Schepens Eye Research Institute
  • Boston, United States
Recent publications
Ocular retinoblastoma malignancies, which develop into metastatic phenotypes, result in poor prognosis and survival for infant and child patients. To improve the prognosis of metastatic retinoblastoma, it is important to identify novel compounds with less toxic side effects and higher therapeutic efficacy compared to existing chemotherapeutics. Piperlongumine (PL), a neuroprotective, plant-derived compound has been explored for its anticancer activities both in vitro and in vivo. Here, we analyze the potential efficacy of PL for metastatic retinoblastoma cell treatment. Our data reveal that PL treatment significantly inhibits cell proliferation in metastatic retinoblastoma Y79 cells compared to the commonly used retinoblastoma chemotherapeutic drugs carboplatin, etoposide, and vincristine. PL treatment also significantly increases cell death compared to treatment with other chemotherapeutic drugs. PL-induced cell-death signaling was associated with significantly higher caspase 3/7 activities and greater loss of mitochondrial membrane potential. PL was also internalized into Y79 cells with an estimated concentration of 0.310pM and expression analysis revealed reduced MYCN oncogene levels. We next examined extracellular vesicles derived from PL-treated Y79 cells. Extracellular vesicles in other cancers are pro-oncogenic, mediating systemic toxicities via the encapsulation of chemotherapeutic drugs. Within metastatic Y79 EV samples, an estimated PL concentration of 0.026pM was detected. PL treatment significantly downregulated Y79 EV cargo of the oncogene MYCN transcript. Interestingly, non-PL-treated Y79 cells incubated with EVs from PL-treated cells exhibited significantly reduced cell growth. These findings indicate that in metastatic Y79 cells, PL exhibits potent anti-proliferation effects and oncogene downregulation. Importantly, PL is also incorporated into extracellular vesicles released from treated metastatic cells with measurable anti-cancer effects on target cells at a distance from the site of primary treatment. The use of PL in the treatment of metastatic retinoblastoma may reduce primary tumor proliferation and inhibit metastatic cancer activity systemically via extracellular vesicle circulation.
In the current literature, clinical registry cohorts related to ocular inflammation are few and far between, and there are none involving multi-continental international data. Many existing registries comprise administrative databases, data related to specific uveitic diseases, or are designed to address a particular clinical problem. The existing data, although useful and serving their intended purposes, are segmented and may not be sufficiently robust to design prognostication tools or draw epidemiological conclusions in the field of uveitis and ocular inflammation. To solve this, we have developed the Ocular Autoimmune Systemic Inflammatory Infectious Study (OASIS) Clinical Registry. OASIS collects prospective and retrospective data on patients with all types of ocular inflammatory conditions from centers all around the world. It is a primarily web-based platform with alternative offline modes of access. A comprehensive set of clinical data ranging from demographics, past medical history, clinical presentation, working diagnosis to visual outcomes are collected over a range of time points. Additionally, clinical images such as optical coherence tomography, fundus fluorescein angiography and indocyanine green angiography studies may be uploaded. Through the capturing of diverse, well-structured, and clinically meaningful data in a simplified and consistent fashion, OASIS will deliver a comprehensive and well organized data set ripe for data analysis. The applications of the registry are numerous, and include performing epidemiological analysis, monitoring drug side effects, and studying treatment safety efficacy. Furthermore, the data compiled in OASIS will be used to develop new classification and diagnostic systems, as well as treatment and prognostication guidelines for uveitis.
Purpose The objective of the study was to analyse the levels of IL-17 and IL-38 in the samples of unstimulated tears, orbital adipose tissues, and sera of patients diagnosed with active forms of TAO. The correlation of the levels of IL-17 and IL-38 with clinical activity score (CAS) was scrutinized. Methods A study was conducted at the Kazakhstan Scientific Research Institute of Eye Diseases (Almaty city, Kazakhstan). Study participants (n = 70) were sub-divided into 3 groups: (1) a group of patients diagnosed with active TAO (n = 25), (2) a group of patients with an inactive form of TAO (n = 28), and (3) a “control group” (patients diagnosed with orbital fat prolapse, n = 17). All patients underwent a clinical assessment and diagnostics. The activity of the disease and its severity were assessed using the CAS and NOSPECS scales. Thyroid function tests were performed, including the study of the levels of thyroid-stimulating hormone, triiodothyronine, free thyroxine, and antibodies to the thyroid-stimulating hormone receptor. IL-17 and IL-38 levels in non-stimulated tear samples, orbital tissue, and patients’ sera were measured using commercial ELISA kits. Results The results showed that the number of former smokers prevailed among patients with active TAO (48%) in comparison with patients with inactive TAO (15.4%), p = 0.001. The concentration of IL-17 significantly increased in the samples of non-stimulated tears, adipose tissues of the orbit and sera of patients with active forms of TAO. The level of IL-38 was reduced in all types of samples (p ≤ 0.05). The results of a histological study of orbital adipose tissues in the group of patients with an active form of TAO showed the presence of focal infiltration with lymphocytes, histiocytes, plasma cells, severe sclerosis and vascular plethora. We observed an association between the CAS of patients with active TAO and the level of IL-17 in sera (r = 0.885; p = 0.001). On the contrary, a negative correlation was detected for the level of IL-38 in sera. Conclusions The results highlighted the systemic effect of IL-17 and the local effect of IL-38 in TAO. We observed a significant increase in the production of IL-17, and a decrease in IL-38 in samples of sera and unstimulated tears (the active form of TAO). Our data indicate a correlation of IL-17 and IL-38 levels with the clinical activity of TAO.
Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies—an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9—to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM.
Epigenetic DNA modification by 5-hydroxymethylcytosine (5hmC), generated by the Ten-eleven translocation (TET) dioxygenases, regulates diverse biological functions in many organ tissues, including the mammalian eye. For example, 5hmC has been shown to be involved in epigenetic regulation of retinal gene expression. However, a functional role of 5hmC in corneal differentiation has not been investigated to date. Here, we examined 5hmC and TET function in the human cornea. We found 5hmC highly expressed in MUC16-positive terminally differentiated cells that also co-expressed the 5hmC-generating enzyme TET2. TET2 knockdown (KD) in cultured corneal epithelial cells led to significant reductions of 5hmC peak distributions and resulted in transcriptional repression of molecular pathways involved in corneal differentiation, as evidenced by downregulation of MUC4, MUC16, and Keratin 12. Additionally, integrated TET2 KD RNA-seq and genome-wide Reduced Representation Hydroxymethylation Profiling revealed novel epigenetically regulated genes expressed by terminally differentiated cells, including KRT78, MYEOV, and MAL. In aggregate, our findings reveal a novel function of TET2 in the epigenetic regulation of corneal epithelial gene expression and identify novel TET2-controlled genes expressed in differentiated corneal epithelial cells. These results point to potential roles for TET2 induction strategies to enhance treatment of corneal diseases associated with abnormal epithelial maturation.
Epithelial-mesenchymal transition (EMT) is a dedifferentiation program in which polarized, differentiated epithelial cells lose their cell-cell adhesions and transform into matrix-producing mesenchymal cells. EMT of retinal pigment epithelial (RPE) cells plays a crucial role in many retinal diseases, including age-related macular degeneration, proliferative vitreoretinopathy, and diabetic retinopathy. This dynamic process requires complex metabolic reprogramming to accommodate the demands of this dramatic cellular transformation. Both transforming growth factor-beta 2 (TGFβ2) and tumor necrosis factor-alpha (TNFα) have the capacity to induce EMT in RPE cells; however, little is known about their impact on the RPE metabolome. Untargeted metabolomics using high-resolution mass spectrometry was performed to reveal the metabolomic signatures of cellular and secreted metabolites of primary human fetal RPE cells treated with either TGFβ2 or TNFα for 5 days. A total of 638 metabolites were detected in both samples; 188 were annotated as primary metabolites. Metabolomics profiling showed distinct metabolomic signatures associated with TGFβ2 and TNFα treatment. Enrichment pathway network analysis revealed alterations in the pentose phosphate pathway, galactose metabolism, nucleotide and pyrimidine metabolism, purine metabolism, and arginine and proline metabolism in TNFα-treated cells compared to untreated control cells, whereas TGFβ2 treatment induced perturbations in fatty acid biosynthesis metabolism, the linoleic acid pathway, and the Notch signaling pathway. These results provide a broad metabolic understanding of the bioenergetic rewiring processes governing TGFβ2- and TNFα-dependent induction of EMT. Elucidating the contributions of TGFβ2 and TNFα and their mechanistic differences in promoting EMT of RPE will enable the identification of novel biomarkers for diagnosis, management, and tailored drug development for retinal fibrotic diseases.
Microglia are dynamic guardians of neural tissue and the resident immune cells of the central nervous system (CNS). The disease-associated microglial signature (DAM), also known as the microglial neurodegenerative phenotype (MGnD), has gained significant attention in recent years as a fundamental microglial response common to various neurodegenerative disease pathologies. Interestingly, this signature shares many features in common with developmental microglia, suggesting the existence of recycled gene programs which play a role both in early neural circuit formation as well as in response to aging and disease. In addition, recent advances in single cell RNA sequencing have revealed significant heterogeneity within the original DAM signature, with contributions from both yolk sac-derived microglia as well as bone marrow-derived macrophages. In this review, we examine the role of the DAM signature in retinal development and disease, highlighting crosstalk between resident microglia and infiltrating monocytes which may critically contribute to the underlying mechanisms of age-related neurodegeneration.
Gene therapy is poised to revolutionize modern medicine, with seemingly unlimited potential for treating and curing genetic disorders. For otherwise incurable indications, including most inherited metabolic liver disorders, gene therapy provides a realistic therapeutic option. In this Review, we discuss gene supplementation and gene editing involving the use of recombinant adeno-associated virus (rAAV) vectors for the treatment of inherited liver diseases, including updates on several ongoing clinical trials that are producing promising results. Clinical testing has been essential in highlighting many key translational challenges associated with this transformative therapy. In particular, the interaction of a patient's immune system with the vector raises issues of safety and the duration of treatment efficacy. Furthermore, several serious adverse events after the administration of high doses of rAAVs suggest greater involvement of innate immune responses and pre-existing hepatic conditions than initially anticipated. Finally, permanent modification of the host genome associated with rAAV genome integration and gene editing raises concerns about the risk of oncogenicity that require careful evaluation. We summarize the main progress, challenges and pathways forward for gene therapy for liver diseases.
Epithelial mesenchymal transition (EMT) plays a vital role in a variety of human diseases including proliferative vitreoretinopathy (PVR), in which retinal pigment epithelial (RPE) cells play a key part. Transcriptomic analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was up-regulated in human RPE cells upon treatment with transforming growth factor (TGF)-β2, a multifunctional cytokine associated with clinical PVR. Stimulation of human RPE cells with TGF-β2 induced expression of p110δ (the catalytic subunit of PI3Kδ) and activation of NFκB/p65. CRISPR-Cas9-mediated depletion of p110δ or NFκB/p65 suppressed TGF-β2-induced fibronectin expression and activation of Akt as well as migration of these cells. Intriguingly, abrogating expression of NFκB/p65 also blocked TGF-β2-induced expression of p110δ, and luciferase reporter assay indicated that TGF-β2 induced NFκB/p65 binding to the promoter of the PIK3CD that encodes p110δ. These data reveal that NFκB/p65-mediated expression of PI3Kδ is essential in human RPE cells for TGF-β2-induced EMT, uncovering hindrance of TGF-β2-induced expression of p110δ as a novel approach to inhibit PVR.
Objective: The narrative description (ND) test objectively measures the ability to understand and describe visual scenes. As subtle differences in speech occur early in cognitive decline, we analyzed linguistic features for their utility in detecting cognitive impairment and predicting downstream decline. Method: Participants (n = 52) with normal cognition to mild dementia performed the ND test (watched twenty 30-s video clips and described the visual content). Cognitive function was followed for up to 5 years. We computed simple linguistic features such as content efficiency, speech rate, and part of speech and unique word counts. We examined (a) relationships between cognitive status and ND score and linguistic features; (b) ability to discriminate early cognitive impairment from normal cognition using ND score and linguistic features; and (c) whether ND score and linguistic features were associated with future cognitive functional decline. Results: Many of the linguistic-feature metrics were related to cognitive status. Many of the linguistic features could distinguish between the cognitively normal group and the mild cognitive impairment (MCI) and Dementia groups. The area under the curve (AUC) for ND score alone was 0.74, with a nonsignificant increase to 0.78 when adding mean word length. Among participants with subjective cognitive decline (SCD) at the first visit, a smaller number of words plus more interjections or a lower ND score at baseline were predictive of future cognitive decline. Conclusions: While many linguistic features were associated with cognitive status, and some were able to detect early cognitive impairment or predictive of future cognitive decline, all the features we tested seem to have been captured by the ND score. Thus, adding linguistic measures to the ND test score did not add to its value in assessing current or predicting future cognitive status. (PsycInfo Database Record (c) 2022 APA, all rights reserved).
Nerve damage following injury has been associated with impaired wound healing. We have previously shown that corneal injury leads to increased activation of ocular surface mast cells. Here, we investigated whether mast cells interact with corneal nerves and contribute to nerve degeneration and neuroinflammation by utilizing trigeminal ganglion-mast cells (TG-MCs) co-culture system and a murine model of corneal injury. Corneas, harvested 6 hours post-injury and immunostained with β-tubulin III and avidin, demonstrated that ocular surface mast cells infiltrate into the injured cornea in close proximity to the damaged corneal nerves. Moreover, the corneal injury resulted in significant activation of TG mast cells and inflammation, as indicated by increased levels of tryptase, CD11b, and SubP (p < 0.01) in the lysates of ipsilateral trigeminal ganglions harvested 6 hours post-injury compared to those from naïve mice. Furthermore, our in vitro data show that mast cells significantly promote neurite degeneration, as indicated by shorter lengths of neurites (p < 0.001) and upregulation of nerve activation markers SubP and CGRP by TGs (p < 0.01) co-cultured with MCs, compared to TGs cultured alone. Lastly, pharmacological inhibition of ocular surface mast cell activation using cromolyn resulted in a significant decline in injury-induced hyperalgesia, as shown by decreased eye wipes in cromolyn-treated groups compared to the control group (p = 0.005). The current investigation on the neuro-immune axis of mast cells and corneal nerves provides novel insights into the function of ocular surface mast cells in exacerbating nerve degeneration and promoting inflammation in the trigeminal ganglion.
Purpose: The aim of the study is to investigate US-based ophthalmologists' preferred corneal transplant techniques and postoperative steroid regimen. Methods: Ophthalmologists attending the 2021 Cornea and Eye Banking Forum and/or Cornea Subspecialty Day were surveyed in person. Results: Ninety-two ophthalmologists with a median of 13 years (range of 1-35; mean of 14.5; ±9.05 mean ± SD) of experience as attending clinicians were surveyed. One hundred percent of the surgeons performed penetrating keratoplasty, which was followed by 96.7% for Descemet stripping endothelial keratoplasty, 90.2% for Descemet membrane endothelial keratoplasty, and 72.8% for deep anterior lamellar keratoplasty. Prednisolone 1% for postoperative care was the preferred choice across all surveyed keratoplasty techniques and postsurgery time intervals. All surgeons reported steroid administration frequency of 4 times a day in the first month and once a day after 12 months postkeratoplasty. To manage ocular hypertension after corneal transplantation, the leading approach was adding a glaucoma medication (44.6%), and beta-adrenergic antagonists were ranked as the most preferred choice by 59 (66.3%) of the respondents. For graft rejection after corneal transplantation, topical steroids (79.8%) were the initial treatment of choice with hourly administration being the most common frequency prescribed (87.4%). Most surgeons either agreed or strongly agreed (78.4%) that a randomized clinical trial evaluating the safety and efficacy of different steroid regimens after corneal transplantation would influence their clinical decision making. Conclusions: Prednisolone remains the predominantly used steroid across different keratoplasties. Steroid regimens are similar for non-high-risk penetrating keratoplasty, Descemet membrane endothelial keratoplasty, Descemet stripping endothelial keratoplasty, and deep anterior lamellar keratoplasty. To treat graft rejection, surgeons tend to initially add a glaucoma medication than to reduce the potency or frequency of the steroid.
Abnormal angiogenesis is associated with myriad human diseases including proliferative diabetic retinopathy. Signaling transduction via phosphoinositide 3-kinases (PI3Ks) plays a critical role in angiogenesis. Herein, we showed that p110δ, the catalytic subunit of PI3Kδ, was highly expressed in pathological retinal vascular endothelial cells (ECs) in a mouse model of oxygen-induced retinopathy (OIR) and in fibrovascular membranes from patients with proliferative diabetic retinopathy. To explore novel intervention with PI3Kδ expression, we developed a recombinant dual adeno-associated viral (rAAV) system for delivering CRISPR/Cas9 in which Streptococcus pyogenes (Sp) Cas9 expression was driven by an endothelial specific promoter of intercellular adhesion molecule 2 (pICAM2) to edit genomic Pik3cd, the gene encoding p110δ. We then demonstrated that infection of cultured mouse vascular endothelial cells with the dual rAAV1s of rAAV1-pICAM2-SpCas9 and rAAV1-SpGuide targeting genomic Pik3cd resulted in 80% DNA insertion/deletion in the locus of genomic Pik3cd and 70% depletion of p110δ expression. Furthermore, we showed that in the mouse model of OIR editing retinal Pik3cd with the dual rAAV1s resulted in not only a significant decrease in p110δ expression, and Akt activation, but also a dramatic reduction in pathological retinal angiogenesis. These findings reveal that Pik3cd editing is a novel approach to treating abnormal retinal angiogenesis.
Aging causes an irreversible, cumulative decline in neuronal function. Using the visual system as a model, we show that astrocytes play a critical role in maintaining retinal ganglion cell health and that deletion of SPP1 (secreted phosphoprotein 1, or osteopontin) from astrocytes leads to increased vulnerability of ganglion cells to age, elevated intraocular pressure, and traumatic optic nerve damage. Overexpression of SPP1 slows the age-related decline in ganglion cell numbers and is highly protective of visual function in a mouse model of glaucoma. SPP1 acts by promoting phagocytosis and secretion of neurotrophic factors while inhibiting production of neurotoxic and pro-inflammatory factors. SPP1 up-regulates transcription of genes related to oxidative phosphorylation, functionally enhances mitochondrial respiration, and promotes the integrity of mitochondrial microstructure. SPP1 increases intracellular ATP concentration via up-regulation of VDAC1.
Background/aims We previously used archetypal analysis (AA) to create a model that quantified patterns (archetypes (ATs)) of visual field (VF) loss that can predict recovery and reveal residual VF deficits from eyes in the Optic Neuritis Treatment Trial (ONTT). We hypothesised that AA could produce similar results for ON VFs collected in clinical practice. Methods We applied AA to a retrospective data set of 486 VFs collected in 1 neuro-ophthalmology service from 141 eyes with acute ON and typical VF defects, to create a clinic-derived AT model. We also used the ONTT-derived AT model to analyse this new dataset. We compared the findings of both models by decomposing VFs into component ATs of varying per cent weight (PW), correlating presentation AT PW with mean deviation (MD) at final visits for each eye and identifying residual deficits in VFs considered normal. Results Both models, each with 16 ATs, decomposed each presentation VF into 0–6 abnormal ATs representative of known patterns of ON-related VF loss. AT1, the normal pattern in both models, correlated strongly with MD for VFs collected at presentation (r=0.82; p<0.001) and the final visit (r=0.81, p<0.001). The presentation AT1 PW was associated with improvement in MD over time. 67% of VFs considered ‘normal’ at final visit had 1.2±0.4 abnormal ATs, and both models revealed similar patterns of regional VF loss. Conclusions AA is a quantitative method to measure change and outcome of ON VFs. Presentation AT features are associated with MD at final visit. AA identifies residual VF deficits not otherwise indicated by MD.
Purpose Clinical registries are increasingly important in research and clinical advancement. This review explores and compares current uveitis registries and recommends future directions on how uveitis registries can complement one another for synergistic effect and benefit. Methods From a systematic search, 861 citations were screened for longitudinal, non-interventional, and multicenter uveitis-specific registries. Additional registries were identified via consultations with uveitis experts. Characteristics of all registries were analyzed and compared. Results Four registries were identified: Treatment Exit Options for Non-infectious Uveitis, AutoInflammatory Disease Alliance International Registry, Ocular Autoimmune Systemic Inflammatory Infectious Study, and Fight Uveitis Blindness!. Despite certain differences, these registries have the overarching goal of collecting large quantities of real-world, high-quality patient data to improve the understanding of uveitis. Conclusion The four uveitis registries share similar goals and collect clinical data from overlapping geographical regions. There is vast potential for collaboration, including data sharing to further augment datasets for analysis.
Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction—a finding potentially relevant to preventing hepatoxicities seen in clinical studies.
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82 members
Daisy Y Shu
  • Department of Ophthalmology
Joan stein-streilein
  • ophthalmology/ Immunology
Yin-Shan Ng
  • Vascular Biology
Petr Baranov
  • Department of Ophthalmology
Boston, United States