulation of autofluorescent spots in Cd59a(-/-) mice, a feature that reflects accumulation of subretinal macrophages and/or microglia. Despite this activation of myeloid cells in the eye, Cd59a(-/-) mice showed normal retinal histology and function as well as normal choroidal microvasculature. With age, they revealed increased expression of activators of the alternative complement pathway (C3, Cfb, Cfd), in particular in the retinal pigment epithelium (RPE)-choroid but less in the retina. This molecular response was not altered by moderately-enhanced light exposure. Cd59a deficiency therefore leads to a preferential age-related dysregulation of the complement system in the RPE-choroid, that alone or in combination with light as a trigger, is not sufficient to cause choroidal vascular changes or retinal degeneration and dysfunction. This data emphasizes the particular vulnerability of the RPE-choroidal complex to dysregulation of the alternative complement pathway during aging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neurobiolaging.2015.05.019DOI Listing
September 2015

Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina.

Stem Cells 2015 Aug 27;33(8):2469-82. Epub 2015 May 27.

Stem Cells and Regenerative Medicine Section, UCL Institute of Child Health, University College London, London, United Kingdom.

Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post-mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(-) facilitates the isolation of photoreceptor precursors from three-dimensional self-forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell-derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation-competent cells for therapeutic application.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.2051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862023PMC
August 2015

Long-term effect of gene therapy on Leber's congenital amaurosis.

N Engl J Med 2015 May 4;372(20):1887-97. Epub 2015 May 4.

From the UCL (University College London) Institute of Ophthalmology (J.W.B.B., M.S.M., V.S., S.J.R., S.E.B., C.R., A.G., F.M.M., S.G.B., P.J.G., V.A.L., K.B., A.V., G.E.H., F.W.F., M.N., A.T.M., M.M., A.S., A.J.S., G.R., R.R.A.) and the Department of Civil, Environmental, and Geomatic Engineering (N.T.), UCL, and Moorfields Eye Hospital (J.W.B.B., M.S.M., V.S., S.J.R., A.G., K.B., G.H., A.M., M.M.), London, and the Department of Psychology, Durham University, Durham (M.N.) - all in the United Kingdom; the College of Veterinary Medicine, Michigan State University, East Lansing (F.M.M., S.M.P.-J.), and the Kellogg Eye Center, University of Michigan Medical School, Ann Arbor (K.L.F., D.A.T., R.R.A.); the Center for Human Genetics, KU Leuven (T.J.L.R.), and the Department of Ophthalmology, UZ Leuven, Campus Sint-Rafaël (I.C.) - both in Leuven, Belgium; Rotterdam Eye Hospital, Rotterdam, the Netherlands (S.Y., L.I.B.); and the Oregon Retinal Degeneration Center, Ophthalmic Genetics Service, Casey Eye Institute, Oregon Health and Science University, Portland (R.G.W.).

Background: Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited.

Methods: We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings.

Results: Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG.

Conclusions: Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1056/NEJMoa1414221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497809PMC
May 2015

Gene therapy restores vision in rd1 mice after removal of a confounding mutation in Gpr179.

Nat Commun 2015 Jan 23;6:6006. Epub 2015 Jan 23.

Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK.

The rd1 mouse with a mutation in the Pde6b gene was the first strain of mice identified with a retinal degeneration. However, AAV-mediated gene supplementation of rd1 mice only results in structural preservation of photoreceptors, and restoration of the photoreceptor-mediated a-wave, but not in restoration of the bipolar cell-mediated b-wave. Here we show that a mutation in Gpr179 prevents the full restoration of vision in rd1 mice. Backcrossing rd1 with C57BL6 mice reveals the complete lack of b-wave in a subset of mice, consistent with an autosomal recessive Mendelian inheritance pattern. We identify a mutation in the Gpr179 gene, which encodes for a G-protein coupled receptor localized to the dendrites of ON-bipolar cells. Gene replacement in rd1 mice that are devoid of the mutation in Gpr179 successfully restores the function of both photoreceptors and bipolar cells, which is maintained for up to 13 months. Our discovery may explain the failure of previous gene therapy attempts in rd1 mice, and we propose that Grp179 mutation status should be taken into account in future studies involving rd1 mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms7006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354202PMC
January 2015

The severity of retinal pathology in homozygous Crb1rd8/rd8 mice is dependent on additional genetic factors.

Hum Mol Genet 2015 Jan 21;24(1):128-41. Epub 2014 Aug 21.

Department of Genetics and NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London EC1V 2PD, UK.

Understanding phenotype-genotype correlations in retinal degeneration is a major challenge. Mutations in CRB1 lead to a spectrum of autosomal recessive retinal dystrophies with variable phenotypes suggesting the influence of modifying factors. To establish the contribution of the genetic background to phenotypic variability associated with the Crb1(rd8/rd8) mutation, we compared the retinal pathology of Crb1(rd8/rd8)/J inbred mice with that of two Crb1(rd8/rd8) lines backcrossed with C57BL/6JOlaHsd mice. Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines. Optical coherence tomography, semithin, ultrastructural morphology and assessment of inflammatory and vascular marker by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction revealed that the lesions were associated with photoreceptor death, Müller and microglia activation and telangiectasia-like vascular remodelling-features that were stable in the inbred, variable in the second, but virtually absent in the third Crb1(rd8/rd8) line, even at 12 months of age. This suggests that the Crb1(rd8/rd8) mutation is necessary, but not sufficient for the development of these degenerative features. By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified. This may carry one or more genetic modifiers for the manifestation of the retinal pathology associated with mutations in Crb1. This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddu424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262495PMC
January 2015

Successful gene therapy in older Rpe65-deficient dogs following subretinal injection of an adeno-associated vector expressing RPE65.

Hum Gene Ther 2013 Oct;24(10):883-93

1 Department of Small Animal Clinical Sciences, Michigan State University , East Lansing, MI 48824.

Young Rpe65-deficient dogs have been used as a model for human RPE65 Leber congenital amaurosis (RPE65-LCA) in proof-of-concept trials of recombinant adeno-associated virus (rAAV) gene therapy. However, there are relatively few reports of the outcome of rAAV gene therapy in Rpe65-deficient dogs older than 2 years of age. The purpose of this study was to investigate the success of this therapy in older Rpe65-deficient dogs. Thirteen eyes were treated in dogs between 2 and 6 years old. An rAAV2 vector expressing the human RPE65 cDNA driven by the human RPE65 promoter was delivered by subretinal injection. Twelve of the 13 eyes had improved retinal function as assessed by electroretinography, and all showed improvement in vision at low lighting intensities. Histologic examination of five of the eyes was performed but found no correlation between electroretinogram (ERG) rescue and numbers of remaining photoreceptors. We conclude that functional rescue is still possible in older dogs and that the use of older Rpe65-deficient dogs, rather than young Rpe65-deficient dogs that have very little loss of photoreceptors, more accurately models the situation when treating human RPE65-LCA patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2013.146DOI Listing
October 2013

Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina.

Nat Biotechnol 2013 Aug 21;31(8):741-7. Epub 2013 Jul 21.

Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL UK.

Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment-bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nbt.2643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826328PMC
August 2013

Assessment and in vivo scoring of murine experimental autoimmune uveoretinitis using optical coherence tomography.

PLoS One 2013 14;8(5):e63002. Epub 2013 May 14.

Department of Genetics, UCL Institute of Ophthalmology, London, United Kingdom.

Despite advances in clinical imaging and grading our understanding of retinal immune responses and their morphological correlates in experimental autoimmune uveoretinitis (EAU), has been hindered by the requirement for post-mortem histology. To date, monitoring changes occurring during EAU disease progression and evaluating the effect of therapeutic intervention in real time has not been possible. We wanted to establish whether optical coherence tomography (OCT) could detect intraretinal changes during inflammation and to determine its utility as a tool for accurate scoring of EAU. EAU was induced in C57BL/6J mice and animals evaluated after 15, 26, 36 and 60 days. At each time-point, contemporaneous Spectralis-OCT scanning, topical endoscopic fundal imaging (TEFI), fundus fluorescein angiography (FFA) and CD45-immunolabelled histology were performed. OCT features were further characterised on retinal flat-mounts using immunohistochemistry and 3D reconstruction. Optic disc swelling and vitreous opacities detected by OCT corresponded to CD45+ cell infiltration on histology. Vasculitis identified by FFA and OCT matched perivascular myeloid and T-cell infiltrates and could be differentiated from unaffected vessels. Evolution of these changes could be followed over time in the same eye. Retinal folds were visible and found to encapsulate mixed populations of activated myeloid cells, T-cells and microglia. Using these features, an OCT-based EAU scoring system was developed, with significant correlation to validated histological (Pearson r(2) = 0.6392, P<0.0001, n = 31 eyes) and TEFI based scoring systems (r(2) = 0.6784, P<0.0001). OCT distinguishes the fundamental features of murine EAU in vivo, permits dynamic assessment of intraretinal changes and can be used to score disease severity. As a result, it allows tissue synchronisation with subsequent cellular and functional assessment and greater efficiency of animal usage. By relating OCT signals with immunohistochemistry in EAU, our findings offer the opportunity to inform the interpretation of OCT changes in human uveitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0063002PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653962PMC
December 2013

Repair of the degenerate retina by photoreceptor transplantation.

Proc Natl Acad Sci U S A 2013 Jan 17;110(1):354-9. Epub 2012 Dec 17.

Department of Genetics, University College London Institute of Ophthalmology, London EC1V 9EL, United Kingdom.

Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2(+/Δ307)), decrease (Crb1(rd8/rd8), Gnat1(-/-), Rho(-/-)), or remain constant (PDE6β(rd1/rd1), Prph2(rd2/rd2)) with disease progression, depending upon the gene defect, with no correlation with severity. Robust integration is possible even in late-stage disease. Glial scarring and outer limiting membrane integrity, features that change with degeneration, significantly affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model with an intact outer limiting membrane, strong gliotic response, and otherwise poor transplantation outcome (Rho(-/-)), leading to an eightfold increase in integration and restoration of visual function. Thus, it is possible to achieve robust integration across a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1212677110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538261PMC
January 2013

Ccl2, Cx3cr1 and Ccl2/Cx3cr1 chemokine deficiencies are not sufficient to cause age-related retinal degeneration.

Exp Eye Res 2013 Feb 8;107:80-7. Epub 2012 Dec 8.

Department of Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, EC1V9EL London, United Kingdom.

Monocytes, macrophages, dendritic cells and microglia play critical roles in the local immune response to acute and chronic tissue injury and have been implicated in the pathogenesis of age-related macular degeneration. Defects in Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling cause enhanced accumulation of bloated subretinal microglia/macrophages in senescent mice and this phenomenon is reported to result in the acceleration of age-related retinal degeneration. The purpose of this study was to determine whether defects in CCL2-CCR2 and CX3CL1-CX3CR1 signalling pathways, alone or in combination, cause age-dependent retinal degeneration. We tested whether three chemokine knockout mouse lines, Ccl2(-/-), Cx3cr1(-/-) and Ccl2(-/-)/Cx3cr1(-/-), in comparison to age-matched C57Bl/6 control mice show differences in subretinal macrophage accumulation and loss of adjacent photoreceptor cells at 12-14 months of age. All mouse lines are derived from common parental strains and do not carry the homozygous rd8 mutation in the Crb1 gene that has been a major confounding factor in previous reports. We quantified subretinal macrophages by counting autofluorescent lesions in fundus images obtained by scanning laser ophthalmoscopy (AF-SLO) and by immunohistochemistry for Iba1 positive cells. The accumulation of subretinal macrophages was enhanced in Ccl2(-/-), but not in Cx3cr1(-/-) or Ccl2(-/-)/Cx3cr1(-/-) mice. We identified no evidence of retinal degeneration in any of these mouse lines by TUNEL staining or semithin histology. In conclusion, CCL2-CCR2 and/or CX3CL1-CX3CR1 signalling defects may differentially affect the trafficking of microglia and macrophages in the retina during ageing, but do not appear to cause age-related retinal degeneration in mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exer.2012.11.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562441PMC
February 2013

Von Hippel-Lindau protein in the RPE is essential for normal ocular growth and vascular development.

Development 2012 Jul 23;139(13):2340-50. Epub 2012 May 23.

Department of Genetics, Institute of Ophthalmology, NIHR Biomedical Research Centre for Ophthalmology, University College London, London EC1V 9EL, UK.

Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.070813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367444PMC
July 2012

Differential modulation of retinal degeneration by Ccl2 and Cx3cr1 chemokine signalling.

PLoS One 2012 24;7(4):e35551. Epub 2012 Apr 24.

Department of Genetics, UCL Institute of Ophthalmology, London, United Kingdom.

Microglia and macrophages are recruited to sites of retinal degeneration where local cytokines and chemokines determine protective or neurotoxic microglia responses. Defining the role of Ccl2-Ccr2 and Cx3cl1-Cx3cr1 signalling for retinal pathology is of particular interest because of its potential role in age-related macular degeneration (AMD). Ccl2, Ccr2, and Cx3cr1 signalling defects impair macrophage trafficking, but have, in several conflicting studies, been reported to show different degrees of age-related retinal degeneration. Ccl2/Cx3cr1 double knockout (CCDKO) mice show an early onset retinal degeneration and have been suggested as a model for AMD. In order to understand phenotypic discrepancies in different chemokine knockout lines and to study how defects in Ccl2 and/or Cx3cr1 signalling contribute to the described early onset retinal degeneration, we defined primary and secondary pathological events in CCDKO mice. To control for genetic background variability, we compared the original phenotype with that of single Ccl2, Cx3cr1 and Ccl2/Cx3cr1 double knockout mice obtained from backcrosses of CCDKO with C57Bl/6 mice. We found that the primary pathological event in CCDKO mice develops in the inferior outer nuclear layer independently of light around postnatal day P14. RPE and vascular lesions develop secondarily with increasing penetrance with age and are clinically similar to retinal telangiectasia not to choroidal neovascularisation. Furthermore, we provide evidence that a third autosomal recessive gene causes the degeneration in CCDKO mice and in all affected re-derived lines and subsequently demonstrated co-segregation of the naturally occurring RD8 mutation in the Crb1 gene. By comparing CCDKO mice with re-derived CCl2(-/-)/Crb1(Rd8/RD8), Cx3cr1(-/-)/Crb1(Rd8/RD8) and CCl2(-/-)/Cx3cr1(-/-)/Crb1(Rd8/RD8) mice, we observed a differential modulation of the retinal phenotype by genetic background and both chemokine signalling pathways. These findings indicate that CCDKO mice are not a model of AMD, but a model for an inherited retinal degeneration that is differentially modulated by Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035551PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335860PMC
August 2012