Selective microRNA expression of exosomes from retinal pigment epithelial cells by oxidative stress.

The function of exosomal miRNAs (miRs) in retinal degeneration is largely unclear. We were aimed to investigate the functions of exosomes as well as their miRs derived from retinal pigment epithelial (RPE) cells following exposure to oxidative stress (OS). After the OS by lipopolysaccharide and rotenone on RPE cells, interleukin-1 beta (IL-1ß), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α) were upregulated, along with the decreased mitochondrial membrane potential and upregulated oxidative damage marker 8-OH-dG in RPE cells. RPE-derived exosomes were then isolated, identified, injected into the subretinal space in mice. After subretinal injection, RPE-exosomes after OS not only induced higher ROS level and apoptotic retinal cells, but also elevated IL-1ß, IL-6 alongside TNF-α expressions among retina/RPE/choroidal complex. Next, miRs inside the exosomes were sequenced by the next generation sequencing (NGS) technology. NGS revealed that certain miRs were abundant in exosomes, while others were selectively kept by RPE cells. Further, downregulated miRs, like miR-125b-5p, miR-125a-5p, alongside miR-128-3p, and upregulated miR, such as miR-7-5p were validated byRT-qPCR. Finally, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to find the possible target genes of those selective exosomal miRs. Our results proved that the RPE-derived exosomes after OS selectively express certain miRs, providing novel insights into the pathogenesis of age-related macular degeneration (AMD) in future.

TSPAN4-positive migrasome derived from retinal pigmented epithelium cells contributes to the development of proliferative vitreoretinopathy.

BACKGROUND: Proliferative vitreoretinopathy (PVR) is a blind-causing disease initiated by the activation of retinal pigmented epithelium (RPE) primarily induced by TGF-ß families. Migrasome is a recently discovered type of extracellular vesicle related to cell migration. RESULTS: Here, we used ex vivo, in vitro, and in vivo models, to investigate the characteristics and functions of migrasomes in RPE activation and PVR development. Results indicated that the migrasome marker tetraspanin-4 (TSPAN4) was abundantly expressed in human PVR-associated clinical samples. The ex vivo model PVR microenvironment is simulated by incubating brown Norway rat RPE eyecups with TGF-ß1. Electron microscope images showed the formation of migrasome-like vesicles during the activation of RPE. Further studies indicated TGF-ß1 increased the expression of TSPAN4 which results in migrasome production. Migrasomes can be internalized by RPE and increase the migration and proliferation ability of RPE. Moreover, TSPAN4-inhibited RPE cells are with reduced ability of initiating experimental PVR. Mechanically, TSPAN4 expression and migrasome production are induced through TGF-ß1/Smad2/3 signaling pathway. CONCLUSION: In conclusion, migrasomes can be produced by RPE under PVR microenvironment. Migrasomes play a pivotal role in RPE activation and PVR progression. Thus, targeting TSPAN4 or blocking migrasome formation might be a new therapeutic method against PVR.

PERFLUOROCARBON LIQUID-ASSISTED INVERTED INTERNAL LIMITING MEMBRANE FLAP TECHNIQUE VERSUS INTERNAL LIMITING MEMBRANE PEELING FOR HIGHLY MYOPIC MACULAR HOLE RETINAL DETACHMENT.

PURPOSE: To compare the efficacy of a modified perfluorocarbon liquid-assisted inverted internal limiting membrane (ILM) flap technique with the standard ILM peeling for the treatment of macular hole retinal detachment in highly myopic eyes. METHODS: This was a retrospective, consecutive, nonrandomized comparative study. Forty-two macular hole retinal detachment eyes of 42 patients were included into either a perfluorocarbon liquid-assisted inverted ILM flap technique group (n = 22, inverted group) or standard ILM removal group (n = 20, peeling group). Outcomes measured were macular hole closure, retinal reattachment, and best-corrected visual acuity at least 6 months after surgery. RESULTS: Macular hole closure was achieved in 20 eyes (90.9%) in the inverted group and in eight eyes (40%) in the peeling group (P < 0.01). Reattachment rates were 100% in the inverted group and 95% in the peeling group (P = 0.476). The mean best-corrected visual acuity improvement from baseline was 27.4 ± 19.9 Early Treatment Diabetic Retinopathy Study letters in the inverted group while the best-corrected visual acuity improvement was 13.6 ± 22.5 Early Treatment Diabetic Retinopathy Study letters in the peeling group (P = 0.044). CONCLUSION: The perfluorocarbon liquid-assisted inverted ILM flap technique was effective in sealing the macular hole, reattaching retina, and improving visual function postoperatively in highly myopic macular hole retinal detachment.

Minimal internal limiting membrane peeling with ILM flap technique for idiopathic macular holes: a preliminary study.

BACKGROUND: Internal limiting membrane (ILM) peeling increases the idiopathic macular hole (IMH) closure rate but causes the inner retina dimplings. This study is to introduce a method to minimally peel the ILM, and with the ILM flap to ensure the IMH closure. METHODS: Twelve consecutive IMH eyes were treated with the minimal ILM peeling with ILM flap technique. The ILM around the MH is peeled off in an annular shape with a width of approximately 200 to 300 µm. A tongue-shape ILM flap is created in the superior retina and the inferior margin of ILM is not peeled off. The ILM flap is then inverted to cover the MH, followed by fluid-air exchange and air or silicon tamponade. Spectral domain-optical coherence tomography (SD-OCT) and en face OCT for morphological assessment, best corrected visual acuity (BCVA) and multifocal electroretinogram (ERG) for functional evaluation were performed at baseline and at each postoperative follow-up. RESULTS: All the 12 eyes achieved macular hole closure on SD-OCT after surgery (100%). At baseline, the mean preoperative BCVA was 0.83 ± 0.33 and it improved to 0.39 ± 0.28 postoperatively (p <  0.001). En face OCT showed the inner retinal dimplings were localized only in superior ILM-free retinas (7 eyes). The mERG response density in the central (R1), para-central (R2), R1/R2 ring ratios were remarkably improved at the last follow-up (p = 0.001, p = 0.033, p = 0.018, respectively). CONCLUSIONS: The minimal ILM peeling with ILM flap technique can achieve favorable MH closure with less inner retinal dimplings and has promising visual recovery for IMH eyes.

Protective effects of microRNA-22-3p against retinal pigment epithelial inflammatory damage by targeting NLRP3 inflammasome.

NLRP3, as a crucial inflammasome component, plays important roles in age-related macular degeneration. Though some activators of NLRP3 have been studied, microRNAs (miRNAs) which potentially regulate NLRP3 messenger RNA (mRNA) have not been fully explored in retinal pigment epithelial (RPE) cells and retinopathy. In this study, by miRNA microarray profiling and bioinformatic analysis, we identified that four miRNAs, miR-4286, miR-223-3p, miR-365a, miR-22-3p, may target NLRP3 mRNA in RPE inflammatory damage in vivo. Further, real-time polymerase chain reaction verified that only miR-22-3p was significantly decreased, which was associated with NLRP3 upregulation in blue-light-induced retinopathy. Mechanistically, the dual-fluorescent reporter suggested miR-22-3p directly binds NLRP3 mRNA. Moreover, overexpression of miR-22-3p could significantly reduce whereas inhibition miR-22-3p could increase the mRNA and protein expressions of NLRP3, Caspase-1, and mature IL-1ß. Collectively, our results indicate that miR-22-3p plays a suppressive role in RPE damage by targeting NLRP3, which provides new insights into the future intervention to retinopathy.

Non-inverted pedicle internal limiting membrane transposition for large macular holes.

PURPOSE: The purpose of this study is to investigate the effectiveness of a new surgical technique of non-inverted pedicle internal limiting membrane (ILM) transposition for the treatment of eyes with large macular hole. METHODS: This is a retrospective, consecutive, interventional case series. Twelve eyes of 12 consecutive patients who underwent vitrectomy for the treatment of a large macular hole (MH size > 400 µm) were treated. ILM was peeled and left with a pedicle attached to the superior temporal retina. The macular hole was covered by transposition of the pedicle ILM in a non-inverted way. Preoperative and postoperative best-corrected visual acuity (BCVA), SD-OCT image, macular sensitivity by microperimetry, and multifocal electroretinogram (mERG) response were evaluated. All of the patients were followed for more than 3 months. RESULTS: Postoperative OCT examination confirmed 11 of 12 macular hole closed (91.7%). Six macular hole filled with silicone oil closed as early as the next day. The postoperative BCVA significantly increased compared with preoperative BCVA (P = 0.002). The improvement of macular sensitivity within 2° and 8° circle was also statistical significant (P = 0.018 and P = 0.017, respectively). Fixation stability, shown as the percentage of fixation point within the 2° circle and 4° circle, was remarkably improved (P = 0.017 and P = 0.018, respectively). The R1/R2 and R1/R4 ring ratios also increased significantly as compared with that of baseline. CONCLUSION: These findings indicate that the non-inverted pedicle ILM transposition results in a high incidence of anatomic closure with good visual outcome for the treatment of large macular hole.