A Novel Mechanism Involved in the Pathogenesis of Graves Ophthalmopathy (GO): Clathrin Is a Possible Targeting Molecule for Inhibiting Local Immune Response in the Orbit

Excessive orbital fibroblast (OF) proliferation and extracellular matrix production, as well as inflammation resulting in the expansion and remodeling of orbital tissue, are characteristic of Graves ophthalmopathy (GO). Our aim was to analyze and inhibit signaling pathways in resident OF that are involved in GO. METHODS/MAIN OUTCOME MEASURES: Primary human OF were obtained from 12 patients with active, severe GO and from 12 healthy control subjects. The cells were characterized by immunofluorescence assay and flow cytometry. Tyrosine phosphorylation of cellular proteins was determined by Western blot techniques, immunoprecipitation, and protein identity with mass spectrometry. Cell proliferation was determined by 5-bromo-2-deoxyuridine incorporation, hyaluronan (HA) production was assessed by a HA-binding protein based assay, and intracellular reactive oxygen species (ROS) were determined by the dichlorofluorescein assay. Clathrin heavy-chain (CHC) expression was inhibited with small interfering RNA technology.