Early ablation of Ccr2 in aggrecan-expressing cells following knee injury ameliorates joint damage and pain during post-traumatic osteoarthritis.

OBJECTIVE: To investigate whether Ccr2 inactivation in aggrecan-expressing cells induced before post-traumatic OA (PTOA) onset or during progression, improves joint structures, synovial thickness and pain. DESIGN: We induced a Ccr2 deletion in aggrecan-expressing cells (CCR2-AggKO) in skeletally mature mice using a tamoxifen-inducible Ccr2 inactivation. We stimulated PTOA changes (destabilization of medial meniscus, DMM) in CCR2-AggKO and CCR2+/+ mice, inducing recombination before DMM or 4 wks after DMM (early-vs late-inactivation). Joint damage was evaluated 2, 4, 8, 12 wks post-DMM using multiple scores: articular-cartilage structure (ACS), Safranin-O, histomorphometry, osteophyte size/maturity, subchondral bone thickness and synovial hyperplasia. Spontaneous (incapacitance meter) and evoked pain (von-Frey filaments) were assessed up to 20 wks. RESULTS: Early aggrecan-Ccr2 inactivation in CCR2-AggKO mice (N=8) resulted in improved ACS score (8-12wk, P=0.002), AC area (4-12wk, P<0.05) and Saf-O score (2wks P=0.004, 4wks P=0.02, 8-12wks P=0.002) compared to CCR2+/+. Increased subchondral bone thickness was delayed only at 2 wks and exclusively following early recombination. Osteophyte size was not affected, but osteophyte maturation (cartilage-to-bone) was delayed (4wks P=0.04; 8 wks P=0.03). Although late aggrecan-Ccr2 deletion led to some cartilage improvement, most data did not reach statistical significance; osteophyte maturity was delayed at 12wks. Early aggrecan-Ccr2 deletion led to improved pain measures of weight bearing compared to CCR2+/+ mice (N = 9, 12wks diff 0.13 [0.01, 0.26], 16wks diff 0.15 [0.05, 0.26], 20wks diff 0.23 [0.14, 0.31]). Improved mechanosensitivity in evoked pain, although less noticeable, was detected. CONCLUSIONS: We demonstrated that deletion of Ccr2 in aggrecan expressing cells reduces the initiation but not progression of OA.

Tibiofemoral articular cartilage composition differs based on serum biochemical profiles following anterior cruciate ligament reconstruction.

OBJECTIVE: Biochemical joint changes contribute to posttraumatic osteoarthritis (PTOA) development following anterior cruciate ligament reconstruction (ACLR). The purpose of this longitudinal cohort study was to compare tibiofemoral cartilage composition between ACLR patients with different serum biochemical profiles. We hypothesized that profiles of increased inflammation (monocyte chemoattractant protein-1 [MCP-1]), type-II collagen turnover (type-II collagen breakdown [C2C]:synthesis [CPII]), matrix degradation (matrix metalloproteinase-3 [MMP-3] and cartilage oligomeric matrix protein [COMP]) preoperatively to 6-months post-ACLR would be associated with greater tibiofemoral cartilage T1ρ relaxation times 12-months post-ACLR. DESIGN: Serum was collected from 24 patients (46% female, 22.1 ± 4.2 years old, 24.0 ± 2.6 kg/m2 body mass index [BMI]) preoperatively (6.4 ± 3.6 days post injury) and 6-months post-ACLR. T1ρ Magnetic Resonance Imaging (MRI) was collected for medial and lateral tibiofemoral articular cartilage at 12-months post-ACLR. A k-means cluster analysis was used to identify profiles based on biomarker changes over time and T1ρ relaxation times were compared between cluster groups controlling for sex, age, BMI, concomitant injury (either meniscal or chondral pathology), and Marx Score. RESULTS: One cluster exhibited increases in MCP-1 and COMP while the other demonstrated decreases in MCP-1 and COMP preoperatively to 6-months post-ACLR. The cluster group with increases in MCP-1 and COMP demonstrated greater lateral tibial (adjusted mean difference = 3.88, 95% confidence intervals [1.97-5.78]) and femoral (adjusted mean difference = 12.71, 95% confidence intervals [0.41-23.81]) T1ρ relaxation times. CONCLUSION: Profiles of increased serum levels of inflammation and matrix degradation markers preoperatively to 6-months post-ACLR are associated with MRI changes consistent with lesser lateral tibiofemoral cartilage proteoglycan density 12-months post-ACLR.

Synovial fluid concentrations of matrix Metalloproteinase-3 and Interluekin-6 following anterior cruciate ligament injury associate with gait biomechanics 6 months following reconstruction.

OBJECTIVE: To compare gait biomechanics 6 months following anterior cruciate ligament (ACL) reconstruction (ACLR) between patients with the highest and lowest concentrations of synovial fluid (SF) interleukin-6 (IL-6) and matrix metalloproteinase-3 (MMP-3), as well as compared to uninjured controls. DESIGN: SF concentrations of IL-6 and MMP-3 were collected 7 ± 4 days post injury in 38 ACL injured patients (55% female, 21±4yrs, 25.3 ± 5.2BMI). ACL injured individuals were stratified into the lowest and highest quartiles based on IL-6 (IL-6Lowest and IL-6Highest) and MMP-3 (MMP-3Lowest and MMP-3Highest) concentrations. Gait biomechanics were collected on the injured limb 6 months post-ACLR and in 38 uninjured controls (50% female, 21±3yrs, 23.8 ± 2.8BMI). Functional analyses of variance were used to compare vertical ground reaction force (vGRF), knee flexion angle (KFA), and internal knee extension moment (KEM) waveforms throughout stance phase of gait to determine the proportions of stance differing between limbs and groups. RESULTS: Compared to uninjured controls, IL-6High and MMP-3High ACL subgroups demonstrated lesser vGRF (largest differences: IL-6, 7.88%BW; MMP-3, 11.05%BW) during early-stance and greater vGRF (largest differences: IL-6, 6.21%BW; MMP-3, 5.85%BW) in mid-stance, lesser KFA (largest differences: IL-6, 3.11°; MMP-3, 3.72°) and lesser KEM (largest differences: IL-6, 0.96%BW•m; MMP-3, 1.07%BW•m) in early-stance, as well as greater KFA in mid-stance (largest differences: IL-6, 1.5°; MMP-3, 2.95°). CONCLUSIONS: High SF concentrations of a proinflammatory cytokine and a degradative enzyme early post-ACL injury are associated with aberrant gait biomechanics in the injured limb at 6 months post-ACLR (i.e., lesser vGRF, KFA and KEM) linked to posttraumatic osteoarthritis development.

Associations between the chemokine biomarker CCL2 and knee osteoarthritis outcomes: the Johnston County Osteoarthritis Project.

OBJECTIVE: Our study analyzes the association between chemokine-ligand-2 (CCL2) serum concentrations at baseline and knee radiographic osteoarthritis (OA) (knee-rOA), knee-rOA progression, individual radiographic features and knee symptomatic OA at 5-year follow-up. DESIGN: OA outcomes were analyzed in a community-based cohort including a baseline enrollment and a 5-year follow-up. Baseline CCL2 serum concentrations were assessed by multiplex assay and associated with presence or progression of individual radiographic features at 5-year follow-up. Separate multiple logistic regression models were used to examine adjusted associations between baseline CCL2 and each of the knee OA variables at follow-up. CCL2 at baseline was modeled as an explanatory variable, whereas each of the knee OA variables at follow-up served as the response variables. Models were adjusted for age, BMI, race, and sex. Trend tests were conducted to assess any linear effect on outcomes across CCL2 tertiles. RESULTS: Participants (n = 168) had a median age of 57-years and median BMI of 29 kg/m2. About 63% of all participants were women, and 58% Caucasian (42% African American). In adjusted logistic models, continuous log-CCL2 was significantly associated with knee-rOA. For each unit increase in log CCL2, the odds of having knee-rOA at follow-up was increased by 72%. CCL2 tertiles showed significant linear associations with presence and progression of knee-rOA and medial joint space narrowing (JSN), but not with presence or progression of osteophytes, bone sclerosis, knee symptoms, or symptomatic knee-rOA. CONCLUSIONS: Serum CCL2 may help to elucidate some mechanisms of joint destruction and identify individuals with higher odds of structural knee changes.

Role of the C-C chemokine receptor-2 in a murine model of injury-induced osteoarthritis.

OBJECTIVE: We previously found in our embryonic studies that proper regulation of the chemokine CCL12 through its sole receptor CCR2, is critical for joint and growth plate development. In the present study, we examined the role of CCR2 in injury-induced-osteoarthritis (OA). METHOD: We used a murine model of injury-induced-OA (destabilization of medial meniscus, DMM), and systemically blocked CCR2 using a specific antagonist (RS504393) at different times during disease progression. We examined joint degeneration by assessing cartilage (cartilage loss, chondrocyte hypertrophy, MMP-13 expression) and bone lesions (bone sclerosis, osteophytes formation) with or without the CCR2 antagonist. We also performed pain behavioral studies by assessing the weight distribution between the normal and arthritic hind paws using the IITS incapacitance meter. RESULTS: Testing early vs delayed administration of the CCR2 antagonist demonstrated differential effects on joint damage. We found that OA changes in articular cartilage and bone were ameliorated by pharmacological CCR2 blockade, if given early in OA development: specifically, pharmacological targeting of CCR2 during the first 4 weeks (wks) following injury, reduced OA cartilage and bone damage, with less effectiveness with later treatments. Importantly, our pain-related behavioral studies showed that blockade of CCR2 signaling during early, 1-4 wks post-surgery or moderate, 4-8 wks post-surgery, OA was sufficient to decrease pain measures, with sustained improvement at later stages, after treatment was stopped. CONCLUSIONS: Our data highlight the potential efficacy of antagonizing CCR2 at early stages to slow the progression of post-injury OA and, in addition, improve pain symptoms.

Geometrical vortex lattice pinning and melting in YBaCuO submicron bridges.

Since the discovery of high-temperature superconductors (HTSs), most efforts of researchers have been focused on the fabrication of superconducting devices capable of immobilizing vortices, hence of operating at enhanced temperatures and magnetic fields. Recent findings that geometric restrictions may induce self-arresting hypervortices recovering the dissipation-free state at high fields and temperatures made superconducting strips a mainstream of superconductivity studies. Here we report on the geometrical melting of the vortex lattice in a wide YBCO submicron bridge preceded by magnetoresistance (MR) oscillations fingerprinting the underlying regular vortex structure. Combined magnetoresistance measurements and numerical simulations unambiguously relate the resistance oscillations to the penetration of vortex rows with intermediate geometrical pinning and uncover the details of geometrical melting. Our findings offer a reliable and reproducible pathway for controlling vortices in geometrically restricted nanodevices and introduce a novel technique of geometrical spectroscopy, inferring detailed information of the structure of the vortex system through a combined use of MR curves and large-scale simulations.

Signaling cross-talk between IGF-binding protein-3 and transforming growth factor-(beta) in mesenchymal chondroprogenitor cell growth.

Cartilage formation is driven by mesenchymal chondroprogenitor cells (MCCs) that proliferate and differentiate into chondrocytes. The molecular mechanisms by which growth factors regulate MCC fate are not well defined. Insulin-like growth factor binding protein-3 (IGFBP-3) has intrinsic bioactivity that is independent of IGF binding. We previously reported that IGFBP-3 has IGF-independent antiproliferative and apoptotic effects in MCCs, and requires STAT-1 activation to mediate its apoptotic effect. Transforming growth factor-beta (TGF-beta) is a key chondroinductive growth factor. The objective of the study is to define the interactions between IGFBP-3 and TGF-beta in MCC growth and their intracellular signaling pathways. We used the RCJ3*1C5*18 mesenchymal chondrogenic cells that without biochemical or oncogenic transformation progress in culture from MCCs to differentiated chondrocytes. Cell proliferation was assessed in MCCs treated with IGFBP-3 or transfected with IGFBP-3, in the presence or absence of TGF-beta. To demonstrate that IGFBP-3 effects were IGF-independent an IGFBP-3 analog that lacks IGF binding was used (GGG-IGFBP-3). To determine the functional roles of the TGF-beta-mediated signaling and the STAT-1 pathway, cells were either stably transfected with a dominant negative TGF-beta type II receptor (MCC-DNTbetaRII) or treated with a STAT-1 morpholino antisense oligonucleotide. We found that in MCCs, TGF-beta antagonized the antiproliferative effect of IGFBP-3. IGFBP-3 increased the cyclin-dependent kinase inhibitor p21 expression and this effect was abolished by TGF-beta. Furthermore, TGF-beta inhibited STAT-1 phosphorylation induced by IGFBP-3. Similarly to TGF-beta, STAT-1 antisense oligonucleotide inhibited the IGFBP-3 antiproliferative action. Although TGF-beta in MCC-DNTbetaRII lacked Smad-mediated signaling, it persistently antagonized the IGFBP-3 antiproliferative action. However, TGF-beta even in MCC-DNTbetaRII cells induced ERK1/2 phosphorylation, and treatment with MEK inhibitor, UO126, inhibited the antagonistic effects of TGF-beta on IGFBP-3. Furthermore, UO126 blocked the TGF-beta inhibition of STAT-1 phosphorylation induced by IGFBP-3. Collectively, these results demonstrate cross-talk between the IGFBP-3-dependent STAT-1 signaling and the TGF-beta-dependent ERK pathway that regulates MCC proliferation.

Macroscopic quantum tunnelling in spin filter ferromagnetic Josephson junctions.

The interfacial coupling of two materials with different ordered phases, such as a superconductor (S) and a ferromagnet (F), is driving new fundamental physics and innovative applications. For example, the creation of spin-filter Josephson junctions and the demonstration of triplet supercurrents have suggested the potential of a dissipationless version of spintronics based on unconventional superconductivity. Here we demonstrate evidence for active quantum applications of S-F-S junctions, through the observation of macroscopic quantum tunnelling in Josephson junctions with GdN ferromagnetic insulator barriers. We show a clear transition from thermal to quantum regime at a crossover temperature of about 100 mK at zero magnetic field in junctions, which present clear signatures of unconventional superconductivity. Following previous demonstration of passive S-F-S phase shifters in a phase qubit, our result paves the way to the active use of spin filter Josephson systems in quantum hybrid circuits.

Differential regulation of three catalytic activities of platelet-activating factor (PAF)-dependent transacetylase.

We have previously established that PAF-dependent transacetylase (TA) purified to apparent homogeneity from rat kidney membranes and cytosol contains three separate catalytic activities, namely PAF lysophospholipid transacetylase (TAL), PAF sphingosine transacetylase (TAS), and PAF acetylhydrolase (AH). In the present investigation, we studied the biochemical factors and mechanism(s) that differentially regulate these three TA activities of the purified enzymes. We found that only the TAS activity of the TA purified from the membranes was stimulated by phosphatidyl-serine (PS) with optimal concentration of activation occurring at 25 microM. Other acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP), are partially effective, while diacylglycerol and free fatty acids had no effect on the TAS activity. PS exerted its effect on the TAS activity through the increases of both Km and Vmax. In addition, N-ethylmalimide (NEM) and dithiobis-(2-nitro-5-thiobenzoic acid) (DTNB) strongly inhibited the TAS activity and partially decreased the TAL and AH activities of the purified membrane enzyme in a dose-dependent manner. The addition of PS, but not by its substrate, sphingosine, could prevented the inhibition by NEM on the basal level of TAS. On the other hand, the inhibition of TAL by NEM and DTNB were partially protected by the substrate, lysoplasmalogens. Furthermore, PAF fully protects the inhibition of AH, partially protects the inhibition of TAL, and does not protect the inhibition of TAS by NEM. These results suggested that the three individual catalytic activities of TA have different dependencies on the thiol-containing residue(s) of the enzyme, i.e., cysteine. Furthermore, the nonresponsiveness of the purified cytosolic TAS to PS activation is consistent with our previous notions that membrane and cytosolic TA are posttranslationally distinct.

Platelet-activating factor (PAF)-dependent transacetylase and its relationship with PAF acetylhydrolases.

Platelet-activating factor (PAF)-dependent transacetylase (TA) is an enzyme that transfers an acetyl group from PAF to acceptor lipids such as lysophospholipids and sphingosine. This enzyme is distributed in membrane and cytosol of the cells. We previously revealed that TA purified from rat kidney membrane showed an amino acid sequence similarity to that of bovine PAF-acetylhydrolase (AH) (II). In the present study, we purified TA from the rat kidney cytosol and analyzed its amino acid sequence. The amino acid sequence of the cytosolic TA is similar to that of bovine PAF-AH (II) and membrane TA. To clarify the relationship between TA and PAF-AH (II), we isolated cDNA of rat PAF-AH (II). The predicted amino acid sequence of rat PAF-AH (II) from isolated cDNA included all the sequences found in TAs purified from the membrane and cytosolic TAs. In addition, monoclonal antibody to recombinant PAF-AH (II) cross-reacted with both cytosolic and membrane TAs. Consistent with sequence identity, recombinant PAF-AH (II) showed TA activity, whereas recombinant PAF-AH Ib, which is a different subtype of intracellular PAF-AHs, did not possess TA activity. Analysis of a series of site-directed mutant PAF-AH (II) proteins showed that TA activity was decreased, whereas PAF-AH activity was not affected in C120S and G2A mutant proteins. Thus, Cys(120) and Gly(2) are implicated in the catalysis of TA reaction in this enzyme. Furthermore, the transfer of acetate from PAF to endogenous acceptor lipids was significantly increased in a time-dependent manner in CHO-K1 cells transfected with PAF-AH (II) gene. These results demonstrate that PAF-AH (II) can function, as a TA in intact cells, and PAF-AH (II) and TA are the same enzyme.