Temporal single cell transcriptome atlas of zebrafish anterior segment development reveals high degree of conservation between the trabecular meshwork and the annular ligament
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ORGANISM(S): Danio rerio
PROVIDER: GSE218068 | GEO | 2022/11/20
REPOSITORIES: GEO
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Project description:One of the undesirable side effects of glucocorticoid use is the potential development of elevated intraocular pressure and glaucoma. The precise mechanisms through which steroid use induces ocular hypertension are unclear. The purpose of this study was to identify gene expression changes induced by steroids in the human trabecular meshwork and the underlying sclerocorneal tissue. The anterior segment of five pairs of adult human donor eyes were maintained in a profusion organ culture system.
2012-10-01 | E-GEOD-37474 | biostudies-arrayexpress
Project description:Temporal single cell transcriptome atlas of zebrafish anterior segment development reveals high degree of conservation between the trabecular meshwork and the annular ligament
| PRJNA902019 | ENA
Project description:PurposeTreatment with corticosteroids can result in ocular hypertension and may lead to the development of steroid-induced glaucoma. The extent to which biomechanical changes in trabecular meshwork (TM) cells and extracellular matrix (ECM) contribute toward this dysfunction is poorly understood.MethodsPrimary human TM (HTM) cells were cultured for either 3 days or 4 weeks in the presence or absence of dexamethasone (DEX), and cell mechanics, matrix mechanics and proteomics were determined, respectively. Adult rabbits were treated topically with either 0.1% DEX or vehicle over 3 weeks, and mechanics of the TM were determined.ResultsTreatment with DEX for 3 days resulted in a 2-fold increase in HTM cell stiffness, and this correlated with activation of extracellular signal-related kinase 1/2 (ERK1/2) and overexpression of ?-smooth muscle actin (?SMA). Further, the matrix deposited by HTM cells chronically treated with DEX is approximately 4-fold stiffer, more organized, and has elevated expression of matrix proteins commonly implicated in glaucoma (decorin, myocilin, fibrillin, secreted frizzle-related protein [SFRP1], matrix-gla). Also, DEX treatment resulted in a 3.5-fold increase in stiffness of the rabbit TM.DiscussionThis integrated approach clearly demonstrates that DEX treatment increases TM cell stiffness concurrent with elevated ?SMA expression and activation of the mitogen-activated protein kinase (MAPK) pathway, stiffens the ECM in vitro along with upregulation of Wnt antagonists and fibrotic markers embedded in a more organized matrix, and increases the stiffness of TM tissues in vivo. These results demonstrate glucocorticoid treatment can initiate the biophysical alteration associated with increased resistance to aqueous humor outflow and the resultant increase in IOP.
| S-EPMC4509060 | biostudies-literature
Project description:The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some, including glaucoma and cataract, are among the most prevalent causes of blindness. To characterize the cell types that comprise these tissues, we generated an anterior segment cell atlas of the human eye using high throughput single-nucleus RNA sequencing (snRNAseq). We profiled 191,992 single nuclei from non-diseased anterior segment tissues from 6 human donors, identifying >60 cell types. Many are discrete, whereas others in lens and cornea form continua, corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment revealing that some cell types are unique to single structure whereas others are shared across tissues. This integrated cell atlas was then used to investigate cell type-specific expression patterns of more than >900 human ocular disease genes identified either through Mendelian inheritance patterns or genome-wide association studies (GWAS).
2022-07-11 | GSE199013 | GEO
Project description:PurposeTo establish a consistent and affordable, high quality porcine anterior segment perfusion and transduction model that allows direct visualization of the trabecular meshwork.MethodsPorcine anterior segments were cultured within 2 hours of death by removing lens and uvea and securing in a specially designed petri dish with a thin bottom to allow direct visualization of the trabecular meshwork with minimal distortion. Twenty-two control eyes (CO) with a constant flow rate were compared to eight gravity perfused eyes (COgr, 15 mm Hg). We established gene delivery to the TM using eGFP expressing feline immunodeficiency virus (FIV) vector GINSIN at 108 transducing units (TU) per eye (GINSIN_8, n = 8) and 107 TU (GINSIN_7, n = 8). Expression was assessed for 14 days before histology was obtained.ResultsPig eyes were a reliable source for consistent and high quality anterior segment cultures with a low failure rate of 12%. Control eyes had an intraocular pressure (IOP) of 15.8 ± 1.9 mm Hg at fixed pump perfusion with 3 μL/min compared to gravity perfused COgr with imputed 3.7 ± 1.6 μL/min. Vector GINSIN_8 eyes experienced a transient posttransduction IOP increase of 44% that resolved at 48 hours; this was not observed in GINSIN_7 eyes. Expression was higher in GINSIN_8 than in GINSIN_7 eyes. Trabecular meshwork architecture was well preserved.ConclusionsCompared with previously used human donor eyes, this inexpensive porcine anterior segment perfusion model is of sufficient, repeatable high quality to develop strategies of TM bioengineering. Trabecular meshwork could be observed directly. Despite significant anatomic differences, effects of transduction replicate the main aspects of previously explored human, feline and rodent models.
| S-EPMC4811178 | biostudies-literature
Project description:Alterations in stiffness of the trabecular meshwork (TM) may play an important role in primary open-angle glaucoma (POAG), the second leading cause of blindness. Specifically, certain data suggest an association between elevated intraocular pressure (IOP) and increased TM stiffness; however, the underlying link between TM stiffness and IOP remains unclear and requires further study. We here first review the literature on TM stiffness measurements, encompassing various species and based on a number of measurement techniques, including direct approaches such as atomic force microscopy (AFM) and uniaxial tension tests, and indirect methods based on a beam deflection model. We also briefly review the effects of several factors that affect TM stiffness, including lysophospholipids, rho-kinase inhibitors, cytoskeletal disrupting agents, dexamethasone (DEX), transforming growth factor-?2 (TGF-?2), nitric oxide (NO) and cellular senescence. We then describe a method we have developed for determining TM stiffness measurement in mice using a cryosection/AFM-based approach, and present preliminary data on TM stiffness in C57BL/6J and CBA/J mouse strains. Finally, we investigate the relationship between TM stiffness and outflow facility between these two strains. The method we have developed shows promise for further direct measurements of mouse TM stiffness, which may be of value in understanding mechanistic relations between outflow facility and TM biomechanical properties.
| S-EPMC6501821 | biostudies-literature
Project description:Glaucoma is the second leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG), the most common form of glaucoma, is often associated with elevation of intraocular pressure (IOP) due to the dysfunction of trabecular meshwork (TM) tissues. Currently, an ex vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors and disease modifying drugs on physiological parameters like aqueous humor (AH) dynamics and IOP homeostasis. This system requires the use of freshly enucleated intact human eyes, which are sparsely available at very high cost. In this study, we explored the feasibility of using human donor corneoscleral segments for modeling morphological and biochemical changes associated with POAG. Among the number of corneas donated each year, many are deemed ineligible for transplantation due to stringent acceptance criteria. These ineligible corneoscleral segments were obtained from the Lions Eye Bank, Tampa, Florida. Each human donor anterior corneoscleral segment was dissected into four equal quadrants and cultured for 7 days by treating with the glaucoma factors dexamethasone (Dex) or recombinant transforming growth factor (TGF) β2 or transduced with lentiviral expression vectors containing wild type (WT) and mutant myocilin. Hematoxylin and Eosin (H&E) staining analysis revealed that the TM structural integrity is maintained after 7 days in culture. Increased TUNEL positive TM cells were observed in corneoscleral quadrants treated with glaucoma factors compared to their respective controls. However, these TUNEL positive cells were mainly confined to the scleral region adjacent to the TM. Treatment of corneoscleral quadrants with Dex or TGFβ2 resulted in glaucomatous changes at the TM, which included increased extracellular matrix (ECM) proteins and induction of endoplasmic reticulum (ER) stress. Western blot analysis of the conditioned medium showed an increase in ECM (fibronectin and collagen IV) levels in Dex- or TGFβ2-treated samples compared to control. Lentiviral transduction of quadrants resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis of conditioned medium revealed decreased secretion of mutant myocilin compared to WT myocilin. Moreover, increased ECM deposition and ER stress induction was observed in the TM of mutant myocilin transduced quadrants. Our findings suggest that the ex-vivo cultured human corneoscleral segment model is cost-effective and can be used as a pre-screening tool to study the effects of glaucoma factors and anti-glaucoma therapeutics on the TM.
| S-EPMC7314024 | biostudies-literature
Project description:Most vertebrate organs are composed of epithelium surrounded by support and stromal tissues formed from mesenchyme cells, which are not generally thought to form organized progenitor pools. Here we use clonal cell labeling with multicolor reporters to characterize individual mesenchymal progenitors in the developing mouse lung. We observe a diversity of mesenchymal progenitor populations with different locations, movements, and lineage boundaries. Airway smooth muscle (ASM) progenitors map exclusively to mesenchyme ahead of budding airways. Progenitors recruited from these tip pools differentiate into ASM around airway stalks; flanking stalk mesenchyme can be induced to form an ASM niche by a lateral bud or by an airway tip plus focal Wnt signal. Thus, mesenchymal progenitors can be organized into localized and carefully controlled domains that rival epithelial progenitor niches in regulatory sophistication. Mesenchyme was microdissected from e11.5 and e12.5 lung branch tips and stalks and profiled directly. Another set of stalk dissections were cultured in either control media or media containing Wnt1. All experiments done in duplicate.
2014-10-04 | E-GEOD-62039 | biostudies-arrayexpress
Project description:Adeno-associated viruses (AAVs) are the vector of choice for gene therapy in the eye, and self-complementary AAVs (scAAVs), which do not require second-strand DNA synthesis, can be transduced into cells of the trabecular meshwork (TM). The scAAV transduction patterns in the anterior segment of normotensive eyes have been investigated previously, but those in ocular hypertensive (OHT) eyes have not. We assessed the transduction efficiencies of AAV serotypes 2, 5, and 8 in the anterior-segment structures of the eyes of Sprague-Dawley rats with OHT by circumlimbal suturing, followed 3 days later by intracameral injection of scAAV serotype 2 (scAAV2), scAAV5, or scAAV8 packaged with EGFP. The transduction of scAAV2 and scAAV5 in the TM of OHT rats was markedly enhanced after 1 month, and transduction of scAAV5 was more efficient than that of scAAV2; transduction of scAAV8 into the TM did not occur. The transduction of scAAV2, scAAV5, and scAAV8 was enhanced in the ciliary body, iris, and corneal endothelium of the OHT eyes for 3 months. The expression levels of receptors for scAAV2 and scAAV5 were significantly increased in the OHT compared with control eyes. The results demonstrated that scAAV2 and scAAV5 target the ciliary body and TM in OHT eyes, and that the OHT-related changes in anterior-segment structures enhance scAAV transduction.
| S-EPMC6685643 | biostudies-literature
Project description:The effect of trabecular meshwork (TM)-targeted minimally invasive glaucoma surgery (MIGS) on the vasculature assessed using anterior segment (AS)-optical coherence tomography angiography (OCTA) has not been established. In this prospective, longitudinal study, we investigated changes in the deep vasculature following TM-targeted MIGS using AS-OCTA for open-angle glaucoma in 31 patients. AS-OCTA images of the sclera and conjunctiva at the nasal corneal limbus were acquired preoperatively and 3 months postoperatively, and the vessel densities (VDs) of the superficial (conjunctival) and deep (intrascleral) layers were calculated. The VDs before and after MIGS were compared, and the factors associated with the change in VD following MIGS were analyzed. The mean deep VD decreased from 11.98 ± 6.80% at baseline to 10.42 ± 5.02% postoperatively (P = 0.044), but superficial VD did not change (P = 0.73). The multivariate stepwise regression analysis revealed that deep VD reduction was directly associated with IOP reduction (P < 0.001) and preoperative IOP (P = 0.007) and inversely associated with preoperative deep VD (P < 0.001). The deep VD reduction following MIGS was significant in the successful group (21 eyes) (P = 0.032) but not in the unsuccessful group (10 eyes) (P = 0.49). The deep VDs assessed using AS-OCTA decreased following TM-targeted MIGS, especially in the eyes with good surgical outcomes.
| S-EPMC9561613 | biostudies-literature