Hydroxytyrosol modulates the levels of microRNA-9 and its target sirtuin-1 thereby counteracting oxidative stress-induced chondrocyte death.

OBJECTIVE: Nutraceutical compounds, such as hydroxytyrosol (HT), have been found to exert protective effects in osteoarthritis (OA) by affecting a variety of key molecular and cellular processes in chondrocytes. However, to our knowledge, no relationship has been reported between nutraceuticals and microRNA (miR) network in OA models. Here, we identified a miR that is implicated in HT-mediated chondroprotection following oxidative stress condition by targeting sirtuin-1 (SIRT-1). METHODS: Human primary and C-28/I2 chondrocytes were pre-treated with 100 µM HT 30 min before 100 µM H2O2 addition. In silico analyses were exploited to select putative candidate miRs able to target SIRT-1 mRNA. Luciferase-based gene reporter assay was employed to demonstrate the direct link between miR-9 and its putative mRNA target. Transient transfection approach was performed to examine the effects of miR-9 levels on caspase activity, cell viability and expression of OA-related genes. RESULTS: MiR-9 was identified and confirmed as a post-transcriptional regulator of SIRT-1. MiR-9 and SIRT-1 levels showed opposite changes in chondrocytes following H2O2 and HT treatment. Moreover mir-9 silencing inhibited cell death induced by H2O2 partly through down-regulation of SIRT-1, whereas miR-9 overexpression markedly reduced the protective effect of HT. The manipulation of miR-9 levels also resulted in the modulation of OA-related gene expression, including MMP-13, VEGF and RUNX-2. CONCLUSIONS: These results show that miR-9 is a critical mediator of the deleterious and OA-related effects of oxidative stress in chondrocytes and that modulation of miR expression may be a crucial mechanism underlying the protective action of HT.

Chondroprotective activity of N-acetyl phenylalanine glucosamine derivative on knee joint structure and inflammation in a murine model of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by joint structure changes and inflammation, both mediated by the IκB kinase (IKK) signalosome complex. The ability of N-acetyl phenylalanine derivative (NAPA) to increase cartilage matrix components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity, has been observed in vitro. The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM). DESIGN: 26 mice were divided into three groups: (1) DMM surgery without treatment; (2) DMM surgery treated after 2 weeks with one intra-articular injection of NAPA (2.5 mM) and (3) no DMM surgery. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and microhardness of subchondral bone (SB) tests. RESULTS: The injection of NAPA significantly improved cartilage thickness (CT) and reduced Chambers and Mankin modified scores and fibrillation index (FI), with weaker MMP13, ADAMTS5, MMP10 and IKKα staining. The microhardness measurements did not shown statistically significant differences between the different groups. CONCLUSIONS: NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity in vivo.

PKCε is a regulator of hypertrophic differentiation of chondrocytes in osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a common and highly debilitating degenerative disease whose complex pathogenesis and the multiplicity of the molecular processes involved, hinder its complete understanding. Protein Kinase C (PKC) novel isozyme PKCε recently proved to be an interesting molecule for further investigations as it can represent an intriguing, new actor in the acquisition of a OA phenotype by the chondrocyte. DESIGN: PKCε was modulated in primary chondrocytes from human OA patient knee cartilage samples by means of short hairpin RNA (ShRNA) and the expression of cartilage specific markers observed at mRNA and protein level. The involvement of Histone deacetylases (HDACs) signaling pathway was also investigated through the use of specific inhibitors MS-275 and Inhibitor VIII. RESULTS: PKCε loss induces up-regulation of Runt-domain transcription factor (RUNX2), Metalloproteinase 13 (MMP13) and Collagen X (COL10) as well as an enhanced calcium deposition in OA chondrocyte cultures. In parallel, PKCε knock-down also leads to SOX9 and Collagen II (COL2) down-modulation and to a lower deposition of glycosaminoglycans (GAGs) in the extracellular matrix (ECM). This novel regulatory role of PKCε over cartilage hypertrophic phenotype is exerted via an HDAC-mediated pathway, as HDAC2 and HDAC4 expression is modulated by PKCε. HDAC2 and HDAC4, in turn, are at least in part responsible for the modulation of the master transcription factors RUNX2 and SOX9, key regulators of chondrocyte phenotype. CONCLUSIONS: PKCε prevents the phenotypic progression of the OA chondrocyte, acting on cartilage specific markers through the modulation of the transcription factors SOX9 and RUNX2. The loss of PKCε enhances, in fact, the OA hypertrophic phenotype, with clear implications in the pathophysiology of the disease.

Cell death in human articular chondrocyte: a morpho-functional study in micromass model.

Chondrocyte death and loss of extracellular matrix are the central features in articular cartilage degeneration during osteoarthritis pathogenesis. Cartilage diseases and, in particular, osteoarthritis are widely correlated to apoptosis but, chondrocytes undergoing apoptosis "in vivo" more often display peculiar features that correspond to a distinct process of programmed cell death termed "chondroptosis". Programmed cell death of primary human chondrocyte has been here investigated in micromasses, a tridimensional culture model, that represents a convenient means for studying chondrocyte biology. Cell death has been induced by different physical or chemical apoptotic agents, such as UVB radiation, hyperthermia and staurosporine delivered at both 1 and 3 weeks maturation. Conventional electron microscopy was used to analyse morphological changes. Occurrence of DNA fragmentation and caspase involvement were also investigated. At Transmission Electron Microscopy, control cells appear rounding or slightly elongated with plurilobated nucleus and diffusely dispersed chromatin. Typically UVB radiation and staurosporine induce chromatin apoptotic features, while hyperthermia triggers the "chondroptotic" phenotype. A weak TUNEL positivity appears in control, correlated to the well known cell death patterns occurring along cartilage differentiation. UVB radiation produces a strong positivity, mostly localized at the micromass periphery. After hyperthermia a higher number of fluorescent nuclei appears, in particular at 3 weeks. Staurosporine evidences a diffuse, but reduced, positivity. Therefore, DNA fragmentation is a common pattern in dying chondrocytes, both in apoptotic and "chondroptotic" cells. Moreover, all triggers induce caspase pathway activation, even if to a different extent, suggesting a fundamental role of apoptotic features, in chondrocyte cell death.

Role of polyamines in hypertrophy and terminal differentiation of osteoarthritic chondrocytes.

Polyamines are naturally occurring, positively charged polycations which are able to control several cellular processes in different cell types, by interacting with negatively charged compounds and structures within the living cell. Functional genomics in rodents targeting key biosynthetic or catabolic enzymes have revealed a series of phenotypic changes, many of them related to human diseases. Several pieces of evidence from the literature point at a role of polyamines in promoting chondrocyte differentiation, a process which is physiological in growth plate maturation or fracture healing, but has pathological consequences in articular chondrocytes, programmed to keep a maturational arrested state. Inappropriate differentiation of articular chondrocytes results in osteoarthritis. Thus, we have studied the effects of exogenously added spermine or spermidine in chondrocyte maturation recapitulated in 3D cultures, to tease out the effects on gene and protein expression of key chondrogenesis regulatory transcription factors, markers and effectors, as well as their posttranscriptional regulation. The results indicate that both polyamines are able to increase the rate and the extent of chondrogenesis, with enhanced collagen 2 deposition and remodeling with downstream generation of collagen 2 bioactive peptides. These were able to promote nuclear localization of RUNX-2, the pivotal transcription factor in chondrocyte hypertrophy and osteoblast generation. Indeed, samples stimulated with polyamines showed an enhanced mineralization, along with increased caspase activity, indicating increased chondrocyte terminal differentiation. In conclusion these results indicate that the polyamine pathway can represent a potential target to control and correct chondrocyte inappropriate maturation in osteoarthritis.

Polyamine biosynthesis as a target to inhibit apoptosis of non-tumoral cells.

Growing evidence suggests a role for polyamines in apoptosis, although the relationship appears to be complex. alpha-Difluoromethylornithine (DFMO), a largely used ornithine decarboxylase inhibitor, is cytostatic, hardly cytotoxic and may even increase the resistance of tumour cells to some apoptotic stimuli. This may represent a problem in cancer therapy, where the killing of tumoral cells would be a desired effect, but could be an advantage in other pathological contexts related to an excess of apoptosis, such as cardiovascular diseases, stem cell transplantation, arthritis and infections. In different cellular models, polyamine depletion following treatment with polyamine biosynthesis inhibitors appears to inhibit mitochondrial and death receptor pathways of apoptosis by affecting key proteins. These studies indicate that inhibition of polyamine biosynthesis may prevent or reduce the apoptotic response triggered by a variety of stimuli in non-tumoral cells, such as cardiac cells, stem cells, chondrocytes, macrophages and intestinal epithelial cells.

Glycogen Synthase Kinase-3ß Inhibition Links Mitochondrial Dysfunction, Extracellular Matrix Remodelling and Terminal Differentiation in Chondrocytes.

Following inflammatory stimuli, GSK3 inhibition functions as a hub with pleiotropic effects leading to cartilage degradation. However, little is known about the effects triggered by its direct inhibition as well as the effects on mitochondrial pathology, that contributes to osteoarthritis pathogenesis. To this aim we assessed the molecular mechanisms triggered by GSK3ß inactivating stimuli on 3-D (micromass) cultures of human articular chondrocytes. Stimuli were delivered either at micromass seeding (long term) or after maturation (short term) to explore "late" effects on terminal differentiation or "early" mitochondrial effects, respectively. GSK3ß inhibition significantly enhanced mitochondrial oxidative stress and damage and endochondral ossification based on increased nuclear translocation of Runx-2 and ß-catenin, calcium deposition, cell death and enhanced remodelling of the extracellular matrix as demonstrated by the increased collagenolytic activity of supernatants, despite unmodified (MMP-1) or even reduced (MMP-13) collagenase gene/protein expression. Molecular dissection of the underlying mechanisms showed that GSK3ß inhibition achieved with pharmacological/silencing strategies impacted on the control of collagenolytic activity, via both decreased inhibition (reduced TIMP-3) and increased activation (increased MMP-10 and MMP-14). To conclude, the inhibition of GSK3ß enhances terminal differentiation via concerted effects on ECM and therefore its activity represents a tool to keep articular cartilage homeostasis.

Flow cytometric analysis of intracellular chemokines in chondrocytes in vivo: constitutive expression and enhancement in osteoarthritis and rheumatoid arthritis.

Chemokines play a key role in modulating leukocyte functions at sites of inflammation. To assess chondrocyte contribution to the chemotactic environment of inflamed joints the intracellular content of CC and CXC chemokines was investigated. IL-8, GROalpha, MCP-1, RANTES, MIP-1alpha and MIP-1beta expression was evaluated by flow cytometric analysis and RT-PCR in chondrocytes isolated from cartilage specimens obtained from patients with osteoarthritis and rheumatoid arthritis and multiorgan donors as normal controls. All the chemokines except RANTES were found in normal chondrocytes, with different degrees of staining intensity. In osteoarthritis and rheumatoid arthritis patients, an enhancement of IL-8, GROalpha, MIP-1alpha and MIP-1beta was observed.

Intracellular Cu/Zn superoxide dismutase levels in T and non-T cells from normal aged subjects.

The decreased immune response associated with aging may, in part, reflect intrinsic age-related biochemical alteration in lymphocytes from older subjects. The 'reactive oxygen species hypothesis' of aging postulates that these molecules are involved in the modifications leading to cellular senescence. Superoxide dismutase (SOD), and in particular the Cu/Zn-dependent intracellular form, plays a critical role in the defense against these species, but it is controversial whether this function declines in lymphocytes in old age. We utilized two different methods to evaluate Cu/Zn SOD levels in T and non-T cells (CD3+, CD3-, CD4+, CD8+, CD16+) from young and old individuals: a specific and sensitive enzyme immunoassay performed on extracts of sorted cells, and a flow cytometry double fluorescence technique with monoclonal antibodies against Cu/Zn SOD and the different lymphocyte subsets. The Cu/Zn SOD cell content was assayed both in basal conditions and after peripheral blood lymphocyte stimulation with Concanavalin A, anti-CD3 monoclonal antibody and phorbol myristate acetate. In basal conditions, and considering the various subsets, no differences were found between young and old individuals, although data analysis revealed high and low responders in both groups. Taking all the subjects together, higher levels of this enzyme were found in CD3+ T lymphocytes, in particular in the CD4+ cells. After peripheral blood lymphocyte stimulation, Cu/Zn SOD concentration was higher than levels in unstimulated cells, both in young and old individuals, and particularly using Concanavalin A with respect to anti-CD3 and phorbol myristate acetate. In conclusion, the synthesis of Cu/Zn SOD does not seem to be affected by aging in proliferating cells. The highest levels of Cu/Zn SOD present in CD4+ cells, both from young and old individuals, may prevent the oxidant stress of these elements which play a major role in the inflammation sites.

Intracellular nucleotides of lymphocytes and granulocytes from normal ageing subjects.

Impaired lymphocyte and granulocyte function in the aged may, in part, reflect intrinsic aged-related biochemical alterations. In this study we compared the ribonucleotide contents of lymphocytes and granulocytes from young and old subjects evaluated by means of an HPLC-anion exchange method. We found that in general both populations from old subjects present higher levels of the various nucleotides, in particular: ATP, UDP, CTP, UDP-glucose in granulocytes, AMP, CTP, UDP-N-acetylglucosamine, UDP-glucose in lymphocytes. These data suggest that these molecules accumulate in aged subjects because of altered biochemical pathways. The increased pool of UDP-sugars, in particular, could be due to a depressed activity of some glycosyltransferases which therefore fail to glycosylate some plasma membrane cell proteins, thus accounting for their functional impairment.

IgG subclass distribution of anti-HBs antibodies following vaccination with cDNA HBsAg.

Serum samples were obtained during follow-up of nine young adults vaccinated over 1 year with cDNA hepatitis B antigen. The absolute amounts of anti-HBs IgG subclass antibodies present in the sera were determined by comparing the optical densities (OD) obtained using an antigen-specific ELISA with those obtained by serial dilutions of known amounts of human IgG1-4. The calibration curves for each IgG subclass were corrected for the corresponding coating efficiency. Our data suggest that HBs antibody responses of vaccinated subjects occur in all IgG subclasses but IgG1 and IgG2 are the major subclasses involved.

A fluorescent in situ hybridization method in flow cytometry to detect HIV-1 specific RNA.

In HIV+ patients, the presence of HIV-RNA in plasma and circulating cells has been reported to be a marker of progression but the percentage of transcriptionally active infected cells remains unclear. We have developed a reliable fluorescent in situ hybridization method for the detection of HIV specific RNA by flow cytometry. The procedure was applied to a panel of chronically infected cell lines and to an acutely infected cell line mimicking normal peripheral blood lymphocytes in susceptibility to HIV-1. The cells were fixed in suspension and hybridized by means of an HIV-1 genomic probe labeled with digoxigenin-11-dUTP. An FITC-labeled anti-digoxigenin antiserum was then applied and the resulting fluorescence signals were analyzed both by flow cytometry and confocal microscopy. Different procedures for double staining HIV-RNA together with virus induced proteins or surface markers were also developed. Flow cytometric detection of in situ hybridization offers the possibility of analyzing thousands of cells in a few seconds and of collecting multiparametric information at the single cell level, thus providing a potential tool for detecting the rare HIV-RNA expressing cells in peripheral blood samples.

Human osteosarcoma cells release matrix degrading enzymes in response to chemokine activation.

Matrix degrading enzymes released upon autocrine and/or paracrine induction exert a key role in modulating tumor cell behavior. Osteosarcoma is a highly metastatic cancer, with a redundancy of autocrine loops. Here we report that human osteosarcoma cells express a wide array of chemokine receptors and respond to chemokine activation with the release of N-acetyl-beta-D-glucosaminidase and gelatinase/collagenase activity. Of the two cell lines studied, the osteoblast-like MG-63 showed a higher responsivity compared to the less differentiated HOS. This suggests that chemokine modulation of matrix degrading enzymes requires the maintaining of the osteoblastic phenotype and of signaling pathways which occur in normal tissue.

Autoantibodies to poly(ADP-ribose)polymerase in autoimmune diseases.

Antibodies to various nuclear proteins are frequently found in sera of patients affected by connective tissue diseases and other autoimmune diseases. We investigated the specificity of circulating autoantibodies to poly(ADP-ribose)polymerase (pADPRP) in different autoimmune and connective tissue diseases: Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Progressive Systemic Sclerosis (PSS), Sjogren's Syndrome (SS), Undifferentiated Connective Tissue Disease (UCTD), Cryptogenic Fibrosing Alveolitis (CFA) and Sarcoidosis. pADPRP was purified from calf thymus. Antibody specificity was detected by ELISA, western blot and enzyme activity precipitation. Positive values (mean O.D. values + 3 S.D. of 36 normal controls) were obtained in 7/15 SLE patients, 1/18 RA patients, 1/30 PSS, 3/14 SS, 0/5 UCTD, 5/21 CFA and 4/25 Sarcoidosis. The positive sera also recognized the pADPRP protein when tested by western blot. Furthermore the enzyme activity was inhibited after immunoprecipitation by some highly positive sera.

Antibodies to topoisomerase II in idiopathic pulmonary fibrosis.

Antibodies to DNA topoisomerase II (anti-topoisomerase II) were detected by ELISA in the sera of 18 out of 41 (44%) patients with idiopathic pulmonary fibrosis (IPF). Follow-up sera were also obtained from 19 of the patients. DNA topoisomerase II binding remained constantly high or low in the majority of follow-up sera, but 2 out of the 8 positive cases became negative while 3 out of the 11 negative cases became positive during follow-up. No association was found between occurrence of anti-topoisomerase II antibodies and any indices of disease severity. Furthermore, individual patient follow-up did not show any correlation between changes in topoisomerase II binding and deterioration or improvement of clinical status. In conclusion our study shows that although anti-topoisomerase II are detectable in a large fraction (approximately 50%) of IPF patients and are useful for diagnostic purposes, they do not provide a measure of clinical activity.

Serum copper/zinc superoxide dismutase levels in patients with rheumatoid arthritis.

Rheumatoid arthritis is characterized by a chronic hypertrophic synovitis leading to destruction of connective tissue and functional damage of cartilage and bone structures. Reactive oxygen species play an important role in tissue injury in this disease. To clarify the role of the cellular antioxidant system in the protection against oxygen free radicals, we examined the levels of copper/zinc superoxide dismutase in the sera of patients with rheumatoid arthritis. We used an enzyme-linked immunosorbent assay which determines the concentration of copper/zinc superoxide dismutase independently from its enzymatic activity. We found that patients with rheumatoid arthritis have higher serum copper/zinc superoxide dismutase levels than control subjects. Copper/zinc superoxide dismutase also correlated positively with serum levels of both neopterin and rheumatoid factor, sensitive markers for disease activity in rheumatoid arthritis. These results support the hypothesis that the increased amount of copper/zinc superoxide dismutase is probably inadequate to exert an effective antioxidant protection but can result in a pro-inflammatory, pathogenic effect enhancing tissue damage. Furthermore, copper/zinc superoxide dismutase might be used as a marker of inflammatory activity in rheumatoid arthritis.

Distribution of IgG subclasses after anti-hepatitis B virus immunization with a recombinant vaccine.

To assess whether a different IgG subclass distribution was elicited in "low" and "high responders" after vaccination with recombinant hepatitis B virus surface antigen, we selected from 360 vaccine recipients 30 "low-responder" subjects, with anti-HBs levels of 10-160 mIU/ml, and 40 "high-responder" subjects, with anti-HBs levels greater than 10,000 mIU/ml. In both groups all IgG subclasses were elicited in the anti-HBs response and the greatest contribution was that of IgG1, followed by IgG2. IgG1 was significantly less represented after the second (58%) and third doses (61%) of vaccine in "low responders" compared with "high responders" (65% and 69%). The relative percentage of IgG2 was significantly higher after the second (33%) and third (30%) doses of vaccine in "low responders" than in "high responders" (29% and 26%). In "low responders" the age of vaccine recipients significantly influenced the anti-HBs IgG subclass distribution: IgG2 and IgG4 production was positively correlated with age, whereas the opposite was observed for IgG1. These data support the evidence that: (1) IgG1 and IgG2 subclasses are mainly involved in the specific anti-HBs response both in "high" and "low responders"; (2) the relative contribution of specific IgG2 to vaccination is higher in low responders and progressively increases with age.

Circulating tumor necrosis factor alpha in rheumatoid arthritis.

Tumor Necrosis Factor alpha is an important mediator of immunity and inflammation, and because of its biologic activities (activation of neutrophils, release of arachidonic acid metabolites from synovial cells, induction of cartilage resorption and inhibition of proteoglycan release in cartilage) is one of the potential mediators of the chronic inflammation in rheumatoid arthritis. A commercially available ELISA was used to evaluate serum levels of Tumor Necrosis Factor alpha (TNF alpha) in patients with rheumatic diseases. We tested sera from patients with rheumatoid arthritis, seronegative arthritis, osteoarthritis, post-traumatic arthritis, systemic lupus erythematosus, progressive systemic sclerosis and normal healthy subjects as controls. Furthermore, we statistically analysed data to investigate whether a correlation exists between serum levels of TNF alpha and some humoral indexes of disease activity. The results show strikingly higher TNF alpha levels in Rheumatoid Arthritis patients when compared both to normal controls and arthritis or connective tissue disease controls. TNF alpha was also found to correlate positively with levels of the rheumatoid factor as measured either by means of the latex agglutination test (LAT) or by nephelometry. These results support the suggestion that TNF alpha plays a central role in the pathogenesis of rheumatoid arthritis.

Human chondrocytes express functional chemokine receptors and release matrix-degrading enzymes in response to C-X-C and C-C chemokines.

OBJECTIVE: Human chondrocytes produce different C-X-C and C-C chemokines under basal conditions and upon activation with proinflammatory cytokines. We investigated whether human chondrocytes also have chemokine receptors and examined the effects of chemokines on chondrocyte activity. METHODS: The expression of chemokine receptors was determined by immunochemical analysis of frozen sections from normal and osteoarthritic cartilage and by flow cytometry of isolated cells. The messenger RNA expression for chemokine receptors was studied by reverse transcriptase-polymerase chain reaction. Isolated chondrocytes were stimulated with different chemokines, and the responses were evaluated by assaying the release of matrix metalloprotease 3 (MMP-3) and of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase in the supernatants. RESULTS: A wide variety of chemokine receptors (CCR-1, CCR-2, CCR-3, CCR-5, CXCR-1, and CXCR-2) was detected on human chondrocytes. Interaction of these receptors with the corresponding ligands induced the release of MMP-3. This response was abrogated by pretreatment of the cells with Bordetella pertussis toxin, demonstrating involvement of G proteins of the Gi type. The response decreased in the presence of cycloheximide, indicating dependence on protein synthesis. Chemokines also induced the exocytosis of N-acetyl-beta-D-glucosaminidase, which was prevented by receptor blockage with anti-CCR-3 and by treatment with B pertussis toxin. Chondrocytes obtained from osteoarthritic tissue showed an increased expression of CCR-3 and possibly of CXCR-1, and an augmented release of matrix-degrading enzymes compared with chondrocytes from normal donors. CONCLUSION: Our findings suggest the existence in human chondrocytes of a novel catabolic pathway, primed by chemokines and their receptors, that leads to the breakdown of cartilage matrix components.