Does metabolic syndrome or its individual components affect pain and function in knee osteoarthritis women?

BACKGROUND: Current studies and research support the role of metabolic syndrome (MetS) in knee osteoarthritis (OA). However, few studies have focused on its impact on knee OA parameters. The aim of this study was to investigate if metabolic syndrome or its individual components affect the intensity of pain, functional disability, and radiographic severity in knee osteoarthritis women. MATERIALS AND METHODS: We conducted a cross sectional study including confirmed radiographic knee osteoarthritis according to Kellgren and Lawrence scale, with and without metabolic syndrome according to the National Cholesterol Education Program Adult Treatment Panel III criteria. The two groups were compared for pain Visual Analogue Scale (VAS), Lequesne index, Womac function, and radiological grade after adjusting for significant covariates. Multiple regression analysis was used to identify the independent effects of each specific component for metabolic syndrome on knee osteoarthritis parameters. RESULTS: One hundred thirty women were included. The mean age was 56.68±8.07 [34-75] years, and the mean BMI was 32.54±2.92 [23-37] kg/m2. The prevalence of metabolic syndrome was 48.5%. Women with and without metabolic syndrome had similar knee osteoarthritis parameters. However, accumulation of MetS components was associated with higher level of pain (OR = 3.7, CI = [1.5-5.9], p=0.001), independently of age and BMI. Multiple regression analyses showed, after adjusting for all covariates, that hyperglycemia had a positive impact on pain (p=0.009), waist circumference was positively associated with Lequesne index (p=0.04), high triglycerides level was significantly associated with increased pain (p=0.04) and higher Lequesne score (p=0.05), and Systolic blood pressure was positively correlated with Lequesne index (p=0.01). CONCLUSION: In addition to weight reduction, appropriate treatment of metabolic syndrome needs to become an important management strategy for knee pain and functional impairment.

Assessment of hemodialysis impact by Polysulfone membrane on brain plasticity using BOLD-fMRI.

PURPOSE: Hemodialysis (HD) is considered the most common alternative for overcoming renal failure. Studies have shown the involvement of HD membrane in the genesis of oxidative stress (OS) which has a direct impact on the brain tissue and is expected to be involved in brain plasticity and also reorganization of brain function control. The goal of this paper was to demonstrate the sensitivity of the blood oxygenation level-dependent functional magnetic resonance imaging (BOLD-fMRI) to characterize the OS before and after the HD session. PATIENTS, MATERIALS AND METHODS: Twelve male patient-volunteers following chronic HD for more than 6months were recruited among 86 HD-patients. All patients underwent identical assessment immediately before and after the full HD-session. This consisted of full biological assessment, including malondialdehyde (MDA) and total antioxidant activity (TAOA); and brain BOLD-fMRI using the motor paradigm in block-design. RESULTS: Functional BOLD-fMRI maps of motor area M1 were obtained from the HD patient before and after the hemodialysis session, important decrease in the intensity of brain activation of the motor area after HD, and important increase of the size of the volume of brain activation were observed, these changes are reflecting brain plasticity that is well correlated to OS levels. Individual patients MDA and TAOA before and after the hemodialysis sessions demonstrated a clear and systematic increase of the OS after HD (P-value=0.03). Correlation of BOLD-fMRI maximal signal intensity and volume of activated cortical brain area behaviors to MDA and total TAOA were close to 1. CONCLUSION: OS is systematically increased in HD-patients after the HD-process. Indeed, the BOLD-fMRI shows a remarkable sensitivity to brain plasticity studied cortical areas. Our results confirm the superiority of the BOLD-fMRI quantities compared to the biological method used for assessing the OS while not being specific, and reflect the increase in OS generated by the HD. BOLD-fMRI is expected to be a suitable tool for evaluating the plasticity process evolution in hemodialysis brain patients.

Melittin inhibits epidermal growth factor-induced protein tyrosine phosphorylation: comparison with phorbol myristate acetate and calcium ionophore A23187.

In HER14 cells, epidermal growth factor (EGF) induces tyrosine phosphorylation of several proteins, including its own receptor. The bee venom peptide, melittin, impaired EGF-dependent protein tyrosine phosphorylation in a calcium-dependent manner. The melittin effect was similarly reproduced by calcium ionophore A23187. The effect of melittin and calcium ionophore A23187 on EGF-dependent protein tyrosine phosphorylation was abolished by treatment of cells with the calcium chelator EGTA. Phorbol-myristate acetate (PMA) inhibited EGF-dependent protein tyrosine phosphorylation, and when compared to melittin or calcium ionophore A23187, only PMA potentiated the EGF-induced tyrosine phosphorylation of two proteins immunologically related to mitogen activated protein (MAP) kinases of 40 kDa and 44 kDa molecular mass. Unlike PMA, the effect of melittin and calcium ionophore A23187 on inhibition of EGF-dependent protein tyrosine phosphorylation was lost neither in protein kinase C-depleted cells nor in cells treated with the protein tyrosine phosphatase inhibitors NaF and Na3VO4. Melittin inhibited high affinity binding of EGF to its receptor in intact cells, but this effect was not prevented by EGTA. It is concluded that melittin and calcium ionophore A23187 differ from PMA in their inhibition of EGF-dependent protein tyrosine phosphorylation in vivo, by acting via a Ca(2+)-dependent pathway, that is independent of protein kinase C, protein tyrosine phosphatase activity and high affinity binding of EGF to its receptor.

Effects of H2O2 on protein tyrosine phosphatase activity in HER14 cells.

Oxidative stress has been implicated in protein phosphorylation and dephosphorylation in cells. In our current studies, H2O2 was shown to reversibly inhibit protein tyrosine phosphatase (PTPase) activity in HER14 cells. H2O2 (150 mM) resulted in 40% inhibition of PTPase activity by 15 min and recovery from inhibition was nearly complete by 60 min. H2O2-induced inhibition or recovery of PTPase activity was not affected by cycloheximide, a protein synthesis inhibitor. L-Buthionine-[S,R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, had no effect on H2O2-induced inhibition of PTPase activity but retarded the recovery of activity. Epidermal growth factor (EGF) and EGTA, a Ca2+ chelator, did not influence H2O2-induced inhibition or recovery of PTPase activity. These results suggest that at least 40% of fibroblast PTPase activity can be regulated by cellular redox activity.

Inhibition of epidermal growth factor-dependent protein tyrosine phosphorylation by phorbol myristate acetate is mediated by protein tyrosine phosphatase activity.

Incubation of HER14 cells with phorbol myristate acetate (PMA) decreases epidermal growth factor (EGF)-dependent protein tyrosine phosphorylation, except for a 40-kDa MAP kinase II-like protein, whose tyrosine phosphorylation is further enhanced. The inhibitory effect of PMA on EGF-dependent protein tyrosine phosphorylation is reversed if cell are pre-incubated with a combination of Na3VO4 and NaF, two known inhibitors of protein tyrosine phosphatase activity. Protein tyrosine phosphatase activity of cell homogenate was measured on immunopurified EGF receptor, and was found to be enhanced in PMA-treated cells. These data suggest that the inhibitory effect of PMA on EGF-dependent protein tyrosine phosphorylation in HER14 cells may be mediated by protein tyrosine phosphatase activity.

Inhibition of protein tyrosine phosphatase activity in HER14 cells by melittin and Ca2+ ionophore A23187.

We investigated the effect of melittin and Ca2+ ionophore A23187 on protein tyrosine phosphatase activity in HER14 cells (NIH-3T3 cells transfected with human epidermal growth factor 'EGF' receptor). Cell fractions were used to measure protein tyrosine phosphatase activity in vitro using 32P-labeled poly(Glu/Tyr) (4:1) peptide as a substrate. Treatment of HER14 cells with melittin or with A23187, inhibited protein tyrosine phosphatase activity in the cell sonicate and homogenate, as well as in cytosolic and particulate fractions of these cells. The inhibitory effect of both drugs was prevented by preincubating cells with EGTA (ethyleneglycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid). The cyclooxygenase inhibitor indomethacin enhanced the inhibitory effect of A23187, but not that of melittin. Addition of arachidonic acid to the cells partially prevented the inhibition of protein tyrosine phosphatase activity by melittin or A23187. Preexposure of cells to EGF enhanced the inhibitory effect of melittin--but not that of A23187. Addition of CaCl2, or MgCl2 to the cell homogenate inhibited protein tyrosine phosphatase activity. These results show that protein tyrosine phosphatase activity in HER14 cells is inhibited by melittin and Ca2+ ionophore A23187 through a Ca(2+)-dependent mechanism, and is regulated by arachidonic acid metabolism and EGF receptor activation.

Characterization of several phospholipase activities and diacylglycerol/2-monoacylglycerol lipases in rat alveolar macrophages.

We measured phospholipase activities in both the microsomal and the cytosolic enriched fractions of rat alveolar macrophages by using exogenous arachidonic acid-labeled phospholipids. The microsomal fractions contain a neutral calcium-independent phospholipase A2 (PLA2) which acts on substrates phosphatidylcholine (PC) and phosphatidylinositol (PI), a calcium-independent PLA2 acting on phosphatidylethanolamine (PE), and a neutral calcium-dependent PI-specific PLC. The cytosolic fractions contain calcium-dependent phospholipase: PLA2 that hydrolyses PC at alkaline pH, and a neutral PI-specific phospholipase C (PLC). The largest release of arachidonic acid from PI occurred with the cytosolic fractions at pH 6 in the presence of calcium. That hydrolysis involved a PLA2, and a PLC followed by the action of a diacyglycerol and 2-monoacylglycerol lipases. The cytosol also contains a calcium-independent PLA2 acting on PE. Our investigation shows that rat alveolar macrophages possess a number of phospholipases, as well as diacylglycerol and 2-monoacylglycerol lipases. The above enzymes could play an essential role in the remodeling of membrane phospholipids in resting cells, and the generation of physiologically active lipids in activated cells.

A purified lipocortin shares the anti-inflammatory effect of glucocorticosteroids in vivo in mice.

1. The injection of a suspension of a polyacrylamide gel (bio gel) into the dorsal subcutaneous area of mice induced an inflammatory reaction and the migration of neutrophils towards the inflamed site. 2. The intravenous administration of anti-inflammatory drugs (dexamethasone, indomethacin and lysine-acetylsalicylate) to polyacrylamide gel-treated mice inhibited the accumulation of neutrophils in the inflamed site. 3. A similar administration of a 36K mouse lipocortin, induced a strong dose-dependent inhibition of neutrophil accumulation in the inflamed site. 4. Dexamethasone and lipocortin inhibited the production of eicosanoids, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the inflamed site of polyacrylamide gel-treated mice. 5. Lipocortin impaired both phospholipase A2 (PLA2) activity and chemotaxis of isolated inflammatory neutrophils. 6. The present studies show an in vivo anti-inflammatory effect of lipocortin similar to that of glucocorticosteroids. In agreement with recent data on the extracellular effects of various lipocortins, these results might implicate lipocortin(s) in the anti-inflammatory effects of glucocorticosteroids.

Inhibition of O2- generation by dexamethasone is mimicked by lipocortin I in alveolar macrophages.

Glucocorticoids inhibit superoxide (O2-) generation by phagocytes through a mechanism that remains unclear. We investigated this effect by using dexamethasone on guinea pig alveolar macrophages. O2- generation was induced either by the calcium ionophore A23187, a potent stimulus of phospholipase A2, or by the protein kinase C activator, phorbol myristate acetate (PMA). Dexamethasone inhibited O2- generation initiated by A23187 by 50-55%. This inhibition required: (a) more than 45 min incubation and was maximal after 2 h; (b) glucocorticoid receptor occupancy; and (c) protein synthesis. The inhibitory effect of dexamethasone could not be explained by an interaction with the respiratory burst enzyme NADPH oxidase since O2- generation was only weakly affected upon PMA stimulation. Lipocortin I, a glucocorticoid inducible and phospholipase A2 inhibitory protein, inhibited O2- generation initiated by A23187 but failed to modulate the respiratory burst activated by PMA. These results were obtained with lipocortin I purified from mouse lungs, human blood mononuclear cells, and with human recombinant lipocortin I. We propose that lipocortin I is capable of inhibiting the activation of NADPH oxidase only when membrane signal transduction involves phospholipase A2. By mimicking the effect of dexamethasone, lipocortin I may extend its potential anti-inflammatory action to the partial control of the formation of oxygen reactive species by phagocytes.

Phospholipase A2 inhibitory activity in thymocytes of dexamethasone-treated mice--possible implication of lipocortins.

The cellular phospholipase A2 activity of mouse thymocytes was estimated in vitro by the release of [3H]-Arachidonic acid from labeled and calcium ionophore A23187-stimulated cells. This activity was decreased in thymocytes from dexamethasone-treated mice. Thus, the presence of phospholipase A2 inhibitory proteins in mouse thymus was investigated. Three main proteins (36 kDa I, 36 kDa II, 73 kDa) were purified. These proteins were able to inhibit both phospholipase A2 in vitro, and the release of [3H]-Arachidonic acid from labeled and stimulated mouse thymocytes. Biochemical analysis revealed that the three proteins were lipocortin-like proteins. Our results show that in vivo dexamethasone treatment induces a phospholipase A2 inhibitory activity in mouse thymus, such an inhibition can be reproduced on isolated thymocytes by purified thymic lipocortins, known as glucocorticosteroid-inducible proteins.

Inhibition of eicosanoid and PAF formation by dexamethasone in rat inflammatory polymorphonuclear neutrophils may implicate lipocortin 's'.

In polymorphonuclear neutrophils, phospholipase A2 activity is the rate-limiting step for platelet-activating factor (PAF) formation and for the biosynthesis of arachidonic acid derivatives, leukotrienes and prostaglandins. Glucocorticosteroids inhibit phospholipase A2 activity by inducing in target cells the synthesis and release of phospholipase A2 inhibitory proteins named 'lipocortins'. Here, we report that rat pleural inflammatory polymorphonuclear neutrophils, treated with dexamethasone, decrease their production of eicosanoids and PAF. Evidence is presented which may implicate lipocortin 's' in these inhibitions since (i) phospholipase A2 inhibitory proteins are found in the supernatant of dexamethasone-treated cells, (ii) this supernatant inhibits the formation of lipid mediators in untreated cells, inhibition being reversed either by incubating the supernatant with a monoclonal antibody against rat lipocortin or by boiling it and (iii) a 36 kDa lipocortin from mice lungs mimics the effects of dexamethasone when added exogenously on untreated cells. Our results favour the hypothesis that the newly formed lipocortin 's' could be responsible for the antiphospholipase A2 activity of glucocorticosteroids.

Inhibition of phospholipase A2 activity of guinea-pig alveolar macrophages by lipocortin-like proteins purified from mice lung.

Guinea-pig alveolar macrophages were harvested by bronchoalveolar lavage and purified by differential adhesion. They were labeled with 14C-Arachidonic acid and then exposed to platelet-activating factor or to the calcium ionophore A23187. The activity of cellular phospholipase A2 was considered as the release of free 14C-Arachidonic acid in the cell supernatant. The pretreatment of guinea-pig alveolar macrophages with two lipocortin-like proteins (36 kDa and 40 kDa) purified from mice lung induced a significant inhibition of their phospholipase A2 activity upon platelet-activating factor and calcium ionophore stimulation. These results indicate that lipocortin-like proteins can modulate the phospholipase A2 activity of isolated cells in vitro.

Rapid purification of two lipocortin-like proteins from mice lung.

We have purified two proteins (40 kDa and 36 kDa) from mice lung by the method of calcium-precipitation/EGTA solubilization and then a separation on a high anion exchanger column (Mono Q HR 5/5. Pharmacia) with a gradient of NaCl. The two proteins were strong inhibitors of phospholipase A2 as assessed in vitro with porcine pancreatic phospholipase A2 and [3H]-oleic acid labeled E. Coli membranes as substrate. The 40 kDa protein had a pI of 5.8 and was found to be immunologically related to human recombinant lipocortin I. The 36 kDa protein had a pI of 4.7 and cross-reacted with a polyclonal antibody raised against a 32 kDa human lipocortin-like protein described in human blood mononuclear cells. We report here a rapid purification of two distinct lipocortin-like proteins from mice lung.

Characterization of a polyacrylamide gel-induced granuloma in mice: involvement of arachidonate metabolites.

The injection of polyacrylamide gel (Bio Gel P-4, 200-400 mesh) into the subcutaneous area of mice, induced an inflammatory reaction which was characterized by the migration of leukocytes (mainly neutrophils) from the blood vessels towards the polyacrylamide gel. A rapid protein accumulation was observed during the migration of cells towards the inflamed site. Neutrophils released some pro-inflammatory lipids (prostaglandins, leukotriene B4) and lysozyme, these products were assayed in the granuloma exudate at various times of the granuloma formation. In our experimental inflammatory model, the results suggest that neutrophils that are attracted by the polyacrylamide gel produce eicosanoids and lysozyme, which could act synergistically to potentiate cell migration and protein accumulation in the inflamed site.

Purification and characterization of a 32-kDa phospholipase A2 inhibitory protein (lipocortin) from human peripheral blood mononuclear cells.

A 32-kDa protein was isolated from human monocytes after calcium precipitation and chromatography. The protein activity was assessed by the inhibition of soluble phospholipase A2 (PLA2). This in vitro inhibitory effect on phospholipases A2 was found only with negatively charged phospholipids. The protein was also able to inhibit cellular PLA2 in mouse thymocytes. The biochemical properties and amino acid composition strongly suggest that the protein shares similarities with endonexin. Using a neutralizing monoclonal antibody against rat lipocortin, we found a cross-reactivity with the 32-kDa protein. According to the biochemical and immunological properties, we propose to relate this PLA2 inhibitory protein from human monocytes to lipocortin.

The presence of lipocortin in human embryonic skin fibroblasts and its regulation by anti-inflammatory steroids.

Human embryonic skin fibroblasts in culture produce pro-inflammatory lipid mediators and all types of prostanoids. When these cells were treated with the anti-inflammatory steroid, dexamethasone, prostaglandin production was inhibited. This phenomenon required glucocorticoid receptor occupancy and mRNA and protein synthesis. The inhibitory effect was prevented by treating the cells with a monoclonal antibody, BF 26, raised against renocortin, a lipocortin-like protein formed in rat kidney medulla interstitial cells in culture. When the proteins present in the supernatants and the cell pellets derived from control and dexamethasone-treated cells were analyzed for their ability to inhibit phospholipase A2, four inhibitory peaks, at 45, 30, 15 kDa and one peak under 12 kDa, were found in the supernatants of control and dexamethasone-treated cells, whereas one single inhibitory peak at 15 kDa was found in the cell pellets. The antiphospholipase activity was much greater in dexamethasone-treated cells than in control cells. These results suggest that preformed lipocortin exists in human cells and that lipocortin is synthesized and released under glucocorticoid treatment.