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Serial analysis of gene expression (SAGE) in normal human trabecular meshwork.

ABSTRACT: To identify the genes expressed in normal human trabecular meshwork tissue, a tissue critical to the pathogenesis of glaucoma.Total RNA was extracted from human trabecular meshwork (HTM) harvested from 3 different donors. Extracted RNA was used to synthesize individual SAGE (serial analysis of gene expression) libraries using the I-SAGE Long kit from Invitrogen. Libraries were analyzed using SAGE 2000 software to extract the 17 base pair sequence tags. The extracted sequence tags were mapped to the genome using SAGE Genie map.A total of 298,834 SAGE tags were identified from all HTM libraries (96,842, 88,126, and 113,866 tags, respectively). Collectively, there were 107,325 unique tags. There were 10,329 unique tags with a minimum of 2 counts from a single library. These tags were mapped to known unique Unigene clusters. Approximately 29% of the tags (orphan tags) did not map to a known Unigene cluster. Thirteen percent of the tags mapped to at least 2 Unigene clusters. Sequence tags from many glaucoma-related genes, including myocilin, optineurin, and WD repeat domain 36, were identified.This is the first time SAGE analysis has been used to characterize the gene expression profile in normal HTM. SAGE analysis provides an unbiased sampling of gene expression of the target tissue. These data will provide new and valuable information to improve understanding of the biology of human aqueous outflow.

SUBMITTER: Liu Y

PROVIDER: S-EPMC3081805 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Serial analysis of gene expression (SAGE) in normal human trabecular meshwork.

Liu Yutao Y Munro Drew D Layfield David D Dellinger Andrew A Walter Jeffrey J Peterson Katherine K Rickman Catherine Bowes CB Allingham R Rand RR Hauser Michael A MA

Molecular vision 20110408

<h4>Purpose</h4>To identify the genes expressed in normal human trabecular meshwork tissue, a tissue critical to the pathogenesis of glaucoma.<h4>Methods</h4>Total RNA was extracted from human trabecular meshwork (HTM) harvested from 3 different donors. Extracted RNA was used to synthesize individual SAGE (serial analysis of gene expression) libraries using the I-SAGE Long kit from Invitrogen. Libraries were analyzed using SAGE 2000 software to extract the 17 base pair sequence tags. The extract ...[more]

PMID: 21528004

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Project description:BackgroundTrabecular meshwork and Schlemm's canal are the tissues appointed to modulate the aqueous humour outflow from the anterior chamber. The impairment of their functions drives to an intraocular pressure increase. The selective laser trabeculoplasty is a laser therapy of the trabecular meshwork able to decrease intraocular pressure. The exact response mechanism to this treatment has not been clearly delineated yet. The herein presented study is aimed at studying the gene expression changes induced in trabecular meshwork cells by selective laser trabeculoplasty (SLT) in order to better understand the mechanisms subtending its efficacy.Methodology/principal findingsPrimary human trabecular meshwork cells cultured in fibroblast medium underwent selective laser trabeculoplasty treatment. RNA was extracted from a pool of cells 30 minutes after treatment while the remaining cells were further cultured and RNA was extracted respectively 2 and 6 hours after treatment. Control cells stored in incubator in absence of SLT treatment were used as reference samples. Gene expression was evaluated by hybridization on miRNA-microarray and laser scanner analysis. Scanning electron microscopic examination was performed on 2 Trabecular meshwork samples after SLT at 4(th) and 6(th) hour from treatment. On the whole, selective laser trabeculoplasty modulates in trabecular meshwork the expression of genes involved in cell motility, intercellular connections, extracellular matrix production, protein repair, DNA repair, membrane repair, reactive oxygen species production, glutamate toxicity, antioxidant activities, and inflammation.Conclusions/significanceSLT did not induce any phenotypic alteration in TM samples. TM is a complex tissue possessing a great variety of function pivotal for the active regulation of aqueous humour outflow from the anterior chamber. SLT is able to modulate these functions at the postgenomic molecular level without inducing damage either at molecular or phenotypic levels.

Project description:PurposeCells in the trabecular meshwork sense and respond to a myriad of physical forces through a process known as mechanotransduction. Whilst the effect of substratum stiffness or stretch on TM cells have been investigated in the context of transforming growth factor (TGF-β), Wnt and YAP/TAZ pathways, the role of Notch signaling, an evolutionarily conserved pathway, recently implicated in mechanotransduction, has not been investigated in trabecular meshwork (TM) cells. Here, we compare the endogenous expression of Notch pathway molecules in TM cells from glaucomatous and non-glaucomatous donors, segmental flow regions, and when subjected to cyclical strain, or grown on hydrogels of varying rigidity.MethodsPrimary TM from glaucomatous (GTM), non-glaucomatous (NTM) donors, and from segmental flow regions [high flow (HF), low flow (LF)], were utilized between passages 2-6. Cells were (i) plated on tissue culture plastic, (ii) subjected to cyclical strain (6 h and 24 h), or (iii) cultured on 3 kPa and 80 kPa hydrogels. mRNA levels of Notch receptors/ligands/effectors in the TM cells was determined by qRT-PCR. Phagocytosis was determined as a function of substratum stiffness in NTM-HF/LF cells in the presence or absence of 100 nM Dexamethasone treatment.ResultsInnate expression of Notch pathway genes were significantly overexpressed in GTM cells with no discernible differences observed between HF/LF cells in either NTM or GTM cells cultured on plastic substrates. With 6 h of cyclical strain, a subset of Notch pathway genes presented with altered expression. Expression of Notch receptors/ligands/receptors/inhibitors progressively declined with increasing stiffness and this correlated with phagocytic ability of NTM cells. Dexamethasone treatment decreased phagocytosis regardless of stiffness or cells isolated from segmental outflow regions.ConclusionsWe demonstrate here that the Notch expression in cultured TM cells differ intrinsically between GTM vs NTM, and by substratum cues (cyclical strain and stiffness). Of import, the most apparent differences in gene expression were observed as a function of substratum stiffness which closely followed phagocytic ability of cells. Interestingly, on soft substrates (mimicking normal TM stiffness) Notch expression and phagocytosis was highest, while both expression and phagocytosis was significantly lower on stiffer substrates (mimicking glaucomatous stiffness) regardless of DEX treatment. Such context dependent changes suggest Notch pathway may play differing roles in disease vs homeostasis. Studies focused on understanding the mechanistic role of Notch (if any) in outflow homeostasis are thus warranted.

Dataset Information

Serial analysis of gene expression (SAGE) in normal human trabecular meshwork.

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Serial analysis of gene expression (SAGE) in normal human trabecular meshwork.

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