Molecular Vision[JOURNAL]
Sun
200 papers
Analysis of interleukin-6 gene polymorphism and its serum levels in Indian age-related macular degeneration patients.
Yadav A, Phogat J, Yadav M … +9 more , Bhardwaj A, Yadav R, Nada M, Bhati M, Goel S, Thakur R, Kumar R, Singh M, Tanwar M
Mol Vis · 2024 · PMID 39959178
BACKGROUND: Age-related macular degeneration (AMD) is a complex condition involving multiple factors. The condition is associated with numerous inflammatory indicators, including cytokines. Single-nucleotide polymorphism...
BACKGROUND: Age-related macular degeneration (AMD) is a complex condition involving multiple factors. The condition is associated with numerous inflammatory indicators, including cytokines. Single-nucleotide polymorphisms in cytokine genes can also modify gene expression, perhaps contributing to the development of the disease. The objective of the present study was to examine the correlation among SNPs (rs1800795, rs1800796, and rs1800797) and the serum levels of IL-6 in AMD patients treated at the Regional Institute of Ophthalmology of Pt. B.D. Sharma PGIMS, Rohtak (Haryana), India. METHODS: This case-control study included 131 patients diagnosed with AMD using precise ophthalmic examinations, such as slit lamp examination, fundoscopy, and ocular coherence tomography. To provide a basis for comparison, we also enlisted 100 healthy individuals as controls. Serum IL-6 protein levels were measured in both patients and controls using an enzyme-linked immunosorbent assay kit (ELISA). Genotyping SNPs was performed using the PCR and DNA Sanger sequencing technique. RESULTS: IL-6 serum levels were considerably elevated in individuals with AMD compared to the control group ( < 0.05). Statistically significant differences were seen in the genotype frequencies of rs1800795 ( = 0.027) and rs1800797 ( = 0.0011) among the AMD patients and the healthy controls. Furthermore, strong correlations were observed between rs1800795 and the likelihood of developing AMD based on the heterozygous (OR = 2.04; = 0.025), dominant (OR = 1.80; = 0.035), and over-dominant models (OR = 2.10; = 0.0094). Additionally, there were notable associations between rs1800797 and vulnerability to AMD through heterozygous (OR = 3.21; = 0.009), dominant (OR = 2.74; = 0.004), and over-dominant (OR = 3.11; = 0.002) models. The rs1800795, rs1800796, and rs1800797 haplotypes C-G-A and G-G-A were linked to an elevated risk of AMD ( = 0.005, = 0.024. respectively). CONCLUSIONS: Our findings indicated a significant elevation in IL-6 serum levels among the AMD patient group compared to the control group. The interleukin-6 gene polymorphisms rs1800795 and rs1800797 were linked to an elevated risk of AMD in our study population.
Caveolin-1 regulates inflammatory mediators in retinal endothelial cells.
Jiang Y, Liu L, Al-Shabrawey M … +1 more , Steinle JJ
Mol Vis · 2024 · PMID 39959177
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Measuring the viability of crystalline lens epithelial cells by triple Hoechst-Ethidium-Calcein-AM staining.
Poinard S, Parveau L, Chapelon G … +9 more , Dorado O, Thomas J, He Z, Perrache C, Ganeau A, Forest F, Mascarelli F, Gain P, Thuret G
Mol Vis · 2024 · PMID 39959176
PURPOSE: To date, the assessment of lens epithelial cell viability has been proposed only in cell cultures or isolated capsule models. This study aimed to develop a method for quantifying the viability of epithelial cell...
PURPOSE: To date, the assessment of lens epithelial cell viability has been proposed only in cell cultures or isolated capsule models. This study aimed to develop a method for quantifying the viability of epithelial cells on whole ex vivo crystalline lenses by triple labeling Hoechst 33342, ethidium homodimer, and calcein-acetoxymethyl (HEC). METHODS: Two models of induced cell death were used to study the performance and potential applications of the technique. First, ten fresh pairs of six-month-old porcine lenses were retrieved. On one lens of each pair, an easily identifiable localized lesion was induced by a calibrated cryo-application, while the other remained intact. Both lenses of each pair were incubated for 1 h at 20 °C in an HEC mixture. Ten other pairs of lenses were used in the second experiment. On one lens of each pair, a diffuse epithelial lesion was induced by incubation in staurosporine (STS) solution (0.5 µM in CorneaMax) for 24 h at 37 °C. The other lens of each pair was incubated in CorneaMax solution without STS for 24 h at 37 °C. The day after, both lenses of each pair were incubated for 1 h at 20 °C in an HEC mixture. Images were acquired with a macroscope (macro zoom) and analyzed with ImageJ. Calcein-AM and ethidium images were used to calculate the area covered by living epithelial cells. Hoescht images allowed us to count cell nuclei per unit area. Viable epithelial cell density (vECD) was defined as the number of viable cells per unit area. Different strategies were developed to reduce background noise. RESULTS: There was no interfering lens autofluorescence for the exposure times used. The vECD median was 2,840 cells/mm [10th-90th percentiles = 2,479-3,494] for cryo-injured lenses versus 3,364 cells/mm [2,919-3,739] for healthy lenses (p = 0.002). The vECD median was 3,804 cells/mm [10th-90th percentiles = 2,922-4,862] for lenses treated with STS versus 3,896 cells/mm [3,169-4,980] for healthy lenses (p = 0.002). CONCLUSIONS: Thanks to simple sample preparation, triple HEC staining allows fluorescence imaging of a large series of a whole lens to respect the architecture of the epithelium. It will be particularly useful for cytotoxicity studies of new therapies targeting the lens.
Conversion of Optineurin into an Hsp72-Inducible protein by C-terminal addition of green fluorescent protein.
Suh W, Sohn S, Kim TE … +1 more , Kee C
Mol Vis · 2024 · PMID 39959175
PURPOSE: Optineurin is known to be associated with glaucoma. This protein has often been investigated as a form of fusion with green fluorescent protein (GFP), but there have been few reports describing any undesired eff...
PURPOSE: Optineurin is known to be associated with glaucoma. This protein has often been investigated as a form of fusion with green fluorescent protein (GFP), but there have been few reports describing any undesired effect of the tag on the normal expression of naïve optineurin. We investigated whether GFP tagging potentially does not influence the characteristics of optineurin expression. METHODS: Wild-type and mutant (E50K and M98K) optineurins were fused with GFP or yellow fluorescent protein (YFP) either at their N-terminus or C-terminus. The fusion constructs were loaded onto an adenoviral vector and analyzed by western blot analysis and immunocytochemistry. RESULTS: In human trabecular meshwork cells, optineurins fused to GFP at their C-terminus (OPTN-GFP) were prone to aggregation and did not fluoresce as brightly as their counterparts fused to YFP did regardless of whether they were wild-type or mutant forms. In addition, their expression was accompanied by the apparent induction of heat shock protein 72 (Hsp72). Furthermore, OPTN-GFP appears to interact with Hsp72 and be co-aggregated into vesicle-like structures scattered throughout the cytoplasm. However, optineurin fused to GFP at its N-terminus was not aggregate and did not induce Hsp72 expression. CONCLUSIONS: It is well-known that misfolded or unfolded proteins are prone to aggregation and, if they are fused with GFP, their chromophores are not fully fluorescent. Additionally, it is also well-known that heat shock response is a key cellular processes against the accumulation of misfolded or unfolded proteins. Therefore, our data suggest that the addition of GFP to the C-terminus of optineurin might convert it into an unfolded or partially folded protein.
A novel gene duplication causing congenital North Carolina macular dystrophy phenotype in a Mexican family.
Chacon-Camacho OF, Flores-Lagunes LL, Small KW … +14 more , Udar N, Udar U, Diaz A, Arce-González R, Molina-Garay C, Martínez-Aguilar A, Montes-Almanza L, Garcia-Martinez F, Gudiño A, Matsui-Serrano R, Fest-Parra S, Alaez-Verson C, Shaya F, Zenteno JC
Mol Vis · 2024 · PMID 39959174
PURPOSE: North Carolina macular dystrophy (NCMD) is a rare autosomal dominantly inherited congenital maculopathy caused by either non-coding point mutations or tandem duplications in the DNase I hypersensitivity site DHS...
PURPOSE: North Carolina macular dystrophy (NCMD) is a rare autosomal dominantly inherited congenital maculopathy caused by either non-coding point mutations or tandem duplications in the DNase I hypersensitivity site DHS6S1, at chromosome 6q16 (MCDR1), or at chromosome 5 (MCDR3). To date, at least 30 NCMD pedigrees from different ethnicities have been genetically identified worldwide. Herein, we report the clinical and genetic features of a newly found NCMD family in Mexico with a novel tandem duplication involving both the DNASE1 site and the gene. METHODS: Seven affected subjects from a Mexican family underwent a complete ophthalmic assessment that included dilated indirect ophthalmoscopy, fundus photography, optical coherence tomography (OCT), fundus autofluorescence (FAF), kinetic and chromatic perimetry, and electroretinography (ERG). Next-generation sequencing (NGS), followed by array-based comparative genomic hybridization (array-CGH) and quantitative polymerase chain reaction (qPCR) analyzes, were employed to demonstrate the causative molecular defect. RESULTS: All seven affected patients had a severe appearing phenotype characterized by symmetric excavated atrophic coloboma-like chorioretinal macular lesions. In addition, using OCT, lacunae in the inner retinal layers and inner retinal loss were observed in all patients. NGS identified a heterozygous tandem duplication of the entire coding sequence of the gene in all seven affected individuals, whereas subsequent array CGH, NGS, and Sanger sequencing allowed for the identification of the precise boundaries of a ~148 kb MCDR1 duplication containing the whole gene and the DNASE1 site. CONCLUSIONS: The phenotypic features in this NCMD pedigree continue to support the concept that this disorder is a congenital macular malformation rather than a progressive dystrophic entity. Unlike most NCMD families, there was no variable expressivity found in this study, possibly due to the relatively small size of the family. The other hypothesis is that the duplication involves genomic segments that are more consistently or tightly bound to other regulatory regions of PRDM13. The identification of a novel causative tandem duplication involving the DNASE1 site and the gene in this family allows for the expansion of the mutational spectrum of the disease.
Oxidation balance scores are positively associated with the occurrence and severity of myopia in adults: results from the National Health and Nutrition Examination Survey 2007-2008.
Wu YL, Liu X, Xu L … +1 more , Ning L
Mol Vis · 2024 · PMID 39959173
PURPOSE: The purpose of this cross-sectional study was to investigate the relationship between oxidation balance scores (OBSs) and myopia. METHODS: Participant information came from the National Health and Nutrition Exam...
PURPOSE: The purpose of this cross-sectional study was to investigate the relationship between oxidation balance scores (OBSs) and myopia. METHODS: Participant information came from the National Health and Nutrition Examination Survey (NHANES) 2007-2008. The relationship between OBSs and myopia was analyzed using a restricted cubic spline, generalized linear regression, trend analysis, and ordinal logistic regression. The important components of the OBS in myopia were analyzed using XGBoost, random forest, and AdaBoost. RESULTS: The data of 5,187 participants from NHANES were collected, and a preliminary analysis was conducted. We found that an association between OBSs and myopia was only found in participants aged ≥ 20 years (n = 4,253). There was a linear relationship between OBSs and the occurrence of myopia in them. The logistic regression showed that OBSs were correlated with an increased incidence of myopia after adjusting for all confounders (OR: 1.01, 95% CI [1.00, 1.02]). The trend test showed that the higher the OBS, the higher the likelihood of developing myopia ( for trend < 0.05). There was a nonlinear relationship between OBSs and myopia severity according to a generalized additive model (β = 0.01, 95% CI [0.00, 0.01], < .01). The ordered logistic regression analysis showed that for every unit increase in OBS, the likelihood of myopia severity increased by 11% after adjusting for all confounders. We also found that calcium was an important OBS component related to the incidence of myopia. CONCLUSIONS: OBS is positively associated with the occurrence and severity of myopia in adults ≥ 20 years of age, and calcium is an important OBS component related to the incidence of myopia.
Genetic implications of CHST6 gene mutations and their corneal microstructural changes in macular corneal dystrophy patients.
Murugan D, Duvesh R, Adsumilli SD … +3 more , Prajna NV, Chermakani P, Sundaresan P
Mol Vis · 2024 · PMID 39959172
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...
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.
The interactions between , , and environmental factors on the risk of age-related macular degeneration.
Altankhuyag A, Ganbold C, Byambadorj B … +4 more , Tumurbaatar S, Sodnomtseren P, Davaatseren U, Jav S
Mol Vis · 2024 · PMID 39959171
BACKGROUND: Age related macular degeneration (AMD) is a multifactorial disease caused by a combination of environmental and genetic factors. The prevalence of allele and genotypeof AMD-related genes is varied throughout...
BACKGROUND: Age related macular degeneration (AMD) is a multifactorial disease caused by a combination of environmental and genetic factors. The prevalence of allele and genotypeof AMD-related genes is varied throughout the world due to racial and ethnic differences. Number of previous studies have shown that the polymorphisms in the , and genes are associated with AMD. In Mongolia, there is a lack of sufficient data on AMD development in its population and thus needs more studies on the topic. Therefore, it needs more studies about AMD development in the population. For this reason, we have investigated several specified polymorphisms in , and genes to reveal a relationship with AMD and determine the prevalence of alleles and genotypes of the genes in Mongolian population. METHODS: Totally 161 AMD patients and 223 controls were enrolled in this case-control study. The polymorphisms in , and - were detected by using the methods of allele-specific polymerase chain reaction (ASPCR) and PCR based restriction fragment length polymorphism (RFLP). Statistical analysis were performed by STATA 13.0, SNPAlyze 9.0 and MDR 3.0.2. RESULTS: According to the study result, the characteristics of hypertension, constant-wearing sunglasses and anticoagulant medications in AMD group were significantly different from those in the control group. As for the dominant model, T allele of rs10490924 (cOR=4.45; 95% CI, 2.44-8.13, p<0.001, aOR=5.08; 95% CI, 2.70-9.59, p<0.001) was more frequent among patients with AMD in comparison with the control group. Also, G/G genotype of rs800292 (cOR=11.61; 95% CI, 3.41-39.51, p<0.001, aOR=12.49; 95% CI, 3.47-44.91, p<0.001) and G/G genotype of rs1065489 (cOR=4.19; 95% CI, 2.53-6.93, p<0.001, aOR=4.67; 95% CI, 2.71-8.05, p<0.001) were significantly higher in AMD group after Bonferroni correction. This result suggests that people who carrying the risk genotypes of these polymorphisms had an increased risk for AMD development. As for the models of three or more SNP interactions, the participants with any combinations of risk genotypes have 6 to 106-fold higher risk for AMD development. This result suggests that there is some positive-additive interaction existing between the genetic variants of , and genes for AMD development. Our study also revealed that the participants with hypertension and carrying G/G for rs1065489 in gene or non G/G for rs10490924 in gene genotypes had 9 to 14 times higher risk for AMD development (cOR=9.05; 95% CI, 4.38-18.68, p<0.001, RERI=4.546; AP=0.502, S=2.298, cOR=13.98; 95% CI, 3.19-61.1, p<0.001, RERI=5.85; AP=0.419, S=1.821) with high level of significance. Moreover, it was found that the participants who avoided wearing sunglasses and had the G/G genotype of ARMS2 rs10490924 or G/G genotype of CFH rs800292 had an extremely higher risk for AMD development (p<.001). CONCLUSIONS: In conclusion, it was observed that the combination of SNPs in , and genes increase the risk for AMD with 6 to 106-fold. Moreover, we found that the participants with hypertension and carrying the non G/G genotype of rs10490924 or the G/G genotype of rs800292 had an extremely higher risk of AMD development.
Analysis of Smad3 in the modulation of stromal extracellular matrix proteins in corneal scarring after alkali injury.
Gupta S, Zhang E, Sinha S … +7 more , Martin LM, Varghese TS, Forck NG, Sinha PR, Ericsson AC, Hesemann NP, Mohan RR
Mol Vis · 2024 · PMID 39959170
PURPOSE: During ocular trauma, excessive proliferation and transdifferentiation of corneal stromal fibroblasts cause haze/fibrosis in the cornea. Transforming growth factor β (TGFβ) plays a key role in corneal fibrosis t...
PURPOSE: During ocular trauma, excessive proliferation and transdifferentiation of corneal stromal fibroblasts cause haze/fibrosis in the cornea. Transforming growth factor β (TGFβ) plays a key role in corneal fibrosis through the Smad signaling pathway. The aberrant activity of TGFβ signaling during ocular trauma (viz. mechanical, infectious, chemical, or surgically altered TGFβ/Smad signaling) leads to regulating the predominant expression of myogenic proteins and the extracellular matrix (ECM). We sought to investigate the functional role of Smad3 in corneal wound repair and stromal ECM assembly using Smad3 wild-type and Smad3 deficient mice. METHODS: Corneal injury was introduced with the topical application of an alkali-soaked 2-mm filter disc on the central cornea in the Smad3 (C57BL/6J) and Smad3 (129-Smad3/J) mouse strains. Slit-lamp and stereo microscopy were used for clinical assessment and corneal haze grading in live animals. Hematoxylin and eosin and Masson's trichrome staining were used to study comparative morphology and collagen level alterations between the groups. Real-time qRT-PCR, western blot, and immunohistochemistry were used to measure changes in profibrotic genes at the mRNA and protein levels. RESULTS: Slit-lamp clinical exams and stereo microscopy detected notably less opaque cornea in the eyes of Smad3 compared with Smad3 mice at 3 weeks (p<0.01) in live animals. Corneal tissue sections of Smad3 mice showed significantly fewer α-smooth muscle actin-positive cells compared with those of the Smad3 animals (p<0.05). The corneas of the Smad3 mice showed significantly lower mRNA levels of pro-fibrotic genes, α-smooth muscle actin, fibronectin, and collagen I (p<0.05, p<0.01, and p<0.001). In addition, the matrix metalloproteinase and tissue inhibitors of metalloproteinase levels were significantly increased (p<0.001) in the corneal tissue during alkali injury in both Smad3 wild-type and Smad3 deficient mice. CONCLUSIONS: The significant changes in profibrotic genes and stromal ECM proteins revealed a direct role of Smad3 in stromal ECM proteins and TGFβ/Smad-driven wound healing. Smad3 appears to be an attractive molecular target for limiting abnormal stroma wound healing to treat corneal fibrosis in vivo.
Global mapping of BMAL1 protein-DNA interactions in human retinal Müller cells.
Luo Q, Sangani N, Abhyankar S … +3 more , Somalraju S, Janga SC, Bhatwadekar AD
Mol Vis · 2024 · PMID 39959169
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,...
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.
Peroxisome proliferator-activated receptor-α affects corneal epithelial wound healing.
Wu Y, Bu J, Zhang R … +3 more , Sun L, Yuan Y, Li W
Mol Vis · 2024 · PMID 39959168
PURPOSE: This study aimed to determine the role of peroxisome proliferator-activated receptor α (PPARα) on corneal epithelial wound healing. METHODS: Ten-week-old PPARα knockout ( ) mice and wild-type (WT) C57BL/6 mice a...
PURPOSE: This study aimed to determine the role of peroxisome proliferator-activated receptor α (PPARα) on corneal epithelial wound healing. METHODS: Ten-week-old PPARα knockout ( ) mice and wild-type (WT) C57BL/6 mice and ex vivo cultured human corneal epithelial cells were used to investigate the function of PPARα on corneal epithelial wound healing. A two-millimeter diameter of the mice's central corneal epithelium was removed to induce corneal epithelial injury. The expression of PPARα during corneal epithelial wound healing was analyzed using immunofluorescent staining and quantitative RT-PCR. Histological and immunostaining techniques were used to evaluate corneal morphology, cell proliferation, and inflammatory response in WT and mice. PPARα agonist fenofibrate was used to determine its effect on corneal epithelial wound healing. RESULTS: PPARα expression was found to significantly increase during corneal epithelial repair. mice exhibited delayed corneal epithelial wound healing compared to WT mice. mice displayed altered proliferative responses and distinct patterns of inflammatory infiltrates. Administration of fenofibrate to WT mice resulted in accelerated corneal epithelial repair and increased PPARα expression and cell proliferation. In vitro studies using human corneal epithelial cells further supported the impact of fenofibrate on promoting corneal epithelial cell wound healing. CONCLUSIONS: PPARα is a regulator of corneal epithelial wound healing, and its absence leads to delayed repair processes in the corneal epithelium.
Molecular genetic analysis of R124H TGFBIp in one family Avellino corneal dystrophy.
Huang Y, Liu M, Lu H … +3 more , Ji Z, Jin T, Lei S
Mol Vis · 2024 · PMID 39959167
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 exp...
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 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.
Human lens epithelial cells induce the inflammatory response when placed into the lens capsular bag model of posterior capsular opacification.
Novo SG, Faranda AP, D'Antin JC … +5 more , Wang Y, Shihan M, Barraquer RI, Michael R, Duncan MK
Mol Vis · 2024 · PMID 39959166
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 transc...
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.
Complex genomic rearrangement with deletion of in a Chinese family with Axenfeld-Rieger syndrome: A case report and literature review.
Jiang Z, Zhang Y, Wang L … +2 more , Yang H, Yu L
Mol Vis · 2024 · PMID 39959165
PURPOSE: This study identified the genetic causes of Axenfeld-Rieger syndrome (ARS) in a Chinese family and evaluated their clinical phenotype and clinical treatment. METHODS: We recruited a Chinese family with ARS. The...
PURPOSE: This study identified the genetic causes of Axenfeld-Rieger syndrome (ARS) in a Chinese family and evaluated their clinical phenotype and clinical treatment. METHODS: We recruited a Chinese family with ARS. The proband presented with bilateral ectopic pupils, periumbilical redundancy, craniofacial abnormalities, and dental abnormalities after birth and was diagnosed with ARS. The symptoms were the same for her younger brother. Blood samples were collected from four family members: the proband, her brother, and her parents. Whole-genome sequencing (WGS) was performed to identify probable genetic variants in the proband. To confirm the identified variants, samples from the other family members were subjected to quantitative polymerase chain reaction (qPCR) and Sanger sequencing. RESULTS: Based on the results of WGS, we suspected a deletion region and an inversion region around the gene. Through qPCR and Sanger sequencing, we identified a complex rearrangement involving a 6.15 Mb deletion on Chromosome 4, including the coding region (Hg38; chr4:110617776-116769011), a 45.71 Mb inversion (Hg38; chr4:116769011-162481408), and a 14-bp deletion (Hg38; chr4:162481409-162481422). Interestingly, the father's copy number was normal, but Sanger sequencing revealed the same breakpoints. This indicated that the father is a balanced rearrangement carrier, and the children are unbalanced rearrangement carriers. While similar deletions and many breakpoints in this region have been reported, this specific rearrangement is novel. CONCLUSIONS: Using WGS, qPCR, and Sanger, we found a complex genomic rearrangement with the deletion of in a Chinese family with ARS. The clinical characteristics of the affected individuals were reported. The current findings broaden our understanding of the phenotype and variant spectrum associated with ARS caused by deletion.
Salidroside ameliorates diabetic retinopathy and Müller cell inflammation via the PI3K/Akt/GSK-3β/NF-𝜅B pathway.
Feng Z, Yang Y, Shi CX … +7 more , Liu AQ, Wu CL, Liu WQ, Yu SX, Yu HD, Zuo ZF, Liu XZ
Mol Vis · 2024 · PMID 39588324
PURPOSE: To determine whether salidroside (SAL) modulates inflammatory cytokines in rat retinal Müller cells (rMC-1) in a hyperglycemic environment by investigating the anti-inflammatory mechanisms of SAL in vitro and in...
PURPOSE: To determine whether salidroside (SAL) modulates inflammatory cytokines in rat retinal Müller cells (rMC-1) in a hyperglycemic environment by investigating the anti-inflammatory mechanisms of SAL in vitro and in vivo. METHODS: A streptozotocin (STZ)-induced diabetic rat model was established to examine the effects of SAL using hematoxylin and eosin (H&E) staining and immunohistochemistry. rMC-1 cells were grown in 50 mM of high-glucose medium. These simulated diabetic conditions were used to evaluate the anti-inflammatory effects of SAL using a Cell Counting Kit-8 (CCK-8) assay, immunofluorescence staining, western blotting, and real-time polymerase chain reaction (qRT‒PCR). H&E staining was used to analyze the number of ganglion cells in the retina. rMC-1 lysates were processed for qRT‒PCR to measure the steady-state mRNA expression levels of inflammatory markers, such as interleukin 6 (IL-6), interleukin 10 (IL-10), and interleukin 1β (IL-1β). Western blot analysis and immunofluorescence staining were performed to determine the levels of these inflammatory markers. RESULTS: Our study showed that SAL reversed retinal ganglion cell loss and attenuated nuclear factor kappa B (NF-𝜅B) p65 translocation to the nucleus in STZ-induced diabetic rats. Incubating rMC-1 in different concentrations of SAL for 24 to 48 h affected cell viability. Furthermore, SAL treatment significantly decreased the protein levels of IL-6, TNF-α, and IL-1β compared with those in cells cultured in high glucose (HG). The mRNA expression levels of IL-6 and IL-1β were considerably reduced after SAL treatment, whereas the mRNA expression levels of IL-10 were significantly increased. Interestingly, the beneficial effects of SAL on HG-treated rMC-1 cells were abolished by the PI3K inhibitor LY294002. CONCLUSIONS: These results indicate that SAL treatment reduces cytokine activation in cultured rMC-1. Furthermore, SAL prevents diabetic retinopathy (DR), in part, by modulating the PI3K/Akt/GSK-3β/NF-kB pathway to inhibit Müller cell activation. Thus, SAL is expected to be a potential agent for ameliorating the progression of DR.
Assessment of anterior scleral thickness in myopes and emmetropes using anterior segment optical coherence tomography.
Li ZL, Xiong Q, Cai SC … +5 more , Fu Y, Li YT, Chen XM, Yang L, Ke M
Mol Vis · 2024 · PMID 39563681
PURPOSE: To investigate the differences in anterior scleral thickness (AST) among the refractive statuses of Chinese adults aged 18-35. METHODS: This study recruited 170 Chinese participants (mean age, 24.06 ± 2.78 years...
PURPOSE: To investigate the differences in anterior scleral thickness (AST) among the refractive statuses of Chinese adults aged 18-35. METHODS: This study recruited 170 Chinese participants (mean age, 24.06 ± 2.78 years), including myopes (spherical equivalent refraction [SER] -1.00 to -12.75 diopters [D]; n = 134), emmetropes (SER ± 0.75 D; n = 36), and AST (superior, inferior, nasal, and temporal), which were investigated via swept-source optical coherence tomography. Semiautomated custom-designed software measured the scleral thickness from the scleral spur to 5 mm along four meridians. RESULTS: The mean axial length and spherical equivalent refractive error were 25.12 ± 1.44 mm and -3.93 ± 3.09 D, respectively. The anterior sclera was thickest in the inferior region and thinnest in the superior region (753.9 ± 88.7 μm versus 613.6 ± 58.4; p < 0.001). The AST in the temporal meridian was significantly thicker than that in the nasal meridian (727.5 ± 60.8, 690.9 ± 55 μm; p < 0.001). There were no significant variations in AST in the myopes and emmetropes along the five latitude lines. AST along the inferior meridian at the 4-mm (r 2 = 0.0992; p < 0.001) and 5-mm (r 2 = 0.0888; p < 0.001) locations decreased significantly with increasing myopia. CONCLUSION: With increased myopia, AST at the 4-mm and 5-mm locations showed significant thinning in the inferior meridian. The results indicate that AST, especially along the inferior meridian, may act as a biologic marker to monitor the progression of myopia.
Sulforaphane inhibits TGF-β-induced fibrogenesis and inflammation in human Tenon's fibroblasts.
Liu Y, Huang Y, Guo Z … +5 more , Yang C, Li Y, Li B, Liu Y, Zheng H
Mol Vis · 2024 · PMID 39563680
PURPOSE: Subconjunctival fibrosis is the main cause of failure after glaucoma filtration surgery. We explored the effects of sulforaphane (SFN) on the conversion of human Tenon's fibroblasts (HTFs) into myofibroblasts, t...
PURPOSE: Subconjunctival fibrosis is the main cause of failure after glaucoma filtration surgery. We explored the effects of sulforaphane (SFN) on the conversion of human Tenon's fibroblasts (HTFs) into myofibroblasts, transforming growth factor (TGF)-β-induced contraction of collagen gel, and inflammation. METHODS: After treatment with the combination of TGF-β and SFN or TGF-β alone, primary HTFs were subjected to a three-dimensional collagen contraction experiment to examine their contractility. Levels of α smooth muscle actin (α-SMA), synthesis of extracellular matrix (ECM), and phosphorylation of various signaling molecules were determined by western blot or quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Fluorescence microscopy was employed to examine stress fiber formation in HTFs. The expressions of interleukin (IL)-6, IL-8, and connective tissue growth factor (CTGF) were determined using RT-qPCR. RESULTS: The contraction of myofibroblasts caused by TGF-β was significantly suppressed by SFN. This suppressive effect was exerted via the differentiation of HTFs into myofibroblasts by inhibiting the production of fibronectin and the expression of α-SMA. Moreover, SFN treatment reduced the expression of TGF-β-promoted integrins β1 and α5, myosin light chain (MLC) phosphorylation, and stress fiber formation, as well as the expression of IL-6, IL-8, and CTGF. Finally, TGF-β-induced Smad2/3 and extracellular signal-regulated kinase (ERK) phosphorylations were attenuated by SFN. CONCLUSIONS: SFN inhibits HTF contractility, differentiation into myofibroblasts, and inflammation caused by TGF-β. These effects are mediated by both classic and non-classic signaling pathways. Our results indicate that SFN has potent anti-fibrotic and anti-inflammatory effects in HTFs and is a potential candidate for subconjunctival fibrosis therapy.
Increased inflammatory mediators in the ocular surface tissue in keratoconus.
Santos A, Filho JAPM, Cenedeze MA … +7 more , Hiyane MI, Amano MT, Cruz MC, Hirai FE, Camara NOS, de Sousa LB, de Oliveira LA
Mol Vis · 2024 · PMID 39563679
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 c...
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 and 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 and in the corneal epithelium was higher in the KC group than in the control group, with 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.
Complement C3 is downregulated following ranibizumab intervention in experimental central retinal vein occlusion.
Cehofski LJ, Kruse A, Mæng MO … +6 more , Kjaergaard B, Sejergaard BF, Schlosser A, Sorensen GL, Honoré B, Vorum H
Mol Vis · 2024 · PMID 39563678
PURPOSE: Ranibizumab is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the treatment of macular edema following central retinal vein occlusion (CRVO). Studying proteins that mediate the benef...
PURPOSE: Ranibizumab is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the treatment of macular edema following central retinal vein occlusion (CRVO). Studying proteins that mediate the beneficial effects of ranibizumab in CRVO can potentially lead to the improved management of macular edema. METHODS: In 14 Danish Landrace pigs, experimental CRVO was induced in the right eyes and treated with either intravitreal ranibizumab (n = 6) or an intravitreal sodium chloride 9 mg/mL solution as a sham injection (n = 8). Successful CRVO was confirmed by fluorescein angiography. Retinal samples were collected 15 days after induced CRVO and analyzed with label-free, quantification, nano-liquid chromatography-tandem mass spectrometry. Validation was performed with western blotting and immunohistochemistry. RESULTS: CRVO was successfully induced and confirmed by fluorescein angiography. A total of 28 proteins were upregulated, and 31 proteins were downregulated following ranibizumab treatment. A high concentration of the ranibizumab component immunoglobulin kappa chain C region was observed in retinas treated with ranibizumab. Complement C3, the Ig lambda chain C region, and nucleobindin-2 were downregulated following ranibizumab intervention. The downregulation of complement C3 was confirmed by western blotting. Modest changes were observed in the remaining significantly regulated proteins. CONCLUSIONS: Retinal complement C3 was downregulated following ranibizumab intervention in CRVO. The decrease in complement C3 may potentially downregulate the inflammatory response in CRVO. A high retinal concentration of ranibizumab was reached 15 days after injection of the compound.
Clinically correlated dose of the amniotic membrane extract is superior to its transplantation in corneal wound healing.
Korkmaz I, Kocamanoglu M, Gurdal M … +6 more , Arici M, Yaman B, Palamar M, Egrilmez S, Yildirim N, Barut-Selver O
Mol Vis · 2024 · PMID 39563677
PURPOSE: This study investigates the superiority of sterile lyophilized amniotic membrane extract (AME) prepared at a clinically correlated dose over amniotic membrane transplantation (AMT) in an experimental corneal wou...
PURPOSE: This study investigates the superiority of sterile lyophilized amniotic membrane extract (AME) prepared at a clinically correlated dose over amniotic membrane transplantation (AMT) in an experimental corneal wound model. METHODS: AME was prepared from a pool of five amniotic membranes. After homogenizing the membranes, they were lyophilized and sterilized by gamma radiation to obtain sterile, lyophilized AME powder. The total protein amount and growth factor levels were measured in the AME samples. AME eye drops were prepared considering the protein concentration of the standard-size amniotic membrane weight used for transplantation, and this total amount was used as the daily dose. For the experimental animal corneal wound model, a full-thickness mechanical corneal epithelial defect was created in 15 eyes of 15 New Zealand rabbits. The rabbits were divided into four groups: Group 1: AME eye drop (n = 4 eyes), Group 2: AMT (n = 4 eyes), Group 3: preservation-free artificial tear (n = 4 eyes), and Group 4: control (n = 3 eyes). Daily anterior segment evaluation and photography were performed to determine the clinical efficacy of the AME. The rabbits were euthanized on day 7, and wound healing was examined histopathologically. RESULTS: The total protein amount in the AME was 0.149 ± 0.01 mg/ml. The growth factor levels were as follows: EGF = 41.19, FGF = 43.11, HGF = 203.67, KGF = 328.03, NGF = 207.92, and TGF-β = 506.93 pg/ml AME. On clinical examination, the mean wound closure times in Groups 1, 3, and 4 were 2.75 ± 0.50 (2-3), 3.5 ± 1.0 (3-5), and 3.33 ± 1.52 (2-5) days, respectively ( > 0.05). Histopathological examination revealed Group 1 corneal epithelium with full thickness, regular healing pattern, and normal anterior stromal keratocytes. In the remaining three groups, there were interruptions in epithelial healing, and loss of anterior stromal keratocytes was evident. Inflammation was more prominent in Group 2. CONCLUSIONS: AME is a liquid product that contains the essence of the amniotic membrane after homogenization and centrifugation. AME has the potential to overcome the disadvantages of AMT, such as surgery requirement and the limitation of postoperative objective clinical observation due to the semi-opaque nature of the amniotic membrane. Although, there are studies showing the advantages of AME over AMT in the literature, the preparation, preservation and sterilization of AME are still controversial. This study is specifically addressing the shortcomings of acquiring AME in the literature, such as minimizing inter-donor variability in AME by pooling amniotic membranes from different donors, lyophilizing AME to preserve its biochemical composition, and preventing infection transmission by using gamma sterilization. Herein, we observed that the AME prepared with this method contains high concentrations of growth factors. In the present study, the dose of AME was correlated with clinical use for the first time, and for the first time, the superiority of sterile lyophilized AME over AMT was clinically demonstrated in a corneal wound model. Furthermore, histopathological findings confirmed that AME seems to not only promote epithelial proliferation during wound healing but also prevent stromal keratocyte loss, inhibit inflammation and accelerate collagen remodeling.
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