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Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid.

Tue, 2020-03-24 15:38
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Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid.

J Exp Med. 2020 Jun 01;217(6):

Authors: Lehmann GL, Hanke-Gogokhia C, Hu Y, Bareja R, Salfati Z, Ginsberg M, Nolan DJ, Mendez-Huergo SP, Dalotto-Moreno T, Wojcinski A, Ochoa F, Zeng S, Cerliani JP, Panagis L, Zager PJ, Mullins RF, Ogura S, Lutty GA, Bang J, Zippin JH, Romano C, Rabinovich GA, Elemento O, Joyner AL, Rafii S, Rodriguez-Boulan E, Benedicto I

Abstract
The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.

PMID: 32196081 [PubMed - in process]

Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-Associated X-Linked Retinitis Pigmentosa.

Tue, 2020-03-17 15:20
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Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-Associated X-Linked Retinitis Pigmentosa.

Hum Gene Ther. 2019 08;30(8):967-974

Authors: Giacalone JC, Andorf JL, Zhang Q, Burnight ER, Ochoa D, Reutzel AJ, Collins MM, Sheffield VC, Mullins RF, Han IC, Stone EM, Tucker BA

Abstract
In a screen of 1,000 consecutively ascertained families, we recently found that mutations in the gene RPGR are the third most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A, are far too large to fit into clinically relevant adeno-associated virus (AAV) vectors, RPGR is an obvious early target for AAV-based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable (i.e., they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector, we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 (i.e., potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5, and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull-down assays, respectively. By making synonymous substitutions within the repetitive region of RPGR, we were able to stabilize the plasmid and subsequently generate a clinical-grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull-down experiments, we were able to show that IA-RPGR protein product retained normal protein binding properties (i.e., analysis revealed normal binding to PDE6D, INPP5E, and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an Investigational New Drug application.

PMID: 31106594 [PubMed - indexed for MEDLINE]

Choriocapillaris Degeneration in Geographic Atrophy.

Tue, 2020-03-17 15:20
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Choriocapillaris Degeneration in Geographic Atrophy.

Am J Pathol. 2019 07;189(7):1473-1480

Authors: Sohn EH, Flamme-Wiese MJ, Whitmore SS, Workalemahu G, Marneros AG, Boese EA, Kwon YH, Wang K, Abramoff MD, Tucker BA, Stone EM, Mullins RF

Abstract
Early age-related macular degeneration (AMD) is characterized by degeneration of the choriocapillaris, the vascular supply of retinal photoreceptor cells. We assessed vascular loss during disease progression in the choriocapillaris and larger vessels in the deeper choroid. Human donor maculae from controls (n = 99), early AMD (n = 35), or clinically diagnosed with geographic atrophy (GA; n = 9, collected from outside the zone of retinal pigment epithelium degeneration) were evaluated using Ulex europaeus agglutinin-I labeling to discriminate between vessels with intact endothelial cells and ghost vessels. Morphometric analyses of choriocapillaris density (cross-sectional area of capillary lumens divided by length) and of vascular lumen/stroma ratio in the outer choroid were performed. Choriocapillaris loss was observed in early AMD (Bonferroni-corrected P = 0.024) with greater loss in GA (Bonferroni-corrected P < 10-9), even in areas of intact retinal pigment epithelium. In contrast, changes in lumen/stroma ratio in the outer choroid were not found to differ between controls and AMD or GA eyes (P > 0.05), suggesting choriocapillaris changes are more prevalent in AMD than those in the outer choroid. In addition, vascular endothelial growth factor-A levels were negatively correlated with choriocapillaris vascular density. These findings support the concept that choroidal vascular degeneration, predominantly in the microvasculature, contributes to dry AMD progression. Addressing capillary loss in AMD remains an important translational target.

PMID: 31051169 [PubMed - indexed for MEDLINE]

APOPTOSIS AND ANGIOFIBROSIS IN DIABETIC TRACTIONAL MEMBRANES AFTER VASCULAR ENDOTHELIAL GROWTH FACTOR INHIBITION: Results of a Prospective Trial. Report No. 2.

Tue, 2020-03-03 09:53
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APOPTOSIS AND ANGIOFIBROSIS IN DIABETIC TRACTIONAL MEMBRANES AFTER VASCULAR ENDOTHELIAL GROWTH FACTOR INHIBITION: Results of a Prospective Trial. Report No. 2.

Retina. 2019 Feb;39(2):265-273

Authors: Jiao C, Eliott D, Spee C, He S, Wang K, Mullins RF, Hinton DR, Sohn EH

Abstract
PURPOSE: We sought to characterize the angiofibrotic and apoptotic effects of vascular endothelial growth factor (VEGF)-inhibition on fibrovascular epiretinal membranes in eyes with traction retinal detachment because of proliferative diabetic retinopathy.
METHODS: Membranes were excised from 20 eyes of 19 patients (10 randomized to intravitreal bevacizumab, 10 controls) at vitrectomy. Membranes were stained with antibodies targeting connective tissue growth factor (CTGF) or VEGF and colabeled with antibodies directed against endothelial cells (CD31), myofibroblasts, or retinal pigment epithelium markers. Quantitative and colocalization analyses of antibody labeling were obtained through immunofluorescence confocal microscopy. Masson trichrome staining, cell counting of hematoxylin and eosin sections, and terminal dUTP nick-end labeling staining were performed.
RESULTS: High levels of fibrosis were observed in both groups. Cell apoptosis was higher (P = 0.05) in bevacizumab-treated membranes compared with controls. The bevacizumab group had a nonsignificant reduction in colocalization in CD31-CTGF and cytokeratin-VEGF studies compared with controls. Vascular endothelial growth factor in extracted membranes was positively correlated with vitreous levels of VEGF; CTGF in extracted membranes was negatively correlated with vitreous levels of CTGF.
CONCLUSION: Bevacizumab suppresses vitreous VEGF levels, but does not significantly alter VEGF or CTGF in diabetic membranes that may be explained by high baseline levels of fibrosis. Bevacizumab may cause apoptosis within fibrovascular membranes.

PMID: 29190236 [PubMed - indexed for MEDLINE]

Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration.

Tue, 2020-02-25 07:16
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Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration.

Cell Rep. 2020 Feb 18;30(7):2209-2224.e5

Authors: Mulfaul K, Ozaki E, Fernando N, Brennan K, Chirco KR, Connolly E, Greene C, Maminishkis A, Salomon RG, Linetsky M, Natoli R, Mullins RF, Campbell M, Doyle SL

Abstract
Retinal degeneration is a form of neurodegenerative disease and is the leading cause of vision loss globally. The Toll-like receptors (TLRs) are primary components of the innate immune system involved in signal transduction. Here we show that TLR2 induces complement factors C3 and CFB, the common and rate-limiting factors of the alternative pathway in both retinal pigment epithelial (RPE) cells and mononuclear phagocytes. Neutralization of TLR2 reduces opsonizing fragments of C3 in the outer retina and protects photoreceptor neurons from oxidative stress-induced degeneration. TLR2 deficiency also preserves tight junction expression and promotes RPE resistance to fragmentation. Finally, oxidative stress-induced formation of the terminal complement membrane attack complex and Iba1+ cell infiltration are strikingly inhibited in the TLR2-deficient retina. Our data directly implicate TLR2 as a mediator of retinal degeneration in response to oxidative stress and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology.

PMID: 32075760 [PubMed - in process]

Single-Cell RNA Sequencing in Human Retinal Degeneration Reveals Distinct Glial Cell Populations.

Tue, 2020-02-25 07:16
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Single-Cell RNA Sequencing in Human Retinal Degeneration Reveals Distinct Glial Cell Populations.

Cells. 2020 Feb 13;9(2):

Authors: Voigt AP, Binkley E, Flamme-Wiese MJ, Zeng S, DeLuca AP, Scheetz TE, Tucker BA, Mullins RF, Stone EM

Abstract
Degenerative diseases affecting retinal photoreceptor cells have numerous etiologies and clinical presentations. We clinically and molecularly studied the retina of a 70-year-old patient with retinal degeneration attributed to autoimmune retinopathy. The patient was followed for 19 years for progressive peripheral visual field loss and pigmentary changes. Single-cell RNA sequencing was performed on foveal and peripheral retina from this patient and four control patients, and cell-specific gene expression differences were identified between healthy and degenerating retina. Distinct populations of glial cells, including astrocytes and Müller cells, were identified in the tissue from the retinal degeneration patient. The glial cell populations demonstrated an expression profile consistent with reactive gliosis. This report provides evidence that glial cells have a distinct transcriptome in the setting of human retinal degeneration and represents a complementary clinical and molecular investigation of a case of progressive retinal disease.

PMID: 32069977 [PubMed - in process]

Molecular characterization of foveal versus peripheral human retina by single-cell RNA sequencing.

Tue, 2020-02-18 07:10
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Molecular characterization of foveal versus peripheral human retina by single-cell RNA sequencing.

Exp Eye Res. 2019 07;184:234-242

Authors: Voigt AP, Whitmore SS, Flamme-Wiese MJ, Riker MJ, Wiley LA, Tucker BA, Stone EM, Mullins RF, Scheetz TE

Abstract
The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells.

PMID: 31075224 [PubMed - indexed for MEDLINE]

Autologous cell replacement: a noninvasive AI approach to clinical release testing.

Mon, 2020-01-27 23:38
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Autologous cell replacement: a noninvasive AI approach to clinical release testing.

J Clin Invest. 2020 Jan 21;:

Authors: Tucker BA, Mullins RF, Stone EM

Abstract
The advent of human induced pluripotent stem cells (iPSCs) provided a means for avoiding ethical concerns associated with the use of cells isolated from human embryos. The number of labs now using iPSCs to generate photoreceptor, retinal pigmented epithelial (RPE), and more recently choroidal endothelial cells has grown exponentially. However, for autologous cell replacement to be effective, manufacturing strategies will need to change. Many tasks carried out by hand will need simplifying and automating. In this issue of the JCI, Schaub and colleagues combined quantitative brightfield microscopy and artificial intelligence (deep neural networks and traditional machine learning) to noninvasively monitor iPSC-derived graft maturation, predict donor cell identity, and evaluate graft function prior to transplantation. This approach allowed the authors to preemptively identify and remove abnormal grafts. Notably, the method is (a) transferable, (b) cost- and time effective, (c) high throughput, and (d) useful for primary product validation.

PMID: 31961338 [PubMed - as supplied by publisher]

The ARMS2 A69S Polymorphism Is Associated with Delayed Rod-Mediated Dark Adaptation in Eyes at Risk for Incident Age-Related Macular Degeneration.

Mon, 2020-01-06 20:09
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The ARMS2 A69S Polymorphism Is Associated with Delayed Rod-Mediated Dark Adaptation in Eyes at Risk for Incident Age-Related Macular Degeneration.

Ophthalmology. 2019 04;126(4):591-600

Authors: Mullins RF, McGwin G, Searcey K, Clark ME, Kennedy EL, Curcio CA, Stone EM, Owsley C

Abstract
PURPOSE: To examine the association between sequence variants in genetic risk factors for age-related macular degeneration (AMD) and delayed rod-mediated dark adaptation (RMDA), the first functional biomarker for incident AMD, in older adults with normal macular health and early AMD.
DESIGN: Cross-sectional.
PARTICIPANTS: Adults 60 years of age or older showing normal macular health (defined as both eyes at step 1 on the Age-Related Eye Disease Study 9-step AMD classification system) and those with AMD in one or both eyes (defined as steps 2-9).
METHODS: Single nucleotide polymorphisms were genotyped in the complement factor H (CFH) and ARMS2 genes using a Taqman assay. Rod-mediated dark adaptation was assessed in 1 eye after photobleach with targets centered at 5° on the inferior vertical meridian. Rate of dark adaptation was defined by rod intercept time (RIT), duration (in minutes) required for sensitivity to reach a criterion sensitivity level in the latter half of the second component of rod recovery. Associations between CFH and ARMS2 polymorphisms and RMDA were adjusted for age and smoking.
MAIN OUTCOME MEASURE: Rod intercept time.
RESULTS: The sample consisted of 543 participants having both genotype and RIT determination; 408 showed normal macular health and 135 demonstrated AMD, most having early AMD (124 of 135). For the combined sample, higher RIT (slower RMDA) was observed for both the A69S variant in ARMS2 and the Y402H variant in CFH (adjusted P = 0.0001 and P = 0.0023, respectively). For healthy participants, the A69S variant in ARMS2 was associated with higher RIT (adjusted P = 0.0011), whereas the Y402H variant in CFH was not (adjusted P = 0.2175). For AMD patients, the A69S variant of ARMS2 and the Y402H variant of CFH were associated with higher RIT (adjusted P = 0.0182 and P = 0.0222, respectively). Those with a larger number of high-risk ARMS2 and CFH alleles showed higher RIT, in both healthy and AMD groups (adjusted P = 0.0002 and P < 0.0001, respectively).
CONCLUSIONS: We report a novel association wherein older adults with high-risk ARMS2 and CFH genotypes are more likely to demonstrate delayed RMDA, the first functional biomarker for incident early AMD. Before the AMD clinical phenotype is present, those showing normal macular health with the ARMS2 A69S allele demonstrate delayed RMDA. Understanding ARMS2 function is a research priority.

PMID: 30389424 [PubMed - indexed for MEDLINE]

Wide-Field Swept-Source OCT and Angiography in X-Linked Retinoschisis.

Mon, 2019-12-23 18:06
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Wide-Field Swept-Source OCT and Angiography in X-Linked Retinoschisis.

Ophthalmol Retina. 2019 02;3(2):178-185

Authors: Han IC, Whitmore SS, Critser DB, Lee SY, DeLuca AP, Daggett HT, Affatigato LM, Mullins RF, Tucker BA, Drack AV, Stone EM

Abstract
PURPOSE: Retinal vascular and structural changes, particularly outside of the central macula, are not well characterized in X-linked retinoschisis (XLRS). We aim to describe wide-field swept-source OCT (SS-OCT) and swept-source OCT angiography (SS-OCTA) findings in XLRS.
DESIGN: Retrospective, cross-sectional study at a tertiary referral center.
PARTICIPANTS: Nine consecutive male patients with molecularly confirmed XLRS.
METHODS: All patients underwent complete ophthalmic examination with multimodal imaging, including SS-OCT with SS-OCTA (PLEX Elite 9000; Carl-Zeiss Meditec Inc., Dublin, CA). Images were then reviewed by 2 retinal specialists as independent graders to determine the frequency and distribution of retinal structural and vascular abnormalities.
MAIN OUTCOME MEASURES: Structural and vascular abnormalities seen on SS-OCT and SS-OCTA in patients with XLRS, with attention to the retinal layers involved, the regional distribution of schitic spaces in the posterior pole, and vascular abnormalities within the superficial and deep capillary plexuses.
RESULTS: Eighteen eyes from 9 male patients (mean age, 20 years; range 9-40) with molecularly confirmed XLRS were included. Median best-corrected visual acuity measured 20/63 (range, 20/25-10/300). A total of 17 of 18 eyes (94.4%) were noted to have schitic spaces on SS-OCT, and these were observed to be predominantly within the inner nuclear layer in all 17 eyes. A regional variation in the distribution of cysts was noted, with schitic spaces within the ganglion cell layer (13/17 eyes; 76.5%) observed to be perifoveal and those within the outer nuclear layer (8/17 eyes, 47.1%) observed to be mostly extramacular. All eyes had vascular abnormalities on SS-OCTA, including an irregular foveal avascular zone and flow loss within the deep capillary plexus corresponding to the distribution of the schisis.
CONCLUSIONS: Wide-field SS-OCT and SS-OCTA provide detailed visualization of structural and vascular changes in XLRS and may be helpful for monitoring disease progression or treatment response in clinical trials for the disease.

PMID: 31014769 [PubMed - indexed for MEDLINE]

Subretinal pseudocysts: A novel OCT finding in diabetic macular edema.

Mon, 2019-12-09 14:27
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Subretinal pseudocysts: A novel OCT finding in diabetic macular edema.

Am J Ophthalmol Case Rep. 2019 Dec;16:100567

Authors: Sacconi R, Lutty GA, Mullins RF, Borrelli E, Bandello F, Querques G

Abstract
Purpose: to report the presence of a new structural optical coherence tomography (OCT) finding, namely subretinal pseudocysts, in a patient affected by diabetic retinopathy (DR).
Observations: A 52-year-old man affected by type 2 diabetes from 10 years was referred to our department complaining of a visual decline in both eyes. Best corrected visual acuity was 20/100 and 20/80 in the right and left eye, respectively. Fundus examination, fluorescein angiography, and structural OCT revealed the presence of a proliferative DR with diabetic macular edema in both eyes. Interestingly, structural OCT showed subretinal pseudocystic spaces inside the subretinal fluid of the macular neuroretinal detachment.
Conclusions and importance: Subretinal pseudocysts are a new structural OCT entity. We reported for the first time the evidence that pseudocysts may develop in the subretinal space in a case of diabetic macular edema.

PMID: 31788575 [PubMed]