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Genome-wide expression profile of human trabecular meshwork cultured cells, non-glaucomatous and POAG tissue

ABSTRACT: The goal of this study was to contrast genome-wide gene expression profiles of cultured human trabecular meshwork (HTM) cells, to that of control and primary open angle glaucoma (POAG) HTM tissues. Total RNA from cultured HTM cells and HTM tissue dissected from control and POAG anterior segments fixed in RNA later™ was linearly amplified with the OvationTM Biotin RNA Amplification and Labeling System and individually hybridized to Affymetrix Human Genome U133 Plus 2.0 high density microarrays. Data analysis was performed using the GeneSpring Software 7.0. Selected genes showing significant differential expression were validated by Quantitative real-time PCR in nonamplified RNA. Cultured HTM cells retained the expression of some genes characteristic of the HTM tissue, including chitinase 3-like 1 and matrix Gla protein, but demonstrated downregulation of physiologically important genes such as myocilin. POAG HTM tissue showed relatively small changes compared to that of control donors. These changes included the statistically significant upregulation of several genes associated with inflammation and acute-phase response, including selectin-E (ELAM-I), as well as the downregulation of the antioxidants, paraoxonase 3 and ceruloplasmin. Downregulation in cultured HTM cells of genes potentially relevant for outflow pathway function highlights the importance of developing new conditions for the culture of TM cells capable of preserving the characteristics of TM cells in vivo. Comparative analysis between control and POAG tissues suggests that the upregulation of inflammation-associated genes might be involved in the progression of glaucoma. Keywords: Cell type comparison

ORGANISM(S): Homo sapiens

PROVIDER: GSE4316 | GEO | 2006/06/21

SECONDARY ACCESSION(S): PRJNA94735

REPOSITORIES: GEO

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Project description:The goal of this study was to contrast genome-wide gene expression profiles of cultured human trabecular meshwork (HTM) cells, to that of control and primary open angle glaucoma (POAG) HTM tissues. Total RNA from cultured HTM cells and HTM tissue dissected from control and POAG anterior segments fixed in RNA laterâ¢ was linearly amplified with the OvationTM Biotin RNA Amplification and Labeling System and individually hybridized to Affymetrix Human Genome U133 Plus 2.0 high density microarrays. Data analysis was performed using the GeneSpring Software 7.0. Selected genes showing significant differential expression were validated by Quantitative real-time PCR in nonamplified RNA. Cultured HTM cells retained the expression of some genes characteristic of the HTM tissue, including chitinase 3-like 1 and matrix Gla protein, but demonstrated downregulation of physiologically important genes such as myocilin. POAG HTM tissue showed relatively small changes compared to that of control donors. These changes included the statistically significant upregulation of several genes associated with inflammation and acute-phase response, including selectin-E (ELAM-I), as well as the downregulation of the antioxidants, paraoxonase 3 and ceruloplasmin. Downregulation in cultured HTM cells of genes potentially relevant for outflow pathway function highlights the importance of developing new conditions for the culture of TM cells capable of preserving the characteristics of TM cells in vivo. Comparative analysis between control and POAG tissues suggests that the upregulation of inflammation-associated genes might be involved in the progression of glaucoma. Experiment Overall Design: Total RNA from three cultured HTM cells and HTM tissue dissected from three pairs of control and two pairs of POAG anterior segments fixed in RNA laterâ¢ was linearly amplified with the OvationTM Biotin RNA Amplification and Labeling System and individually hybridized to Affymetrix Human Genome U133 Plus 2.0 high density microarrays.

Project description:Glaucoma is a neurodegenerative disease in which vision is lost as a result of the apoptosis of retinal ganglion cells. When the trabecular meshwork cells, that regulate aqueous humor outflow, are stressed as they are in some forms of glaucoma, they considerably up-regulated secretion of a protein called myocilin into the aqueous. The function of this protein is unclear; but since some aqueous humor flows to the posterior of the eye, the effects of myocilin will also be felt by the retinal cells. We have a transgenic mouse that secretes large amounts of myocilin into aqueous humor. In these mice, there is a deposition of myocilin on membranes of certain cells suggesting myocilin might have some signaling functions. This signaling function could explain why stresses that increase intraocular pressures can increase the likelihood for glaucoma, cause the myocilin to be secreted at very high levels by the trabecular meshwork. Myocilin may be important in treating glaucoma. This experiment will determine if myocilin is altering gene expression in the retina. The results should show variations in expression levels and will give us some indication of the pathways that are important to preserve retinal ganglion cells after a diagnosis of glaucoma. Our hypothesis is that myocilin is acting like a signaling protein in the eye. It acts not only in the anterior segment but also in retina as a result of the flow of some aqueous to the back of the eye. This signaling function is protective to certain ocular cells. This function of myocilin may influence cells in the retina and may counter the apoptotic signals in the retinal ganglion cells that occur during glaucoma. Retinas from transgenic mice and from mice of the same strain that do not have the transgene will be dissected. The mice will be three weeks of age. Because of the small size of the retina, samples will be pooled to obtain the RNA necessary to run the microarray. Three control and three transgenic samples will be run and compared with each other. We have data indicating that myocilin is at high levels in the aqueous humor of the transgenic animals. Keywords: dose response

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Genome-wide expression profile of human trabecular meshwork cultured cells, non-glaucomatous and POAG tissue

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