Inhibition Of The Acid Sphingomyelinase/ceramide System Prevents Hallmarks Of Graves Ophthalmopathy

Abstract

Purpose: : To study mechanisms of pathologic proliferation of orbital fibroblasts and the transformation of these cells to adipocytes, excessive production of extracellular matrix, and inflammation which are hallmarks in the pathogenesis of Graves ophthalmopathy (GO). These processes finally result in remodeling of the orbital tissue with the typical symptoms of lid retraction, periorbital swelling, disfiguring proptosis, optic nerve compression, and impaired ocular motility caused by fibrotic changes in the extraocular muscles.

Methods: : Human orbital fibroblasts 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. Expression of acid sphingomyelinase (ASM) was determined by Western blot techniques, ASM activity was measured by degradation of [14C]-labeled sphingomyelin to [14C]-labeled phosphorylcholine, and Ceramide-levels were detected by DAG kinase assay and immunofluorescence in orbital fibroblasts. Cell proliferation was determined by BrdU-incorporation, hyaluronan (HA) production was assessed by a HA-binding protein based assay, intracellular reactive oxygen species (ROS) were determined by the dichlorofluorescein assay, and cell viability was measured by the MTT-assay.

Results: : ASM activity was inhibited with amitryptiline and with siRNA technology. We demonstrate that a stable change in the phenotype of orbital fibroblasts from GO patients results in a constitutive overactivity of ASM and a high release of ceramide in these cells. High activity of the acid sphingomyelinase/ceramide system in GO orbital fibroblasts results in a constitutive increase of proliferation, release of reactive oxygen species, and formation of hyaluronan. These hallmarks of GO are corrected upon pharmacologic or siRNA-mediated inhibition of acid sphingomyelinase activity.

Conclusions: : Our findings identify the acid sphingomyelinase/ceramide system as a key mechanism in the pathophysiological changes affecting orbital fibroblasts during GO and provide a rationale for inhibiting acid sphingomyelinase activity as a novel treatment for GO.

Keywords: autoimmune disease • signal transduction: pharmacology/physiology • inflammation