The signs and symptoms of Graves' ophthalmopathy (GO) result from increased volume of the orbital contents, including adipose, connective, and extraocular muscle tissues. We wanted to determine whether the expanded adipose tissue volume might be in part attributable to de novo adipogenesis. We measured levels of mRNA encoding leptin, adiponectin, peroxisome proliferator-activated receptor gamma (PPAR gamma), preadipocyte factor-1, and TSH receptor (TSHr) genes in orbital adipose tissues from GO patients (n = 22) and normal individuals (n = 18) and in orbital preadipocyte cultures derived from GO patients (n = 6) and normal subjects (n = 3) using quantitative real-time RT PCR. We found increased leptin, adiponectin, PPAR gamma, and TSHr expression in GO compared with normal orbital tissue samples, with positive correlations in the GO tissues between TSHr and leptin, adiponectin and PPAR gamma. In vitro differentiation of GO and normal preadipocytes resulted in enhanced adiponectin, leptin, and TSHr expression, with greater expression of the latter two genes in the GO cultures. These results suggest that de novo adipogenesis within orbital tissues with parallel enhanced expression of TSHr may be important in the pathogenesis of GO, and that potential therapies for GO might include inhibition of the adipogenic pathway.

Graves' ophthalmopathy (GO) is characterized by expanded volume of the orbital tissues associated with elevated serum levels of TSH receptor (TSHR) autoantibodies. Because previous studies have demonstrated evidence of adipogenesis within the GO orbit, we sought to determine whether M22, a human monoclonal antibody directed against TSHR, enhances adipogenesis in orbital fibroblasts from patients with GO and, if so, to identify signaling mechanisms involved. GO orbital fibroblast cultures (n=10) were treated for 10 days with bovine TSH (1 or 10·0 U/l) or M22 (1 or 10 ng/ml) in serum-free adipocyte differentiation medium. Some cultures also received a phosphoinositide 3-kinase (PI3K) inhibitor or an inhibitor of cAMP production. In other experiments, confluent cultures (n=8) were treated for between 1 and 30 min with TSH (0·1–10·0 U/l) or M22 (0·1–100 ng/ml) with measurement of cAMP production or levels of phosphorylated AKT (pAKT). We found levels of adiponectin, leptin, and TSHR mRNA to be increased in GO cultures treated for 10 days with either M22 (2·6 mean fold ±0·7; P=0·03) or TSH (13·2±5·8-fold, P=0·048). In other studies, M22 and TSH stimulated cAMP production and pAKT levels in GO cells. Inhibition of PI3K activity during 10 days in culture decreased the levels of M22-stimulated mRNA encoding adiponectin (67±12%; P=0·021), as well as adiponectin and CCAAT/enhancer-binding protein α protein levels. In conclusion, M22 is a pro-adipogenic factor in GO orbital preadipocytes. This antibody appears to act via the PI3K signaling cascade, suggesting that inhibition of PI3K signaling may represent a potential novel therapeutic approach in GO.

The first therapeutic application of messenger RNA (mRNA) was suggested more than two decades ago. However, its application was constrained by the ability of mRNA to activate the innate immune response, cytotoxicity, and poor potency. We and others recently demonstrated that these undesirable properties of mRNA may be overcome by alterating its structure. In this study, we developed a new chemically modified mRNA coding for BMP-2 with improved osteogenic features. To develop this new construct, we removed from the mRNA sequence the following undesirable elements: an upstream open reading frame in the 5'-untranslated region (UTR) and a polyadenylation element together with an AU-rich tract in the 3'UTR. In addition, a translation initiator of short UTRs (TISU) was introduced together with 5-iodo modified pyrimidine nucleotides. The new TISU BMP-2 chemically modified RNA (cmRNA) showed robust BMP-2 production in vitro in cell lines (HEK293 and MC3T3) and primary cells (muscle-derived mesenchymal stem cells). Stem cells additionally showed upregulation of osteogenic and angiogenic genes as a result of the TISU BMP-2 cmRNA transfection. The in vivo osteogenic properties of TISU BMP-2 cmRNA were explored in a critical-sized femoral defect in the rat. For this, the TISU BMP-2 cmRNA was loaded into collagen sponges to form transcript-activated matrices. Animals treated with TISU BMP-2 cmRNA showed superior bone formation that seemed to recapitulate endochondral ossification. The higher of the two doses examined in this model showed more robust new tissue formation. Finally, improved vascularization was detected in the healing area for animals treated with TISU BMP-2 cmRNA.

These results support the concept that orbital adipogenesis is enhanced in GO and suggest that elevated levels of sFRP-1 in the GO orbit may be involved in stimulating this pathogenic process.