Role Of Osteopontin In The Pathogenesis Of Thyroid Associated Ophthalmopathy

ObjectiveThyriod associated ophalmopathy(TAO)is an autoimmune inflammatory disorder involving the orbit,which is one of the most common extra-thyroidal complication of Graves’ disease(GD).The specific pathogenesis of TAO is very complicated and still instinct,accordingly,the treatment for TAO is very limited.Current evidence suggests the orbital fibroblast(OFs)is the key effector cell in TAO,which participate in the orbital inflammation,tissue remodeling and fibrosis and other pathological processes.As a secreted phosphorylated glycoprotein,osteopontin(OPN)has many biological effects and play important roles in various autoimmune diseases.OPN can induce fibroblast proliferation,and is closely related with tissue fibrosis.Recent studies found that the level of serum OPN in GD patients was significantly increased.In order to explore the relationship between OPN and the incidence of TAO,we examined the relative quantification of the expression of OPN mRNA and protein levels in orbital adipose connective tissue from patients with TAO,as well as some related cytokine genes.The relationships of mRNA levels between these cytokines and OPN was also tested.Then the vitro culture studies were performed to observe the effect of OPN on TAO derived OFs proliferation,migration and the expression of VEGF and Collagen I mRNA,and the related mechanisms were also explored.Methods1.The expression of OPN in orbital adipose connective tissue from patients with TAOOrbital adipose connective tissue specimens were obtained from six patients with active TAO who underwent orbital decompression surgery and from six patients with inactive TAO.Normal orbital tissues were retrieved from six patients who undergoing orbital plastic surgeries for noninflammatory conditions.We used Real-time PCR(RT-PCR)and Western blotting to detect OPN mRNA and protein expression respectively in all of the orbital adipose connective tissue specimens.Then RT-PCR was performed using fluorescent probes and primers for cytokines including CD40 L,CollagenⅠ,HAS2,NF-κB,CCL20,CD44,IFN-α and IFN-γ.We also conducted correlation analysis of mRNA expression levels between these cytokines and OPN.2.The effect of OPN on OFs in patients with TAO and and the mechanisms.OFs were cultivated with tissue culturing method from orbital adipose connective tissue under sterile conditions during decompression surgery for patients with TAO who were exclusion of other autoimmune and inflammatory diseases.After preprocessing with OPN(5uM),the proliferation and cell migration rates of OFs were measured by MTT and cell scratch test respectively.RT-PCR was applied for measuring the mRNA levels of VEGF and CollagenⅠin OFs.Pretreated OFs with OPN(5uM),OPN combined LY294002 or PD98059(5uM+20uM),which were signal pathway inhibitors of PI3K/Akt and MAPK/ERK respectively,OPN combined LY294002 and PD98059(5uM+20uM+20uM)and OPN combined with 1A12(5uM+20μg/ml).Using MTT,cell scratch test and RT-PCR to measure the proliferation and cell migration rates of OFs,and mRNA levels of VEGF and CollagenⅠin OFs.Results1.The mRNA and protien levels of OPN are increased in orbital adipose connective tissue from patients with active TAOThe mRNA levels of OPN increased in orbital adipose connective tissues from patients with active TAO,the differences were statistically significant(P< 0.05).The protien levels of OPN were also increased in patients with active TAO,the difference between active and inactive TAO patients were statistically significant(P<0.05),while was not significant between active TAO patients and normal patients(P<0.05).The mRNA levels of CD40 L,CollagenⅠand HAS2 increased in patients with active TAO(P < 0.05),while the differences of HAS2 mRNA levels was not statistically significant in any group.The CD44 mRNA levels were significantly lower in active and inactive TAO patients compared with normal patients(P<0.05).There existed significant linear correlations between OPN and CD40 L,OPN and CD44,OPN and HAS2 mRNA levels in patients with TAO(P<0.05).2.OPN promotes proliferation and migration rate of OFs and induces VEGF and Collagen I mRNA expression.The proliferation and migration rate of OFs was accelerated with OPN,the effects were inhibited by 1A12 or LY294002 rather than PD98059.OPN induces VEGF and Collagen I mRNA expression,the mRNA levels were decreased after treatment with 1A12 or LY294002,PD98059 had no effect on it.ConclusionsThe mRNA and protein levels of OPN in orbital adipose connective tissues were significantly increased in patients with active TAO,as well as CD40 L,collagen I and HAS2 mRNA.There existed significant linear correlation between OPN and CD40 L,OPN and CD44,OPN and HAS2 mRNA levels in patients with TAO.OPN could promote proliferation and migration of OFs and induce VEGF and Collagen I mRNA expression in OFs through PI3K/Akt signaling pathway.Indicating that OPN may play an important role in the pathogenesis of TAO through the activation of OFs by combinding with CD44 to promote orbital inflammation and the remodeling and fibrosis in orbital tissue,which was synergistically by CD40 L and HAS2.