Atsttrin, an engineered protein derived from progranulin growth factor, is therapeutic in osteoarthritis

Purpose: Progranulin (PGRN) is a multifunctional growth factor which is composed of seven-and-a-half repeats of a cysteine-rich motif in the order P-G-F-B-A-C-D-E. PGRN directly binds to TNF-α receptors (TNFR), and inhibits TNF-α activity. Atsttrin(A50), an engineered protein composed of three PGRN fragments (1/2F-1/2A-1/2C), exhibited selective TNFR binding, and potently prevented inflammation in multiple arthritis mouse models (Tang, et al, Science, 2011). In addition, recent studies demonstrated that local injection of PGRN significantly prevented OA progression (Zhao et al, Annals of the Rheumatic Diseases, 2015). These previous data promoted us to determine whether Atsttrin also has therapeutic effects in preventing OA, and if so what is the molecular mechanism involved. Methods: Mice models were established to determine the role A50 in OA pathogenesis. Primary human cartilage and chondrocyte were used to determine the anti-catabolic and anabolic effect of A50. Results: 1. Atsttrin effectively prevents osteoarthritis in surgically-induced mice model in vivo. ACL (anterior cruciate ligament) transection model in wild type mice was established to determine the preventative effect of A50. 6μg A50 was intra-articular injected weekly for 4 weeks once the model was established. As shown in Fig.1A, Safranin O staining indicated A50 effectively prevents cartilage loss in OA progression. Additionally, as demonstrated in Fig.1B&C, histological grading analysis showed significant improvement of OA score and attenuation of proteoglycan loss in cartilage with treatment of A50. Furthermore, as shown in Fig.1D, immunochemistry staining demonstrated that the A50 decreased the degradation of cartilage matrix molecules (e.g. COMP), and reduced the hypertrophic markers of chondrocyte (e.g. Col X), and suppressed the expression of matrix-degrading enzymes (e.g. MMP13), slowing down OA progression. Furthermore, OA is primarily characterized by cartilage destruction but also involves other pathological changes, including synovial inflammation and subchondral bone sclerosis, in all tissues of joints. As indicated in Fig.1E, H&E staining revealed that A50 effectively prevented subchondral bone sclerosis and synovial inflammation. 2. Atsttrin inhibits TNF-α-induced catabolic metabolism and promotes anabolic metabolism. It is well established that inflammatory cytokine TNF-α plays an important role in cartilage degeneration in OA. The finding that A50 prevented cartilage destruction in inflammatory arthritis mediated by TNF-α (Tang, et al, Science, 2011) prompted us to investigate the potential association between A50 and TNF-α in OA. Cartilage samples from patients with OA were isolated and cultured with 10 ng/mL TNF-α in presence or absence of 200 ng/mL A50 for 7 days. As revealed in Fig.2A&B, Safranin O staining of the cartilage samples indicated that TNF-α markedly enhanced loss of proteoglycan, while the additional use of A50 largely rescued this effect of TNF-α. Moreover, primary human chondrocytes were treated in with 10 ng/mL TNF-α in presence or absence of 200 ng/mL A50 for indicated time points. As shown in Fig.2C, A50 dramatically inhibited TNF-α-mediated pro-inflammatory signaling(P38, JNK pathways). In addition, chondrocytes were cultured for 72 hours, as demonstrated in Fig.2D, A50 dose-dependently inhibited expression of TNF-α-induced matrix-degrading enzymes (e.g. MMP-3, MMP-13, ADAMTS-4) and inflammatory severity marker, NOS-2. More importantly, as revealed in Fig.2E, A50 remarkably promoted anabolism in primary human cartilage. As indicated in Fig.2F&G, anabolic effect of A50 was further confirmed by the increased gene expression of aggrecan and Col II in human primary chondrocytes. Conclusions: These findings not only provide novel insights into the role of Atsttrin in cartilage homeostasis and arthritis in vivo, but may also lead to the development of novel therapeutic intervention strategies for osteoarthritis.Fig.2 Atsttrin inhibits TNF-α-induced catabolic metabolism and promotes anabolic metabolism. (A) Safranin O staining of primary human cartilage in presence or absence of 200ng/ml Atsttrin with or without 10ng/ml TNF-α.(B) Statistic analysis of proteoglycan area based on Safranin O staining. (C) Primary human chondrocytes were incubated with TNF-α in the presence or absence of Atsttrin, and phosphorylation and expression of the indicated signaling molecules at various time points were determined by immunoblotting. Tubulin is shown as a loading control. (D) The protein level of MMP-3, MMP-13, ADAMTS-4 and NOS-2 in primary human chondrocytes with 10ng/ml TNF-α in absence or presence of Atsttrin. (E) Atsttrin promoted anabolism in primary human cartilage, assayed by Safranin O staining. (F-G) Levels of anabolic biomarkers in chondrocyte, including Acn and Col II, as measured by real-time PCR. Values are the normalised mean±SEM; *p<0.05, **p<0.01 versus control group. Six cartilages were used in each group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)