Molecular Vision最新文献

The circadian clock, a conserved biologic timekeeping mechanism, is pivotal in orchestrating rhythmic physiologic processes. While extensively studied in the central clock, the involvement of BMAL1 in peripheral clocks, particularly in human Müller cells, remains underexplored. Müller cells, critical for retinal homeostasis, may unveil novel insights into circadian regulation. Employing ChIP-sequencing, we comprehensively mapped BMAL1 binding sites in human Müller cells. The analysis identified 275 reproducible peaks, with predominant distribution across promoters (26.6%), intronic (26.3%), and intergenic (22.1%) regions, with 80% of these confident peaks linked to protein-coding genes. Differential peak analysis revealed 89 unique genes significantly enriched with BMAL1 sites in their promoters, while functional enrichment of the associated genes indicated key biologic processes such as circadian regulation of gene expression, photoperiodism, and glucocorticoid receptor signaling pathway regulation. Motif analysis revealed a highly conserved 6-nucleotide motif, CACGTG, appearing in 89.09% of the peaks. Analysis of the binding sites across genomic regions highlighted the robust BMAL1 binding, further confirmed by qPCR validation of circadian targets such as G6PC3, CIART, PER1, and TXNIP, which are critical for Müller cell health, along with SHMT2 and MALAT1, which have emerged as novel genes that may have implications for Müller cell health. Our findings unveil the regulatory landscape of BMAL1 in Müller cells, contributing to a broader understanding of the clock-mediated mechanism in ocular tissues. These insights hold therapeutic potential for circadian-related retinal diseases, presenting avenues for chronotherapeutic interventions.

Purpose: To explore the role of cytokines during the progression process of cataract patients with pathologic myopia (PMC) and simple high myopia (SHMC).

Methods: A total of 63 cataract patients who underwent cataract surgery were classified into a PMC group (22 eyes), an SHMC group (21 eyes), and an age-related cataract (ARC) group (20 eyes), based on axial length (AL) and International Myopia Institute (IMI)'s classification. Aqueous humor samples were extracted before surgery. Cytometric bead array (CBA) was employed to measure the level of interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), transforming growth factor-β1 (TGF-β1), basic fibroblast growth factor (bFGF), interleukin-10 (IL-10), interleukin-17a (IL-17a), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor -α (TNF-α), intercellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM). Additionally, the correlations between cytokines and the AL or myopic maculopathy categories were examined.

Results: VEGF, IL-6, MCP-1, ICAM, and VCAM (all p<0.001), TGF-β1 (p=0.018), and IL-8 (p=0.008) were statistically different among the three groups. In parallel, the levels of VCAM (r=0.718), MCP-1 (r=0.591), ICAM (r=0.584), IL-8 (r=0.435), IL-6 (r=0.396), and TNF-α (r=0.280) were positively associated with myopic maculopathy, while VEGF (r=-0.542), TGF-β1 (r=-0.381), and IL-17a (r=-0.284) were correlated inversely with myopic maculopathy (all p<0.05). Furthermore, a significant positive correlation was observed between AL and levels of VCAM (r=0.726), MCP-1 (r=0.644), ICAM (r=0.573), IL-6 (r=0.386), and IL-8(r=0.376). VEGF (r=-0.610), TGF-β1 (r=-0.361), and IL-17a (r=-0.319) were inversely associated with AL (all p<0.05). Further analysis using multiple regression indicated that, after adjusting for confounding factors, lower VEGF and higher VCAM were significantly associated with AL. However, the limitations of this study were reflected in the inability to determine whether the changes in cytokines were the consequences or causes of the formation of high myopia.

Conclusions: The pathogeneses of PMC and SHMC may differ, and there are significant changes associated with inflammation and the immune response in eyes with PMC.

Purpose: Neovascular age-related macular degeneration (nAMD) is now a major cause of central vision loss in older adults worldwide. The primary characteristic of nAMD is the formation of macular neovascularization (MNV), which is a pathologic form of angiogenesis. Epigenetics plays a role in multiple pathological physiologic processes. N6-methyladenosine (m6A) modification is the most common, abundant, and reversible modification in eukaryotic mRNAs, and it plays a role in various pathological angiogenesis processes. This study intends to reveal the expression and functions of m6A during the macular neovascularization (MNV) process.

Methods: A laser-induced MNV mouse model was used in this study. m6A quantitative analysis was performed to detect the expression of m6A. Subsequently, the expression of various m6A writers and erasers was detected using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Immunohistochemistry was used to detect Wilms' tumor 1-associating protein (WTAP) expression in the MNV lesions. Intravitreal injection of WTAP siRNA in MNV mice to silence the WTAP gene. Hematoxylin and eosin (H&E) were used to determine the thickness and length of the MNV. Fundus fluorescein angiography (FFA) and indocyanine green angiography (ICGA) were examined to measure the leakage area of the MNV. Proliferating cell nuclear antigen (PCNA) expression was detected with a western blot. The mRNA and protein levels of β-catenin were tested with qRT-PCR and western blot.

Results: We found increased m6A modification levels after laser induction compared with the normal control group. Subsequently, the expression of various m6A writers and erasers was detected. The results showed that WTAP increased in the MNV model in mice. After the injection of WTAP siRNA into the vitreous body, the expression of WTAP significantly decreased, subsequently decreasing the m6A modification levels. The width, breadth, and leakage area of MNV damage markedly decreased, and endothelial cell proliferation was inhibited. After laser-induced MNV, the expression of β-catenin increased, and that of β-catenin significantly decreased after WTAP knockout.

Conclusions: In conclusion, this study suggests that WTAP-mediated m6A methylation can regulate pathological angiogenesis during MNV and that WTAP may participate in the formation of MNV through the wingless-related integration site (Wnt) pathway. WTAP may be a potential target for MNV treatment.

Purpose: Cataracts are typically treated by phacoemulsification followed by intraocular lens implantation. Studies of mouse models of cataract surgery have revealed that lens epithelial cells rapidly remodel their transcriptome to express proinflammatory cytokines after lens fiber cell removal, but it is currently unknown whether this response is conserved in human lenses. This study seeks to fill this knowledge gap.

Methods: Human cadaver eyes from 70 to 89 year old individuals were prepared for the human capsular bag model of cataract surgery. The central epithelium was preserved in RNAlater during culture preparation, then the equatorial epithelium was either immediately preserved in RNAlater after the culture was created, or 24 h later. Gene expression profiles were generated by bulk sequencing of RNA isolated from these tissue samples. The transcriptomic response of human cadaver-derived lens epithelial cells to culture in this "capsular bag" model was characterized by bioinformatic analysis. The human response was directly compared to that of 24-month-old mouse lens epithelial cells subjected to fiber cell removal surgery.

Results: Human lens epithelial cells remodel approximately a third of their transcriptome by 24 h after surgery, and like mice, this response consists of induction of proinflammatory cytokine genes, upregulation of fibrotic markers and downregulation of genes controlling the lens epithelial phenotype.

Conclusions: These observations demonstrate that humans and mice have similar responses to cataract surgery and support the use of mice to study the response of lens epithelial cells to cataract surgery, suggesting that identified injury response mechanisms can be leveraged to elucidate new approaches to improve the outcomes of cataract surgery.

Purpose: To collectively investigate the carbohydrate sulfotransferase 6 (CHST6) mutation spectrum and corneal morphological alterations of macular corneal dystrophy (MCD) patients using in vivo confocal microscopy (IVCM), histochemistry, immunohistochemistry, and further ascertaining the immunophenotype using an enzyme-linked immunosorbent assay (ELISA).

Methods: Sanger sequencing-based CHST6 gene screening was performed for 112 study participants (MCD patients, n = 68; family members, n = 44). Twenty-seven MCD patients underwent IVCM analyses, and corneal buttons were analyzed with histochemistry Alcian blue (AB) staining and immunohistochemistry anti-keratan sulfate (KS) monoclonal antibody, 5D4MoAb. An ELISA was used to determine serum KS levels. Quantitative analysis of the central corneal thickness (CCT), epithelial cell thickness, epithelial cell count, and stromal keratocyte cell count was performed using a one-way ANOVA.

Results: Eighteen distinct CHST6 mutations, including one novel (p.L129V), were identified. MCD patients with predominant immunophenotype IA (n = 15) harboring major p.Q182Rfs199 deletion, p.194_R196delinsRC (delins), and open reading frame (ORF) mutations displayed AB positivity corresponding to loss of Bowman's layer, interlamellar glycosaminoglycan (GAG) depositions, and faint KS expression (5D4-MoAb) only in stromal keratocytes. Notably, IVCM imaging revealed BL loss due to confluent clumps of hyper-reflective, granular deposits together with scar tissue seen only in this group. Eight patients (with missense mutations) displayed immunophenotype I with positive GAG deposits and negative KS expression. Patients with immunophenotype II (n = 4) with no mutations showed both positive GAG deposits and KS expression. A quantitative analysis revealed a statistically significant decrease in CCT (p-value < 0.001), epithelial cell thickness, epithelial cell count, and stromal keratocyte cell count among the patients with truncation mutations compared to the control group.

Conclusions: In this current study, the combinational findings of MCD-related corneal morphological alterations, immunophenotypes, and mutation spectrum are presented first, which indicated a severe phenotype in patients identified with truncation (deletion, delins, and deletion of ORF) mutations. However, additional studies with a larger number of patients would help highlight these findings and reinforce the possible correlation between genotypes and immunophenotypes in MCD pathogenesis.

Purpose: The mutation of R124H in TGFBIp causes Avellino corneal dystrophy (ACD, GCD II). However, the molecular mechanisms of ACD caused by the p. R124H mutation are not well understood. In our research, we aimed to explain the molecular mechanisms of ACD caused by the R124H mutation.

Methods: The whole blood of a three-generation family having ACD was studied with the whole exome sequencing. Sanger sequencing was used to identify the mutation gene. The mutant structure of R124H TGFBIp was visualized in Pymol, using the PISA server, Coot and the HDOCK automated docking program. The TGFBIp was expressed in mammalian expression system. And size exclusion chromatography (SEC) was used to identify the aggregate state of TGFBIp.

Results: The whole exome sequencing results showed that there was a c.371G>A mutation in the TGFBI gene in one family, including three patients. In biochemical assays, the purified soluble wild-type TGFBIp and R124H TGFBIp formed a homodimer through a novel interface distinct from the previously proposed FAS1-1: FAS1-4 dimer (interface I). R124H TGFBIp is likely to have formed more severe cross-links and aggregation. Therefore, R124H TGFBIp causes homozygous patients to have more serious symptom than heterozygous patients.

Conclusions: In our study, one family having ACD harboring the mutation of R124H TGFBIp was identified. A new homodimerization interface was determined for wild-type TGFBIp and R124H TGFBIp. Besides, we provided a possible molecular explanation for why the symptom of homozygous patients was more severe than those of heterozygous patients. The possible molecular explanation can provide a new insight into the treatment of ACD.

Purpose: This study aimed to characterize the inflammatory mediators present in the tear film of patients with keratoconus (KC). It also aimed to investigate the gene expression of these mediators in corneal epithelial cells and their immune activity in conjunctival epithelial cells in patients with KC compared to a control group.

Methods: This transversal study included 30 patients with KC and 23 control group participants. Tear samples were collected by washing the ocular surface with 60 μL of sterile buffered saline solution. The levels of interleukin IL-5, IL-13, IL-2, IL-6, IL-10, interferon-gamma, tumor necrosis factor-alpha, and IL-4 were measured using a LEGEND plex HU Th1/Th2 panel kit and analyzed using flow cytometry. Corneal epithelial samples were obtained via manual keratectomy from KC patients scheduled for corneal crosslinking and from individuals scheduled for photorefractive keratectomy (control group). These samples were immediately stored at -70 °C for mRNA extraction and subsequent reverse transcription polymerase chain reaction analysis to measure IL-5 and IL-6 gene expression. Conjunctival epithelium samples were collected using impression cytology and analyzed using immunohistochemistry and confocal microscopy to detect IL-5 and IL-6 immunoreactions.

Results: Our study found no statistically significant differences in the tear film cytokine concentrations between the two groups. In addition, the gene expression of IL-5 and IL-6 in the corneal epithelium was higher in the KC group than in the control group, with IL-5 showing a 50% increase and IL-6 showing a 20% increase. Immunohistochemical analysis revealed a greater immunostaining of IL-5 and IL-6 in the conjunctival epithelium of patients with KC compared to the control group.

Conclusions: In this study, despite higher levels of IL-5 and IL-6 in the tear film of patients with KC, there was no statistically significant difference compared to the control group. However, there was heightened immune activity in the corneal and conjunctival epithelial cells of patients with KC based on IL-5 and IL-6 gene expression and their immunodetection, respectively.