Elevated bone morphogenic protein 4 expression implicated in site-specific adipogenesis in thyroid associated orbitopathy.

Periorbital adipose tissue expansion is a key pathological change in thyroid associated orbitopathy (TAO). Bone morphogenic protein 4 (BMP4) is instrumental in adipogenesis. We compared site-specific BMP4 expression and its effect on adipogenesis using donor-matched adipose tissue-derived stromal cells (ADSC) from TAO patients. In this study, ADSC were generated from periorbital (eyelid, orbital) and subcutaneous (abdominal) adipose tissue. BMP4 expression was characterized by RT-PCR and immunofluorescent staining and compared among ADSC from the three anatomic depots. Effects on adipogenesis after knocking down endogenous BMP4 were quantified by adipogenic markers PPARγ and perilipin. Exogenous BMP4 protein was added after BMP4 knockdown to study the role of BMP4 in adipogenesis. Our results showed that BMP4 staining in periorbital adipose tissue was stronger than those in subcutaneous. BMP4 mRNA expression was higher in eyelid (4.4-2489.4-fold) and orbital (6.9-1811-fold) than that of subcutaneous ADSC, whereas expression fell during induced adipogenesis. After BMP4 knockdown, both adipogenic markers PPARγ (eyelid: 1.7-fold, p = 0.038; orbital: 1.4-fold, p = 0.126) and perilipin (eyelid:1.7-fold, p = 0.001; orbital:2.6-fold, p = 0.066) increased in periorbital ADSC upon induction. These increased expression fell after adding exogenous BMP4 protein. Our findings demonstrated higher BMP4 expression was found in periorbital ADSC and adipose tissue compared to donor-matched subcutaneous counterparts, which fell during adipogenic induction. Knocking down BMP4 expression further enhanced adipogenesis in periorbital ADSC. This effect was reversed by adding exogenous BMP4 protein. We suggested a novel role of BMP4 in modulating site-specific adipogenesis in TAO patients.

Depot-specific characteristics of adipose tissue-derived stromal cells in thyroid-associated orbitopathy.

BACKGROUND: Thyroid-associated orbitopathy (TAO) causes inflammatory fibroproliferation of periocular connective tissues. We compared adipose tissue-derived stem/stromal cells (ADSCs) from three adipose depots of each patient with TAO on mesenchymal, myofibrogenic, adipogenic properties and associated hyaluronan (HA) synthesis. METHODS: ADSCs were generated from periocular (eyelid, orbital) and subcutaneous (abdominal) adipose tissues of three patients with TAO. Mesenchymal markers were characterised by reverse transcription-PCR and immunofluorescent staining. A 3-week adipogenic induction was evaluated by Nile red staining and quantitative PCR (qPCR) of peroxisome proliferator-activated receptor (PPARγ), adiponectin and hyaluronan synthase (HAS)-2. A 7-day myofibrogenic induction was assayed by immunofluorescent staining and qPCR of α-smooth muscle actin (α-SMA). RESULTS: ADSCs from all depots expressed similar levels of mesenchymal markers CD44, CD90 and CD105 (p=0.288, p=0.43 and p=0.837, respectively). After adipogenic induction, intracellular lipid increased for more than 32% and PPARγ mRNA showed more than twofold increase from all three depots. However, adiponectin and HAS-2 mRNA levels were significantly higher in the eyelid and orbital ADSCs than those from the subcutaneous ADSCs after induction (2.4×107, 3.9×106 folds vs below detection limit; 63.3-fold, 26.1-fold, vs 33% reduction, respectively; all p=0.002). Significantly more myofibroblasts and higher mRNA level of α-SMA were obtained from the orbital and eyelid compared with the subcutaneous ADSCs during myofibrogenic induction (80.2%, 70.6% vs 29.3%; 30.2-fold, 24.2-fold vs 1.7-fold, respectively; all p=0.002). CONCLUSION: ADSCs from different adipose depots of the same donors exhibited similar mesenchymal phenotypes but differed significantly in adipogenic, myofibrogenic potentials and associated HA synthesis. These depot-specific characteristics of ADSCs may contribute to site-specific adipose tissue involvement in TAO.

Antagonist of GH-releasing hormone receptors alleviates experimental ocular inflammation.

Disruptions in immunity and occurrence of inflammation cause many eye diseases. The growth hormone-releasing hormone-growth hormone-insulin-like growth factor-1 (GHRH-GH-IGF1) axis exerts regulatory effects on the immune system. Its involvement in ocular inflammation remains to be investigated. Here we studied this signaling in endotoxin-induced uveitis (EIU) generated by LPS. The increase in GHRH receptor (GHRH-R) protein levels was parallel to the increase in mRNA levels of pituitary-specific transcription factor-1, GHRH-R splice variant 1, GHRH, and GH following LPS insult. Elevation of GHRH-R and GH receptor was localized on the epithelium of the iris and ciliary body, and GHRH-R was confined to the infiltrating macrophages and leukocytes in aqueous humor but not to those in stroma. Treatment with GHRH-R antagonist decreased LPS-stimulated surges of GH and IGF1 in aqueous humor and alleviated inflammation by reducing the infiltration of macrophages and leukocytes and the production of TNF-α, IL-1ß, and monocyte chemotactic protein-1. Our results indicate that inflammation in the iris and ciliary body involves the activation of GHRH signaling, which affects the recruitment of immune cells and the production of proinflammatory mediators that contribute to EIU pathogenesis. Moreover, the results suggest that GHRH-R antagonists are potential therapeutic agents for the treatment of acute ocular inflammation.