The Effects of an Osteoarthritic Joint Environment on ACL Damage and Degeneration: A Yucatan Miniature Pig Model.

Posttraumatic osteoarthritis (PTOA) arises secondary to joint injuries and is characteristically driven by inflammatory mediators. PTOA is often studied in the setting of ACL tears. However, a wide range of other injuries also lead to PTOA pathogenesis. The purpose of this study was to characterize the morphological changes in the uninjured ACL in a PTOA inflammatory environment. We retrospectively reviewed 14 ACLs from 13 Yucatan minipigs, 7 of which had undergone our modified intra-articular drilling (mIAD) procedure, which induced PTOA through inflammatory mediators. Seven ACLs were harvested from mIAD minipigs (PTOA) and seven ACLs from control minipigs with no cartilage degeneration (non-PTOA). ACL degeneration was evaluated using histological scoring systems. IL-1ß, NF-κB, and TNF-α mRNA expression in the synovium was measured using qRT-PCR. PTOA minipigs demonstrated significant ACL degeneration, marked by a disorganized extracellular matrix, increased vascularity, and changes in cellular shape, density, and alignment. Furthermore, IL-1ß, NF-κB, and TNF-α expression was elevated in the synovium of PTOA minipigs. These findings demonstrate the potential for ACL degeneration in a PTOA environment and emphasize the need for anti-inflammatory disease-modifying therapies following joint injury.

A novel large animal model of posttraumatic osteoarthritis induced by inflammation with mechanical stability.

OBJECTIVES: Animal models are needed to reliably separate the effects of mechanical joint instability and inflammation on posttraumatic osteoarthritis (PTOA) pathogenesis. We hypothesized that our modified intra-articular drilling (mIAD) procedure induces cartilage damage and synovial changes through increased inflammation without causing changes in gait. METHODS: Twenty-four Yucatan minipigs were randomized into the mIAD (n=12) or sham control group (n=12). mIAD animals had two osseous tunnels drilled into each of the tibia and femur adjacent to the anterior cruciate ligament (ACL) attachment sites on the left hind knee. Surgical and contralateral limbs were harvested 15 weeks post-surgery. Cartilage degeneration was evaluated macroscopically and histologically. Synovial changes were evaluated histologically. Interleukin-1 beta (IL-1ß), nuclear factor kappa B (NF-κB), and tumor necrosis factor alpha (TNF-α) mRNA expression levels in the synovial membrane were measured using quantitative real-time polymerase chain reaction. IL-1ß and NF-κB levels in chondrocytes were assessed using immunohistochemistry. Load asymmetry during gait was recorded by a pressure-sensing walkway system before and after surgery. RESULTS: The mIAD surgical knees demonstrated greater gross and histological cartilage damage than contralateral (P<.01) and sham knees (P<.05). Synovitis was present only in the mIAD surgical knee. Synovial inflammatory marker (IL-1ß, NF-κB, and TNF-α) expression was three times higher in the mIAD surgical knee than the contralateral (P<.05). Chondrocyte IL-1ß and NF-κB levels were highest in the mIAD surgical knee. In general, there were no significant changes in gait. CONCLUSIONS: The mIAD model induced PTOA through inflammation without affecting gait mechanics. This large animal model has significant applications for evaluating the role of inflammation in PTOA and for developing therapies aimed at reducing inflammation following joint injury.

A2M inhibits inflammatory mediators of chondrocytes by blocking IL-1ß/NF-κB pathway.

A hallmark of osteoarthritis (OA) is cartilage degeneration, which has been previously correlated with dramatic increases in inflammatory enzymes. Specifically, interleukin-1ß (IL-1ß) and subsequent upregulation of nuclear factor kappa B (NF-κB) is implicated as an important player in the development of posttraumatic osteoarthritis (PTOA). Alpha 2-macroglobulin (A2M) can inhibit this inflammatory pathway, making it a promising therapy for PTOA. Herein, we demonstrate that A2M binds and neutralizes IL-1ß, blocking downstream NF-κB-induced catabolism seen in in vitro. Human chondrocytes (cell line C28) were incubated with A2M protein and then treated with IL-1ß. A2M was labeled with VivoTag™ 680 to localize the protein postincubation. The degree of binding between A2M and IL-1ß was evaluated through immunoprecipitation (IP). Catabolic proteins, including IL-1ß and NF-kB, were detected by Western blot. Pro-inflammatory and chondrocyte-related gene expression was examined by qRT-PCR. VivoTag™ 680-labeled A2M was observed in the cytoplasm of C28 human chondrocytes by fluorescence microscopy. IP experiments demonstrated that A2M could bind IL-1ß. Additionally, western blot analysis revealed that A2M neutralized IL-1ß and NF-κB in a dose-dependent manner. Moreover, A2M decreased levels of MMPs and TNF-α and increased the expression of cartilage protective genes Col2, Type2, Smad4, and aggrecan. Mostly importantly, A2M was shown to directly neutralize IL-1ß to downregulate the pro-inflammatory responses mediated by the NF-kB pathway. These results demonstrate a mechanism by which A2M reduces inflammatory catabolic activity and protects cartilage after joint injury. Further in vivo studies are needed to fully understand the potential of A2M as a novel PTOA therapy.

Inhibition of lipid phosphatase SHIP1 expands myeloid-derived suppressor cells and attenuates rheumatoid arthritis in mice.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease of unknown cause, characterized by infiltration and accumulation of activated immune cells in the synovial joints where cartilage and bone destructions occur. Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) was shown to be involved in the regulation of MDSC differentiation. The purpose of the present study was to investigate the effect of inhibition of SHIP1 on the expansion of MDSCs in RA using a collagen-induced inflammatory arthritis (CIA) mouse model. In DBA/1 mice, treatment with a small molecule-specific SHIP1 inhibitor 3α-aminocholestane (3AC) induced a marked expansion of MDSCs in vivo. Both pretreatment with 3AC of DBA/1 mice prior to CIA induction and intervention with 3AC during CIA progression significantly reduced disease incidence and severity. Adoptive transfer of MDSCs isolated from 3AC-treated mice, but not naïve MDSCs from normal mice, into CIA mice significantly reduced disease incidence and severity, indicating that the 3AC-induced MDSCs were the cellular mediators of the observed amelioration of the disease. In conclusion, inhibition of SHIP1 expands MDSCs in vivo and attenuates development of CIA in mice. Small molecule-specific inhibition of SHIP1 may therefore offer therapeutic benefit to patients with RA and other autoimmune diseases.

Monolayer Amorphous Carbon-Bridged Nanosheet Mesocrystal: Facile Preparation, Morphosynthetic Transformation, and Energy Storage Applications.

Self-assembly of nanoscale building units into mesoscopically ordered superstructures opens the possibility for tailored applications. Nonetheless, the realization of precise controllability related specifically to the atomic scale has been challenging. Here, first, we explore the key role of a molecular surfactant in adjusting the growth kinetics of two-dimensional (2D) layered SnS2. Experimentally, we show that high pressure both enhances the adsorption energy of the surfactant sodium dodecylbenzene sulfonate (SDBS) on the SnS2(001) surface at the initial nucleation stage and induces the subsequent oriented attachment (OA) growth of 2D crystallites with monolayer thickness, leading to the formation of a monolayer amorphous carbon-bridged nanosheet mesocrystal. It is notable that such a nanosheet-coalesced mesocrystal is metastable with a flowerlike morphology and can be turned into a single crystal via crystallographic fusion. Subsequently, direct encapsulation of the mesocrystal via FeCl3-induced pyrrole monomer self-polymerization generates conformal polypyrrole (PPy) coating, and carbonization of the resulting nanocomposites generates Fe-N-S-co-doped carbons that are embedded with well-dispersed SnS/FeCl3 quantum sheets; this process skillfully integrated structural phase transformation, pyrolysis graphitization, and self-doping. Interestingly, such an integrated design not only guarantees the flowerlike morphology of the final nanohybrids but also, more importantly, allows the thickness of petalous carbon and the size of the nanoconfined particles to be controlled. Benefiting from the unique structural features, the resultant nanohybrids exhibited the brilliant electrochemical performance while simultaneously acting as a reliable platform for exploring the structure-performance correlation of a Li-ion battery (LIB).

Lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development.

SH2-containing inositol-5'-phosphatase-1 (SHIP-1) controls the phosphatidylinositol-3'-kinase (PI3K) initiated signaling pathway by limiting cell membrane recruitment and activation of Akt. Despite the fact that many of the growth factors important to cartilage development and functions are able to activate the PI3K signal transduction pathway, little is known about the role of PI3K signaling in chondrocyte biology and its contribution to mammalian skeletogenesis. Here, we report that the lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development through its expression in osteochondroprogenitor cells. Global SHIP-1 knockout led to accelerated chondrocyte hypertrophy and premature formation of the secondary ossification center in the bones of postnatal mice. Drastically higher vascularization and greater number of c-kit + progenitors associated with sinusoids in the bone marrow also indicated more advanced chondrocyte hypertrophic differentiation in SHIP-1 knockout mice than in wild-type mice. In corroboration with the in vivo phenotype, SHIP-1 deficient PDGFRα + Sca-1 + osteochondroprogenitor cells exhibited rapid differentiation into hypertrophic chondrocytes under chondrogenic culture conditions in vitro. Furthermore, SHIP-1 deficiency inhibited hypoxia-induced cellular activation of Akt and extracellular-signal-regulated kinase (Erk) and suppressed hypoxia-induced cell proliferation. These results suggest that SHIP-1 is required for hypoxia-induced growth signaling under physiological hypoxia in the bone marrow. In conclusion, the lipid phosphatase SHIP-1 regulates skeletal development by modulating chondrogenesis and the hypoxia response of the osteochondroprogenitors during endochondral bone formation.

Loss of lipid phosphatase SHIP1 promotes macrophage differentiation through suppression of dendritic cell differentiation.

SH2-containing inositol 5'-phosphatase-1 (SHIP1) deficiency in mice results in abnormal myeloid expansion, and proinflammatory conditions in the lung. However, the mechanisms involved in SHIP1-mediated regulation of myeloid differentiation remain unclear. Here we show that SHIP1 is a key regulator of early differentiation for dendritic cells (DCs). We also provide critical evidence to modify the function of SHIP1 in in vitro development of BMDCs using the recent framework of defining DCs. We found that loss of SHIP1 suppresses GM-CSF-induced formation of bone marrow-derived DC (BMDC) colonies, leading to reduced BMDC number in BM cell culture. The number of maturated BMDCs decreased in SHIP1-KO culture, due to reduction of immature BMDCs, suggesting SHIP1 is critical for lineage commitment rather than for maturation from myeloid precursors to DCs. We further showed that F4/80+/MHCIIlow BM macrophage-like cells (BMMs) were the main population of SHIP1-KO BM culture. Treatment of wild-type BM culture with 3 α-aminocholestane (3AC), a specific inhibitor for functional activity of SHIP1, caused a similar developmental defect in BMDCs as seen in SHIP1-KO cells, resulting in the absence of BMDC colony, and increased number of BMMs in BM culture. In conclusion, our results suggest that differentiation of BMDCs are markedly impaired under SHIP1 deficient condition, which causes skewed development of myeloid lineage cells manifested as pathological conditions associated with an excess of macrophage population.

Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages.

BACKGROUND: Gout is an inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystals' joint deposition. MSU phagocytosis by resident macrophages is a key step in gout pathogenesis. MSU phagocytosis triggers nuclear factor kappa B (NFκB) activation and production of cytokines and chemokines. Proteoglycan-4 (PRG4) is a glycoprotein produced by synovial fibroblasts and exerts an anti-inflammatory effect in the joint mediated by its interaction with cell surface receptor CD44. PRG4 also binds and antagonizes TLR2 and TLR4. The objective of this study is to evaluate the efficacy of recombinant human PRG4 (rhPRG4) in suppressing MSU-induced inflammation and mechanical allodynia in vitro and in vivo. METHODS: THP-1 macrophages were incubated with MSU crystals ± rhPRG4 or bovine submaxillary mucin (BSM), and crystal phagocytosis, cytokines and chemokines expression and production were determined. NFκB p65 subunit nuclear translocation, NLRP3 induction, caspase-1 activation and conversion of proIL-1ß to mature IL-1ß were studied. MSU phagocytosis by Prg4+/+ and Prg4-/- peritoneal macrophages was determined in the absence or presence of rhPRG4, BSM, anti-CD44, anti-TLR2, anti-TLR4 and isotype control antibodies. Rhodamine-labeled rhPRG4 was incubated with murine macrophages and receptor colocalization studies were performed. Lewis rats underwent intra-articular injection of MSU crystals followed by intra-articular treatment with PBS or rhPRG4. Weight bearing and SF myeloperoxidase activities were determined. RESULTS: rhPRG4 reduced MSU crystal phagocytosis at 4 h (p < 0.01) and IL-1ß, TNF-α, IL-8 and MCP-1 expression and production at 6 h (p < 0.05). BSM did not alter MSU phagocytosis or IL-1ß production in human and murine macrophages. rhPRG4 treatment reduced NFκB nuclear translocation, NLRP3 expression, caspase-1 activation and generation of mature IL-1ß (p < 0.05). MSU-stimulated IL-1ß production was higher in Prg4-/- macrophages compared to Prg4+/+ macrophages (p < 0.001). rhPRG4, anti-CD44, anti-TLR2 and anti-TLR4 antibody treatments reduced MSU phagocytosis and IL-1ß production in murine macrophages (p < 0.05). rhPRG4 preferentially colocalized with CD44 on Prg4-/- peritoneal macrophages compared to TLR2 or TLR4 (p < 0.01). rhPRG4 normalized weight bearing and reduced SF myeloperoxidase activity compared to PBS in vivo. CONCLUSION: rhPRG4 inhibits MSU crystal phagocytosis and exhibits an anti-inflammatory and anti-nociceptive activity in vitro and in vivo. rhPRG4's anti-inflammatory mechanism may be due to targeting CD44 on macrophages.

The Effects of Indian Hedgehog Deletion on Mesenchyme Cells: Inducing Intermediate Cartilage Scaffold Ossification to Cause Growth Plate and Phalange Joint Absence, Short Limb, and Dwarfish Phenotypes.

The endochondral ossification plays a critical role in vertebrate limb development and skeletal homeostasis, where limb mesenchyme cells form an intermediate cartilage scaffold that develops into growth plates and then replaced by bone. Although Indian hedgehog (Ihh) is known to control the hypertrophic differentiation process of chondrocytes, its role from the mesenchyme cells to the early stages of chondrogenesis is unclear. To define the function of Ihh in the mesenchymal cell's early stages of chondrogenesis, we specifically delete Ihh in Prx1-expressed mesenchyme cells at E9.5 using Prx1-Cre;Ihhfl/fl;Rosa26-ZsGreen1 mice. We found that deleting Ihh in the mesenchyme cells results in an early and quick ossification of the intermediate cartilage scaffold, causing the growth plate and phalange joint absence, short limbs, and dwarfishness. The green fluorescent protein (GFP)-positive cells derived from deleted Ihh mesenchyme cells overlap with von Kossa- and osteocalcin-positive staining area. These deleted Ihh/GFP-positive cells isolated from Prx1-Cre;Ihhfl/fl;Rosa26-ZsGreen1 newborn mice had osteogenic differentiation by showing a positive Alizarin red and von Kossa staining, as well as an enhanced Col1a1, osteocalcin, and Runx2 expression. Our findings demonstrate that deleting Ihh in mesenchyme cells during early limb development promotes intermediate cartilage scaffold ossification, which prevents growth plate formation that causes phalange joint absence, short limb, and dwarfish phenotype.

M1 Macrophage-Induced Endothelial-to-Mesenchymal Transition Promotes Infantile Hemangioma Regression.

Infantile hemangiomas are benign tumors of vascular endothelial cells (ECs), characterized by three distinct stages: proliferating phase, involuting phase, and involuted phase. The mechanisms that trigger involution of hemangioma into fibro-fatty tissue remain unknown. We report a novel mechanism by which M1-polarized macrophages induce endothelial-to-mesenchymal transition (EndMT) and promote hemangioma regression. M1- but not M2-polarized macrophages induced EndMT in ECs. Tumor necrosis factor-α and, to a lesser extent, IL-1ß and interferon-γ were the most potent cytokines produced by the M1 macrophages that induce in vitro EndMT. Western blot analysis and gene expression profiling showed that ECs treated with M1 macrophages, tumor necrosis factor-α, or IL-1ß decreased the expression of endothelial markers, whereas mesenchymal markers increased concomitantly. Immunohistochemical staining of patient samples revealed that a significant perivascular infiltration of M1, but not M2, macrophages coincides with endothelial expression of the critical EndMT transcription factors Snail/Slug in involuting hemangiomas. Most strikingly, M1 macrophage-treated ECs isolated from patient hemangiomas (HemECs) but not untreated HemECs readily differentiated into adipocytes on adipogenic induction. Thus, in vitro EndMT and adipogenesis of HemECs have, in part, recapitulated the natural history of hemangioma regression. In conclusion, our findings indicate that EndMT induced by M1 macrophages promotes infantile hemangioma regression and may lead to novel therapeutic treatments for this vascular tumor.

Decreased histone deacetylase 4 is associated with human osteoarthritis cartilage degeneration by releasing histone deacetylase 4 inhibition of runt-related transcription factor-2 and increasing osteoarthritis-related genes: a novel mechanism of human osteoarthritis cartilage degeneration.

INTRODUCTION: To investigate if decreased histone deacetylase 4 (HDAC4) is associated with human osteoarthritis (OA) cartilage degeneration by releasing HDAC4 inhibition of runt-related transcription factor-2 (Runx2) resulting in increase of OA cartilage degeneration-related genes. METHODS: The mRNA and protein levels of HDAC4, Runx2, matrix metalloproteinase (MMP)-13, Indian hedgehog (Ihh) and type X collagen were detected by performing real-time PCR (RT-PCR), western blotting and immunohistochemistry on specimens from human OA and normal cartilage. To further explore the mechanism of regulation of Runx2 and OA-related genes by HDAC4, changes in these OA-related genes were further quantified by RT-PCR after overexpression of HDAC4 and knockdown of HDAC4 by siRNA. Runx2 and MMP-13 promoter activities were measured by dual luciferase assays. RESULTS: The levels of HDAC4 in the cartilage from OA patients and healthy 40- to 60-year-old donors were decreased to 31% and 65% compared with specimens from 20- to 40-year-old healthy donors, respectively (P <0.05). Decreased HDAC4 was associated with increased Runx2 and other OA-related genes in human OA cartilage, specifically: MMP-13, Ihh and type X collagen. Exogenous HDAC4 decreased the mRNA levels of Runx2, MMP1, MMP3, MMP-13, type X collagen, Ihh, ADAMTS-4 and -5, and increased the mRNA of type II collagen. In addition, the data also shows that overexpression of HDAC4 not only decreased the expression of interleukin (IL)-1ß, Cox2 and iNos and increased the expression of aggrecan, but also partially blocked the effect of IL-1ß on expression of catabolic events in human OA chondrocytes. HDAC4 also inhibited Runx2 promoter activity and MMP13 promotor activity in a dose-dependent manner. In contrast, inhibition of HDAC4 by TSA drug had an opposite effect. CONCLUSIONS: Our study is the first to demonstrate that decreased HDAC4 contributes, at least in part, to the pathogenesis of OA cartilage degeneration. Thus, HDAC4 may have chondroprotective properties by inhibiting Runx2 and OA-related genes.

Matrilin-3 inhibits chondrocyte hypertrophy as a bone morphogenetic protein-2 antagonist.

Increased chondrocyte hypertrophy is often associated with cartilage joint degeneration in human osteoarthritis patients. Matrilin-3 knock-out (Matn3 KO) mice exhibit these features. However, the underlying mechanism is unknown. In this study, we sought a molecular explanation for increased chondrocyte hypertrophy in the mice prone to cartilage degeneration. We analyzed the effects of Matn3 on chondrocyte hypertrophy and bone morphogenetic protein (Bmp) signaling by quantifying the hypertrophic marker collagen type X (Col X) gene expression and Smad1 activity in Matn3 KO mice in vivo and in Matn3-overexpressing chondrocytes in vitro. The effect of Matn3 and its specific domains on BMP activity were quantified by Col X promoter activity containing the Bmp-responsive element. Binding of MATN3 with BMP-2 was determined by immunoprecipitation, solid phase binding, and surface plasmon resonance assays. In Matn3 KO mice, Smad1 activity was increased more in growth plate chondrocytes than in wild-type mice. Conversely, Matn3 overexpression in hypertrophic chondrocytes led to inhibition of Bmp-2-stimulated, BMP-responsive element-dependent Col X expression and Smad1 activity. MATN3 bound BMP-2 in a dose-dependent manner. Multiple epidermal growth factor (EGF)-like domains clustered together by the coiled coil of Matn3 is required for Smad1 inhibition. Hence, as a novel BMP-2-binding protein and antagonist in the cartilage extracellular matrix, MATN3 may have the inherent ability to inhibit premature chondrocyte hypertrophy by suppressing BMP-2/Smad1 activity.

MicroRNA-1 regulates chondrocyte phenotype by repressing histone deacetylase 4 during growth plate development.

MicroRNAs (miRs) are noncoding RNAs (17-25 nt) that control translation and/or mRNA degradation. Using Northern blot analysis, we identified that miR-1 is specifically expressed in growth plate cartilage in addition to muscle tissue, but not in brain, intestine, liver, or lung. We obtained the first evidence that miR-1 is highly expressed in the hypertrophic zone of the growth plate, with an 8-fold increase compared with the proliferation zone; this location coincides with the Ihh and Col X expression regions in vivo. MiR-1 significantly induces chondrocyte proliferation and differentiation. We further identified histone deacetylase 4 (HDAC4) as a target of miR-1. HDAC4 negatively regulates chondrocyte hypertrophy by inhibiting Runx2, a critical transcription factor for chondrocyte hypertrophy. MiR-1 inhibits both endogenous HDAC4 protein by 2.2-fold and the activity of a reporter gene bearing the 3'-untranslated region (UTR) of HDAC4 by 3.3-fold. Conversely, knockdown of endogenous miR-1 relieves the repression of HDAC4. Deletion of the miR-1 binding site in HDAC4 3'-UTR or mutated miR-1 abolishes miR-1-mediated inhibition of the reporter gene activity. Overexpression of HDAC4 reverses miR-1 induction of chondrocyte differentiation markers Col X and Ihh. HDAC4 inhibits Runx2 promoter activity in a dosage-dependent manner. Thus, miR-1 plays an important role in the regulation of the chondrocyte phenotype during the growth plate development via direct targeting of HDAC4.

Chloroplast SecA and Escherichia coli SecA have distinct lipid and signal peptide preferences.

Like prokaryotic Sec-dependent protein transport, chloroplasts utilize SecA. However, we observe distinctive requirements for the stimulation of chloroplast SecA ATPase activity; it is optimally stimulated in the presence of galactolipid and only a small fraction of anionic lipid and by Sec-dependent thylakoid signal peptides but not Escherichia coli signal peptides.

Pericellular matrilins regulate activation of chondrocytes by cyclic load-induced matrix deformation.

UNLABELLED: Pericellular matrix is at the ideal location to be involved in transmitting mechanical signals from the microenvironment to a cell. We found that changes of the content of matrilins that link various pericellular molecules surrounding chondrocytes affect mechanical stimulation of chondrocyte proliferation and gene expression. Thus, pericellular matrilins may play a role in chondrocyte mechanotransduction. INTRODUCTION: Chondrocytes reside in a capsule of pericellular matrix (chondron), which has been hypothesized to play a critical role in transducing mechanical signals to the cell. In this study, we test the hypothesis that the levels of matrilin (MATN)-1 and -3, major components of the chondrocyte pericellular matrix network, regulate activation of chondrocyte proliferation and differentiation by cyclic load-induced matrix deformation. MATERIALS AND METHODS: Functional matrilins were decreased by expressing a dominant negative mini-MATN in primary chondrocytes or by using MATN1-null chondrocytes. The abundance of matrilins was also increased by expressing a wildtype MATN1 or MATN3 in chondrocytes. Chondrocytes were cultured in a 3D sponge subjected to cyclic deformation at 1 Hz. Chondrocyte gene expression was quantified by real-time RT-PCR and by Western blot analysis. Matrilin pericellular matrix assembly was examined by immunocytochemistry. RESULTS: Elimination of functional matrilins from pericellular matrix abrogated mechanical activation of Indian hedgehog signaling and abolished mechanical stimulation of chondrocyte proliferation and differentiation. Excessive or reduced matrilin content decreased mechanical response of chondrocytes. CONCLUSIONS: Normal content of matrilins is essential to optimal activation of chondrocytes by mechanical signals. Our data suggest that the sensitivity of chondrocytes to the changes in the microenvironment can be adjusted by altering the content of matrilins in pericellular matrix. This finding supports a critical role of pericellular matrix in chondrocyte mechano-transduction and has important implications in cartilage tissue engineering and mechanical adaptation.

Chondrocyte death induced by pathological concentration of chemokine stromal cell-derived factor-1.

OBJECTIVE: It had been found that the concentration of chemokine stromal cell-derived factor-1 (SDF-1) was significantly higher in synovial fluid (SF) of patients with osteoarthritis (OA; > or = 200 ng/ml) and rheumatoid arthritis (RA; > or = 700 ng/ml) compared to controls (< or = 100 ng/ml). Our aim was to determine whether the pathological concentration of SDF-1 induces chondrocyte death and to investigate mechanisms underlying such death. METHODS: Human OA chondrocytes were treated with different doses of SDF-1, or in combination with SF from patients with arthritis. Apoptotic and necrotic cells were labeled by annexin V and propidium iodide, respectively, and quantified by FACS analysis. Caspase-3 activity was quantified by a plate absorbance assay, and matrix metalloproteinase 13 mRNA levels were determined by RT-PCR. The release of high mobility group box chromatin protein 1, a specific marker of cell necrosis, and the activities of chondrocyte mitogen-activated protein kinases (MAPK) including ERK, JNK, and p38 in response to SDF-1 treatment were quantified by Western blot analysis. RESULTS: Pathological concentrations of SDF-1 (> or = 200 ng/ml) in SF or in recombinant form induced death of human chondrocytes in a necrosis-dependent manner. Chondrocyte death was inhibited by the treatment of cells with anti-CXCR4, an antibody blocking the interaction between SDF-1 and its receptor CXCR4. However, the rate of chondrocyte apoptosis and the level of caspase-3, a key apoptotic enzyme, were not affected by the treatment with anti-CXCR4. SDF-1 stimulated p38 MAPK activity in a dose- and time-dependent manner. The presence of the p38 MAPK inhibitor SB203580 during SDF-1 treatment abolished the induction of chondrocyte death by SDF-1. CONCLUSION: Our findings suggest a novel pathological mechanism by which high concentrations of SDF-1 in SF induce chondrocyte death during OA and RA.

Physical properties of phosphatidylcholine vesicles containing small amount of sodium cholate and consideration on the initial stage of vesicle solubilization.

The effects of sub-solubilizing concentrations of sodium cholate (Na-chol) on several physicochemical properties of phosphatidylcholine (PC) small unilamellar vesicles (SUV) were considered in connection with the initial stage of membrane solubilization. ESR spectra of 12-doxylstearic acid (12-DS) in phosphatidylcholine from egg yolk (EPC) or dimyristoylphosphatidylcholine (DMPC) SUV at low concentrations (insufficient to destroy the vesicles) of Na-chol were composed of two (a strongly immobilized and an additional weakly immobilized) immiscible components. The origin of the additional bands was phase separation which occurred in the hydrophobic parts of PC SUV in the presence of Na-chol. Differential scanning calorimetry measurements demonstrated that the mixed DMPC/Na-chol SUV possessed two (a sharp low-temperature and a broad high-temperature) endothermic peaks, which is consistent with the coexistence of two immiscible phases in the vesicular membranes. zeta Potentials of the EPC/Na-chol SUV revealed that high anionic densities appeared on the surfaces of the SUV at a Na-chol concentration slightly below the upper boundary of the vesicle region. Thus, the initial stage of the solubilization of PC SUV by Na-chol was caused by the aggregation of hydrophobic parts of PC membranes, followed by the occurrence of high anionic densities on the surfaces of the vesicles. The fact that removal of Na-chol from PC/Na-chol mixed systems preferentially resulted in the formation of small vesicles might originate from these anionic charges.