Molecular Vision[JOURNAL]
Sun
200 papers
Neural differentiation of human retinal pigment epithelial cells on alginate/gelatin substrate.
Shamsnajafabadi H, Soheili ZS, Samiee S … +3 more , Ahmadieh H, Pirmardan ER, Haghighi M
Mol Vis · 2022 · PMID 36601411
PURPOSE: The development of biomaterials provides potent promise for the regeneration of neuroretinal cells in degenerative eye diseases and retinal tissue engineering. Biomimetic three-dimensional (3D) microenvironments...
PURPOSE: The development of biomaterials provides potent promise for the regeneration of neuroretinal cells in degenerative eye diseases and retinal tissue engineering. Biomimetic three-dimensional (3D) microenvironments and specific growth factors motivate the differentiation of human retinal pigment epithelial (hRPE) cells toward a retinal neural lineage. In this study, we evaluated alginate/gelatin (A/G) as a substrate for the culture of hRPE cells. METHODS: hRPE cells were isolated from neonatal human cadaver globes and cultivated on A/G substrate under different culture conditions, including 30% human amniotic fluid (HAF), 10% fetal bovine serum (FBS), and serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12). The proliferation of cells in different culture conditions was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and a cell proliferation assay. Immunocytochemistry and real-time PCR were performed to evaluate the effect of the substrate on hRPE cell differentiation. RESULTS: A significant increase in the cell proliferation rate was observed in hRPE cells cultivated on an A/G substrate. Continuous observations demonstrated that hRPE cells formed densely packed, suspended spheroids in DMEM/F12 culture conditions, with dominant transdifferentiation into amacrine cells. Small adherent clusters of hRPE cells in HAF- and FBS-treated cultures represented dedifferentiation toward retinal progenitor cells. These cultures generated amacrine, rod photoreceptors, and bipolar cells. CONCLUSIONS: These findings indicated that A/G substrate induced neural retinal cell propagation in cultures and would therefore be promising for RPE-based tissue engineering studies.
In vitro induction and intraocular application in oxygen-induced retinopathy of adipose-derived mesenchymal stem cells.
Zhou L, Zhang H, Wu S … +2 more , He Y, Guo K
Mol Vis · 2022 · PMID 36601410
PURPOSE: We designed a study to find theoretical evidence for the induction, movement, fusion, proliferation, and safety of human adipose mesenchymal stem cells (hADSCs) in intraocular application. METHODS: HADSCs were i...
PURPOSE: We designed a study to find theoretical evidence for the induction, movement, fusion, proliferation, and safety of human adipose mesenchymal stem cells (hADSCs) in intraocular application. METHODS: HADSCs were induced to confirm that they can express the characteristics of endothelial cells (ECs) in vitro. HADSCs were intraocularly injected into oxygen-induced retinopathy (OIR) mice to check the movement, fusion, proliferation, and prognosis in vivo. Electron microscopy was used to check retinal changes to confirm the safety of hADSCs in intraocular application. RESULTS: After induction, hADSCs expressed von Willebrand Factor (vWF), the cell marker of ECs. The hADSCs were distributed above the retina after an intravitreal injection in the OIR mice. The injected cells did not fuse with the retina and gathered in the central and peripheral areas, which is the lesion area of the OIR model. Five days after the hADSC intravitreal injection, the area of neovascularization was reduced by 94.83% compared with that of the OIR group. Hematologic staining and electron microscopy did not show noticeable proliferation and degeneration of the retina. CONCLUSIONS: This study provides evidence for the intraocular application of hADSCs.
Lipoxin A4 alleviates inflammation in stimulated human corneal epithelial cells by Nrf2/HO-1 signaling pathway.
Peng X, Zhu X, Luan J … +4 more , Lin J, Zhang Y, Wang Q, Zhao G
Mol Vis · 2022 · PMID 36601409
PURPOSE: To investigate the therapeutic effect of lipoxin A4 (LXA4) on -stimulated human corneal epithelial cells (HCECs). METHODS: The cell counting kit-8 (CCK-8) was performed in HCECs to evaluate the toxicity of LXA4....
PURPOSE: To investigate the therapeutic effect of lipoxin A4 (LXA4) on -stimulated human corneal epithelial cells (HCECs). METHODS: The cell counting kit-8 (CCK-8) was performed in HCECs to evaluate the toxicity of LXA4. A cell scratch test was used to assess the impact of LXA4 on the migration of HCECs. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot were applied to examine the expression of inflammatory mediators in -stimulated HCECs. The nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and expression in HCECs were detected by immunofluorescence staining. RESULTS: LXA4 at 0-10 nmol·L (nM) had no significant cytotoxic effect on HCECs. LXA4 at a concentration of 1 nM and 10 nM significantly promoted the migration rate of HCECs. The mRNA and protein levels of pro-inflammatory mediators, including IL-1β, TNF-α, and IL-6, were remarkably lower in the LXA4-treated group. LXA4 promoted the expression of Nrf2 and heme oxygenase 1 (HO-1) in -stimulated HCECs compared with the PBS control group. Pretreatment with brusatol (BT, Nrf2 inhibitor) or Zine Protoporphyrin (Znpp, HO-1 inhibitor) receded the anti-inflammatory ability of LXA4. CONCLUSIONS: LXA4 plays a protective role in -stimulated HCECs by inhibiting the expression of pro-inflammatory mediators through the Nrf2/HO-1 signaling pathway.
αA and αB peptides from human cataractous lenses show antichaperone activity and enhance aggregation of lens proteins.
Srivastava O, Wilson L, Barnes S … +2 more , Srivastava K, Joseph R
Mol Vis · 2022 · PMID 36540064
PURPOSE: To identify and characterize properties of αA- and αB-crystallins' low molecular weight peptides (molecular weight [Mr] < 5 kDa) that were present in a 62-year-old human nuclear cataract, but not in normal 62-ye...
PURPOSE: To identify and characterize properties of αA- and αB-crystallins' low molecular weight peptides (molecular weight [Mr] < 5 kDa) that were present in a 62-year-old human nuclear cataract, but not in normal 62-year-old human lenses. METHODS: Low molecular weight peptides (< 5 kDa) were isolated with a trichloroacetic acid (TCA) solubilization method from water-soluble (WS) and water-insoluble (WI) proteins of nuclear cataractous lenses of a 62-year-old donor and normal human lenses from an age-matched donor. Five commercially synthesized peptides (found only in cataractous lenses and not in normal lenses) were used to determine their chaperone and antichaperone activity and aggregation properties. RESULTS: Mass spectrometric analysis showed 28 peptides of αA-crystallin and 38 peptides of αB-crystallin were present in the cataractous lenses but not in the normal lenses. Two αA peptides (named αAP1 and αAP2; both derived from the αA N-terminal domain (NTD) region) and three αB peptides (named αBP3, αBP4, and αBP5, derived from the αB NTD-, core domain (CD), and C-terminal extension (CTE) regions, respectively) were commercially synthesized. αAP1 inhibited the chaperone activity of αA- and αB-crystallins, but the other four peptides (αAP2, αBP3, αBP4, and αBP5) exhibited mixed effects on chaperone activity. Upon incubation with human WS proteins and peptides in vitro, the αBP4 peptide showed higher aggregation properties relative to the αAP1 peptide. During in vivo experiments, the cell-penetrating polyarginine-labeled αAP1 and αBP4 peptides showed 57% and 85% aggregates, respectively, around the nuclei of cultured human lens epithelial cells compared to only 35% by a scrambled peptide. CONCLUSIONS: The antichaperone activity of the αAP1 peptide and the aggregation property of the αBP4 peptide with lens proteins could play a potential role during the development of lens opacity.
Morphological, biochemical, and transcriptomic characterization of iPSC-derived human RPE cells from normal and Smith-Lemli-Opitz syndrome patients.
Farkas MH, Skelton LA, Ramachandra-Rao S … +2 more , Au E, Fliesler SJ
Mol Vis · 2022 · PMID 36540063
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A five-year follow-up of carriers showing deterioration of retinal function and increased structural changes.
Kjellström U, Andréasson S
Mol Vis · 2022 · PMID 36338671
PURPOSE: To investigate whether the reduced retinal function and morphological retinal changes previously demonstrated in carriers had remained stationary or had deteriorated over time at 5-year follow-up to further exp...
PURPOSE: To investigate whether the reduced retinal function and morphological retinal changes previously demonstrated in carriers had remained stationary or had deteriorated over time at 5-year follow-up to further explore if carriers of an autosomal recessive trait also express a weak phenotype, although this is not expected for an autosomal recessive disorder. METHODS: Thirteen carriers from a previous study that included parents to patients with well known genetically verified -associated retinal degenerations were reexamined 5 years after the initial examination. As novel genes and new variants in already established genes are continuously reported, all subjects underwent renewed genetic testing with a next-generation sequencing (NGS) panel that included 288 genes associated with retinal dystrophies and an analysis of deep intronic mutations and copy number variations in the gene. Moreover, to evaluate any changes in retinal function and/or structure over time, clinical reassessment with Goldmann perimetry, visual acuity testing, fundus photography, fundus autofluorescence (FAF) imaging, optical coherence tomography (OCT), full-field electroretinography (ffERG), and multifocal ERG (mfERG) were performed 5 years after the initial investigation. The values of the ffERG parameters were compared between the two time points (the measurements obtained in the initial study versus the measurements at 5-year follow-up) and with the controls. The mfERG results of the carriers were compared with those of the controls. RESULTS: The renewed genetic testing confirmed the previously established mutations but also revealed the hypomorph variant c.5603A>T in five carriers. In three of them, the variant was found to be associated with known disease-causing alleles that always carry the c.5603A>T in cis. According to recent publications, the subjects could still be considered carriers because both variants are on the same allele. In the remaining two subjects, c.5603A>T could be in trans with the previously known variant, and the subjects were therefore excluded from the study since they could no longer be considered as carriers only. Statistical comparison of ffERG parameters showed significant reduction of the isolated rod, -as well as the combined rod-cone amplitudes over the five years of follow-up, but not compared with the controls. Concerning macular function, mfERG amplitudes were reduced for all rings in the carriers compared with the controls. Fundus photographs demonstrated morphological changes in 64% of the carriers, and 36% of them had further changes at follow-up. FAF images showed alterations in 55% of the carriers, with increased changes in 36% of them. Abnormalities on OCT were observed in 82% of the carriers, of whom 9% had newly found abnormalities at follow-up. CONCLUSIONS: At 5-year follow-up, the carriers, who previously demonstrated reduced macular function, presented with deterioration of general retinal function, including reduced isolated rod and mixed rod-cone ffERG responses combined with a slight increase in morphological changes in some subjects. This indicates that carriership of at least some variants may cause a condition similar to a subgroup of dry age-related macular degeneration (AMD). In the long run, this might be of importance concerning the possibilities to also treat this subgroup of AMD patients with future gene-based and pharmacological drugs targeting -associated disorders.
Sulforaphane recovers cone function in an Nrf2-dependent manner in middle-aged mice undergoing RPE oxidative stress.
Qi X, Walton DA, Plafker KS … +2 more , Boulton ME, Plafker SM
Mol Vis · 2022 · PMID 36338670
PURPOSE: Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables that has therapeutic efficacy in numerous animal models of human disease, including mouse models of retinal degeneration. However, desp...
PURPOSE: Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables that has therapeutic efficacy in numerous animal models of human disease, including mouse models of retinal degeneration. However, despite dozens of clinical trials, the compound remains to be tested as a clinical treatment for ocular disease. Numerous cellular activities of SFN have been identified, including the activation of Nrf2, a transcription factor that induces a battery of target gene products to neutralize oxidative and xenobiotic stresses. As Nrf2 expression and function reportedly decrease with aging, we tested whether the loss of the transcription factor limits the therapeutic efficacy of SFN against retinal degeneration. METHODS: Six- to 8-month-old wild-type and Nrf2 knockout mice were treated with SFN beginning 1 month after ribozyme-mediated knockdown of superoxide dismutase 2 (SOD2) mRNA in the RPE. The impacts of MnSOD (the protein product of SOD2) knockdown and the efficacy of SFN were evaluated using a combination of electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT), and postmortem histology. RESULTS: SFN restored the ERG photopic b-wave suppressed by MnSOD loss in wild-type mice, but not in the Nrf2 knockout mice. In contrast, ERG scotopic a- and b-wave loss was not restored for either genotype. SFN significantly improved retinal thickness in the Nrf2 knockout mice with MnSOD knockdown, but this was not observed in the wild-type mice. In both genotypes, SFN treatment reduced morphological markers of RPE atrophy and degeneration, although these improvements did not correlate proportionally with functional recovery. CONCLUSIONS: These findings highlight the capacity of SFN to preserve cone function, as well as the potential challenges of using the compound as a standalone treatment for age-related retinal degeneration under conditions associated with reduced Nrf2 function.
Autosomal dominant retinitis pigmentosa with incomplete penetrance due to an intronic mutation of the gene.
Ali-Nasser T, Zayit-Soudry S, Banin E … +2 more , Sharon D, Ben-Yosef T
Mol Vis · 2022 · PMID 36338669
PURPOSE: To identify the molecular mechanisms of the development of autosomal dominant retinitis pigmentosa (adRP) with incomplete penetrance in an Israeli Muslim Arab family. METHODS: Two patients with adRP underwent a...
PURPOSE: To identify the molecular mechanisms of the development of autosomal dominant retinitis pigmentosa (adRP) with incomplete penetrance in an Israeli Muslim Arab family. METHODS: Two patients with adRP underwent a detailed ophthalmic evaluation, including funduscopic examination, visual field testing, optical coherence tomography, and electroretinography. Genetic analysis was performed using a combination of whole exome sequencing (WES) and Sanger sequencing. The pathogenicity of the identified intronic variant was evaluated in silico using several web-based tools, in vitro using a minigene-based assay, and in vivo using reverse transcription PCR analysis of lymphocyte-derived RNA. The relative abundance of alternatively spliced transcripts was evaluated using amplicon-based next-generation sequencing. The relative expression levels of and were measured using quantitative PCR (qPCR) analysis. RESULTS: The two patients recruited in this study had childhood-onset RP, with night blindness as the initial symptom, followed by concentric restriction of the visual field. The funduscopic findings included narrowed retinal blood vessels and peripheral bone spicule pigmentation. By the third decade of life, the full-field electroretinography findings had been remarkably attenuated. In these patients, we identified a novel heterozygous intronic variant at position +5 of intron 11 (c.1146+5G>T). The same variant was also detected in one asymptomatic family member. Through in silico analysis, the variant was predicted to alter the splicing of intron 11. An in vitro splicing assay and a reverse transcription PCR analysis of lymphocyte-derived RNA revealed that the mutant allele yielded mainly a shorter transcript in which exon 11 was skipped. The skipping of exon 11 was expected to cause a frameshift and an aberrant truncated protein (p.Tyr359Ser*29). The qPCR analysis revealed reduced expression levels in the mutation carriers, without a significant difference between the affected patient and his asymptomatic brother. We evaluated several factors that have been suggested to correlate with non-penetrance of mutations, including the number of cis-acting MSR1 elements adjacent to the core promoter, expression level, and rs4806718 single-nucleotide polymorphism. None of these factors correlated with non-penetrance in the family in this study. CONCLUSIONS: We report a novel intronic mutation in underlying adRP. This report expands the spectrum of pathogenic mutations in and further demonstrates the importance of intronic mutations. Moreover, it demonstrates the phenomenon of incomplete penetrance previously associated with mutations. The fact that the non-penetrance in the family in this study could not be explained by any of the known mechanisms suggests the possible contribution of a novel modifier of penetrance.
Oxidative stress alters transcript localization of disease-associated genes in the retinal pigment epithelium.
Kaczynski TJ, Au ED, Farkas MH
Mol Vis · 2022 · PMID 36338668
PURPOSE: Nuclear retention is a mechanism whereby excess RNA transcripts are stored in the event that a cell needs to quickly respond to a stimulus; maintaining proper nuclear-to-cytoplasmic balance is important for cell...
PURPOSE: Nuclear retention is a mechanism whereby excess RNA transcripts are stored in the event that a cell needs to quickly respond to a stimulus; maintaining proper nuclear-to-cytoplasmic balance is important for cellular homeostasis and cell function. There are many mechanisms that are employed to determine whether to retain a transcript or export it to the cytoplasm, although the extent to which tissue or cell type, internal and external stressors, and disease pathogenesis affect this process is not yet clear. As the most biochemically active tissue in the body, the retina must mitigate endogenous and exogenous stressors to maintain cell health and tissue function. Oxidative stress, believed to contribute to the pathogenesis or progression of age-related macular degeneration (AMD) and inherited retinal dystrophies (IRDs), is produced both internally from biochemical processes as well as externally from environmental insult. Here, we evaluate the effect of oxidative stress on transcript localization in the retinal pigment epithelium (RPE), with specific focus on transcripts related to RPE function and disease. METHODS: We performed poly(A) RNA sequencing on nuclear and cytoplasmic fractions from human induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) cells exposed to hydrogen peroxide (HO), as well as on untreated controls. RESULTS: Under normal conditions, the number of mRNA transcripts retained in the nucleus exceeded that found in studies on other tissues. Further, the nuclear-to-cytoplasmic ratio of transcripts was altered following oxidative stress, as was the retention of genes associated with AMD and IRDs, as well as those that are important for RPE physiology. CONCLUSIONS: These results provide a localization catalog of all expressed mRNA in iPSC-RPE under normal conditions and after exposure to HO, shedding light on the extent to which HO alters transcript localization and potentially offering insight into one mechanism through which oxidative stress may contribute to the progression of visual disorders.
Structural and functional analysis of SNP rs76740365 G>A in exon-3 of the alpha A-crystallin gene in lens epithelial cells.
Zhao Z, Sun Y, Fan Q … +2 more , Jiang Y, Lu Y
Mol Vis · 2022 · PMID 36338667
PURPOSE: To clarify the effect of a previously identified single nucleotide polymorphism (SNP; rs76740365 G>A) in the exon-3 of the gene on the properties of and to investigate its function in human lens epithelial cel...
PURPOSE: To clarify the effect of a previously identified single nucleotide polymorphism (SNP; rs76740365 G>A) in the exon-3 of the gene on the properties of and to investigate its function in human lens epithelial cells (HLECs). METHODS: The human recombinant wild-type and mutant (E156K) were constructed, and the molecular weight was measured by mass spectrometry. The structural changes induced by E156K mutation were analyzed by UV circular dichroism spectra and intrinsic tryptophan fluorescence and were predicted using Schrödinger software. The chaperone-like ability of wild-type and E156K mutant was invested against the heat-induced aggregation of βL-crystallin and the DTT-induced aggregation of insulin. HLECs expressing wild-type and mutated were subjected to quantitative PCR (qPCR) and western blot. Cell apoptosis was determined using flow cytometry analysis, and the expression of apoptosis-related proteins were determined using western blot. RESULTS: The mass spectrometric detection revealed that E156K mutation had no significant effect on the apparent molecular mass of the oligomeric complex. Evaluation of the structures of the indicated that E156K mutation did not significantly affect the secondary structures, while causing perturbations of the tertiary structure. The mutant displayed an increase in chaperone-like activity, which might be related to the increase of the surface hydrophobicity. We also predicted that E156K mutation would induce a change from negatively charged surface to positively charged, which was the possible reason for the disturbance to the surface hydrophobicity. Transfection studies of HLECs revealed that the E156K mutant induced anti-apoptotic function in HLECs, which was possibly associated with the activation of the p-AKT signal pathway and downregulation of Casepase3. CONCLUSIONS: Taken together, our results for the first time showed that E156K mutation in associated with ARC resulted in enhanced chaperone-like function by inducing its surface hydrophobicity, which was directly related to the activation of its anti-apoptotic function.
Clinical and genetic studies of 17 Han Chinese pedigrees and 31 sporadic patients with blepharophimosis-ptosis-epicanthus inversus syndrome.
Wang Y, Wu Q, Cao W … +4 more , Huang L, Liu W, Li C, Li N
Mol Vis · 2022 · PMID 36338666
PURPOSE: To investigate the molecular pathogenesis of a large group of Han Chinese patients with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), and to evaluate the correlation between the phenotype and geno...
PURPOSE: To investigate the molecular pathogenesis of a large group of Han Chinese patients with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), and to evaluate the correlation between the phenotype and genotype for these patients. METHODS: Seventy-six affected individuals, including 45 patients from 17 pedigrees and 31 sporadic patients, were recruited with their family members. All participants underwent complete clinical examinations and were classified as having type I or II based on whether they had premature ovarian failure. The patients' genomic DNA was extracted. A genetic test was performed with direct sequencing of the coding regions of the () gene. Variations were analyzed using online databases and programs. Genotype-phenotype correction was investigated. RESULTS: Seventy-six affected and 75 unaffected individuals underwent clinical evaluations and genetic testing. Only one family was diagnosed with type I; the others could not be classified because of a lack of female patients or a definite history of premature ovarian failure. Twenty-seven variations were identified, including 12 novel and 15 previously reported variations. Six variations were detected repeatedly in different nonconsanguineous pedigrees. Four indel variations, located in the alanine/proline-rich region of the gene, presented with a relatively higher frequency. Two rare double variations were detected in two sporadic patients. gene variations were not detected in five sporadic patients. The phenotype varied among different families and patients, although they carried the same variations. CONCLUSIONS: We identified 12 novel variations in the gene that would expand the spectrum of the variation database. In addition, we found that the alanine/proline-rich region is a variation hotspot in the gene. The genotype-phenotype correlation is not easy to establish due to clinical and genetic heterogeneity.
Association of polymorphisms with exotropia in a Pakistani cohort.
Zehra Z, Khan N, Nadeem M … +4 more , Siddiqui SN, von Bartheld CS, Azam M, Qamar R
Mol Vis · 2022 · PMID 36338665
PURPOSE: Strabismus (STBMS) is a multifactorial ocular disorder in children that leads to misalignment of the eyes. Insulin-like growth factor 1 () has been shown to be involved in the development of extraocular muscles...
PURPOSE: Strabismus (STBMS) is a multifactorial ocular disorder in children that leads to misalignment of the eyes. Insulin-like growth factor 1 () has been shown to be involved in the development of extraocular muscles and myopia; however, data are limited on the genetic associations of with STBMS in Pakistan. METHODS: Two hundred seventy-four STBMS cases and 272 unaffected controls were recruited, and their DNA was extracted. Two single nucleotide polymorphisms, rs6214 and rs5742632, were genotyped using PCR-restriction fragment length polymorphism. Univariate logistic regression analysis was performed to determine the association of these single nucleotide polymorphisms with STBMS, and the results were adjusted for age and sex. In addition, 26 extraocular muscle tissues were collected from patients with STBMS undergoing squint correction surgery, along with 3 deceased control samples. mRNA expression was measured by quantitative PCR; the Mann-Whitney U test was applied, and fold change was calculated. Logistic regression analysis was applied to determine the association of RNA expression and fold change with genotype. RESULTS: Multivariate logistic regression analysis revealed that rs5742632 (odds ratio [95% confidence interval] = 1.05[1.01-1.06], p = 0.03) is associated with STBM. Moreover, rs6214 (1.03[1.01-1.05], p = 0.03) and rs5742632 (1.09[1.04-1.11], p = 0.04) were associated with exotropia. Statistically, no significant difference in mRNA expression in the extraocular muscles between the STBMS cases and the controls was observed. CONCLUSIONS: polymorphisms rs5742632 (A>G) and rs6214 (C>T) are plausible risk factors for the development of exotropia. However, the physiologic mechanism requires further evaluation.
Isolation efficiency of collagenase and EDTA for the culture of corneal endothelial cells.
Santerre K, Proulx S
Mol Vis · 2022 · PMID 36338664
PURPOSE: Tissue engineering of the corneal endothelium, as well as cell therapy, has been proposed as an alternative approach for the treatment of corneal endotheliopathies. These approaches require in vitro amplificatio...
PURPOSE: Tissue engineering of the corneal endothelium, as well as cell therapy, has been proposed as an alternative approach for the treatment of corneal endotheliopathies. These approaches require in vitro amplification of functional corneal endothelial cells (CECs). The goal of this study was to compare two common isolation methods, collagenase A and EDTA (EDTA), and determine whether they influence cell viability, morphology, and barrier function. METHODS: Human eye bank research-grade corneas were used to isolate and cultivate CECs. All donors were more than 40 years old. Two Descemet membranes from the same donor were used separately to compare the collagenase A and EDTA cell isolation methods. The number of isolated cells, cell viability, morphology, and barrier functionality were compared. RESULTS: A higher isolation efficiency of viable CECs and a higher circularity index (endothelial morphology) were obtained using collagenase A. Passage 3 cells presented similar barrier functionalities regardless of the isolation method. CONCLUSIONS: This study showed that isolation of CECs using collagenase A yields higher isolation efficiency than EDTA, delaying the loss of endothelial morphology for early passage cells.
Using optical coherence tomography angiography as a biomarker of retinopathy severity and treatment for diabetic retinopathy.
Scheive M, Reinhart KL, Hajrasouliha AR
Mol Vis · 2022 · PMID 36284673
PURPOSE: The goal was to evaluate optical coherence tomography angiography (OCT-A) as a biomarker to correlate retinal vessel density (VD) with diabetic retinopathy (DR) severity and visual acuity, as well as track antiv...
PURPOSE: The goal was to evaluate optical coherence tomography angiography (OCT-A) as a biomarker to correlate retinal vessel density (VD) with diabetic retinopathy (DR) severity and visual acuity, as well as track antivascular endothelial growth factor (VEGF) treatment efficacy. METHODS: This retrospective cohort study analyzed the automatically quantified VDs of the superficial vascular complex (SVC) and deep vascular complex (DVC), including the whole, foveal, and parafoveal VDs, on quality OCT-A scans in patients diagnosed with DR. A multivariate linear regression and analysis of variance (ANOVA) analysis compared VDs to DR severity, visual acuity, and demographic factors. A linear mixed analysis determined the effects of VD by whether anti-VEGF therapy was given to patients with OCT-A scans at multiple time points. RESULTS: There was a positive correlation of the VDs in both the SVC whole and parafoveal VD and DVC parafoveal VD with decreased DR severity and increased visual acuity (p≤0.001). The DVC whole VD was also positively correlated with increased visual acuity (p<0.001). There was no difference in the VDs associated with anti-VEGF treatment over time. CONCLUSIONS: OCT-A VD shows promise for diagnosing and monitoring DR using DR severity and visual acuity. Anti-VEGF treatment had no significant effect (p=0.063) on vascular density in diabetic retinopathy.
Hyposmotic stress causes ATP release in a discrete zone within the outer cortex of rat lens.
Suzuki-Kerr H, Walker KL, Han MH … +2 more , Lim JC, Donaldson PJ
Mol Vis · 2022 · PMID 36284672
PURPOSE: Purinergic signaling pathways activated by extracellular ATP have been implicated in the regulation of lens volume and transparency. In this study, we investigated the location of ATP release from whole rat lens...
PURPOSE: Purinergic signaling pathways activated by extracellular ATP have been implicated in the regulation of lens volume and transparency. In this study, we investigated the location of ATP release from whole rat lenses and the mechanism by which osmotic challenge alters such ATP release. METHODS: Three-week-old rat lenses were cultured for 1 h in isotonic artificial aqueous humor (AAH) with no extracellular Ca, hypotonic AAH, or hypertonic AAH. The hypotonic AAH-treated lenses were also cultured in the absence or presence of connexin hemichannels and the pannexin channel blockers carbenoxolone, probenecid, and flufenamic acid. The ATP concentration in the AAH was determined using a Luciferin/luciferase bioluminescence assay. To visualize sites of ATP release induced by hemichannel and/or pannexin opening, the lenses were cultured in different AAH solutions, as described above, and incubated in the presence of Lucifer yellow (MW = 456 Da) and Texas red-dextran (MW = 10 kDa) for 1 h. Then the lenses were fixed, cryosectioned, and imaged using confocal microscopy to visualize areas of dye uptake from the extracellular space. RESULTS: The incubation of the rat lenses in the AAH that lacked Ca induced a significant increase in the extracellular ATP concentration. This was associated with an increased uptake of Lucifer yellow but not of Texas red-dextran in a discrete region of the outer cortex of the lens. Hypotonic stress caused a similar increase in ATP release and an increase in the uptake of Lucifer yellow in the outer cortex, which was significantly reduced by probenecid but not by carbenoxolone or flufenamic acid. CONCLUSIONS: Our data suggest that in response to hypotonic stress, the intact rat lens is capable of releasing ATP. This seems to be mediated via the opening of pannexin channels in a specific zone of the outer cortex of the lens. Our results support the growing evidence that the lens actively regulates its volume and therefore, its optical properties, via puerinergic signaling pathways.
Untargeted metabolomic analysis of aqueous humor in diabetic macular edema.
Chu KO, Chan TI, Chan KP … +5 more , Yip YW, Bakthavatsalam M, Wang CC, Pang CP, Brelen ME
Mol Vis · 2022 · PMID 36284671
BACKGROUND: The mechanism of diabetic macular edema (DME) was explored by comparing the intraocular metabolite profiles of the aqueous humor of patients with DME to those of diabetic patients without DME using untargeted...
BACKGROUND: The mechanism of diabetic macular edema (DME) was explored by comparing the intraocular metabolite profiles of the aqueous humor of patients with DME to those of diabetic patients without DME using untargeted metabolomic analysis. METHODS: Aqueous samples from 18 type 2 diabetic patients with DME and 18 type 2 diabetic patients without DME used as controls were analyzed using liquid chromatography-mass spectrometry (LCMS). The two groups of patients were age and gender matched and had no systemic diseases other than diabetes mellitus (DM). The metabolites were analyzed using orthogonal partial least square discriminant analysis. RESULTS: The metabolite profiles in DME patients differed from those in DM controls. This indicates the following metabolic derangements in DME: (a) a higher amount of oxidized fatty acids but a lower amount of endogenous antioxidants (oxidative stress); (b) higher levels of β-glucose and homocysteine but a lower level of sorbitol (hyperglycemia); (c) a higher amount of prostaglandin metabolites (inflammation); (d) higher amounts of acylcarnitines, odd-numbered fatty acids, and 7,8-diaminononanoate (respiration deterioration); (e) a higher amount of neurotransmitter metabolites and homovanillic acid (neuronal damage); (f) a lower amount of extracellular matrix (ECM) constituents (ECM deterioration); and (g) a higher amount of di-amino peptides (microvascular damage). CONCLUSIONS: The change in the metabolic profiles in the aqueous humor of DME patients compared to DM controls without DME indicates that DME patients may have less capability to resist various stresses or damaging pathological conditions, such as oxidative stress, mitochondrial insufficiency, inflammation, and ECM deterioration.
Identification of numerous novel disease-causing variants in patients with inherited retinal diseases, combining careful clinical-functional phenotyping with systematic, broad NGS panel-based genotyping.
Gupta PR, Kheir W, Peng B … +3 more , Duan J, Chiang JP, Iannaccone A
Mol Vis · 2022 · PMID 36284670
PURPOSE: The widespread consensus is that genotyping is essential for patients with inherited retinal disease (IRD). Given the numerous ongoing gene therapy clinical trials for IRDs, identifying the pathogenic mutation i...
PURPOSE: The widespread consensus is that genotyping is essential for patients with inherited retinal disease (IRD). Given the numerous ongoing gene therapy clinical trials for IRDs, identifying the pathogenic mutation in these patients has potential important therapeutic implications. In this study, we demonstrate how we identified with a high degree of confidence numerous novel disease-causing mutations, deletions, and duplications in a large consecutive IRD case series by using a judicious combination of careful, in-depth clinical-functional phenotyping to guide and integrate our genotyping approach. METHODS: We conducted a retrospective analysis of data between November 2016 and March 2018 from the Duke Center for Retinal Degenerations and Ophthalmic Genetic Diseases IRD patient database, which encompassed 378 IRD cases that had not yet been previously genotyped. With the exception of some patients who presented with classical clinical-functional phenotypes that allowed for targeted gene testing, all other subjects systematically underwent next-generation sequencing-based broad, IRD-focused panel testing. Most cases were also tested for parental allele phase. Results were reviewed vis-à-vis the clinical-functional phenotypes for reconciliation and potential addition of supplemental testing such as deletion/duplication microarrays or copy number variant (CNV) analysis. Supplemental testing was driven by an IRD specialist-laboratory consensus, and decisions were clinically or genetically driven or both. RESULTS: By judiciously using this two-way approach and leveraging to its full potential the benefits of careful, in-depth clinical-functional phenotyping by an experienced IRD specialist, more than 80% of the cases in this series were successfully genotyped. We also identified with a high degree of confidence 52 novel disease-causing mutations, deletions, and duplications. CONCLUSIONS: The combination of meticulous, expert clinical-functional phenotyping studies with systematic next-generation sequencing panel-based genotyping and microarray deletion/duplication testing or CNV analysis as applicable in accordance with the above-mentioned consensus was extremely effective at the diagnostic end, reduced costs, and saved time. IRD specialist-laboratory two-way interactions and case discussions would augment the efficacy of this approach and improve the diagnostic yield in successfully solving and genotyping IRD cases.
Nestin contributes to laser choroidal and retinal neovascularization.
Miloudi S, Valensi M, El Sanharawi M … +3 more , Abitbol MM, Behar-Cohen F, Versaux-Botteri C
Mol Vis · 2022 · PMID 36284669
PURPOSE: Choroidal and retinal neovascularization plays an essential role in various ocular diseases. In this study, we examined the role of nestin in this process. Nestin is an intermediate filament protein known to pla...
PURPOSE: Choroidal and retinal neovascularization plays an essential role in various ocular diseases. In this study, we examined the role of nestin in this process. Nestin is an intermediate filament protein known to play several roles, including as a marker of neural progenitor and proliferating endothelial cells. METHODS: We used Brown Norway rats, in which choroidal and retinal neovascularization was induced using intraocular laser impacts. The role of nestin was examined using angiography, western blot from the second to the 14th day after laser impacts, and intraocular injection of nestin siRNA. The localization of the protein was specified by co-immunoreactivity with glial fibrillary protein (GFAP), glutamine synthetase (GS), and von Willebrand factor (vWF). RESULTS: In the control retina, nestin was found principally in glial structures in the ganglion cell layer, as confirmed by nestin/GFAP immunolabeling. Two days after the laser impacts, the nestin expression extended to numerous radial processes at the site of the impacts. With Bruch's membrane ruptured, these processes penetrated into the choroid. Nestin immunolabeling remained high from the third to the seventh day but appeared reduced on the 14th day. The nature of these processes was not clearly defined, but co-immunolabeling with GFAP suggested that they were principally in activated Müller cells from the third day after the laser impacts. However, the co-immunoreactivity of nestin and GS, a marker of mature functional Müller cells, could be observable only from the seventh day. Nestin was also observed in some vascular cells, as demonstrated by the co-immunoreactivity of the protein with vWF in the choroid and retina. As observed on angiography, the numbers of choroidal and retinal blood vessels were significantly increased (principally on the seventh day) after the laser impacts. An intraocular injection of nestin siRNAs led to a significant decrease in the number of blood vessels. CONCLUSIONS: Our results confirmed the presence of nestin in glial (e.g., astrocytes), reactive Müller, and endothelial cells. They demonstrated their critical involvement in a rat model of retinal and choroidal neovascularization experimentally induced using ocular laser impacts.
The long noncoding RNA MALAT1/microRNA-598-3p axis regulates the proliferation and apoptosis of retinoblastoma cells through the PI3K/AKT pathway.
Lin X, Huang X, Wang L … +1 more , Liu W
Mol Vis · 2022 · PMID 36284668
PURPOSE: This study was designed to dissect the role of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in retinoblastoma (RB) and its underlying mechanism. METHODS: Gain- and loss-of-f...
PURPOSE: This study was designed to dissect the role of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in retinoblastoma (RB) and its underlying mechanism. METHODS: Gain- and loss-of-function experiments were adopted to explore the effects of MALAT1 and microRNA (miR)-598-3p on the biologic behaviors of RB cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression of MALAT1 and miR-598-3p in Y79 and HXO-RB44 cells. The proliferation of RB cells was determined with the cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) staining. Flow cytometry was employed for the measurement of the apoptotic rate, western blotting for examination of the expression of apoptosis-related proteins (Bax and Bcl-2) and phosphoinositide 3-kinase/protein kinase-B (PI3K/AKT) pathway-related factors (PI3K, AKT, p-PI3K, and p-AKT), and the luciferase reporter assay for assessment of the interaction between MALAT1 and miR-598-3p. RESULTS: High expression of MALAT1 and low expression of miR-598-3p were noticed in Y79 and HXO-RB44 cells. MALAT1 upregulation or miR-598-3p downregulation facilitated RB cell proliferation and inhibited cell apoptosis, as evidenced by the increased proliferation rate and Bcl-2 expression, as well as diminished Bax expression and apoptotic rate, in the RB cells after transfection with pcDNA3.1-MALAT1 or miR-598-3p inhibitor. MALAT1 bound to and negatively regulated miR-598-3p. The PI3K/AKT pathway activation occurred with MALAT1 overexpression. MALAT1 promoted RB cell proliferation and repressed cell apoptosis by repressing miR-598-3p to activate the PI3K/AKT pathway. CONCLUSIONS: MALAT1 repressed miR-598-3p to activate the PI3K/AKT pathway, thus facilitating cell proliferation and inhibiting cell apoptosis in RB.
The phenotypic spectrum of associated ectopia lentis: Additional cases, complications, and review of literature.
Knight LSW, Mullany S, Taranath DA … +9 more , Ruddle JB, Barnett CP, Sallevelt SCEH, Berry EC, Marshall HN, Hollitt GL, Souzeau E, Craig JE, Siggs OM
Mol Vis · 2022 · PMID 36284667
PURPOSE: associated ectopia lentis is a rare autosomal recessive condition that is primarily associated with crystalline lens displacement. However, the prevalence of other ocular and systemic manifestations of this cond...
PURPOSE: associated ectopia lentis is a rare autosomal recessive condition that is primarily associated with crystalline lens displacement. However, the prevalence of other ocular and systemic manifestations of this condition is poorly understood. In this study, we summarize the ocular and systemic phenotypic spectrum of this condition. METHODS: A cross-sectional case study series of four individuals with biallelic pathogenic or likely pathogenic variants was performed alongside a literature review of individuals with -associated ectopia lentis on September 29, 2021. Ocular and systemic findings, complications, and genetic findings of all four individuals were collected and summarized. RESULTS: The phenotypic spectrum across 91 individuals sourced from literature and four individuals from this case study series was highly variable. The main ocular phenotypes included ectopia lentis (95/95, 100%), ectopia lentis et pupillae (18/95, 19%), iris transillumination (13/95, 14%), iridodonesis (12/95, 13%), persistent pupillary membrane (12/95, 13%), and early-onset cataract or lens opacities (12/95, 13%). Anterior segment features other than ectopia lentis appeared to be exclusively associated with biallelic loss of function variants (p<0.001). Pupillary block glaucoma had a prevalence of 1%. Post-lensectomy complications included retinal detachment (6/41, 15%), elevated intraocular pressure (4/41, 10%), and aphakic glaucoma (1/41, 2%). Most individuals were not reported to have had systemic features (69/95, 73%). CONCLUSIONS: The clinical phenotype of -associated ectopia lentis was summarized and expanded. Clinicians should be aware of the varied ocular phenotype and the risks of retinal detachment, ocular hypertension, and glaucoma in the diagnosis and management of this condition.
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