Explore the vast range of titles available.

Retinal pigment epithelial (RPE) cells maintain the health and functional integrity of both photoreceptors and the choroidal vasculature. Loss of RPE differentiation has long been known to play a critical role in numerous retinal diseases, including inherited rod-cone degenerations, inherited macular degeneration, age-related macular degeneration, and proliferative vitreoretinopathy. Recent studies in post-mortem eyes have found upregulation of critical epithelial-mesenchymal transition (EMT) drivers such as TGF-β, Wnt, and Hippo. As RPE cells become less differentiated, they begin to exhibit the defining characteristics of mesenchymal cells, namely, the capacity to migrate and proliferate. A number of preclinical studies, including animal and cell culture experiments, also have shown that RPE cells undergo EMT. Taken together, these data suggest that RPE cells retain the reprogramming capacity to move along a continuum between polarized epithelial cells and mesenchymal cells. We propose that movement along this continuum toward a mesenchymal phenotype be defined as RPE Dysfunction. Potential mechanisms include impaired tight junctions, accumulation of misfolded proteins and dysregulation of several key pathways and molecules, such as TGF-β pathway, Wnt pathway, nicotinamide, microRNA 204/211 and extracellular vesicles. This review synthesizes the evidence implicating EMT of RPE cells in post-mortem eyes, animal studies, primary RPE, iPSC-RPE and ARPE-19 cell lines.

BackgroundAnti-GD2 monoclonal antibody immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. However, 40% of patients fail to respond or develop resistance to treatment, and the molecular mechanisms by which this occurs remain poorly understood. Tumor-derived small extracellular vesicles (sEVs) have emerged as critical regulators in modulating the response to immunotherapy. In this study, we investigated the role of neuroblastoma-derived sEVs in promoting resistance to the anti-GD2 monoclonal antibody dinutuximab. Moreover, to determine whether pharmacologic inhibition of sEV secretion sensitizes tumors to dinutuximab treatment, we combined dinutuximab with tipifarnib, a farnesyltransferase inhibitor that inhibits sEV secretion.MethodsWe investigated the role of neuroblastoma-derived sEVs in modulating the response to dinutuximab by utilizing the syngeneic 9464D-GD2 mouse model. The effect of neuroblastoma-derived sEVs in modulating the tumor microenvironment (TME) and host immune system were evaluated by RNA-sequencing and flow cytometry. Importantly, we used this mouse model to investigate the efficacy of tipifarnib in sensitizing neuroblastoma tumors to dinutuximab. The effect of tipifarnib on both the TME and host immune system were assessed by flow cytometry.ResultsWe demonstrated that neuroblastoma-derived sEVs significantly attenuated the efficacy of dinutuximab in vivo and modulated tumor immune cell infiltration upon dinutuximab treatment to create an immunosuppressive TME that contains more tumor-associated macrophages and fewer tumor-infiltrating NK cells. In addition, we demonstrated that neuroblastoma-derived sEVs suppress splenic NK cell maturation in vivo and dinutuximab-induced NK cell-mediated antibody-dependent cellular cytotoxicity in vitro. Importantly, tipifarnib drastically enhanced the efficacy of dinutuximab-mediated inhibition of tumor growth and prevented the immunosuppressive effects of neuroblastoma-derived sEVs in vivo.ConclusionsThese preclinical findings uncover a novel mechanism by which neuroblastoma-derived sEVs modulate the immune system to promote resistance to dinutuximab and suggest that tipifarnib-mediated inhibition of sEV secretion may serve as a viable treatment strategy to enhance the antitumor efficacy of anti-GD2 immunotherapy in high-risk neuroblastoma patients.

Neurodegenerative retinal diseases, such as glaucoma and diabetic retinopathy, involve a gradual loss of neurons in the retina as the disease progresses. Central nervous system neurons are not able to regenerate in mammals, therefore, an often sought after course of treatment for neuronal loss follows a neuroprotective or regenerative strategy. Neuroprotection is the process of preserving the structure and function of the neurons that have survived a harmful insult; while regenerative approaches aim to replace or rewire the neurons and synaptic connections that were lost, or induce regrowth of damaged axons or dendrites. In order to test the neuroprotective effectiveness or the regenerative capacity of a particular agent, a robust experimental model of retinal neuronal damage is essential. Zebrafish are being used more often in this type of study because their eye structure and development is well-conserved between zebrafish and mammals. Zebrafish are robust genetic tools and are relatively inexpensive to maintain. The large array of functional and behavioral tests available in zebrafish makes them an attractive model for neuroprotection studies. Some common insults used to model retinal disease and study neuroprotection in zebrafish include intense light, chemical toxicity and mechanical damage. This review covers the existing retinal neuroprotection and regeneration literature in the zebrafish and highlights their potential for future studies.

Background:Age-related macular degeneration (AMD) is the most common cause of vision loss in people above the age of 50, affecting approximately 10% of the population worldwide and the incidence is rising. Hyperreflective foci (HRF) are a major predictor of AMD progression. The purpose of this study was to use the sodium iodate mouse model to study HRF formation in retinal degeneration.Methods:Sodium iodate (NaIO3) treated rodents were studied to characterize HRF. 3-month-old male wild-type (WT) C57Bl/6J mice were injected with phosphate-buffered saline (PBS) or varying doses of NaIO3 (15–60 mg/kg). Optical Coherence Tomography (OCT) images were collected at baseline and several days post-NaIO3 injection. Retinal thicknesses were measured using Bioptigen software. Seven days post-injection, eyes were prepared for either transmission electron microscopy (TEM), Hematoxylin & Eosin (H&E), or immunofluorescence.Results:OCT imaging of the mice given higher doses of NaIO3 revealed HRF formation in the neural retina (n = 4). The amount of HRF correlated with the degree of retinal tissue loss. H&E and TEM imaging of the retinas seven days post-NaIO3 injection revealed several pigmented bodies in multiple layers of the retina (n = 3–5). Immunofluorescence revealed that some pigmented bodies were positive for macrophage markers and an epithelial-to-mesenchymal transition marker, while all were retinal pigment epithelium (RPE) 65-negative (n = 4).Conclusions:The data suggest that NaIO3 induces the formation of HRF in the outer retina and their abundance correlates with retinal tissue loss. The experiments in this study highlight NaIO3 as a clinically relevant model of intermediate AMD that can be used to study HRF formation and to discover new treatment targets.

Tight junctions (TJs) are dynamic cellular structures that are critical for compartmentalizing environments within tissues and regulating transport of small molecules, ions, and fluids. Phosphorylation-dependent binding of the transmembrane protein occludin to the structural organizing protein ZO-1 contributes to the regulation of barrier properties; however, the details of their interaction are controversial. Using small angle X-ray scattering (SAXS), NMR chemical shift perturbation, cross-saturation, in vitro binding, and site-directed mutagenesis experiments. we define the interface between the ZO-1 PDZ3-SH3-U5-GuK (PSG) and occludin coiled-coil (CC) domains. The interface is comprised of basic residues in PSG and an acidic region in CC. Complex formation is blocked by a peptide (REESEEYM) that corresponds to CC residues 468–475 and includes a previously uncharacterized phosphosite, with the phosphorylated version having a larger effect. Furthermore, mutation of E470 and E472 reduces cell border localization of occludin. Together, these results localize the interaction to an acidic region in CC and a predominantly basic helix V within the ZO-1 GuK domain. This model has important implications for the phosphorylation-dependent regulation of the occludin∶ZO-1 complex.

We have shown previously that expression of R345W‐Fibulin‐3 induces epithelial‐mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W‐Fibulin‐3 mutation are sufficient to induce EMT in recipient cells. ARPE‐19 cells were infected with luciferase‐tagged wild‐type (WT)‐ Fibulin‐3 or luciferase‐tagged R345W‐Fibulin‐3 (R345W) using lentiviruses. EVs were isolated from the media by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC‐MS/MS based proteomics. EV‐associated transforming growth factor beta 1 (TGFβ1) protein was measured by enzyme‐linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT‐ or R345W‐EVs. The role of EV‐bound TGFβ was determined by pre‐incubation of EVs with a pan‐TGFβ blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W‐ EVs, while EMT drivers were enriched. Additionally, R345W‐EVs had higher concentrations of TGFβ1 compared to control. Critically, treatment with R345W‐EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV‐increased cell migration was significantly inhibited by pre‐incubation of EVs with pan‐TGFβ‐neutralising antibody. In conclusion, the expression of R345W‐Fibulin‐3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFβ dependent manner. These results suggest that EV‐bound TGFβ plays a critical role in the induction of EMT in RPE cells.

Extracellular vesicles (EVs) are lipid bilayer particles released by virtually all cells, with prominent roles in both physiological and pathological processes. The size, number, and molecular composition of released EVs correlate to the cells of origin, modulated by the cell’s environment and pathologic state. The proteins, DNA, RNA, and protein cargo carried by EVs are protected by degradation, with a prominent role in targeted intercellular signaling. These properties make EVs salient targets as both carriers of biomarkers and potential therapeutic delivery vehicles. The majority of EV research has focused on blood, urine, saliva, and cerebrospinal fluid due to easy accessibility. EVs have also been identified and studied in all ocular biofluids, including the vitreous humor, the aqueous humor, and the tear film, and the study of EVs in ocular disease is a new, promising, and underexplored direction with unique challenges and considerations. This review covers recent advances in the diagnostic and therapeutic use of ocular EVs, with a focus on human applications and key preceding in vitro and in vivo animal studies. We also discuss future directions based on the study of EVs in other organ systems and disease sates.

Neuroblastoma is a highly metastatic cancer, and thus is one of the leading causes of cancer-related mortalities in pediatric patients. More than 50% of NB cases exhibit 17q21-ter partial chromosomal gain, which is independently associated with poor survival, suggesting the clinical importance of genes at this locus in NB. IGF2BP1 is one such proto-oncogene located at 17q locus, and was found to be upregulated in patients with metastatic NBs. Here, utilizing multiple immunocompetent mouse models, along with our newly developed highly metastatic NB cell line, we demonstrate the role of IGF2BP1 in promoting NB metastasis. Importantly, we show the significance of small extracellular vesicles (EVs) in NB progression, and determine the pro-metastatic function of IGF2BP1 by regulating the NB-EV-protein cargo. Through unbiased proteomic analysis of EVs, we discovered two novel targets (SEMA3A and SHMT2) of IGF2BP1, and reveal the mechanism of IGF2BP1 in NB metastasis. We demonstrate that IGF2BP1 directly binds and governs the expression of SEMA3A/SHMT2 in NB cells, thereby modulating their protein levels in NB-EVs. IGF2BP1-affected levels of SEMA3A and SHMT2 in the EVs, regulate the formation of pro-metastatic microenvironment at potential metastatic organs. Finally, higher levels of SEMA3A/SHMT2 proteins in the EVs derived from NB-PDX models indicate the clinical significance of the two proteins and IGF2BP1-SEMA3A/SHMT2 axis in NB metastasis.

To investigate the role of protein misfolding in retinal pigment epithelial (RPE) cell dysfunction, the effects of R345W-Fibulin-3 expression on RPE cell phenotype were studied. Primary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of vascular endothelial growth factor (VEGF), was measured by Enzyme-linked immunosorbent assay (ELISA). Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells, and conversion to an epithelial-mesenchymal transition (EMT) phenotype was assessed by a migration assay. Compared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345W-Fibulin-3. In cells expressing mutant Fibulin-3, VEGF secretion was attenuated basally but not in the apical direction, whereas Fibulin-3 secretion was reduced in both the apical and basal directions. Retinal pigment epithelial signature genes were downregulated and multiple genes associated with EMT were upregulated in the mutant group. Migration assays revealed a faster recovery rate in ARPE-19 cells overexpressing R345W-Fibulin-3 compared to WT. The results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells.

Bardet–Biedl syndrome (BBS), an autosomal recessive ciliopathy with pleiotropic effects, manifests as a spectrum of anomalies involving multiple genes and affects fewer than 3,000 individuals in the USA. Due to its rarity and phenotypic variability, early diagnosis of BBS poses a significant challenge. Therefore, we aim to shed light on the intrafamilial phenotypic variation of BBS resulting from a BBS1 variant by delineating the clinical presentation in two siblings.