ABSTRACT
BACKGROUND: The long-term success of fistulating therapies for the treatment of glaucoma is essentially limited by excessive scarring reactions (fibrosis). Cytostatic agents such as mitomycin C can prevent fibrosis, but are often associated with side effects. Specific antifibrotics are not currently in clinical use. Therefore, this study describes a systems biology approach using a dedicated bioinformatics technology platform, with which active substances can be identified and repositioned as antifibrotics. MATERIALS AND METHODS: Differential gene expression data of human Tenon fibroblasts (hTF) were collected from untreated hTF and from hTF stimulated with TGF-ß1 ("fibrotic fibroblasts") by next-generation sequencing (NGS) and were used as the basis for the drug identification process. These data were filtered with the bioinformatic tool "FocusHeuristics". In comparison with the Connectivity Map database, antifibrotic agents were identified. The evaluation of a potentially promising drug as an antifibrotic was performed at hTF by indirect immunofluorescence in vitro. RESULTS: The analysis of the gene expression data led to the identification of several interaction networks of genes or proteins involved in fibrotic processes. One of these networks contains the cytokine bone morphogenic protein 6 (BMP6), interleukin 6 (IL6) and fibroblast growth factor 1 (FGF1). Another relevant network has been identified around the cluster of differentiation 34 (CD34) gene. The comparison of these data with those of the Connectivity Map allowed the identification of an inhibitory drug. Its evaluation in the fibrotic cell culture model in vitro using indirect immunofluorescence led to a significant reduction in the expression of the fibrotic marker proteins fibronectin and alpha-smooth muscle actin (α-SMA), which confirmed the predicted antifibrotic effect. CONCLUSION: Systems biological approaches can be used for the identification of antifibrotic drug candidates for the prevention of postoperative fibrosis and should be transferable by the investigating differential gene expression data of further ocular cells or tissues to other ophthalmological fields of application.
Subject(s)
Fibrosis , Ophthalmologic Surgical Procedures , Ophthalmology , Systems Biology , Actins , Fibroblasts , Fibrosis/etiology , Fibrosis/prevention & control , Humans , Mitomycin , Ophthalmologic Surgical Procedures/adverse effects , Postoperative ComplicationsABSTRACT
MicroRNAs (miRNAs) are small non-coding RNA molecules acting as post-transcriptional regulators of gene expression. They are involved in many biological processes, and their dysregulation is implicated in various diseases, including multiple sclerosis (MS). Interferon-beta (IFN-beta) is widely used as a first-line immunomodulatory treatment of MS patients. Here, we present the first longitudinal study on the miRNA expression changes in response to IFN-beta therapy. Peripheral blood mononuclear cells (PBMC) were obtained before treatment initiation as well as after two days, four days, and one month, from patients with clinically isolated syndrome (CIS) and patients with relapsing-remitting MS (RRMS). We measured the expression of 651 mature miRNAs and about 19,000 mRNAs in parallel using real-time PCR arrays and Affymetrix microarrays. We observed that the up-regulation of IFN-beta-responsive genes is accompanied by a down-regulation of several miRNAs, including members of the mir-29 family. These differentially expressed miRNAs were found to be associated with apoptotic processes and IFN feedback loops. A network of miRNA-mRNA target interactions was constructed by integrating the information from different databases. Our results suggest that miRNA-mediated regulation plays an important role in the mechanisms of action of IFN-beta, not only in the treatment of MS but also in normal immune responses. miRNA expression levels in the blood may serve as a biomarker of the biological effects of IFN-beta therapy that may predict individual disease activity and progression.
