Abstract Diabetic retinopathy (DR) is a leading cause of blindness in the working population of the Western world. Advances in our understanding of specific variables contributing to this condition is restricted by the lack of characterization available with in vitro model systems. In this study we establish a retinal porcine model and investigate the effect of hyperglycemia on the level of hypoxia inducible factor type 1 alpha in the porcine retina. Porcine retinae were excised from porcine eyes and cultured in 5 or 25 mM D-glucose in Dulbecco’s Minimal essential Media (DMEM), without a matrix (2D) or within a matrix (3D) consisting of a co-gel (0.5% agarose plus collagen 0.25mg/ml) in DMEM and maintained in a humidified atmosphere with 5% CO2 at 37 °C. This process took 30 min. Retinal integrity was monitored by visualization with a light microscope followed by hematoxylin and eosin staining and Gomori Trichrome. Matrix oxygen saturation (% O2) was recorded with a Microx TX3 oxygen microsensor at time zero and 24 h following incubation. Immunostaining for HIF-1α was performed using primary mouse anti-HIF-1α (1:20) followed by anti-mouse DyLight 488 goat secondary antibody (1:200) and visualized with an inverted fluorescent microscope. Retinal tissue and vessel integrity were morphologically preserved on 3D matrices versus 2D control evidenced by hematoxylin and eosin staining. A time dependent increase in collagen incorporation within the explant was seen with the Gomori Trichrome stain. Oxygen levels significantly increased within the explant during the 23h test period from 15.5% to 22.3% (p<0.001) in media alone and from 10% to 13% (p<0.05) with the matrix (n=6) with all values relative to atmospheric pressure. HIF-1α was detected by immune-histochemical analysis in explants at t=0 in 5 and 25mM glucose concentration, with staining of serial sections with DAPi. HIF-1α levels only remained elevated at 24 h in 25 mM D-glucose and was undetected in 5 mM D-glucose. This study presents a novel model of retinal explants demonstrating that retinal tissue can be cultured in non-hypoxic conditions and that the 3-dimensional matrix was able to sustain retinal structure. Retinal structure was, at least partially sustained by the incorporation of collagen into the retina. Differential expression of protein was identified in the different matrix environment; specifically, hyperglycemia sustained non-hypoxia induced HIF-1α expression in isolated retina.
Abstract Purpose Inflammatory processes in the aged retina may be exacerbated by systemic infection with viruses and bacteria, potentially aggravating age-related macular degeneration (AMD). Toll-like receptors (TLR) are key mediators in detecting pathogen-associated molecular patterns (PAMPs) and host-derived damage-associated molecular patterns (DAMPs). This study aimed to investigate TLR gene expression in cultured retinal pigment epithelial (RPE) cells, focusing particularly on TLR9. Methods TLR gene expression was assessed using semiquantitative qPCR, while protein expression was evaluated through western blotting, immunocytochemistry, and ELISA. Hypoxic conditions were simulated using CoCl2 or by incubation in a 0.1% O2 atmosphere. RPE cell proliferation and viability were examined using bromodeoxyuridine (BrdU) and MTT assays. Cell necrosis and apoptosis were analyzed with a cellular DNA fragmentation ELISA. Results Among the TLRs analyzed, TLR9 showed the most pronounced upregulation under hypoxic conditions. TLR9 protein expression was similarly induced by CoCl2 as well as in a 0.1% O2 atmosphere. Hypoxia increased the protein levels of γH2A.X, a marker for DNA damage and cellular senescence. The TLR9 agonist ODN 1826 induced the gene expression of downstream signaling molecules (MYD88, IRF7, RELA), inflammatory factors (IFNA, COX2), and MMP9 as well as the secretion of TGF-β1. These effects were reversed by TLR9 inhibitors (ODN 2088 and ODN INH-18). Transforming growth factor (TGF)-β1 significantly suppressed the TLR9 expression at both the mRNA and protein levels. This suppressive effect was reversed at the RNA level by inhibiting components of the TGF-β1 signaling pathway, including activin receptor-like kinase (ALK), Smad3, p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and phosphatidylinositol 3-kinase (PI3K) under hypoxic conditions. Conclusions TLR9 activation under hypoxic conditions initiates pro-inflammatory pathways in RPE cells. In the aging retina, viral and bacterial infections and/or DAMPs may further amplify these responses, worsening retinal degeneration. TGF-β1 may exert protective, anti-inflammatory effects by downregulating TLR9 expression.
Abstract Purpose Dry age-related macular degeneration (DAMD) is the leading cause of vision loss in developed countries, yet there are no FDA-approved treatments currently available. Mitochondria play a significant role in the pathology of DAMD; the retinal pigment epithelium cells of patients with DAMD exhibit mitochondrial dysfunction, elevated levels of mitochondrial DNA lesions, and increased mitochondrial reactive oxygen species. Investigations into the mitochondrial contributions to DAMD are complex as human tissue is challenging to acquire, and animal models do not fully recapitulate disease phenotypes. Cytoplasmic hybrid (cybrid) cells, formed by depleting the mitochondria of an immortalized cell line and fusing with patient platelets, are a possible model for mitochondrial studies on DAMD. This study evaluates if cybrid models of DAMD recapitulate the mitochondrial hallmarks of the disease, including mitochondrial dysfunction, decreased mitochondrial protein levels, lipid accumulation, and mitochondrial DNA lesions. Methods The mitochondrial functions of five healthy and five DAMD cybrid cell lines were compared based on mitochondrial oxygen consumption rates, membrane potential, and protein expression. Secondary factors of mitochondrial dysfunction, including lipid accumulation and mitochondrial DNA stress response, were also examined. Results Compared to healthy control cybrid lines, we found no alterations in bioenergetics, protein levels, and lipid accumulation in DAMD cybrid lines. Mitochondrial DNA stress responses were aberrant in DAMD cybrids compared to healthy controls, suggesting some conserved mitochondrial dysfunction. Conclusions Taken together, this study suggests that these DAMD cybrids do not fully recapitulate DAMD mitochondrial pathology, though this is limited to the study population of males with the H mtDNA haplogroup. However, there may be a niche for cybrid cell lines in investigating mitochondrial DNA phenotypes in patients with DAMD. This is likely because DAMD is a multifactorial disease, dependent upon an individual’s genetics and the retinal microenvironment.
Abstract Purpose To develop and characterize a xeno-free, two-dimensional (2D) differentiation protocol for directing human induced pluripotent stem cells (hIPSCs) toward photoreceptor (PhR)–like cells, using a live-reporter system and transcriptomic analysis to evaluate lineage fidelity and maturation compared to the three-dimensional (3D) culture paradigm. Methods A CRISPR/Cas9-engineered PGP1 hIPSC line expressing fluorescent reporters for retinal markers (VSX2, BRN3B, and RCVRN) was differentiated in adherent culture using chemically defined media supplemented with small molecules (T3, DAPT, taurine, and retinoic acid). Differentiation was assessed over time by immunocytochemistry, flow cytometry, reverse transcription quantitative polymerase chain reaction, ultrastructural imaging (transmission electron microscopy and scanning electron microscopy), and bulk RNA sequencing. Comparative transcriptomic analysis with 3D retinal organoid data was conducted to evaluate developmental kinetics and pathway enrichment. Results The 2D protocol reproducibly generated PhR-like cells expressing PhR-associated protein markers, including CRX, NR2F2, RCVRN, THRβ OPSIN-S, OPSN-M/L, and ARR3. Flow cytometric analysis demonstrated 93.6%, 96.2%, and 70.3% of RCVRN, OPSN-M/L, and OPSN-S positive populations at day (D) 42, while up to 97.4% of cells stained positive for RCVRN by D52. Early commitment to a PhR lineage was evident by D30, supported by transcriptional profiles consistent with PhR-like ontogeny. Ultrastructural analyses revealed features of putative inner and outer segments, including developing cilia and disc-like “whorls” supported by expression of gap junction protein markers and cilium markers (TMEM138 and CX36). Bulk RNA sequencing demonstrated faithful temporal regulation of PhR gene networks and highlighted accelerated differentiation compared to 3D cultures. Conclusions This pilot xeno-free 2D differentiation protocol offers a timely and scalable method for generating PhR-like cells from hIPSCs comparable to standard 3D culture systems. The results validate its downstream utility for retinal cell therapy development, high-throughput screening, and transplantation outputs pending further investigation, while supporting the transcriptome dominance model as a framework for evaluating photoreceptor fate acquisition in this culture paradigm.
Objective: To test whether topical thymoquinone and black cumin seed oil formulations improve signs of experimental dry eye in rats, and to compare their histopathology with standard treatment of dry eye disease (DED) Methods: Seventy-two adult male Wistar-Albino rats were included in this study randomly divided into 12 groups with 6 subjects in each group. DED models were induced for all groups except the control group, using the most accepted cage method. One group remained untreated, the remaining groups were treated with balanced salt solution (BSS), cyclosporine 0.05% (CYC), CYC 0.05%+hyaluronic acid (CYC+HA), fluorometholone 0.01% (FML), TQ solution, TQ emulsion, TQ hydrogel, CO solution, CO emulsion. The Schirmer test for 1-min and Oxford corneal staining tests performed by a single masked grader, were conducted at the end of the first and third weeks. All tests were performed on each of the 12 groups of six rabbits at the end of the first and third weeks. Results: There was no statistically significant difference in the ocular surface tests during the first-week among the groups. When the degree of ocular staining at third week obtained it was found to be significantly higher in the cases treated with BSS and CYC+HA compared to controls (respectively, Z=-2.298, p=0.022; Z=-2.298, p=0.022). TQ solution and CO hydrogel showed a significant decrease compared to week 1 in staining score (respectively, t=5.0, p=0.004; t=3.162, p=0.025). In the third week, ocular surface tests revealed a statistically significant improvement in the group treated with TQ emulsion compared to the control group (Z=-2.127, p=0.033). Conclusions: Thymoquinone solution improved tear and staining metrics in this model. Emulsions and hydrogels showed mixed efficacy and higher histopathologic injury. These findings support reformulation and vehicle-controlled testing before translation. The observed improvement in ocular surface parameters may be attributed to the anti-inflammatory properties of TQ and cytotoxic effects may be ameliorated by different formulation types or doses.
Purpose: To assess the diagnostic value of corneal epithelial thickness parameters measured by RTVue XR OCT in different stages of keratoconus. Methods: This is a cross-sectional study of 64 patients (64 eyes) with keratoconus who visited from January 2021 to December 2023. The control group randomly had 46 patients (46 eyes) with refractive errors. The RTVue XR OCT was used to measure the distribution and parameter indicators of corneal epithelial thickness in various regions of the normal corneal group. We measured parameters with statistically significant differences and calculated cutoff points, sensitivity, specificity, the area under the receiver operating characteristic curve, and the Youden index for the parameter indicators in the preclinical, mild, moderate, and severe keratoconus groups during the clinical period and in the normal control group. Results: The normal corneal epithelial thickness was unevenly distributed in various regions within the range of 0 to 5 mm. The cornea was thinner above and below the nose, as well as thicker above and below the temporal area, and the central area of the cornea was the thinnest. The Min-Max parameter showed the highest diagnostic value for preclinical keratoconus. For mild, moderate, and severe keratoconus, parameters including superior-inferior (S-I), supranasal (SN)infranasal (IT), Max, and standard deviation showed high diagnostic value. SN-IT and standard deviation demonstrated high specificity and sensitivity in distinguishing the clinical keratoconus groups from the normal control group. Conclusions: Corneal epithelial thickness parameters are valuable for diagnosing various stages of keratoconus. The Min-Max parameter is particularly valuable for preclinical diagnosis, while SN-IT and standard deviation are highly valuable for diagnosing clinical stages.
Purpose: To characterize structural and biomechanical changes following ex vivo treatment of rabbit sclera with the bacteriochlorophyll derivative WST11 and near-infrared (NIR) light. Methods: Enucleated left scleras of 10 rabbits were cross-linked by a 20-min topical application of WST11 solution, followed by 30-min NIR irradiation (10 mW/cm(2)). Paired right eyes served as untreated controls. Scleral tangent modulus and ultimate tensile strength were obtained from tensile strip testing. Collagen molecular-level nanostructure was measured using wide-angle X-ray scattering (WAXS). Scleral fibrillar-level microstructure was assessed using second harmonic generation (SHG) and two-photon fluorescence (TPF) imaging. Results: WST11/NIR treatment increased scleral tangent modulus by an average of 79% for strains between 1.5% and 3% (p < 0.05), while ultimate tensile strength increased by 140% (control: 1.719 MPa, treated: 4.134 MPa, p < 0.01). WAXS indicated that collagen molecular spacing (control: 1.485 nm/treated: 1.480 nm, p = 0.65), spatial disorder (47.6 a.u./47.6 a.u., p > 0.99), and anisotropy (0.935 a.u./1.157 a.u., p = 0.09) were not significantly affected by treatment. No marked treatment effects on collagen fibril bundle organization were observable by SHG imaging. TPF imaging revealed additional crimped fibrous structures in the outer half of the tissue depth in treated specimens, indicative of enhanced collagen cross-linking. Conclusions: WST11/NIR treatment significantly increased the stiffness of the ex vivo rabbit sclera. The origin of the stiffening effect is consistent with the formation of collagen cross-links that are predominantly extrafibrillar in nature. Further research is needed to establish the full mechanism, efficacy, and safety of the method as a potential myopia treatment.
This study aimed to investigate factors affecting visual outcomes after phacoemulsification in highly myopic patients with cataracts, based on the detection of nanobacteria (NB) in aqueous humor. Fifty highly myopic patients with cataracts who underwent phacoemulsification surgery at The First People's Hospital of Ziyang from December 2022 to June 2023 were enrolled. Aqueous humor samples were gathered from patients before surgery, and NB were isolated and cultured from the aqueous humor. They were identified using scanning electron microscopy and transmission electron microscopy. The calcium-phosphorus ratios in NB cultures (NB group) and nanohydroxyapatite (nHA) cultures (nHA group) were determined by energy-dispersive X-ray spectroscopy. Cell inhibition was compared using a CCK-8 assay. The expressions of calcification-related proteins, bone morphogenetic protein 2, osteopontin, apoptosis-related proteins, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein in cells were examined by western blot. Based on the best-corrected visual acuity (VA) 3 months after surgery, patients were classified into a normal vision (NV) group (best-corrected VA ≥0.3) and an abnormal vision (AV) group (best-corrected VA <0.3). The factors influencing postoperative VA and efficacy were analyzed. Results revealed that the characteristics of the aqueous humor cultures were consistent with the features of NB described in the literature, with NB measuring approximately 90 to 340 nm in transmission electron microscopy. Energy-dispersive X-ray results indicated no remarkable difference in calcium-phosphorus ratios between the NB and nHA groups (p > 0.05), but the NB group exhibited remarkably stronger cell inhibition relative to the nHA group (p < 0.05). Western blot results revealed obviously higher levels of bone morphogenetic protein 2, osteopontin, and Bcl-2-associated X proteins in the NB group compared to the nHA group (p < 0.05), while Bcl-2 expression was sharply lower, with a great difference in the NB group (p > 0.05). The gap in best-corrected VA 1 month after surgery between the NV group (68%) and the AV group (32%) was obvious (p < 0.05). With increasing recovery time, the number of patients with best-corrected VA ≥0.3 at 6 months postoperatively substantially increased (86%). The NB-positive rate in the aqueous humor samples was observably lower in the NV group than the rate in the AV group (p < 0.05). Univariate logistic regression analysis revealed great differences in age, duration of high myopia, axial length (AXL), corneal astigmatism, incidence of macular disease, and NB-positive rate (p < 0.05). Multivariate logistic regression analysis indicated that AXL and the presence of NB in the aqueous humor were independent factors influencing the postoperative visual prognosis of highly myopic patients with cataracts (p < 0.05). The experimental results signified that NB cultures suppressed cells and, at the cellular level, influenced the visual recovery after surgery by regulating the expression of calcification-related proteins and mitochondrial apoptosis-related proteins. Multivariate logistic regression analysis further confirmed that AXL and the presence of NB in the aqueous humor were independent factors affecting the postoperative visual prognosis. This finding offered strong support for the future development of relevant treatment modalities.
Purpose: Retinal and inner ear ribbon synapses are specialized sensory synapses characterized by synaptic ribbons, electron-dense and protein-rich structures that enable rapid and sustained neurotransmitter release. This review aims to examine the molecular architecture of ribbon synapses with a particular focus on the potential involvement of Tiam1, a guanine nucleotide exchange factor implicated in neuronal development and synaptic plasticity. Methods:A comprehensive review of the available literature was conducted to summarize current knowledge on the structural organization and molecular components of ribbon synapses. Particular attention was given to studies investigating Tiam1 expression, function, and its possible role in cytoskeletal remodeling and synaptic regulation. Results:Evidence supports the central role of RIBEYE as the primary structural component of ribbon synapses; however, the regulatory mechanisms governing ribbon formation and function remain incompletely understood. Recent studies suggest a potential contribution of Tiam1 in modulating synaptic organization and function through Rac1 activation and cytoskeletal regulation, although direct experimental evidence in ribbon synapses is still limited. Conclusions:Ribbon synapses are critical for sustained neurotransmission in sensory systems, yet their molecular regulation remains incompletely defined. Tiam1 emerges as a promising candidate molecule that may influence ribbon synapse function. Future experimental studies are needed to clarify its localization, molecular interactions, and contribution to synaptic organization and plasticity.
Purpose: Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal diseases characterized by progressive photoreceptor degeneration. The early growth response-1 gene (Egr1) is an immediate-early gene implicated in neurodegenerative and stress responses in the retina, among many other tissues. While its expression is induced in the retina across various RP models, its functional role in the degenerative process remains unclear. This study aimed to investigate the contribution of Egr1 to photoreceptor degeneration in vivo. Methods: We used adeno-associated virus (AAV)-mediated RNA interference and transgenic overexpression to modify Egr1 levels in rod and cone photoreceptors of wild-type and Rho(P23H/+) mice. Rod-and cone-specific promoters enabled cell-specific expression. Exposure to high levels of white light was used to induce retinal degeneration in wild-type mice. We assessed retinal structure and transgene expression through funduscopy, optical coherence tomography (OCT), immunofluorescence, and histological analysis. We measured Egr1 mRNA expression levels via real-time PCR and assessed the effects of Egr1 modulation on the retina by determining the thickness of the outer nuclear layer (ONL) and the number of surviving cones. Results: Similar to other models of retinal degeneration, Egr1 was induced in the retina after light exposure and in the Rho(P23H/+) mouse during degeneration. AAV-mediated down-or upregulation ofEgr1 in rods or cones did not affect retinal morphology in wild-type mice. In Rho(P23H/+) mice, Egr1 knockdown in rods modestly preserved ONL thickness up to 12 weeks after AAV injection. Overexpression did not accelerate degeneration beyond controls. Egr1 modulation in cones of wild-type or Rho(P23H/+) mice did not affect cone survival. Conclusions: Egr1 upregulation is a consistent early marker of photoreceptor stress, independent of the nature of the underlying stimulus. Since moderate support for cell survival and preservation of retinal morphology was achieved through the downregulation of Egr1 expression in rods, but not in cones of the Rho(P23H/+) mouse, the function of EGR1 in degenerative processes may be cell type specific. Although Egr1 may contribute to disease progression, it is unlikely to be a causative factor for degeneration. Our findings underscore the complexity of the transcriptional response in retinal degeneration and suggest that Egr1 is a secondary effector of degenerative processes in rods.
Purpose:Retinal ganglion cells (RGCs) are the principal conduits responsible for propagating visual stimuli from the retina to visual centers in the brain. The loss of RGCs leads to visual deficits following trauma or in diseases such as glaucoma. Mouse models are consistently used to investigate root causes for RGC loss. This study quantifies the total number of RGCs and selected RGC subtypes across six strains of inbred mice used in ophthalmic research. Methods:Six mouse strains (C57BL/6J, BALB/cByJ, 129X1/SvJ, A/J, CBA/CaJ, and CAST/EiJ) were selected to represent genetic diversity across the mouse genome. Normal retinas were immunostained for POU6F2, BRN3A, SATB2, OPN4, SMI32, and TO-PRO-3. Cells positively labeled for POU6F2, BRN3A, and SATB2 were quantified using an automated deep learning tool, RGCode. Cells labeled with OPN4, SMI32, and TO-PRO-3 were quantified using the Fiji software. Results:We found statistically significant differences in the quantity (Mean±SEM) and percentage of different RGCs across the inbred mouse strains. The total number of RGCs per retina ranged from 39,961±838 in CAST/EiJ to 53,872±1864 in 129X1/SvJ (p<0.005). Global BRN3A counts ranged from 34,572±494 in CAST/EiJ to 44,253±798 in C57BL/6J (p<0.005). SATB2 counts ranged from 9944±384 in BALB/cByJ to 15,872±1196 in CBA/CaJ (p<0.005). OPN4 density ranged from 110±7 cells/mm2 in CAST/EiJ to 164±13 cells/mm2 in 129X1/SvJ (p<0.05). Differences in SMI32 density were not significant across all strains, with densities ranging from 183±14 cells/mm2 in A/J to 279±12 cells/mm2 in C57BL/6J (not significant). Conclusions:There is a significant variation in total RGC counts and RGC subtypes across the different mouse strains. When working with different strains of mice, it is important to consider this strain-based variation before drawing conclusions from experimental data.
Purpose: The aim of this study was to gain insight into the molecular spectrum of anophthalmia and microphthalmia (A/M) in the Egyptian population. Methods: We studied a cohort of 34 patients from 31 unrelated families affected by the A/M spectrum. All patients underwent a thorough clinical examination, ophthalmological assessment, and genetic testing including conventional karyotyping and exome sequencing (ES). Results: Chromosomal anomalies were identified in six patients. ES was performed on the remaining cases, revealing potentially causative variants in 13 families. The implicated genes were SOX2, OTX2, CHD7, HMX1, PRR12, ATOH7, ZBTB11, B3GALNT2, GCNT2, DPH1, GJA8, FRAS1 and UBE3B. Among the variants, six were classified as pathogenic, five as likely pathogenic, and two as variants of uncertain significance. Notably, a DPH1 pathogenic variant was identified in a patient with bilateral severe microphthalmia, representing a novel phenotype. Additionally, we report the fifth family diagnosed with oculo-auricular syndrome. Conclusions: Our findings confirm that genetic factors are a predominant cause of both syndromic and non-syndromic A/M and underscore the value of ES in uncovering the molecular basis of this spectrum. By reporting novel variants and unusual phenotypes within our cohort, we contribute to expanding both the mutational landscape and the phenotypic spectrum of A/M associated syndromes.
Purpose:Uveitis (inflammation inside the eye) is a disabling manifestation of the post-Ebola syndrome that affects 10% to 35% of individuals who survive the infection. Post-Ebola uveitis presents with diverse clinical features but frequently involves the posterior segment of the eye, where the retinal pigment epithelium plays a key role in directing immune responses. Our previous work shows that this epithelium is relatively susceptible to infection with Zaire ebolavirus (EBOV), the strain responsible for most Ebola outbreaks. In addition to production roles, viral proteins may act to alter the molecular responses of host cells. Methods:We investigated the activity of EBOV viral protein 24 (VP24) in human retinal pigment epithelial cells. An EBOV VP24 expression plasmid was constructed in-house. Multiple primary cell isolates were lipofectamine-transfected, first with VP24 or control expression plasmids and then with polyinosinic-polycytidylic acid (poly I:C) to simulate viral RNA. A type I interferon (IFN) response to transfection was confirmed by an IFN-β enzyme-linked immunosorbent assay. Cellular immune responses after 4- and 24-h exposures to poly I:C were characterized by reverse transcription-quantitative polymerase chain reaction. Results:Multidimensional scaling, drawing on 19 immune response-related gene transcripts, covering antiviral, immunomodulatory, and proinflammatory molecules, demonstrated changes in gene expression profiles following transfection. Analysis of individual cell isolates showed a range of changes, including upregulation and downregulation of different gene transcripts across the two investigated time points. Conclusions:Our findings suggest VP24 elicits variable immune responses from human retinal pigment epithelial cells, potentially contributing to the variation in clinical presentations of uveitis in Ebola survivors.
Purpose:Inherited diseases characterized by abnormal corneal morphology include cornea plana, megalocornea, keratoconus and brittle cornea syndrome. This study aims to investigate genes responsible for these diseases. Methods:Variants in genes responsible for cornea plana, megalocornea, keratoconus and brittle cornea syndrome were analyzed and characterized by multistep bioinformatics approach based on three large data sets, including our in-house exome sequencing database from patients with inherited eye diseases, literature review, and gnomAD database. Additionally, the phenotypes of patients carrying these variants were collected. Results:125 variants in six genes, namely KERA (Keratocan; OMIM: 603288), CHRDL1 (Chordin-like 1; OMIM: 300350), VSX1 (Visual system homeobox 1; OMIM: 605020), TUBA3D (Tubulin, alpha-3d; OMIM: 617878), ZNF469 (Zinc finger protein 469; OMIM: 612078), and PRDM5 (PR domain-containing protein 5; OMIM: 614161), have been reported in 244 families by literature review, of which 78 with cornea plana, 38 with megalocornea, 67 with keratoconus, and 61 with brittle cornea syndrome. Three variants in KERA were identified in 2 families with cornea plana in our cohort. Moreover, all reported variants in VSX1 were reclassified as likely benign or benign based on several major evidence, including high allelic frequency in gnomAD, presence in unaffected individuals in in-house data set, and relatively tolerated by multiple computational prediction tools. Misinterpreted variants in VSX1 has been detected in up to 3.13% of the general population. Conclusions:This study delineates the genetic and clinical landscape of cornea plana, megalocornea, keratoconus and brittle cornea syndrome for the first time. The pathogenicity of VSX1 variants could not be confirmed, making VSX1 an unlikely candidate gene for keratoconus. Correct classification of genes like VSX1 is critical in the era of genomic medicine.
Fibrosis is strictly a histopathological term that refers to the replacement of functional tissue with permanent deposition of nonfunctional extracellular matrix (ECM), following an injury or disease, and can occur in any tissue in the body. In this histological setting, there is no overt difference between fibrosis and scarring. However, in the clinical context, fibrosis tends to be associated with chronic disease, as it ensures ongoing ECM deposition. This could be considered "excessive" when compared to ECM deposition in acute or end-stage chronic disease. This perspective highlights how fibrosis in neovascular age-related macular degeneration follows a stereotypical tissue repair process similar to that seen in other tissues and how salient biologic processes, such as aging and metabolic health, impact fibrosis development. In addition, we highlight the emerging and pivotal profibrogenic role played by progressive endothelial cell (EC) dysfunction, together with secondary blood flow impedance in the neovasculature. This results in abnormal vascular permeability, endothelial-to-mesenchymal transition, and EC senescence-associated secretory phenotype, processes that could potentially be targeted therapeutically. Finally, the dramatic impact of anti-vascular endothelial growth factor therapy, by primarily targeting permeability of the nascent microvasculature, on the natural history of fibrosis in neovascular age-related macular degeneration indirectly highlights the potentially significant role of ECs in fibrosis development and points toward how novel future targeting of these cells could further modulate the development of fibrosis.
Diabetic retinopathy (DR) is a common and severe complication of diabetes, which poses a serious threat to vision, and its pathogenesis is complex. Inflammatory response plays a crucial role in the progression of DR, but currently, anti-vascular endothelial growth factor therapy, the preferred treatment for DR, only targets the vascular part. Therefore, determining a treatment method targeting the inflammatory response in DR is an important step in addressing DR. As immune cells within the retina, microglia play a key role in the inflammatory response in DR, which is a crucial link in its pathogenic mechanism. By regulating the inflammatory response of microglia, the progression of DR can be effectively slowed down. This review deeply explores the mechanism of action of microglia in DR by reviewing relevant research achievements and evaluates the potential of treatment strategies targeting microglia in slowing down the progression of DR. The study focuses on how to further optimize the treatment regimen for DR by regulating different pathways such as the release of inflammatory factors, the occurrence of oxidative stress, phenotypic transformation, intercellular interactions, and phagocytic activity of microglia, providing important clues for the development of novel treatment methods for DR. Through a comprehensive analysis and evaluation of the effectiveness and safety of these treatment strategies, the study aims to provide more precise and effective treatment regimens for patients with DR, so as to improve their visual prognosis and quality of life.
Purpose ABCA4 retinopathies are a group of ABCA4 gene-associated disorders with phenotypic heterogeneity and variable disease severity. The genotype-phenotype correlation is a great challenge for exact disease diagnosis because of symptom variations even within the same family members and the same gene variants giving altered disease phenotypes. This study describes the molecular epidemiology of 10 Pakistani families segregating pathogenic variants of the ABCA4 gene and summarizes ABCA4-associated genetic findings from Pakistani families reported until October 2024. Methods We enrolled consanguineous Pakistani families having at least one child affected with retinal dystrophy. DNA was extracted from blood samples. Probands were analyzed using capture panel sequencing of 344 known genes for retinal dystrophies. Sanger sequencing was used to perform family segregation testing. To review previously published ABCA4 disease-causing variants from Pakistan, data were extracted by retrieving articles through online sources—specifically, PubMed and Google Scholar. Results Out of 72 families, 10 revealed a total of five reported (c.6658C>T, c.214G>A, c.6088C>T, c.6729+5_6729+19del, and c.6218G>C) and two novel (c.2790C>A and c.1099+5G>A) variants in the ABCA4 gene were segregating in each respective family. Furthermore, one of the novel variants, c.1099+5G>A, was segregating in a compound heterozygous manner along with a c.6658C>T stop-gain variant of the ABCA4 gene in one family. All identified ABCA4 variants were segregated in an autosomal recessive manner. Conclusions The variant c.6658C>T was detected in 50% of families analyzed in this study, but previous reports from Pakistan highlight the c.214G>A variant as a frequent ABCA4 mutation in cases of Pakistani decent. Identification of two novel pathogenic variants in the present study reaffirms the allelic and genetic heterogeneity of ABCA4 retinopathies in Pakistani patients. The clinical variability or discordance among individuals carrying the same pathogenic variant may be due to other factors influencing the phenotype, including variables such as sex of the individual or role of modifiers that have yet to be identified.
Purpose A trinucleotide repeat expansion in TCF4 is thought to cause Fuchs endothelial corneal dystrophy (FECD) in ~70% of European patients. In addition, strong evidence exists for the involvement of rare variants in COL8A2 and SLC4A11 in a small number of FECD cases, and more controversially, it has been suggested that variants in ZEB1, AGBL1, and LOXHD1 may also be involved. We screened patients without a TCF4 repeat expansion for causative variants in the other candidate FECD genes. Methods Genomic DNA from blood was genotyped for expansion of the CTG18.1 repeat in intron 2 of TCF4 using short-tandem repeat PCR, followed by triplet-repeat primed PCR (STR/TP-PCR). Single-molecule molecular inversion probes (smMIPs) were designed to amplify the coding exons and splice recognition sites of FECD candidate genes COL8A2, SLC4A11, ZEB1, AGBL1, and LOXHD1 using MIPGEN, and the libraries generated were sequenced on a NextSeq 2000. FASTQ-formatted sequence reads were aligned to the human reference genome with MIPVAR, and identified variants were annotated using Annovar. Rare potentially pathogenic variants (minor allele frequency ≤0.01 for assumed dominant inheritance, combined annotation-dependent depletion ≥15) were confirmed by Sanger sequencing and interpreted according to American College of Medical Genetics and Genomics criteria using the Franklin by Genoox interface. Results Analysis of 114 FECD cases by STR/TP-PCR stratified the patients into FECD expansion-negative cases that had <50 trinucleotide repeats on both alleles at the CTG18.1 locus (n = 33 probands and three additional family members) and FECD expansion-positive (n = 78) cases with at least one allele harboring ≥50 repeats in size. All 36 expansion negative cases were then analyzed by smMIP targeted capture and short-read sequencing of the five other genes implicated in FECD causation. For comparison, two control groups were similarly analyzed: a subset of 29 of the expansion-positive cases whose FECD was assumed to be caused by the repeat expansion and 29 expansion-negative unaffected individuals. Across all groups, 13 variants passed filtration criteria: 1 in COL8A2, 2 in SLC4A11, 4 in AGBL1, and 6 in LOXHD1. No variants were identified in ZEB1. Eight of the variants identified were found in seven FECD expansion-negative cases, five in four FECD expansion-positive cases, and three in two non-FECD controls, with three variants appearing in both expansion-positive and expansion-negative patient groups. Statistical analysis indicated no significant enrichment of variants in expansion-negative cases compared to the other two groups (p = 0.7612 and 0.3275). Only one variant (LOXHD1 NM_144612.7, c.5545G>A, p.(Gly1849Arg)), found in an expansion-negative case, was classified as likely pathogenic, while others were classed as variant of unknown significance (n = 4), likely benign (n = 4), or benign (n = 4). Conclusions smMIPs were used for targeted screening of candidate genes implicated in FECD and proved a versatile, economic approach for prescreening before whole-exome or genome sequencing. This study confirmed the well-documented enrichment of the TCF4 repeat expansion in cases over controls but found a paucity of evidence for the involvement of variants in COL8A2, SLC4A11, ZEB1, AGBL1, or LOXHD1 in this set of expansion-negative cases, implying knowledge of the causes of FECD remains incomplete.
BACKGROUND:Proteostasis impairment is central to cellular dysfunction in protein aggregation disorders such as Alzheimer disease, Parkinson disease, and age-related macular degeneration. Pseudoexfoliation (PEX), a systemic age-related disorder and a leading cause of secondary glaucoma, is increasingly recognized as a protein aggregation disease. It is characterized by the deposition of pseudoexfoliative material (PEXM) in ocular tissues, leading to elevated intraocular pressure and optic neuropathy. OBJECTIVE:This review synthesizes current evidence on the role of proteostasis failure in PEX pathogenesis, with a focus on molecular mechanisms, stress response pathways, and potential therapeutic interventions. METHODS:We conducted a comprehensive literature review of studies examining proteostasis mechanisms in PEX. Emphasis was placed on cellular pathways regulating protein synthesis, folding, and degradation, including the unfolded protein response (UPR), ubiquitin-proteasome system (UPS), and autophagy, as well as environmental and aging-related triggers of proteotoxic stress. RESULTS:Evidence indicates that chronic proteotoxic stress, arising from aging, oxidative damage, and environmental influences, disrupts the proteostasis network (PN). Dysregulation of ER stress signaling, cytosolic stress responses, and protein degradation pathways contributes to the accumulation of misfolded proteins and extracellular matrix deposits in ocular tissues. These molecular alterations underlie disease onset and progression in PEX syndrome (PEXS) and PEX glaucoma (PEXG). CONCLUSIONS:Proteostasis dysfunction plays a pivotal role in PEX pathogenesis by promoting protein misfolding, aggregation, and extracellular deposition. Targeting the proteostasis network, through modulation of stress responses and enhancement of degradation pathways, represents a promising therapeutic strategy for PEXS and PEXG.
Purpose:To characterize the distribution of reticular fibers in the human sclera and explore their potential role in the pathogenesis of pathologic myopia and posterior staphyloma. Methods:Central sagittal sections of 13 globes (6 right eyes and 7 left eyes) were obtained from 12 East Asian cadavers aged 38-94 years (mean age: 74.9 years). Three scleral regions were examined: the pars plana, 3:00-3:30 clock-hour position, and 5:00-5:30 clock-hour position. Specimens were fixed in 10% formalin and stained with silver nitrate. ImageJ software was used for image processing and quantification of fiber density. Results:Reticular fiber density exhibited considerable inter- and intraindividual variability. The average densities at the pars plana, 3:00-3:30, and 5:00-5:30 were 31.07%, 26.10%, and 22.70%, respectively. Density ranges were 23.46%-58.51% (pars plana), 19.18%-43.07% (3:00-3:30), and 13.48%-50.95% (5:00-5:30). In 10 eyes, the pars plana showed the highest density, followed by 3:00-3:30 and then 5:00-5:30. Two eyes had the lowest density at 3:00-3:30, while one eye exhibited the highest density in this region. Conclusions:Reticular fiber density in the sclera exhibits considerable interindividual variability. Our findings suggest that regions of structural vulnerability within the sclera may extend beyond the posterior pole, potentially offering new insights into the pathogenesis of posterior staphyloma. A reduction in reticular fiber density may be implicated in the progression of pathologic myopia and the development of posterior staphylomas, although further investigation is warranted to substantiate this association.
