A Pilot Application of an iTRAQ-Based Proteomics Screen Estimates the Effects of Cigarette Smokers' Serum on RPE Cells With AMD High-Risk Alleles.

PURPOSE: The aim of this study was to explore whether there are interactions between genetic (ARMS2/HTRA1) and environmental factors (cigarette smoking) in the pathogenesis of age-related macular degeneration (AMD). METHODS: Primary human retinal pigment epithelial (hRPE) cells were obtained from four donors' eyes with AMD high-risk ARMS2/HTRA1 alleles, and two donors' eyes with wild-type alleles were used as controls. The pooled serum from 32 smokers and 35 nonsmokers were collected and used separately to treat hRPE cells. The isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics was used to identify associated proteins and comparing the differences between AMD high-risk and low-risk HTRA1/ARMS2 alleles after exposure to smokers' serum. RESULTS: After stimulation with the smokers' serum, 400 differentially expressed proteins (DEPs) were detected in the high-risk allele cells. Several DEPs are involved in neuronal protein degeneration and oxidative stress pathways. The smokers' serum stimulation or HTRA1 overexpression can both upregulate caveolin-1, which was one of the DEPs. Besides, the smokers' serum enhanced the phagocytosis of cultured human RPE cells. CONCLUSIONS: The study confirmed the AMD high-risk alleles, HTRA1, and cigarette smoking can promote AMD development by regulating caveolin-1 expression. TRANSLATIONAL RELEVANCE: AMD high-risk alleles and environmental risk factors can promote the occurrence and development of AMD by regulating caveolin-1 expression, upregulation of which will induce apoptotic cell death in response to cellular stress in early AMD conditions.

HMGB1 and Caveolin-1 related to RPE cell senescence in age-related macular degeneration.

Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is considered a major cause of RPE dysfunction and senescence in age-related macular degeneration (AMD), and N-retinylidene-N-retinylethanolamine (A2E) is the main fluorophore identified in lipofuscin from aged human eyes. Here, human-induced pluripotent stem cell (iPSC)-RPE was generated from healthy individuals to reveal proteomic changes associated with A2E-related RPE cell senescence. A novel RPE cell senescence-related protein, high-mobility group box 1 (HMGB1), was identified based on proteomic mass spectrometry measurements on iPSC-RPE with A2E treatment. Furthermore, HMGB1 upregulated Caveolin-1, which also was related RPE cell senescence. To investigate whether changes in HMGB1 and Caveolin-1 expression under A2E exposure contribute to RPE cell senescence, human ARPE-19 cells were stimulated with A2E; expression of HMGB1, Caveolin-1, tight junction proteins and senescent phenotypes were verified. HMGB1 inhibition alleviated A2E induced cell senescence. Migration of RPE cells was evaluated. Notably, A2E less than or equal to 10µM induced both HMGB1 and Caveolin-1 protein upregulation and HMGB1 translocation, while Caveolin-1 expression was downregulated when there was more than 10µM A2E. Our data indicate that A2E-induced upregulation of HMGB1、Caveolin-1 and HMGB1 release may relate to RPE cell senescence and play a role in the pathogenesis of AMD.

Novel mutations in the OPN1LW and NR2R3 genes in a patient with blue cone monochromacy.

BACKGROUND: To clarify the diagnosis of a Chinese patient with novel double heterozygous in the NR2E3 and OPN1LW genes and describe the clinical features. MATERIALS AND METHODS: A 47-year-old man presented with an 8-year history of decreased vision and poor night vision. Based on his clinical phenotype, we focused on 36 genes associated with these characteristics. Possible pathogenic mutation sites were screened by next-generation sequencing (NGS), which showed novel mutations in the NR2E3 and OPN1LW genes. These mutations were confirmed in the patient's sister and daughter by Sanger sequencing. To clarify the diagnosis, the clinical symptoms of the patient were observed and analyzed in combination with comprehensive ophthalmologic examinations. RESULTS: Genetic analysis identified the presence of novel double heterozygous of c.361G>A; p.E121K in NR2E3, a gene responsible for enhanced S-cone syndrome (ESCS; OMIM #268100) and c.244A>G; p.K82E in OPN1LW, a gene responsible for blue cone monochromacy (BCM; OMIM#303700). No typical clinical presentation or fundus features were found. The differential diagnosis of ESCS was excluded by electroretinography (ERG) due to the lack of characteristic abnormalities associated with ESCS. Based on the clinical manifestations and comprehensive ophthalmologic examinations, the patient was diagnosed with BCM. CONCLUSIONS: The novel mutations of c.244A>G; p.K82E in the OPN1LW gene and c.361G>A; p.E121K in the NR2E3 gene both cause BCM, but OPN1LW gene mutation dominated the retinal degeneration, resulting in the clinical features observed in this patient. These novel double heterozygous may be helpful for future genetic diagnosis and treatment for BCM.

Application of CRISPR/Cas9 technologies combined with iPSCs in the study and treatment of retinal degenerative diseases.

Retinal degeneration diseases, such as age-related macular degeneration and retinitis pigmentosa, affect millions of people worldwide and are major causes of irreversible blindness. Effective treatments for retinal degeneration, including drug therapy, gene augmentation or transplantation approaches, have been widely investigated. Nevertheless, more research should be dedicated to therapeutic methods to improve future clinical treatments. Recently, with the rapid development of genome-editing technology, gene therapy has become a potentially effective treatment for retinal degeneration diseases. A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been developed as a powerful genome-editing tool in ophthalmic studies. The CRISPR/Cas9 system has been widely applied in basic research to develop animal models and gene therapies in vivo. With the ability to self-renew and the potential to differentiate into different types of cells, induced pluripotent stem cells (iPSCs) have already been used as a promising tool for understanding disease pathophysiology and evaluating the effect of drug and gene therapeutics. iPSCs are also a cell source for autologous transplantation. In this review, we compared genome-editing strategies and highlighted the advantages and concerns of the CRISPR/Cas9 system. Moreover, the latest progress and applications of the CRISPR/Cas9 system and its combination with iPSCs for the treatment of retinal degenerative diseases are summarized.

Bringing the age-related macular degeneration high-risk allele age-related maculopathy susceptibility 2 into focus with stem cell technology.

Age-related macular degeneration (AMD) is a major cause of blindness in older adults in developed countries. It is a multifactorial disease triggered by both environmental and genetic factors. High-temperature requirement A serine peptidase 1 (HTRA1) and age-related maculopathy susceptibility 2 (ARMS2) are two genes that are strongly associated with AMD. Because ARMS2 is an evolutionarily recent primate-specific gene and because the ARMS2/HTRA1 genes are positioned at a locus on chromosome 10q26 in a region with strong linkage disequilibrium, it is difficult to distinguish the functions of the individual genes. Therefore, it is necessary to bring these genes into focus. Patient-specific induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) provides direct access to a patient's genetics and allows for the possibility of identifying the initiating events of RPE-associated degenerative diseases. In this paper, a review of recent epidemiological studies of AMD is offered. An argument for a definite correlation between the ARMS2 gene and AMD is presented. A summary of the use of ARMS2 genotyping for medical treatment is provided. Several ARMS2-related genetic models based on such stem cells as iPSCs are introduced. The possibility of applying gene-editing techniques and stem-cell techniques to better explore the mechanisms of the ARMS2 high-risk allele, which will lead to important guidance for treatment, is also discussed.

Comparison of the efficacy of intravitreal ranibizumab for choroidal neovascularization due to pathological myopia with and without a dome-shaped macula.

Ranibizumab injection in the treatment of choroidal neovascularization (CNV) secondary to pathologic myopia (PM) with and without a dome-shaped macula (DSM).Prospective observational study.A total of 24 patients (24 eyes) with angiographic evidence of CNV secondary to PM were divided into 2 groups: eyes with a DSM and eyes without DSM. All patients received a baseline intravitreal ranibizumab injection. Additional injections were considered at each follow-up visit. Best-corrected visual acuity (BCVA) and optical coherence tomography were tested monthly through 12 months of follow-up. The mean changes in BCVA, central retinal thickness (CRT, including retinal and CNV thickness), and the number of injections were evaluated.There were no significant differences in visual outcomes between the groups over 12 months (P > .05). Patients with a DSM had a mean change in BCVA of +8.7 letters compared with +14.2 letters in patients without a DSM (P = .68). However, there were more patients without a DSM who gained at least 15 letters from baseline compared with patients with a DSM. By the end of the follow-up, there was no significant difference in the mean change in baseline CRT between patients with and without a DSM (-65.0 and -90.7, respectively, P = .42). The mean number of injections was 8.83 in the patients with DSM and 8.17 in the patients without a DSM (P > .05).For the pathological myopia patients who had CNV with a DSM, the DSM did not alter the effect of the ranibizumab treatment. There was no difference in the visual improvement, anatomic benefit and number of treatments between the 2 groups.