R.E. Miller

CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis

Osteoarthritis is one of the leading causes of chronic pain, but almost nothing is known about the mechanisms and molecules that mediate osteoarthritis-associated joint pain. Consequently, treatment options remain inadequate and joint replacement is often inevitable. Here, we use a surgical mouse model that captures the long-term progression of knee osteoarthritis to longitudinally assess pain-related behaviors and concomitant changes in the innervating dorsal root ganglia (DRG). We demonstrate that monocyte chemoattractant protein (MCP)-1 (CCL2) and its high-affinity receptor, chemokine (C-C motif) receptor 2 (CCR2), are central to the development of pain associated with knee osteoarthritis. After destabilization of the medial meniscus, mice developed early-onset secondary mechanical allodynia that was maintained for 16 wk. MCP-1 and CCR2 mRNA, protein, and signaling activity were temporarily up-regulated in the innervating DRG at 8 wk after surgery. This result correlated with the presentation of movement-provoked pain behaviors, which were maintained up to 16 wk. Mice that lack Ccr2 also developed mechanical allodynia, but this started to resolve from 8 wk onwards. Despite severe allodynia and structural knee joint damage equal to wild-type mice, Ccr2-null mice did not develop movement-provoked pain behaviors at 8 wk. In wild-type mice, macrophages infiltrated the DRG by 8 wk and this was maintained through 16 wk after surgery. In contrast, macrophage infiltration was not observed in Ccr2-null mice. These observations suggest a key role for the MCP-1/CCR2 pathway in establishing osteoarthritis pain.

Osteoarthritis joint pain: the cytokine connection.

Osteoarthritis is a chronic and painful disease of synovial joints. Chondrocytes, synovial cells and other cells in the joint can express and respond to cytokines and chemokines, and all of these molecules can also be detected in synovial fluid of patients with osteoarthritis. The presence of inflammatory cytokines in the osteoarthritic joint raises the question whether they may directly participate in pain generation by acting on innervating joint nociceptors. Here, we first provide a systematic discussion of the known proalgesic effects of cytokines and chemokines that have been detected in osteoarthritic joints, including TNF-α, IL-1, IL-6, IL-15, IL-10, and the chemokines, MCP-1 and fractalkine. Subsequently, we discuss what is known about their contribution to joint pain based on studies in animal models. Finally, we briefly discuss limited data available from clinical studies in human osteoarthritis.

Effect of Self-assembling Peptide, Chondrogenic Factors, and Bone Marrow Derived Stromal Cells on Osteochondral Repair

OBJECTIVE The goal of this study was to test the ability of an injectable self-assembling peptide (KLD) hydrogel with or without chondrogenic factors (CF) and allogeneic bone marrow stromal cells (BMSCs) to stimulate cartilage regeneration in a full-thickness, critically-sized, rabbit cartilage defect model in vivo. We used CF treatments to test the hypotheses that CF would stimulate chondrogenesis and matrix production by cells migrating into acellular KLD (KLD+CF) or by BMSCs delivered in KLD (KLD+CF+BMSCs). DESIGN Three groups were tested against contralateral untreated controls: KLD, KLD+CF, and KLD+CF+BMSCs, n=6-7. Transforming growth factor-β1 (TGF-β1), dexamethasone, and insulin-like growth factor-1 (IGF-1) were used as CF pre-mixed with KLD and BMSCs before injection. Evaluations included gross, histological, immunohistochemical and radiographic analyses. RESULTS KLD without CF or BMSCs showed the greatest repair after 12 weeks with significantly higher Safranin-O, collagen II immunostaining, and cumulative histology scores than untreated contralateral controls. KLD+CF resulted in significantly higher aggrecan immunostaining than untreated contralateral controls. Including allogeneic BMSCs+CF markedly reduced the quality of repair and increased osteophyte formation compared to KLD-alone. CONCLUSIONS These data show that KLD can fill full-thickness osteochondral defects in situ and improve cartilage repair as shown by Safranin-O, collagen II immunostaining, and cumulative histology. In this small animal model, the full-thickness critically-sized defect provided access to the marrow, similar in concept to abrasion arthroplasty or spongialization in large animal models, and suggests that combining KLD with these techniques may improve current practice.

Engineering insulin-like growth factor-1 for local delivery

Insulin‐like growth factor‐1 (IGF‐1) is a small protein that promotes cell survival and growth, often acting over long distances. Although for decades IGF‐1 has been considered to have therapeutic poten tial, systemic side effects of IGF‐1 are significant, and local delivery of IGF‐1 for tissue repair has been a long‐standing challenge. In this study, we designed and purified a novel protein, heparin‐binding IGF‐1 (Xp‐ HB‐IGF‐1), which is a fusion protein of native IGF‐1 with the heparin‐binding domain of heparin‐binding epidermal growth factor‐like growth factor. Xp‐HB‐ IGF‐1 bound selectively to heparin as well as the cell surfaces of 3T3 fibroblasts, neonatal cardiac myocytes and differentiating ES cells. Xp‐HB‐IGF‐1 activated the IGF‐1 receptor and Akt with identical kinetics and dose response, indicating no compromise of biological activ ity due to the heparin‐binding domain. Because carti lage is a proteoglycan‐rich environment and IGF‐1 is a known stimulus for chondrocyte biosynthesis, we then studied the effectiveness of Xp‐HB‐IGF‐1 in cartilage. Xp‐HB‐IGF‐1 was selectively retained by cartilage ex plants and led to sustained chondrocyte proteoglycan biosynthesis compared to IGF‐1. These data show that the strategy of engineering a “long‐distance” growth factor like IGF‐1 for local delivery may be useful for tissue repair and minimizing systemic effects.— Tokunou, T., Miller, R., Patwari, P., Davis, M. E., Segers, V. F. M., Grodzinsky, A. J., Lee, R. T. Engineering insulin‐like growth factor‐1 for local delivery. FASEB J. 22, 1886–1893 (2008)

Translational development of an ADAMTS-5 antibody for osteoarthritis disease modification

OBJECTIVE/METHOD Aggrecanase activity, most notably ADAMTS-5, is implicated in pathogenic cartilage degradation. Selective monoclonal antibodies (mAbs) to both ADAMTS-5 and ADAMTS-4 were generated and in vitro, ex vivo and in vivo systems were utilized to assess target engagement, aggrecanase inhibition and modulation of disease-related endpoints with the intent of selecting a candidate for clinical development in osteoarthritis (OA). RESULTS Structural mapping predicts the most potent mAbs employ a unique mode of inhibition by cross-linking the catalytic and disintegrin domains. In a surgical mouse model of OA, both ADAMTS-5 and ADAMTS-4-specific mAbs penetrate cartilage following systemic administration, demonstrating access to the anticipated site of action. Structural disease modification and associated alleviation of pain-related behavior were observed with ADAMTS-5 mAb treatment. Treatment of human OA cartilage demonstrated a preferential role for ADAMTS-5 inhibition over ADAMTS-4, as measured by ARGS neoepitope release in explant cultures. ADAMTS-5 mAb activity was most evident in a subset of patient-derived tissues and suppression of ARGS neoepitope release was sustained for weeks after a single treatment in human explants and in cynomolgus monkeys, consistent with high affinity target engagement and slow ADAMTS-5 turnover. CONCLUSION This data supports a hypothesis set forth from knockout mouse studies that ADAMTS-5 is the major aggrecanase involved in cartilage degradation and provides a link between a biological pathway and pharmacology which translates to human tissues, non-human primate models and points to a target OA patient population. Therefore, a humanized ADAMTS-5-selective monoclonal antibody (GSK2394002) was progressed as a potential OA disease modifying therapeutic.

Growth Factor Delivery Through Self-assembling Peptide Scaffolds

BackgroundThe best strategy for delivering growth factors to cells for the purpose of cartilage tissue engineering remains an unmet challenge. Tethering biotinylated insulin-like growth factor-1 (bIGF-1) to the self-assembling peptide scaffold (RADA)4 effectively delivers bioactive bIGF-1 to cardiac tissue.Questions/purposesWe therefore asked whether: (1) soluble bIGF-1 could stimulate proteoglycan production by chondrocytes; (2) bIGF-1 could be adsorbed or tethered to the self-assembling peptide scaffold (KLDL)3; (3) adsorbed or tethered bIGF-1 could stimulate proteoglycan production; and (4) transforming growth factor-β1 (TGF-β1) could be adsorbed or tethered and stimulate proteoglycan production by bone marrow stromal cells (BMSCs).MethodsChondrocytes or BMSCs were encapsulated in (KLDL)3. The growth factors were (1) delivered solubly in the medium; (2) adsorbed to (KLDL)3; or (3) tethered to (KLDL)3 through biotin-streptavidin bonds. Fluorescently tagged streptavidin was used to determine IGF-1 kinetics; sGAG and DNA content was measured.ResultsSoluble bIGF-1 stimulated comparable sGAG accumulation as soluble IGF-1. Tethering IGF-1 to (KLDL)3 increased retention of IGF-1 in (KLDL)3 compared with adsorption, but neither method increased sGAG or DNA accumulation above control. Adsorbing TGF-β1 increased proteoglycan accumulation above control, but tethering did not affect sGAG levels.ConclusionsAlthough TGF-β1 can be effectively delivered by adsorption to (KLDL)3, IGF-1 cannot. Additionally, although tethering these factors provided long-term sequestration, tethering did not stimulate proteoglycan production.Clinical RelevanceTethering growth factors to (KLDL)3 results in long-term delivery, but tethering does not necessarily result in the same bioactivity as soluble delivery, indicating presentation of proteins is vital when considering a delivery strategy.

Intraarticular injection of heparin-binding insulin-like growth factor 1 sustains delivery of insulin-like growth factor 1 to cartilage through binding to chondroitin sulfate

OBJECTIVE Insulin-like growth factor 1 (IGF-1) stimulates cartilage repair but is not a practical therapy due to its short half-life. We have previously modified IGF-1 by adding a heparin-binding domain and have shown that this fusion protein (HB-IGF-1) stimulates sustained proteoglycan synthesis in cartilage. This study was undertaken to examine the mechanism by which HB-IGF-1 is retained in cartilage and to test whether HB-IGF-1 provides sustained growth factor delivery to cartilage in vivo and to human cartilage explants. METHODS Retention of HB-IGF-1 and IGF-1 was analyzed by Western blotting. The necessity of heparan sulfate (HS) or chondroitin sulfate (CS) glycosaminoglycans (GAGs) for binding was tested using enzymatic removal and cells with genetic deficiency of HS. Binding affinities of HB-IGF-1 and IGF-1 proteins for isolated GAGs were examined by surface plasmon resonance and enzyme-linked immunosorbent assay. RESULTS In cartilage explants, chondroitinase treatment decreased binding of HB-IGF-1, whereas heparitinase had no effect. Furthermore, HS was not necessary for HB-IGF-1 retention on cell monolayers. Binding assays showed that HB-IGF-1 bound both CS and HS, whereas IGF-1 did not bind either. After intraarticular injection in rat knees, HB-IGF-1 was retained in articular and meniscal cartilage, but not in tendon, consistent with enhanced delivery to CS-rich cartilage. Finally, HB-IGF-1 was retained in human cartilage explants but IGF-1 was not. CONCLUSION Our findings indicate that after intraarticular injection in rats, HB-IGF-1 is specifically retained in cartilage through its high abundance of CS. Modification of growth factors with heparin-binding domains may be a new strategy for sustained and specific local delivery to cartilage.

PCSK6-mediated corin activation is essential for normal blood pressure.

Hypertension is the most common cardiovascular disease, afflicting >30% of adults. The cause of hypertension in most individuals remains unknown, suggesting that additional contributing factors have yet to be discovered. Corin is a serine protease that activates the natriuretic peptides, thereby regulating blood pressure. It is synthesized as a zymogen that is activated by proteolytic cleavage. CORIN variants and mutations impairing corin activation have been identified in people with hypertension and pre-eclampsia. To date, however, the identity of the protease that activates corin remains elusive. Here we show that proprotein convertase subtilisin/kexin-6 (PCSK6, also named PACE4; ref. 10) cleaves and activates corin. In cultured cells, we found that corin activation was inhibited by inhibitors of PCSK family proteases and by small interfering RNAs blocking PCSK6 expression. Conversely, PCSK6 overexpression enhanced corin activation. In addition, purified PCSK6 cleaved wild-type corin but not the R801A variant that lacks the conserved activation site. Pcsk6-knockout mice developed salt-sensitive hypertension, and corin activation and pro-atrial natriuretic peptide processing activity were undetectable in these mice. Moreover, we found that CORIN variants in individuals with hypertension and pre-eclampsia were defective in PCSK6-mediated activation. We also identified a PCSK6 mutation that impaired corin activation activity in a hypertensive patient. Our results indicate that PCSK6 is the long-sought corin activator and is important for sodium homeostasis and normal blood pressure.

Damage-associated molecular patterns generated in osteoarthritis directly excite murine nociceptive neurons through Toll-like receptor 4.

To determine whether selected damage‐associated molecular patterns (DAMPs) present in the osteoarthritic (OA) joints of mice excite nociceptors through Toll‐like receptor 4 (TLR‐4).

Effects of Dexamethasone on Mesenchymal Stromal Cell Chondrogenesis and Aggrecanase Activity: Comparison of Agarose and Self-Assembling Peptide Scaffolds.

Objective: Dexamethasone (Dex) is a synthetic glucocorticoid that has pro-anabolic and anticatabolic effects in cartilage tissue engineering systems, though the mechanisms by which these effects are mediated are not well understood. We tested the hypothesis that the addition of Dex to chondrogenic medium would affect matrix production and aggrecanase activity of human and bovine bone marrow stromal cells (BMSCs) cultured in self-assembling peptide and agarose hydrogels. Design: We cultured young bovine and adult human BMSCs in (RADA)4 self-assembling peptide and agarose hydrogels in medium containing TGF-β1±Dex and analyzed extracellular matrix composition, aggrecan cleavage products, and the effects of the glucocorticoid receptor antagonist RU-486 on proteoglycan content, synthesis, and catabolic processing. Results: Dex improved proteoglycan synthesis and retention in agarose hydrogels seeded with young bovine cells but decreased proteoglycan accumulation in peptide scaffolds. These effects were mediated by the glucocorticoid receptor. Adult human BMSCs showed minimal matrix accumulation in agarose, but accumulated ~50% as much proteoglycan and collagen as young bovine BMSCs in peptide hydrogels. Dex reduced aggrecanase activity in (RADA)4 and agarose hydrogels, as measured by anti-NITEGE Western blotting, for both bovine and human BMSC-seeded gels. Conclusions: The effects of Dex on matrix production are dependent on cell source and hydrogel identity. This is the first report of Dex reducing aggrecanase activity in a tissue engineering culture system.