Proteome Landscape of Epithelial-to-Mesenchymal Transition (EMT) of Retinal Pigment Epithelium Shares Commonalities With Malignancy-Associated EMT.

Stress and injury to the retinal pigment epithelium (RPE) often lead to dedifferentiation and epithelial-to-mesenchymal transition (EMT). These processes have been implicated in several retinal diseases, including proliferative vitreoretinopathy, diabetic retinopathy, and age-related macular degeneration. Despite the importance of RPE-EMT and the large body of data characterizing malignancy-related EMT, comprehensive proteomic studies to define the protein changes and pathways underlying RPE-EMT have not been reported. This study sought to investigate the temporal protein expression changes that occur in a human-induced pluripotent stem cell-based RPE-EMT model. We utilized multiplexed isobaric tandem mass tag labeling followed by high-resolution tandem MS for precise and in-depth quantification of the RPE-EMT proteome. We have identified and quantified 7937 protein groups in our tandem mass tag-based MS analysis. We observed a total of 532 proteins that are differentially regulated during RPE-EMT. Furthermore, we integrated our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into RPE-EMT mechanisms. To validate these results, we have performed a label-free single-shot data-independent acquisition MS study. Our integrated analysis indicates both the commonality and uniqueness of RPE-EMT compared with malignancy-associated EMT. Our comparative analysis also revealed that multiple age-related macular degeneration-associated risk factors are differentially regulated during RPE-EMT. Together, our integrated dataset provides a comprehensive RPE-EMT atlas and resource for understanding the molecular signaling events and associated biological pathways that underlie RPE-EMT onset. This resource has already facilitated the identification of chemical modulators that could inhibit RPE-EMT, and it will hopefully aid in ongoing efforts to develop EMT inhibition as an approach for the treatment of retinal disease.

Genetic mechanisms and age-related macular degeneration: common variants, rare variants, copy number variations, epigenetics, and mitochondrial genetics.

Age-related macular degeneration (AMD) is a complex and multifaceted disease involving contributions from both genetic and environmental influences. Previous work exploring the genetic contributions of AMD has implicated numerous genomic regions and a variety of candidate genes as modulators of AMD susceptibility. Nevertheless, much of this work has revolved around single-nucleotide polymorphisms (SNPs), and it is apparent that a significant portion of the heritability of AMD cannot be explained through these mechanisms. In this review, we consider the role of common variants, rare variants, copy number variations, epigenetics, microRNAs, and mitochondrial genetics in AMD. Copy number variations in regulators of complement activation genes (CFHR1 and CFHR3) and glutathione S transferase genes (GSTM1 and GSTT1) have been associated with AMD, and several additional loci have been identified as regions of potential interest but require further evaluation. MicroRNA dysregulation has been linked to the retinal pigment epithelium degeneration in geographic atrophy, ocular neovascularization, and oxidative stress, all of which are hallmarks in the pathogenesis of AMD. Certain mitochondrial DNA haplogroups and SNPs in mitochondrially encoded NADH dehydrogenase genes have also been associated with AMD. The role of these additional mechanisms remains only partly understood, but the importance of their further investigation is clear to elucidate more completely the genetic basis of AMD.

Epigenetics in ocular diseases.

Epigenetics pertains to heritable alterations in gene expression that do not involve modification of the underlying genomic DNA sequence. Historically, the study of epigenetic mechanisms has focused on DNA methylation and histone modifications, but the concept of epigenetics has been more recently extended to include microRNAs as well. Epigenetic patterning is modified by environmental exposures and may be a mechanistic link between environmental risk factors and the development of disease. Epigenetic dysregulation has been associated with a variety of human diseases, including cancer, neurological disorders, and autoimmune diseases. In this review, we consider the role of epigenetics in common ocular diseases, with a particular focus on DNA methylation and microRNAs. DNA methylation is a critical regulator of gene expression in the eye and is necessary for the proper development and postmitotic survival of retinal neurons. Aberrant methylation patterns have been associated with age-related macular degeneration, susceptibility to oxidative stress, cataract, pterygium, and retinoblastoma. Changes in histone modifications have also been observed in experimental models of diabetic retinopathy and glaucoma. The expression levels of specific microRNAs have also been found to be altered in the context of ocular inflammation, retinal degeneration, pathological angiogenesis, diabetic retinopathy, and ocular neoplasms. Although the complete spectrum of epigenetic modifications remains to be more fully explored, it is clear that epigenetic dysregulation is an important contributor to common ocular diseases and may be a relevant therapeutic target.

Chronic interleukin-13 expression in mouse olfactory mucosa results in regional aneuronal epithelium.

BACKGROUND: Olfactory dysfunction is highly associated with chronic rhinosinusitis with nasal polyps (CRSwNP), and the severity of loss has been linked with biomarkers of type 2 inflammation. The ability of dupilumab to rapidly improve the sense of smell prior to improvement in polyp size suggests a direct role of IL-4/IL-13 receptor signaling in the olfactory epithelium (OE). METHODS: We created a transgenic mouse model in which IL-13 is inducibly expressed specifically within the OE. Gene expression analysis and immunohistology were utilized to characterize the effect of IL-13 on the structure of the OE. RESULTS: After induction of olfactory IL-13 expression, there is a time-dependent loss of neurons from OE regions, accompanied by a modest inflammatory infiltrate. Horizontal basal cells undergo morphologic changes consistent with activation and demonstrate proliferation. Mucus production and increased expression of eotaxins is observed, with marked expression of Ym2 by sustentacular cells. DISCUSSION: Chronic IL-13 exposure has several effects on the OE that are likely to affect function. The neuronal loss is in keeping with other models of allergic type 2 nasal inflammation. Future studies are needed to correlate cellular and molecular alterations in olfactory cell populations with findings in human CRSwNP, as well as to assess olfactory function in behavioral model systems.

Republished review: Gene therapy for ocular diseases.

The eye is an easily accessible, highly compartmentalised and immune-privileged organ that offers unique advantages as a gene therapy target. Significant advancements have been made in understanding the genetic pathogenesis of ocular diseases, and gene replacement and gene silencing have been implicated as potentially efficacious therapies. Recent improvements have been made in the safety and specificity of vector-based ocular gene transfer methods. Proof-of-concept for vector-based gene therapies has also been established in several experimental models of human ocular diseases. After nearly two decades of ocular gene therapy research, preliminary successes are now being reported in phase 1 clinical trials for the treatment of Leber congenital amaurosis. This review describes current developments and future prospects for ocular gene therapy. Novel methods are being developed to enhance the performance and regulation of recombinant adeno-associated virus- and lentivirus-mediated ocular gene transfer. Gene therapy prospects have advanced for a variety of retinal disorders, including retinitis pigmentosa, retinoschisis, Stargardt disease and age-related macular degeneration. Advances have also been made using experimental models for non-retinal diseases, such as uveitis and glaucoma. These methodological advancements are critical for the implementation of additional gene-based therapies for human ocular diseases in the near future.

Laryngotracheal Mucosal Surface Expression of Candidate Biomarkers in Idiopathic Subglottic Stenosis.

OBJECTIVES: Idiopathic subglottic stenosis (iSGS) is an inflammatory process leading to fibrosis and narrowing of the laryngotracheal airway. There is variability in patient response to surgical intervention, but the mechanisms underlying this variability are unknown. In this pilot study, we measure expression of candidate targets at the mucosal surface of the subglottis in iSGS patients. We aim to identify putative biomarkers for iSGS that provide insights into the molecular basis of disease progression, yield a gene signature for the disease, and/or predict a response to therapy. STUDY DESIGN: In vitro comparative study of human cells. METHODS: Levels of candidate transcripts and proteins were measured in healthy and stenotic laryngotracheal tissue specimens taken from the mucosal surface in 16 iSGS patients undergoing endoscopic balloon dilation. Pre- and post-operative pulmonary function test and patient reported voice and breathing outcomes were also assessed. Unsupervised clustering was used to define patient subgroups based on expression profile. RESULTS: Pulmonary function and voice and breathing outcome metrics demonstrated significant post-operative improvement. Transcript levels of αSMA, CCL2, COL1A1, COL3A1, FN1, IFNG, and TGFB1 and protein levels of CCL2, IFNG, and IL-6 were significantly upregulated in stenotic as compared to healthy tissues. Marked heterogeneity was observed in the patterns of expression of candidate markers across individuals and tissue types. Patient subgroups defined by expression profile did not show a statistically significant difference in dilation interval. CONCLUSION: Pro-inflammatory and pro-fibrotic pathways are significantly upregulated along the mucosal surface of stenotic laryngotracheal tissues, and CCL2 and IFNG merit further investigation as potential iSGS biomarkers. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:342-349, 2021.

Transcriptome Landscape of Epithelial to Mesenchymal Transition of Human Stem Cell-Derived RPE.

Purpose: RPE injury often induces epithelial to mesenchymal transition (EMT). Although RPE-EMT has been implicated in a variety of retinal diseases, including proliferative vitroretinopathy, neovascular and atrophic AMD, and diabetic retinopathy, it is not well-understood at the molecular level. To contribute to our understanding of EMT in human RPE, we performed a time-course transcriptomic analysis of human stem cell-derived RPE (hRPE) monolayers induced to undergo EMT using 2 independent, yet complementary, model systems. Methods: EMT of human stem cell-derived RPE monolayers was induced by either enzymatic dissociation or modulation of TGF-ß signaling. Transcriptomic analysis of cells at different stages of EMT was performed by RNA-sequencing, and select findings were confirmed by reverse transcription quantitative PCR and immunostaining. An ingenuity pathway analysis (IPA) was performed to identify signaling pathways and regulatory networks associated with EMT. Results: Proteocollagenolytic enzymatic dissociation and cotreatment with TGF-ß and TNF-α both induce EMT in human stem cell-derived RPE monolayers, leading to an increased expression of mesenchymal factors and a decreased expression of RPE differentiation-associated factors. Ingenuity pathway analysis identified the upstream regulators of the RPE-EMT regulatory networks and identified master switches and nodes during RPE-EMT. Of particular interest was the identification of widespread dysregulation of axon guidance molecules during RPE-EMT progression. Conclusions: The temporal transcriptome profiles described here provide a comprehensive resource of the dynamic signaling events and the associated biological pathways that underlie RPE-EMT onset. The pathways defined by these studies may help to identify targets for the development of novel therapeutic targets for the treatment of retinal disease.

Detection of Age-Related Macular Degeneration by Portable Optical Coherence Tomography Operated by Nonexpert Personnel: Potential Use for Screenings.

PURPOSE: The purpose of this study is to determine the sensitivity and specificity of detecting age-related macular degeneration (AMD) using portable optical coherence tomography (OCT) operated by nonexpert photographers on undilated patients. METHODS: In this case-control study, 92 individuals were recruited from the glaucoma and retina clinics at the Wilmer Eye Institute (Johns Hopkins University, Baltimore, MD). Using the portable iVue (Optovue, Inc, Fremont, CA) spectral-domain OCT (SD-OCT), 2 nonexpert photographers acquired retina map scans on undilated eyes of all participants. In total, 33 AMD eyes and 105 control eyes were evaluated and graded by ophthalmologists masked to the diagnoses. RESULTS: Detection of AMD on the portable OCT by ophthalmologists exhibited sensitivities of 0.91 and 0.94 and specificities of 0.88 and 0.89, for graders 1 and 2, respectively. A strong intergrader agreement was observed (κ = 0.87). CONCLUSIONS: Nonexpert photographers can use a portable OCT on undilated eyes to acquire images for the detection of AMD. These findings present the potential utility of implementing a portable OCT in community screenings for earlier detection and treatment of disease.

Use of Optical Coherence Tomography by Nonexpert Personnel as a Screening Approach for Glaucoma.

PURPOSE: This pilot study was conducted to assess optical coherence tomography (OCT) as a screening tool for glaucoma when used by nonexpert personnel. METHODS: This prospective case-control study included 54 patients with open-angle glaucoma and 54 age-matched comparison individuals. Optovue iVue SD-OCT imaging was performed by nonprofessional photographers on undilated patients. The sensitivity, specificity, negative predictive value, and positive predictive value of iVue scan parameters for detecting open-angle glaucoma were evaluated. RESULTS: The iVue cup to disc vertical ratio had a sensitivity of 0.96 [95% confidence interval (CI), 0.90-1.00] at 90% specificity and was strongly correlated with both the Cirrus HD-OCT cup to disc vertical ratio (Pearson coefficient=0.84) and the cup to disc ratio observed on dilated clinical examination by faculty ophthalmologists (Pearson coefficient=0.80). The retinal nerve fiber layer (RNFL) parameters performed robustly, but the ganglion cell complex parameters showed limited diagnostic value. The inferior quadrant thickness was among the best performing RNFL parameters, with a sensitivity of 0.87 (95% CI, 0.78-0.96) and a specificity of 0.88 (95% CI, 0.80-0.97) using the iVue normative database thresholds for abnormality. CONCLUSIONS: OCT imaging may be performed by nonprofessional photographers on undilated patients, and quantitative parameters derived from the resultant images, particularly the vertical cup to disc ratio and the RNFL inferior quadrant thickness, demonstrate sensitivities and specificities that may be adequately robust for glaucoma screening in the community setting.

De novo assembly and annotation of the retinal transcriptome for the Nile grass rat (Arvicanthis ansorgei).

Cone photoreceptors are required for color vision and high acuity vision, and they die in a variety of retinal degenerations, leading to irreversible vision loss and reduced quality of life. To date, there are no approved therapies that promote the health and survival of cones. The development of novel treatments targeting cones has been challenging and impeded, in part, by the limitations inherent in using common rodent model organisms, which are nocturnal and rod-dominant, to study cone biology. The African Nile grass rat (Arvicanthis ansorgei), a diurnal animal whose photoreceptor population is more than 30% cones, offers significant potential as a model organism for the study of cone development, biology, and degeneration. However, a significant limitation in using the A. ansorgei retina for molecular studies is that A. ansorgei does not have a sequenced genome or transcriptome. Here we present the first de novo assembled and functionally annotated transcriptome for A. ansorgei. We performed RNA sequencing for A. ansorgei whole retina to a depth of 321 million pairs of reads and assembled 400,584 Trinity transcripts. Transcriptome-wide analyses and annotations suggest that our data set confers nearly full length coverage for the majority of retinal transcripts. Our high quality annotated transcriptome is publicly available, and we hope it will facilitate wider usage of A. ansorgei as a model organism for molecular studies of cone biology and retinal degeneration.

Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells.

Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. Stem Cells Translational Medicine 2017;6:1972-1986.

Comparison of time- and spectral-domain optical coherence tomography in management of diabetic macular edema.

PURPOSE: Some clinical trials that proved the benefits of anti-VEGF therapy for diabetic macular edema (DME) based retreatment decisions on visual acuity and time-domain ocular coherence tomography (TD-OCT) central subfield thickness changes since the last treatment. This study assessed the impact of TD-OCT followed by spectral domain (SD)-OCT on as needed treatment decision-making in the management of DME with anti-VEGF medications. METHODS: Patients previously treated for DME with anti-VEGF medications in the Retina Division of the Wilmer Eye Institute, following an institutional review board-approved informed consent process, underwent clinical examination, TD-, and SD-OCT imaging. Their retina specialists recorded whether additional anti-VEGF therapy was recommended and their level of certainty in the decision after performing a clinical examination and reviewing a TD-OCT, and then again after reviewing a SD-OCT. RESULTS: Data were collected for 129 treatment decision pairs involving 67 eyes from 46 subjects. Nonconcordant decisions occurred in 9 (7%) treatment decision pairs. In 7 of these (5%, 95% confidence interval [CI]: 2%-11%), the addition of SD-OCT changed the retina specialist's decision from not recommending to recommending retreatment. The addition of SD-OCT increased the certainty of the retina specialist in 36% (95% CI: 27%-45%) of all treatment decision pairs. CONCLUSIONS: Spectral-domain OCT does not appear to change the ultimate treatment decision or increase the level of certainty of the retina specialist relative to TD-OCT in most cases of DME under anti-VEGF management in clinical practice. The few nonconcordant decisions appear to trend toward recommending more anti-VEGF therapy following SD-OCT.

Gene therapy for ocular diseases.

The eye is an easily accessible, highly compartmentalised and immune-privileged organ that offers unique advantages as a gene therapy target. Significant advancements have been made in understanding the genetic pathogenesis of ocular diseases, and gene replacement and gene silencing have been implicated as potentially efficacious therapies. Recent improvements have been made in the safety and specificity of vector-based ocular gene transfer methods. Proof-of-concept for vector-based gene therapies has also been established in several experimental models of human ocular diseases. After nearly two decades of ocular gene therapy research, preliminary successes are now being reported in phase 1 clinical trials for the treatment of Leber congenital amaurosis. This review describes current developments and future prospects for ocular gene therapy. Novel methods are being developed to enhance the performance and regulation of recombinant adeno-associated virus- and lentivirus-mediated ocular gene transfer. Gene therapy prospects have advanced for a variety of retinal disorders, including retinitis pigmentosa, retinoschisis, Stargardt disease and age-related macular degeneration. Advances have also been made using experimental models for non-retinal diseases, such as uveitis and glaucoma. These methodological advancements are critical for the implementation of additional gene-based therapies for human ocular diseases in the near future.

The first Western-style hospital in China.

Peter Parker (1804-1888), a Yale University--trained missionary and physician, founded the first Western-style hospital in China, the Ophthalmic Hospital in Canton (now known as Guangzhou), on November 4, 1835. During its first 3 months, Parker treated 1061 patients, of whom 1020 (96.1%) had ocular illnesses. Since then, the Ophthalmic Hospital in Canton has become a comprehensive institution that is affiliated with Sun Yat-sen University and is one of the largest hospitals in China. In 1965, the Department of Ophthalmology, which originally employed only 2 ophthalmologists, expanded to become an ophthalmic hospital. In 1983, it joined the Eye Research Institute and the Office of Prevention of Blindness to form the Zhongshan Ophthalmic Center. The center currently employs nearly 800 staff members and provides care to more than 500 000 patients annually. The first Western-style hospital in China has survived and thrived; it is now one of the most prestigious ophthalmic institutes in the world.

Copy number variations in candidate genes in neovascular age-related macular degeneration.

PURPOSE: The pathogenesis of age-related macular degeneration (AMD) is strongly influenced by genetic factors, and single nucleotide polymorphisms have been consistently linked to AMD. Copy number variation (CNV), or variation in the number of copies of a particular segment of DNA, may also contribute to AMD pathogenesis. This study evaluated CNVs in candidate genes that have been reported to be linked to AMD. METHODS: Study participants were 131 patients with neovascular AMD and 103 elderly persons without AMD who were evaluated by retinal specialists at the National Eye Institute. DNA was collected from peripheral whole blood, and duplex RT-PCR based copy number (CN) assays were performed for the genes CCR3, CFH, CX3CR1, ERCC6, HTRA1, and VEGF. Quantitative CNs (CN = 0, 1, 2, or 3+) were determined. RESULTS: Novel CNVs were discovered in CCR3, CX3CR1, and ERCC6. The unadjusted data suggested that CN = 3+ for CX3CR1 might be mildly protective against AMD, but this trend did not persist after adjustment for age. AMD patients appeared to have an elevated mean CFH CN relative to controls (2.13 [95% confidence interval (CI), 2.05-2.21] vs. 2.01 [95% CI, 1.92-2.09 copies]; P = 0.05). No significant associations between CNV and AMD were observed for the remaining genes. CONCLUSIONS: The methods described are suitable for quantitative characterization of CNV in candidate genes. The authors identified CNVs in AMD-associated genes but did not find strong evidence for a link with neovascular AMD.

Ocular manifestations and pathology of adult T-cell leukemia/lymphoma associated with human T-lymphotropic virus type 1.

The human T-cell lymphotropic virus type 1 (HTLV-1), endemic in defined geographical areas around the world, is recognized as the etiologic agent of adult T-cell leukemia/lymphoma (ATL), or HTLV-1. ATL is a rare adult onset T-cell malignancy that is characterized by the presence of ATL flower cells with T-cell markers, HTLV-1 antibodies in the serum, and monoclonal integration of HTLV-1 provirus in affected cells. Ocular manifestations associated with HTLV-1 virus infection have been reported and include HTLV-1 uveitis and keratoconjunctivitis sicca, but reports of ocular involvement in ATL are exceedingly rare. This article describes the ocular manifestations and pathology of ATL. We also report for the first time a case of a 34-year-old male with systemic ATL and prominent atypical lymphoid cell infiltration in the choroid. To our knowledge, this is the first report defining prominent choroidal involvement as a distinct ocular manifestation of ATL. ATL may masquerade as a variety of other conditions, and molecular techniques involving microdissection and PCR have proven to be critical diagnostic tools. International collaboration will be needed to better understand the presentation and diagnosis of this rare malignancy.