Dataset Information

Comparison of extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells

ABSTRACT: Extracellular matrix (ECM) materials accumulate in the trabecular meshwork (TM) tissue of patients with glaucoma, which is associated with a decrease in aqueous humor outflow and therefore an increase in intraocular pressure. To explore a potential mechanism for ECM regulation in the TM, we purified extracellular vesicles (EVs) from conditioned media of differentiated TM cells in culture isolated from non-glaucomatous and glaucomatous human donor eyes. EVs were purified using the double cushion ultracentrifugation gradient method. Fractions containing EV markers CD9 and TSG101 were analyzed using nanoparticle tracking analysis to determine their size and concentration. We then determined their proteomic cargo by mass spectrometry and compared protein profiles of EVs between normal and glaucomatous TM cells using PANTHER. Key protein components from EV preparations were validated with Western blotting. Results showed changes in the percentage of ECM proteins associated with EVs from glaucomatous TM cells compared to non-glaucomatous TM cells (5.7% vs 13.1% respectively). Correspondingly, we found that two ECM-related cargo proteins found across all samples, fibronection and EDIL3 were significantly less abundant in glaucomatous EVs (<0.3 fold change across all groups) compared to non-glaucomatous EVs. Overall, these data establish that ECM materials are prominent cargo in EVs from TM cells, and their binding to EVs is diminished in glaucoma

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Epithelial Cell

DISEASE(S): Disease Free

SUBMITTER: Nik Skiba

LAB HEAD: W. Daniel Stamer

PROVIDER: PXD044913 | Pride | 2024-01-26

REPOSITORIES: Pride

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Publications

Comparison of the extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells.

McDonnell Fiona S FS Riddick Bre'Ida J BJ Roberts Haven H Skiba Nikolai N Stamer W Daniel WD

Frontiers in ophthalmology 20231006

<h4>Introduction</h4>Extracellular matrix (ECM) materials accumulate in the trabecular meshwork (TM) tissue of patients with glaucoma, which is associated with a decrease in aqueous humor outflow and therefore an increase in intraocular pressure. To explore a potential mechanism for ECM regulation in the TM, we purified extracellular vesicles (EVs) from conditioned media of differentiated TM cells in culture isolated from non-glaucomatous and glaucomatous human donor eyes.<h4>Methods</h4>EVs wer ...[more]

PMID: 38223904

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Project description:Dr. Panjwani's laboratory is focusing on the mechanism by which galectins-3 and 7 mediate corneal epithelial cell migration. We are currently performing studies to: (i) identify and characterize the corneal epithelial cell surface and extracellular matrix (ECM) molecules which serve as counterreceptors of galectin-3 and -7, to establish whether the lectins modulate corneal epithelial cell migration by binding to well known integrins, growth factor receptors, and/or ECM molecules and (ii) determine whether galectin-3 mediates corneal epithelial cell migration indirectly by modulating the expression of key adhesion and/or signal transduction molecules by using small interfering RNA, cDNA microarrays and glycogene arrays. This study seeks the role of carbohydrate-based recognition system in the pathogenesis of glaucoma. In this study, RNA preparations of four normal and five glaucoma TM samples harvested over the last year from cadavers were hybridized and analyzed using the GLYCOv2 array. We have an NIH RO3 grant award to study the role of carbohydrate-based recognition system in the pathogenesis of glaucoma. In particular, it is known that a carbohydrate-binding protein, ELAM, serves as a marker for glaucoma. Other preliminary studies in my lab have shown that galectin-8 plays a role in the adhesion and spreading of trabecular meshwork cells (TM; the cells which modulate ocular pressure) and galectin-3 influences phagocytic capacity of TM cells. These studies suggest that galectins as well as ELAM and their counterreceptors may contribute to the pathogenesis of glaucoma. We would therefore like to compare glycogene expression patterns of normal and glaucomatous TM tissue. To this end, last month we submitted RNA preparations of five each of normal and glaucoma TM tissue harvested over the last year from cadavers. The data of these samples are now being analyzed. Meanwhile, we have analyzed RNA preparations of TM cells grown in culture from five normal and six glaucoma eyes. It is our hope that data from the two projects will enable us conclusively identify glycogenes which are differentially expressed in normal and glaucoma TM cell. Overall, the goal is to shed light on the role of carbohydrate-based recognition system and the carbohydrate-binding proteins in the pathogenesis of glaucoma.

Project description:Purpose: The optic nerve head (ONH) is the likely site of initial damage in the glaucomatous eye. Despite the recognition of elevated intraocular pressure (IOP) as a leading risk factor for the development of glaucoma, ocular hypertension (OHT) eyes displaying consistently elevated IOP do not experience ONH damage. This study aims to identify global gene expression variations in glaucomatous ONHs and their relationship to those identified in OHT derived ONHs in order to improve our understanding of IOP-induced ONH damage. Methods: (N=6) ONHs were collected from clinically confirmed glaucoma, OHT and age-matched control donor eyes. Total RNA extracted from ONHs was reverse transcribed and assayed using the Affymetrix Human Exon 1.0 ST array. Differentially expressed genes in glaucoma versus control and OHT derived ONHs were identified using an ANOVA analysis with a 1.25 fold change limit and P-value < 0.05. Quantitative RT-PCR was performed to validate selected differentially expressed genes. Results: Microarray analysis revealed 149 under under-expressed genes in POAG versus control ONHs, many of which are involved in ion transport, axonogenesis and macromolecule catabolic processes. 297 genes were over expressed in OHT versus glaucoma derived ONHs. Mediators of oxidation-reduction and chemical homeostasis were among the most prominent gene groups identified. The over expression of prostaglandin-endoperoxide synthase 2, integrin, beta-like 1 and fibulin 5 in glaucomatous ONHs was confirmed by qRT-PCR. Conclusions: Our data demonstrates marked alteration in global gene expression patterns in the glaucomatous ONH, likely due to extensive tissue injury. The observed overlapping of several differentially expressed genes in glaucoma and OHT derived ONHs suggests the induction of common mechanisms in response to elevated IOP. Preferential over-expression of certain gene groups in OHT but not glaucoma derived ONHs may confer possible protection against IOP-induced ONH damage, which remains to be investigated in future studies. (N=6) Glaucoma, ocular hypertension and age-matched control ONHs were assayed to investigate and compare global gene expression patterns in each sample group.

Dataset Information

Comparison of extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells

Publications

Comparison of the extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells.

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