Introduction: One of the most promising potential targets for new treatments in Graves ophthalmopathy (GO) is the insulin-like growth factor 1 receptor (IGF1R). However, a significant impediment to the translational pathway in developing new treatments for GO is the lack of any animal or in vitro model.
Aims: To characterise dysregulation of IGF1 pathway related gene expression in GO. To develop a new in vitro model for tissue expansion in GO using 3D collagen constructs.
Methods: RNA extraction and expression analysis was performed using the gold-standard Affymetrix GeneChip Human Genome U133 Plus 2.0 genome level microarray platform according to manufacturers protocols using 5 GO and 5 Control samples of orbital fat. Microarray findings were validated with qPCR. Cell lines of orbital fibroblasts were derived from 3 GO and 3 control patients. Cells were seeded in compressed 3D collagen cultures under several conditions including recombinant IGF1, GO patient serum and IGF1R blocking antibody. Gel thickness (expansion) was measured over 7 days using confocal microscopy. Parallel experiments were conducted using 2D cell monolayers. Hyaluronic acid (HA) synthesis was measures using ELISA.
Results: Dysregulation of numerous IGF1 signalling genes, including: IGF1, IGFBP6, SOCS3, IRS2, SGK and c-JUN were identified and validated with qPCR. HA synthesis was increased in the presence of both rIGF1 and GO patient serum. HA synthesis was significantly attenuated in the presence of IGF1R antibody. Corresponding changes in gel thickness were found to correlate with HA concentrations measured with ELISA.
Conclusions: Our microarray data is the first data to demonstrate abnormal IGF1 signalling in human GO tissue and our in vitro model for tissue expansion may be of use as a functional assay for testing potential new treatments and accelerate the move to clinical trials.