Search Results (18)

Glaucoma represents a social and economic burden due to both its increasing incidence and the lack of knowledge about its physiopathology and treatment strategies. The main factor hindering progress in glaucoma research is the disease’s heterogeneity, which depends on both genetic and environmental factors. This limitation directly affects glaucoma research, posing obstacles to the elucidation of risk factors, disease mechanisms, and treatment strategies. Therefore, the need emerges to integrate pre-clinical experimental observations from different experimental models to recapitulate different aspects of the disease and achieve a successful translation to clinics. Here, we reviewed the glaucoma models that are currently available for basic and translational research, with a specific focus on models based on rodents. Regarding genetic glaucoma models, we considered the main hallmarks and limitations of DBA/2J, glutamate/aspartate transporter/excitatory amino acid carrier 1, myocilin, connective tissue growth factor, optineurin, purinergic receptor 2Y, caveolin 1, and endothelin-1 mice. Regarding other glaucoma models, we considered rodent models based on intraocular pressure elevation via perturbation of aqueous humor dynamics or on direct degeneration of retinal ganglion cells via physical or chemical damage. Full article

Glaucoma is the leading cause of irreversible blindness globally, with the commonest subtype being primary open angle glaucoma (POAG). POAG is characterised by an increase in intraocular pressure (IOP), optic nerve damage and irreversible visual field loss. People of African descent (AD) are significantly more susceptible to POAG when compared to people of European descent (ED), and the reasons for this are complex and multifaceted. The vast level of genetic diversity in AD populations has allowed, through genome-wide association studies (GWAS), for the identification of several single nucleotide polymorphisms (SNPs) as well as differences in mitochondrial haplogroups, which could explain the pathophysiology underlying the increased susceptibility of AD populations to POAG. The altered expression of genes such as MYOC as well as the expression of inflammatory mediators influencing reactive astrocytes have also been implicated. There are also several differences in morphology between AD and ED eyes which must be considered, including differences in central corneal thickness (CCT) and corneal hysteresis (CH) as well as variation in properties of optic discs. The link between all the aforementioned factors and the increased prevalence of POAG in AD populations will be explored in this review. Full article

Glaucoma is a group of optic neuropathies characterized by the degeneration of retinal ganglion cells and the loss of their axons in the optic nerve. The only approved therapies for the treatment of glaucoma are topical medications and surgical procedures aimed at lowering intraocular pressure. Gene therapy involves the insertion, removal, or modification of genetic material within cells to repair or compensate for the loss of a gene’s function. It describes a process or technology that enables the genetic modification of cells to produce a therapeutic effect. However, changing the genetic material alone does not extend the duration of overexpression of proteins that combat disease, nor does it facilitate the production of new proteins for this purpose. We reviewed the literature concerning the use of gene therapy in the treatment of glaucoma and explored the future directions that this innovation may offer. Three genes associated with glaucoma have been identified within these loci: myocilin/trabecular meshwork glucocorticoid response (TIGR) (GLC1A), optineurin (GLC1E), and WDR36 (GLC1G). Among these, the most extensively studied glaucoma gene is myocilin (a TM-inducible glucocorticoid response gene). Building on previous successes, researchers have begun to apply genetic therapeutic approaches to alleviate or reduce symptoms associated with ocular hypertension (OHT) and glaucoma-like optic neuropathy (GON). It is evident that several therapeutic strategies exist that modulate aqueous humor production and flow, thereby regulating intraocular pressure (IOP) and protecting retinal ganglion cells (RGCs) from apoptosis. With the emergence of gene therapy as a potentially viable approach to preserving vision, new methods for managing glaucoma may soon become available. Genomic therapy is a promising treatment option for glaucoma patients and has significant potential for widespread clinical application. Full article

Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, β-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis. Full article

Childhood glaucoma, a significant cause of global blindness, represents a heterogeneous group of disorders categorized into primary or secondary forms. Primary childhood glaucoma stands as the most prevalent subtype, comprising primary congenital glaucoma (PCG) and juvenile open-angle glaucoma (JOAG). Presently, multiple genes are implicated in inherited forms of primary childhood glaucoma. This comprehensive review delves into genetic investigations into primary childhood glaucoma, with a focus on identifying causative genes, understanding their inheritance patterns, exploring essential biological pathways in disease pathogenesis, and utilizing animal models to study these mechanisms. Specifically, attention is directed towards genes such as CYP1B1 (cytochrome P450 family 1 subfamily B member 1), LTBP2 (latent transforming growth factor beta binding protein 2), TEK (TEK receptor tyrosine kinase), ANGPT1 (angiopoietin 1), and FOXC1 (forkhead box C1), all associated with PCG; and MYOC (myocilin), associated with JOAG. Through exploring these genetic factors, this review aims to deepen our understanding of the intricate pathogenesis of primary childhood glaucoma, thereby facilitating the development of enhanced diagnostic and therapeutic strategies. Full article

Glaucoma is a chronic optic neuropathy that leads to irreversible vision loss. Aging and family history are the two most important risk factors of glaucoma. One of the most studied genes involved in the onset of open-angle glaucoma is myocilin (MYOC). About 105 germline mutations within MYOC are known to be associated with glaucoma and result in endoplasmic reticulum (ER) stress, which leads to trabecular meshwork (TM) cell death and subsequent intraocular pressure (IOP) elevation. However, only about 4% of the population carry these mutations. An analysis of MYOC somatic cancer-associated mutations revealed a notable overlap with pathogenic glaucoma variants. Because TM cells have the potential to accumulate somatic mutations at a rapid rate due to ultraviolet (UV) light exposure, we propose that an accumulation of somatic mutations within MYOC is an important contributor to the onset of glaucoma. Full article

Deep neural network-based programs can be applied to protein structure modeling by inputting amino acid sequences. Here, we aimed to evaluate the AlphaFold2-modeled myocilin wild-type and variant protein structures and compare to the experimentally determined protein structures. Molecular dynamic and ligand binding properties of the experimentally determined and AlphaFold2-modeled protein structures were also analyzed. AlphaFold2-modeled myocilin variant protein structures showed high similarities in overall structure to the experimentally determined mutant protein structures, but the orientations and geometries of amino acid side chains were slightly different. The olfactomedin-like domain of the modeled missense variant protein structures showed fewer folding changes than the nonsense variant when compared to the predicted wild-type protein structure. Differences were also observed in molecular dynamics and ligand binding sites between the AlphaFold2-modeled and experimentally determined structures as well as between the wild-type and variant structures. In summary, the folding of the AlphaFold2-modeled MYOC variant protein structures could be similar to that determined by the experiments but with differences in amino acid side chain orientations and geometries. Careful comparisons with experimentally determined structures are needed before the applications of the in silico modeled variant protein structures. Full article

Glaucoma, where increased intraocular pressure (IOP) leads to damage to the optic nerve and loss of sight, is amongst the foremost causes of irreversible blindness worldwide. In primary open angle glaucoma, the increased IOP is a result of the malfunctioning human trabecular meshwork (HTM) cells’ inability to properly regulate the outflow of aqueous humor from the eye. A potential future treatment for glaucoma is to replace damaged HTM cells with a tissue-engineered substitute, thus restoring proper fluid outflow. Polycaprolactone (PCL) is a versatile, biodegradable, and implantable material that is widely used for cell culture and tissue engineering. In this work, PCL scaffolds were lithographically fabricated using a sacrificial process to produce submicron-thick scaffolds with openings of specific sizes and shapes (e.g., grid, hexagonal pattern). The HTM cell growth on gelatin-coated PCL scaffolds was assessed by scanning electron microscopy, tetrazolium metabolic activity assay, and cytoskeletal organization of F-actin. Expression of HTM-specific markers and ECM deposition were assessed by immunocytochemistry and qPCR analysis. Gelatin-coated, micropatterned, ultrathin, porous PCL scaffolds with a grid pattern supported proper HTM cell growth, cytoskeleton organization, HTM-marker expression, and ECM deposition, demonstrating the feasibility of using these PCL scaffolds to tissue-engineer implantable, healthy ocular outflow tissue. Full article

Trabecular meshwork (TM) tissue is highly specialized, and its structural integrity is crucial for maintaining homeostatic intraocular pressure (IOP). The administration of glucocorticoids, such as dexamethasone (DEX), can perturb the TM structure and significantly increase IOP in susceptible individuals, resulting in ocular diseases such as steroid-induced glaucoma, a form of open-angle glaucoma. Although the exact mechanism involved in steroid-induced glaucoma remains elusive, increasing evidence suggests that DEX may act through various signaling cascades in TM cells. Despite uncertainty surrounding the specific process by which steroid-induced glaucoma occurs, there is growing evidence to indicate that DEX can impact multiple signaling pathways within TM cells. In this study, we examined the impact of DEX treatment on the Wnt signaling pathway in TM cells, given that Wnt signaling has been reported to play a crucial role in regulating extracellular matrix (ECM) levels in the TM. To further elucidate the role of Wnt signaling in the glaucomatous phenotype, we examined mRNA expression patterns between Wnt signaling markers AXIN2 and sFRP1 and DEX-mediated induction of myocilin (MYOC) mRNA and protein levels over 10 days in DEX-treated primary TM cells. We observed a sequential pattern of peak expression between AXIN2, sFRP1, and MYOC. Based on the study, we propose that sFRP1 upregulation could be a result of a negative feedback mechanism generated by stressed TM cells to suppress abnormal Wnt signaling activities. Full article

Obesity is already accompanied by adipose tissue (AT) dysfunction and metabolic disease in children and increases the risk of premature death. Due to its energy-dissipating function, brown AT (BAT) has been discussed as being protective against obesity and related metabolic dysfunction. To analyze the molecular processes associated with BAT development, we investigated genome-wide expression profiles in brown and white subcutaneous and perirenal AT samples of children. We identified 39 upregulated and 26 downregulated genes in uncoupling protein 1 (UCP1)-positive compared to UCP1-negative AT samples. We prioritized for genes that had not been characterized regarding a role in BAT biology before and selected cordon-bleu WH2 repeat protein (COBL), mohawk homeobox (MKX) and myocilin (MYOC) for further functional characterization. The siRNA-mediated knockdown of Cobl and Mkx during brown adipocyte differentiation in vitro resulted in decreased Ucp1 expression, while the inhibition of Myoc led to increased Ucp1 expression. Furthermore, COBL, MKX and MYOC expression in the subcutaneous AT of children is related to obesity and parameters of AT dysfunction and metabolic disease, such as adipocyte size, leptin levels and HOMA-IR. In conclusion, we identify COBL, MKX and MYOC as potential regulators of BAT development and show an association of these genes with early metabolic dysfunction in children. Full article

Although elevated TGFβ2 levels appear to be a causative factor in glaucoma pathogenesis, little is known about how TGFβ2 expression is regulated in the trabecular meshwork (TM). Here, we investigated if activation of the cytokine regulator NFATc1 controlled transcription of TGFβ2 in human TM cells by using dexamethasone (DEX) to induce NFATc1 activity. The study used both proliferating and cell cycle arrested quiescent cells. Cell cycle arrest was achieved by either cell–cell contact inhibition or serum starvation. β-catenin staining and p21 and Ki-67 nuclear labeling were used to verify the formation of cell–cell contacts and activity of the cell cycle. NFATc1 inhibitors cyclosporine A (CsA) or 11R-VIVIT were used to determine the role of NFATc1. mRNA levels were determined by RT-qPCR. DEX increased TGFβ2 mRNA expression by 3.5-fold in proliferating cells but not in quiescent cells or serum-starved cells, and both CsA and 11R-VIVIT inhibited this increase. In contrast, the expression of other DEX/NFATc1-induced mRNAs (myocilin and β3 integrin) occurred regardless of the proliferative state of the cells. These studies show that NAFTc1 regulates TGFβ2 transcription in TM cells and reveals a previously unknown connection between the TM cell cycle and modulation of gene expression by NFATc1 and/or DEX in TM cells. Full article

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-year-old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation. Full article

Elevated intraocular pressure (IOP) is a major risk factor in developing primary open angle glaucoma (POAG), which is the most common form of glaucoma. Transforming growth factor-beta 2 (TGFβ2) is a pro-fibrotic cytokine that plays an important role in POAG pathogenesis. TGFβ2 induced extracellular matrix (ECM) production, deposition and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) contribute to increased aqueous humor (AH) outflow resistance and IOP elevation. Drugs which alter the glaucomatous fibrotic changes and ER stress in the TM may be effective in reducing ocular hypertension. Astragaloside IV (AS.IV), a novel saponin isolated from the roots of Astragalus membranaceus, has demonstrated antifibrotic and ER stress lowering effects in various tissues during disease conditions. However, the effect of AS.IV on glaucomatous TM fibrosis, ER stress and ocular hypertension has not been studied. Primary human TM cells treated with AS.IV decreased TGFβ2 induced ECM (FN, Col-I) deposition and ER stress (KDEL, ATF4 and CHOP). Moreover, AS.IV treatment reduced TGFβ2 induced NF-κB activation and αSMA expression in TM cells. We found that AS.IV treatment significantly increased levels of matrix metalloproteases (MMP9 and MMP2) and MMP2 enzymatic activity, indicating that the antifibrotic effects of AS.IV are mediated via inhibition of NF-κB and activation of MMPs. AS.IV treatment also reduced ER stress in TM3 cells stably expressing mutant myocilin. Interestingly, the topical ocular AS.IV eye drops (1 mM) significantly decreased TGFβ2 induced ocular hypertension in mice, and this was associated with a decrease in FN, Col-1 (ECM), KDEL (ER stress) and αSMA in mouse TM tissues. Taken together, the results suggest that AS.IV prevents TGFβ2 induced ocular hypertension by modulating ECM deposition and ER stress in the TM. Full article

Glaucoma is the leading cause of irreversible blindness worldwide, with elevated intraocular pressure (IOP) as the only known modifiable risk factor. Trabecular meshwork (TM)-inducible myocilin (the MYOC gene) was the first to be identified and linked to juvenile and primary open-angle glaucoma. It has been suggested that mutations in the MYOC gene and the aggregation of mutant myocilin in the endoplasmic reticulum (ER) of TM may cause ER stress, resulting in a reduced outflow of aqueous humor and an increase in IOP. We selected 20 MYOC mutations with experimentally determined melting temperatures of mutated myocilin proteins. We included 40 published studies with at least one glaucoma patient with one of these 20 MYOC mutations and information on age at glaucoma diagnosis. Based on data from 458 patients, we found that a statistically significant but weak correlation was present between age and melting temperature based on various assumptions for age. We therefore conclude that genetic analysis of MYOC mutations alone cannot be used to accurately predict age at glaucoma diagnosis. However, it might be an important prognostic factor combined with other clinical factors for critical and early detection of glaucoma. Full article

Myocilin (MYOC) is a glycoprotein encoded by a gene associated with glaucoma pathology. In addition to the eyes, it also expresses at high transcription levels in the heart and skeletal muscle. MYOC affects the formation of the murine gastrocnemius muscle and is associated with the differentiation of mouse osteoblasts, but its role in the differentiation of C2C12 cells has not yet been reported. Here, MYOC expression was found to increase gradually during the differentiation of C2C12 cells. Overexpression of MYOC resulted in enhanced differentiation of C2C12 cells while its inhibition caused reduced differentiation. Furthermore, immunoprecipitation indicated that MYOC binds to Caveolin-1 (CAV1), a protein that influences the TGF-β pathway. Laser confocal microscopy also revealed the common sites of action of the two during the differentiation of C2C12 cells. Additionally, CAV1 was upregulated significantly as C2C12 cells differentiated, with CAV1 able to influence the differentiation of the cells. Furthermore, the Western blotting analysis demonstrated that the expression of MYOC affected the TGF-β pathway. Finally, MYOC was overexpressed while CAV1 was inhibited. The results indicate that reduced CAV1 expression blocked the promotion of C2C12 cell differentiation by MYOC. In conclusion, the results demonstrated that MYOC regulates TGF-β by influencing CAV1 to promote the differentiation of C2C12 cells. Full article