Dataset Information

Ocular fibroblast types differ in their mRNA profiles - implications for fibrosis prevention after aqueous shunt implantation

ABSTRACT: Surgical glaucoma therapy is characterized by implantation of an aqueous shunt either draining into the extraocular Tenon’s space or the intraocular suprachoroidal space. In both cases the long term drainage is hampered by fibrotic reactions around the outflow region of the shunt. The prevention of fibrosis should extend the operating life of the shunt. For an aqueous shunt draining from the anterior chamber into the choroidal space fibroblasts from the choroidea and/or the sclera are most likely responsible for a fibrotic response around the outflow region of such a shunt. A detailed characterization of fibroblasts derived from choroidea and sclera should provide information whether a fibrosis reaction can be inhibited by cell type specific agents. Therefore, we have decided to generate mRNA profiles of fibroblasts from the choroidea, sclera and Tenon’s space in order to look for potential pharmacological targets for fibrosis prevention. The three fibroblast types investigated share fibroblast specific gene expression patterns, concerning extracellular matrix proteins as collagens and fibronectin, but also show distinct mRNA patterns, which we plan to search for targets responsible for fibrotic processes which hopefully can be targeted by specific antifibrotic drugs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE40929 | GEO | 2012/09/25

SECONDARY ACCESSION(S): PRJNA175384

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:Surgical glaucoma therapy is characterized by implantation of an aqueous shunt either draining into the extraocular Tenon’s space or the intraocular suprachoroidal space. In both cases the long term drainage is hampered by fibrotic reactions around the outflow region of the shunt. The prevention of fibrosis should extend the operating life of the shunt. For an aqueous shunt draining from the anterior chamber into the choroidal space fibroblasts from the choroidea and/or the sclera are most likely responsible for a fibrotic response around the outflow region of such a shunt. A detailed characterization of fibroblasts derived from choroidea and sclera should provide information whether a fibrosis reaction can be inhibited by cell type specific agents. Therefore, we have decided to generate mRNA profiles of fibroblasts from the choroidea, sclera and Tenon’s space in order to look for potential pharmacological targets for fibrosis prevention. The three fibroblast types investigated share fibroblast specific gene expression patterns, concerning extracellular matrix proteins as collagens and fibronectin, but also show distinct mRNA patterns, which we plan to search for targets responsible for fibrotic processes which hopefully can be targeted by specific antifibrotic drugs. Three human fibroblast cell type cultures from different ocular tissues were established: sclera fibroblasts (hSF), choroidea fibroblasts (hCF), and Tenon’s space fibroblasts (hTF). For the gene expression analysis n = 5 for hCF, n = 4 for hSF, and n = 5 for hTF donor cells were cultivated from different donors. After appropriate cultivation, cells were harvested, RNA was extracted, purified and quantity and quality was assessed. All total RNA samples were analyzed by Affymetrix' Whole-Transcript Expression Analysis & Profiling Human Gene ST Arrays, respectively. In this set-up, we run = 5 arrays for hCF, n = 4 arrays for hSF, and n = 5 arrays for hTF i.e. one array per biological replicate. No technical replication was carried out. Microarray data analysis was carried out by using the Rosetta Resolver® system for gene expression data analysis (Rosetta Biosoftware, Seattle, WA, USA). In brief, the raw signals of the probes were summarized using RMA thereby generating probe set specific signal intensities. Chips were normalized by using quantile normalization. To compare RNA expression levels of genes in hCF, hSF and hTF, normalized expression signals of genes from corresponding samples were averaged and fold changes were calculated. To assess differences in mean signal intensities between experimental groups, ANOVA (analysis of variance, with Benjamini Hochberg test correction) and a post-hoc Scheffe test was performed. Rosetta Resolver ratio built statistics to correct for possible signal intensity bias were also considered. Only genes (1) an absolute fold change of ≥ 1.5 together with a Scheffe test p value ≤ 0.05 in at least one of the three pairwise comparisons hCF vs. hTF, hSF vs. hTF and hCF vs. hSF, resp., as well as (2) a ratio built p value ≤ 0.05 were deemed differentially expressed genes (DEG) and considered for further evaluation.

Project description:Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated contractile activity, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active RhoA (RhoAV14). Organ cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 revealed strong contractile cell morphology, increased actin stress fibers and focal adhesions, along with increased levels of phosphorylated myosin II, and collagen IV, fibronectin and laminin. cDNA microarray analysis of RNA extracted from RhoAV14 expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of myosin II, paxillin and focal adhesion kinase, and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the contractile force, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells. Keywords: Gene Expression Two condition experiment: Human trabecular mesh work cells infected with Adenivirus expressing GFP Vs Adenovirus expressing GFP and constitutively active RhoAV14

Dataset Information

Ocular fibroblast types differ in their mRNA profiles - implications for fibrosis prevention after aqueous shunt implantation

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure