CITED2 mediates the cross-talk between mechanical loading and IL-4 to promote chondroprotection.

Osteoarthritis (OA) pathogenesis is mediated largely through the actions of proteolytic enzymes such as matrix metalloproteinase (MMP) 13. The transcriptional regulator CITED2, which suppresses the expression of MMP13 in chondrocytes, is induced by interleukin (IL)-4 in T cells and macrophages, and by moderate mechanical loading in chondrocytes. We tested the hypothesis that CITED2 mediates cross-talk between IL-4 signaling and mechanical loading-induced pathways that result in chondroprotection, at least in part, by downregulating MMP13. IL-4 induced CITED2 gene expression in human chondrocytes in a dose- and time-dependent manner through JAK/STAT signaling. Mechanical loading combined with IL-4 resulted in additive effects on inducing CITED2 expression and downregulating of MMP13 in human chondrocytes in vitro. In vivo, IL-4 gene knockout (KO) mice exhibited reduced basal levels of CITED2 expression in chondrocytes. While moderate treadmill running induced CITED2 expression and reduced MMP13 expression in wild-type mice, these effects were blunted (for CITED2) or abolished (for MMP13) in chondrocytes of IL-4 gene KO mice. Moreover, intra-articular injections of mouse recombinant IL-4 combined with regular cage activity mitigated post-traumatic OA to a greater degree compared to immobilized mice treated with IL-4 alone. These data suggest that using moderate loading to enhance IL-4 may be a potential therapeutic strategy for chondroprotection in OA.

Correction to: Green tea polyphenol treatment is chondroprotective, anti-inflammatory and palliative in a mouse posttraumatic osteoarthritis model.

Following publication of the original article [1], the authors reported an error in Figs. 2C and 5C.

Procyanidins Mitigate Osteoarthritis Pathogenesis by, at Least in Part, Suppressing Vascular Endothelial Growth Factor Signaling.

Procyanidins are a family of plant metabolites that have been suggested to mitigate osteoarthritis pathogenesis in mice. However, the underlying mechanism is largely unknown. This study aimed to determine whether procyanidins mitigate traumatic injury-induced osteoarthritis (OA) disease progression, and whether procyanidins exert a chondroprotective effect by, at least in part, suppressing vascular endothelial growth factor signaling. Procyanidins (extracts from pine bark), orally administered to mice subjected to surgery for destabilization of the medial meniscus, significantly slowed OA disease progression. Real-time polymerase chain reaction revealed that procyanidin treatment reduced expression of vascular endothelial growth factor and effectors in OA pathogenesis that are regulated by vascular endothelial growth factor. Procyanidin-suppressed vascular endothelial growth factor expression was correlated with reduced phosphorylation of vascular endothelial growth factor receptor 2 in human OA primary chondrocytes. Moreover, components of procyanidins, procyanidin B2 and procyanidin B3 exerted effects similar to those of total procyanidins in mitigating the OA-related gene expression profile in the primary culture of human OA chondrocytes in the presence of vascular endothelial growth factor. Together, these findings suggest procyanidins mitigate OA pathogenesis, which is mediated, at least in part, by suppressing vascular endothelial growth factor signaling.

Curcumin slows osteoarthritis progression and relieves osteoarthritis-associated pain symptoms in a post-traumatic osteoarthritis mouse model.

BACKGROUND: Curcumin has been shown to have chondroprotective potential in vitro. However, its effect on disease and symptom modification in osteoarthritis (OA) is largely unknown. This study aimed to determine whether curcumin could slow progression of OA and relieve OA-related pain in a mouse model of destabilization of the medial meniscus (DMM). METHODS: Expression of selected cartilage degradative-associated genes was evaluated in human primary chondrocytes treated with curcumin and curcumin nanoparticles and assayed by real-time PCR. The mice subjected to DMM surgery were orally administered curcumin or topically administered curcumin nanoparticles for 8 weeks. Cartilage integrity was evaluated by Safranin O staining and Osteoarthritis Research Society International (OARSI) score, and by immunohistochemical staining of cleaved aggrecan and type II collagen, and levels of matrix metalloproteinase (MMP)-13 and ADAMTS5. Synovitis and subchondral bone thickness were scored based on histologic images. OA-associated pain and symptoms were evaluated by von Frey assay, and locomotor behavior including distance traveled and rearing. RESULTS: Both curcumin and nanoparticles encapsulating curcumin suppressed mRNA expression of pro-inflammatory mediators IL-1ß and TNF-α, MMPs 1, 3, and 13, and aggrecanase ADAMTS5, and upregulated the chondroprotective transcriptional regulator CITED2, in primary cultured chondrocytes in the absence or presence of IL-1ß. Oral administration of curcumin significantly reduced OA disease progression, but showed no significant effect on OA pain relief. Curcumin was detected in the infrapatellar fat pad (IPFP) following topical administration of curcumin nanoparticles on the skin of the injured mouse knee. Compared to vehicle-treated controls, topical treatment led to: (1) reduced proteoglycan loss and cartilage erosion and lower OARSI scores, (2) reduced synovitis and subchondral plate thickness, (3) reduced immunochemical staining of type II collagen and aggrecan cleavage epitopes and numbers of chondrocytes positive for MMP-13 and ADAMTS5 in the articular cartilage, and (4) reduced expression of adipokines and pro-inflammatory mediators in the IPFP. In contrast to oral curcumin, topical application of curcumin nanoparticles relieved OA-related pain as indicated by reduced tactile hypersensitivity and improved locomotor behavior. CONCLUSION: This study provides the first evidence that curcumin significantly slows OA disease progression and exerts a palliative effect in an OA mouse model.

Consequences of metabolic and oxidative modifications of cartilage tissue.

A hallmark of chronic metabolic diseases, such as diabetes and metabolic syndrome, and oxidative stress, as occurs in chronic inflammatory and degenerative conditions, is the presence of extensive protein post-translational modifications, including glycation, glycoxidation, carbonylation and nitrosylation. These modifications have been detected on structural cartilage proteins in joints and intervertebral discs, where they are known to affect protein folding, induce protein aggregation and, ultimately, generate microanatomical changes in the proteoglycan-collagen network that surrounds chondrocytes. Many of these modifications have also been shown to promote oxidative cleavage as well as enzymatically-mediated matrix degradation. Overall, a general picture starts to emerge indicating that biochemical changes in proteins constitute an early event that compromises the anatomical organization and viscoelasticity of cartilage, thereby affecting its ability to sustain pressure and, ultimately, impeding its overall bio-performance.

Green tea polyphenol treatment is chondroprotective, anti-inflammatory and palliative in a mouse post-traumatic osteoarthritis model.

INTRODUCTION: Epigallocatechin 3-gallate (EGCG), a polyphenol present in green tea, was shown to exert chondroprotective effects in vitro. In this study, we used a post-traumatic osteoarthritis (OA) mouse model to test whether EGCG could slow the progression of OA and relieve OA-associated pain. METHODS: C57BL/6 mice were subjected to surgical destabilization of the medial meniscus (DMM) or sham surgery. EGCG (25 mg/kg) or vehicle control was administered daily for four or eight weeks by intraperitoneal injection starting on the day of surgery. OA severity was evaluated by Safranin O staining and Osteoarthritis Research Society International (OARSI) score, and by immunohistochemical analysis to detect cleaved aggrecan and type II collagen, and expression of proteolytic enzymes matrix metalloproteinase (MMP)-13 and A Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS5). Real-time polymerase chain reaction (PCR) was performed to characterize the expression of genes critical for articular cartilage homeostasis. During the course of the experiments, tactile sensitivity testing (von Frey test) and open field assays were used to evaluate pain behaviors associated with OA, and expression of pain expression markers and inflammatory cytokines in the dorsal root ganglion (DRG) were determined by real-time PCR. RESULTS: Four and eight weeks after DMM surgery, the cartilage in EGCG-treated mice exhibited less Safranin O loss and cartilage erosion, and lower OARSI scores compared to vehicle-treated controls, which was associated with reduced staining for aggrecan and type II collagen cleavage epitopes, and reduced staining for MMP-13 and ADAMTS5 in the articular cartilage. Articular cartilage in the EGCG-treated mice also exhibited reduced levels of MMP-1, -3, -8, -13, ADAMTS5, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α mRNA and elevated gene expression of the MMP regulator Cbp/p300 Interacting Transactivator 2 (CITED2). Compared to vehicle controls, mice treated with EGCG exhibited reduced OA-associated pain, as indicated by higher locomotor behavior (i.e. distance traveled). Moreover, expression of chemokine receptor (CCR2), and pro-inflammatory cytokines IL-1ß and TNF-α in the DRG were significantly reduced to levels similar to sham-operated animals. CONCLUSIONS: This study provides the first evidence in an OA animal model that EGCG significantly slows OA disease progression and exerts a palliative effect.

Nutraceuticals: potential for chondroprotection and molecular targeting of osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease and a leading cause of adult disability. There is no cure for OA, and no effective treatments which arrest or slow its progression. Current pharmacologic treatments such as analgesics may improve pain relief but do not alter OA disease progression. Prolonged consumption of these drugs can result in severe adverse effects. Given the nature of OA, life-long treatment will likely be required to arrest or slow its progression. Consequently, there is an urgent need for OA disease-modifying therapies which also improve symptoms and are safe for clinical use over long periods of time. Nutraceuticals-food or food products that provide medical or health benefits, including the prevention and/or treatment of a disease-offer not only favorable safety profiles, but may exert disease- and symptom-modification effects in OA. Forty-seven percent of OA patients use alternative medications, including nutraceuticals. This review will overview the efficacy and mechanism of action of commonly used nutraceuticals, discuss recent experimental and clinical data on the effects of select nutraceuticals, such as phytoflavonoids, polyphenols, and bioflavonoids on OA, and highlight their known molecular actions and limitations of their current use. We will conclude with a proposed novel nutraceutical-based molecular targeting strategy for chondroprotection and OA treatment.

Age-related carbonylation of fibrocartilage structural proteins drives tissue degenerative modification.

Aging-related oxidative stress has been linked to degenerative modifications in different organs and tissues. Using redox proteomic analysis and illustrative tandem mass spectrometry mapping, we demonstrate oxidative posttranslational modifications in structural proteins of intervertebral discs (IVDs) isolated from aging mice. Increased protein carbonylation was associated with protein fragmentation and aggregation. Complementing these findings, a significant loss of elasticity and increased stiffness was measured in fibrocartilage from aging mice. Studies using circular dichroism and intrinsic tryptophan fluorescence revealed a significant loss of secondary and tertiary structures of purified collagens following oxidation. Collagen unfolding and oxidation promoted both nonenzymatic and enzymatic degradation. Importantly, induction of oxidative modification in healthy fibrocartilage recapitulated the biochemical and biophysical modifications observed in the aging IVD. Together, these results suggest that protein carbonylation, glycation, and lipoxidation could be early events in promoting IVD degenerative changes.

Mechanotransduction and cartilage integrity.

Osteoarthritis (OA) is characterized by the breakdown of articular cartilage that is mediated in part by increased production of matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS), enzymes that degrade components of the cartilage extracellular matrix. Efforts to design synthetic inhibitors of MMPs/ADAMTS have only led to limited clinical success. In addition to pharmacologic therapies, physiologic joint loading is widely recommended as a nonpharmacologic approach to improve joint function in osteoarthritis. Clinical trials report that moderate levels of exercise exert beneficial effects, such as improvements in pain and physical function. Experimental studies demonstrate that mechanical loading mitigates joint destruction through the downregulation of MMPs/ADAMTS. However, the molecular mechanisms underlying these effects of physiologic loading on arthritic joints are not well understood. We review here the recent progress on mechanotransduction in articular joints, highlighting the mediators and pathways in the maintenance of cartilage integrity, especially in the prevention of cartilage degradation in OA.

Dendritic cell-mediated in vivo bone resorption.

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo, de novo osteoclastogenesis is observed but it is currently unknown whether, besides increased osteoclast differentiation from undifferentiated precursors, other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study, an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment, the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally, chromosomal painting identified nuclei from female DCs, previously injected into a male recipient, among the nuclei of giant cells at sites of osteolysis. Finally, osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r(-/-) mice, which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether, our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases.

Vitamin D does not modulate NF-kappaB activity in Jurkat T cells.

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], has been reported to influence the functioning of the immune system by targeting the activities of cellular signaling pathways, in addition to its direct genomic effects. One of the signaling pathways reported to be targeted by vitamin D is the NF-kappaB pathway, which is highly active in most immune cell types, including T cells. However, the effects of vitamin D on the NF-kappaB pathway in T cells are not fully understood. Therefore, we examined the effects of 1alpha,25(OH)(2)D(3) on the NF-kappaB pathway in the Jurkat cell line, a human T cell line that constitutively expresses endogenous vitamin D receptor. We found that 1alpha,25(OH)(2)D(3) does not inhibit the induction of IkappaBalpha degradation and the expression of an NF-kappaB-dependent reporter gene in Jurkat cells following treatment with PMA/ionomycin. Also, 1alpha,25(OH)(2)D(3) did not suppress the activation of NF-kappaB by TNFalpha or PHA. Furthermore, we demonstrate that 1alpha,25(OH)(2)D(3) does not block the induction of CD69, which is an NF-kappaB target gene and an early T cell activation marker. Therefore, we conclude that vitamin D does not modulate the activity of the NF-kappaB pathway in Jurkat cells.

The impact of vitamin D on dendritic cell function in patients with systemic lupus erythematosus.

BACKGROUND: Excessive activity of dendritic cells (DCs) is postulated as a central disease mechanism in Systemic Lupus Erythematosus (SLE). Vitamin D is known to reduce responsiveness of healthy donor DCs to the stimulatory effects of Type I IFN. As vitamin D deficiency is reportedly common in SLE, we hypothesized that vitamin D might play a regulatory role in the IFNalpha amplification loop in SLE. Our goals were to investigate the relationship between vitamin D levels and disease activity in SLE patients and to investigate the effects of vitamin D on DC activation and expression of IFNalpha-regulated genes in vitro. METHODOLOGY/PRINCIPAL FINDINGS: In this study, 25-OH vitamin D (25-D) levels were measured in 198 consecutively recruited SLE patients. Respectively, 29.3% and 11.8% of African American and Hispanic SLE patient had 25-D levels <10 ng/ml. The degree of vitamin D deficiency correlated inversely with disease activity; R = -.234, p = .002. In 19 SLE patients stratified by 25-D levels, there were no differences between circulating DC number and phenotype. Monocyte-derived DCs (MDDCs) of SLE patients were normally responsive to the regulatory effects of vitamin D in vitro as evidenced by decreased activation in response to LPS stimulation in the presence of 1,25-D. Additionally, vitamin D conditioning reduced expression of IFNalpha-regulated genes by healthy donor and SLE MDDCs in response to factors in activating SLE plasma. CONCLUSIONS/SIGNIFICANCE: We report on severe 25-D deficiency in a substantial percentage of SLE patients tested and demonstrate an inverse correlation with disease activity. Our results suggest that vitamin D supplementation will contribute to restoring immune homeostasis in SLE patients through its inhibitory effects on DC maturation and activation. We are encouraged to support the importance of adequate vitamin D supplementation and the need for a clinical trial to assess whether vitamin D supplementation affects IFNalpha activity in vivo and, most importantly, improves clinical outcome.

PMA induces expression from the herpes simplex virus thymidine kinase promoter via the activation of JNK and ERK in the presence of adenoviral E1A proteins.

The herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) promoter contains elements involved in both constitutive and induced expression. We determined that phorbol 12-myristate 13-acetate (PMA) induces the HSV-1 TK promoter in HEK293 cells. However, PMA did not induce expression from the promoter in HeLa cells and did not result in a globally increased gene expression in HEK293 cells. Induction of HSV-1 TK promoter required activation of both of JNK and ERK pathways. However, activation of the two pathways alone was not sufficient for induction of HSV-1 TK promoter. By transiently transfecting into HeLa cells the adenoviral E1A gene, which exists as an integrant in HEK293 genome, we demonstrated that E1A proteins are necessary for induction of HSV-1 TK promoter by PMA. We propose mechanisms by which signaling pathways activated by the tumor-promoter PMA cooperate with the oncogene E1A to stimulate a eukaryotic promoter, namely the HSV-1 TK promoter.

Overwhelming leukoencephalopathy as the only sign of neuropsychiatric lupus.

We describe a patient with diffuse leukoencephalopathy, a rare central nervous system complication of systemic lupus erythematosus, who died of brain herniation despite aggressive management. Brain magnetic resonance imaging revealed diffuse white matter hyperintensities consistent with vasogenic edema. Autopsy revealed only widespread cerebral edema. Early recognition and persistent, aggressive treatment will be required to avoid this fatal and rare manifestation of neuropsychiatric lupus.

A critical evaluation of enzyme immunoassay kits for detection of antinuclear autoantibodies of defined specificities. III. Comparative performance characteristics of academic and manufacturers' laboratories.

OBJECTIVE: To analyze the performance of different commercial enzyme immunoassay (EIA) kits for measuring antinuclear antibodies (ANA) specific for dsDNA, SSB/La, Sm, and Scl-70. METHODS: EIA kits for detection of ANA from 9 commercial manufacturers were evaluated. The manufacturers were advised that they would be sent coded sera containing mixtures of the Arthritis Foundation/Centers for Disease Control reference reagents, and that they were to use their own test kits to analyze the antibody specificities of these sera and to report the data, in optical density (OD) units or their equivalent. Independently, 12 investigators in academic institutions who have done research in this field agreed to participate in a parallel study. The concentration of the antibodies and the specificities were blinded to the analysts and the coefficients of variation (CV) were computed for each participant. RESULTS: There were statistically significant differences between laboratories in terms of CV for all 9 kits tested. With the exception of one kit, there were no significant CV differences between the various autoantibody kits provided by each manufacturer and, with the exception of kits from 2 manufacturers, there were no significant differences between the various antibody kits in terms of reproducibility (CV). From the point of view of interlaboratory variability, manufacturers could be separated into either a high or low performance group. CONCLUSION: We found a disconcertingly large range of performance characteristics in the various laboratories, which could be quite detrimental in routine utilization of EIA ANA kits. Clinicians should be aware of the performance issues raised in our study, and should know and be involved in how their service laboratory assesses its own performance and the performance of commercial testing systems utilized. Manufacturers and clinical laboratories need to exercise constant quality assurance and surveillance of kit performance in the hands of medical laboratory technologists involved in routine testing.

A critical evaluation of enzyme immunoassay kits for detection of antinuclear autoantibodies of defined specificities. II. Potential for quantitation of antibody content.

OBJECTIVE: To analyze the performance of different commercial enzyme immunoassay (EIA) kits for measuring antibody levels of antinuclear antibodies (ANA) specific for double stranded (ds) DNA, SSB/La, Sm, and Scl-70. METHODS: Twenty companies that were known major purveyors of EIA kits for detection of ANA were approached to determine their interest and willingness to participate in this study. The manufacturers were advised that they would be sent coded sera containing mixtures of the Arthritis Foundation/Centers for Disease Control reference reagents, and that they were to use their own test kits to analyze the antibody specificities of these sera and to report the data, in optical density (OD) units, or their equivalent. The analysts were blinded to the concentration of the antibodies and the specificities. RESULTS: Initially, 11 manufacturers out of 20 agreed to participate, but 2 subsequently withdrew. The commercial EIA kits have the potential of being able to quantitate specific autoantibody content to ds-DNA, SSB/La, Sm, and Scl-70. However, certain deficiencies in these kits were also detected, the most obvious being lack of uniformly good performance, with kits of certain manufacturers showing exceptional accuracy in 3 out of 4 of their antibody-specific kits and poor accuracy for a 4th kit. CONCLUSION: It is important for clinicians to appreciate that there is marked inter-manufacturer variation in the performance of EIA kits used as an aid in the diagnosis of systemic rheumatic diseases. Manufacturers need to exercise constant surveillance of kit performance and to provide assurance that such is being done. Improved EIA kits would lend themselves to reliable quantitation of antibody levels in human sera and help to determine whether serial measurement of antibody levels might be useful in monitoring disease activity.