The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRα displayed chondrogenic activity in the presence of TGFβ3 and expressed cartilage-specific genes in 7 to 16 day pellet cultures. Depositions of cartilage matrix and type II collagen were detected by day 14. TGFβ-stimulated chondrogenesis was synergistically enhanced by PDGF-BB, resulting in a larger cartilage particle filled with a cartilaginous area containing type II collagen, with a surface cell layer expressing type I collagen. In contrast, noggin inhibited both the TGFβ-and TGFβ+PDGF-stimulated cartilage formation, suggesting that a BMPdependent pathway is involved. In fact, replacement of TGFβ3 with BMP4 on days 10 to 12 markedly elevated the cartilage matrix deposition during the following 7 to 8 days. Moreover, culture with TGFβ3 and PDGF-BB, followed by the incubation with BMP4 alone, resulted in a cartilage particle lacking type I collagen in the matrix and the surface layer, which suggests hyaline cartilage formation. Furthermore, such hyaline cartilage particles were mineralized. These studies indicate that the PDGFRα + and/or flk-1 + cells derived from embryonic stem cells possess the full developmental potential toward chondrocytes, in common with embryonic mesenchymal cells.

We have identified a novel chordin-like protein, CHL2, which is structurally most homologous to CHL/neuralin/ventroptin. When injected into Xenopus embryos, CHL2 RNA induced a secondary axis. Recombinant CHL2 protein interacted directly with BMPs in a competitive manner to prevent binding to the type I BMP receptor ectodomain, and inhibited BMP-dependent induction of alkaline phosphatase in C2C12 cells. Thus, CHL2 behaves as a secreted BMP-binding inhibitor. In situ hybridization revealed that CHL2 expression is restricted to chondrocytes of various developing joint cartilage surfaces and connective tissues in reproductive organs. Adult mesenchymal progenitor cells expressed CHL2, and its levels decreased during chondrogenic differentiation. Addition of CHL2 protein to a chondrogenic culture system reduced cartilage matrix deposition. Consistently,CHL2 transcripts were weakly detected in normal adult joint cartilage. However, CHL2 expression was upregulated in middle zone chondrocytes in osteoarthritic joint cartilage (where hypertrophic markers are induced). CHL2 depressed chondrocyte mineralization when added during the hypertrophic differentiation of cultured hyaline cartilage particles. Thus, CHL2 may play negative roles in the (re)generation and maturation of articular chondrocytes in the hyaline cartilage of both developing and degenerated joints.

Introduction Rats with adjuvant-induced arthritis (AIA) were necropsied on 14 occasions during preclinical, acute clinical and chronic clinical stages of AIA progression to characterize local (joint protein extracts) and systemic (serum) levels of mediators regulating inflammation and bone erosion in conjunction with lymphoid tissue-specific leukocyte kinetics. Results Systemic increases in alpha1 acid glycoprotein, tumor necrosis factor-α (TNFα), interleukin (IL)-17, transforming growth factor beta (TGFβ), and chemokine (C-C motif) ligand 2 (CCL2) together with local IL-1α/β and TGFβ enrichment and local lymphoid hyperplasia preceded the onset of clinical disease and joint damage. Systemic upregulation of TNFα, IL-6, IL-17, TGFβ, IL-18, CCL2, receptor activator of nuclear factor-κβ ligand (RANKL), and prostaglandin E 2 during acute and/or chronic AIA coincided with systemic leukocytosis and CD4+ T cell increase in blood and spleen. In contrast, progression of joint erosions during clinical AIA was associated with intraarticular increases in IL-1α/β, IL-6, RANKL, IL-17, TGFβ, CCL2, and KC/GRO and also a dramatic decline in osteoprotegerin. Conclusion These data indicate that systemic and local events in inflammatory arthritis are discrete processes, driven by multiple cellular and humoral mediators with distinct kinetic profiles.

Objective. To assess the capacities of the cytokine inhibitors interleukin-1 receptor antagonist (IL-1Ra; anakinra) and PEGylated soluble tumor necrosis factor receptor I (PEG sTNFRI; pegsunercept) to suppress neovascularization.Methods. A corneal angiogenesis assay was performed by implanting nylon discs impregnated with an angiogenic stimulator (basic fibroblast growth factor or vascular endothelial growth factor) into one cornea of female Sprague-Dawley rats. Animals were treated with IL-1Ra or PEG sTNFRI for 7 days, after which new vessels were quantified. In a parallel study, male Lewis rats with mycobacteria-induced adjuvant-induced arthritis were treated with IL-1Ra or PEG sTNFRI for 7 days beginning at disease onset, after which scores for inflammation and bone erosion as well as capillary counts were acquired from sections of arthritic hind paws.Results. Treatment with IL-1Ra yielded a dosedependent reduction in growth factor-induced corneal angiogenesis, while PEG sTNFRI did not. IL-1Ra, but not PEG sTNFRI, significantly reduced the number of capillaries in arthritic paws, even though both anticytokines reduced inflammation and bone erosion to a similar degree.Conclusion. These data support a major role for IL-1, but not TNF␣, in angiogenesis and suggest that an additional antiarthritic mechanism afforded by IL-1 inhibitors, but not anti-TNF agents, is the suppression of the angiogenic component of pannus.

IntroductionRat adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) feature bone loss and systemic increases in TNFα, IL-1β, and receptor activator of NF-κB ligand (RANKL). Anti-IL-1 or anti-TNFα therapies consistently reduce inflammation in these models, but systemic bone loss often persists. RANKL inhibition consistently prevents bone loss in both models without reducing joint inflammation. Effects of these therapies on systemic markers of bone turnover and inflammation have not been directly compared.MethodsLewis rats with established AIA or CIA were treated for 10 days (from day 4 post onset) with either PBS (Veh), TNFα inhibitor (pegsunercept), IL-1 inhibitor (anakinra), or RANKL inhibitor (osteoprotegerin (OPG)-Fc). Local inflammation was evaluated by monitoring hind paw swelling. Bone mineral density (BMD) of paws and lumbar vertebrae was assessed by dual X-ray absorptiometry. Markers and mediators of bone resorption (RANKL, tartrate-resistant acid phosphatase 5b (TRACP 5B)) and inflammation (prostaglandin E2 (PGE2), acute-phase protein alpha-1-acid glycoprotein (α1AGP), multiple cytokines) were measured in serum (day 14 post onset).ResultsArthritis progression significantly increased paw swelling and ankle and vertebral BMD loss. Anti-TNFα reduced paw swelling in both models, and reduced ankle BMD loss in AIA rats. Anti-IL-1 decreased paw swelling in CIA rats, and reduced ankle BMD loss in both models. Anti-TNFα and anti-IL-1 failed to prevent vertebral BMD loss in either model. OPG-Fc reduced BMD loss in ankles and vertebrae in both models, but had no effect on paw swelling. Serum RANKL was elevated in AIA-Veh and CIA-Veh rats. While antiTNFα and anti-IL-1 partially normalized serum RANKL without any changes in serum TRACP 5B, OPG-Fc treatment reduced serum TRACP 5B by over 90% in both CIA and AIA rats. CIA-Veh and AIA-Veh rats had increased serum α1AGP, IL-1β, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), and AIA-Veh rats also had significantly greater serum PGE2, TNFα and IL-17. Anti-TNFα reduced systemic α1AGP, CCL2 and PGE2 in AIA rats, while anti-IL-1 decreased systemic α1AGP, IL-8 and PGE2. In contrast, RANKL inhibition by OPG-Fc did not lessen systemic cytokine levels in either model.ConclusionsAnti-TNFα or anti-IL-1 therapy inhibited parameters of local and systemic inflammation, and partially reduced local but not systemic bone loss in AIA and CIA rats. RANKL inhibition prevented local and systemic bone loss without significantly inhibiting local or systemic inflammatory parameters.