Functional Characterization of Lysophospholipids by Proteomic and Lipidomic Analysis of Fibroblast-like Synoviocytes.

Synovial fluid (SF) from human knee joints with osteoarthritis (OA) has elevated levels of lysophosphatidylcholine (LPC) species, but their functional role is not well understood. This in vitro study was designed to test the hypothesis that various LPCs found elevated in OA SF and their metabolites, lysophosphatidic acids (LPAs), modulate the abundance of proteins and phospholipids (PLs) in human fibroblast-like synoviocytes (FLSs), with even minute chemical variations in lysophospholipids determining the extent of regulation. Cultured FLSs (n = 5-7) were treated with one of the LPC species, LPA species, IL-1ß, or a vehicle. Tandem mass tag peptide labeling coupled with LC-MS/MS/MS was performed to quantify proteins. The expression of mRNA from regulated proteins was analyzed using RT-PCR. PL synthesis was determined via ESI-MS/MS, and the release of radiolabeled PLs was determined by means of liquid scintillation counting. In total, 3960 proteins were quantified using multiplexed MS, of which 119, 8, and 3 were significantly and reproducibly regulated by IL-1ß, LPC 16:0, and LPC 18:0, respectively. LPC 16:0 significantly inhibited the release of PLs and the synthesis of phosphatidylcholine, LPC, and sphingomyelin. Neither LPC metabolite-LPA 16:0 nor LPA 18:0-had any reproducible effect on the levels of each protein. In conclusion, small chemical variations in LPC species can result in the significantly altered expression and secretion of proteins and PLs from FLSs. IL-1ß influenced all proteins that were reproducibly regulated by LPC 16:0. LPC species are likely to modulate FLS protein expression only in more advanced OA stages with low IL-1ß levels. None of the eight proteins being significantly regulated by LPC 16:0 have been previously reported in OA. However, our in vitro findings show that the CD81 antigen, calumenin, and B4E2C1 are promising candidates for further study, focusing in particular on their potential ability to modulate inflammatory and catabolic mechanisms.

Interleukin-1 Induces the Release of Lubricating Phospholipids from Human Osteoarthritic Fibroblast-like Synoviocytes.

(1) Background: Synovial fluid (SF) from knee joints with osteoarthritis (OA) has increased levels of phospholipids (PL). We have reported earlier that TGF-ß and IGF-1 stimulate fibroblast-like synoviocytes (FLS) to synthesize increased amounts of PLs. The current study examined whether IL-1ß induces the release of PLs in FLS and the underlying mechanism. (2) Methods: Cultured human OA FLS were treated with IL-1ß alone and with pathway inhibitors or with synthetic liver X receptor (LXR) agonists. Cholesterol hydroxylases, ABC transporters, apolipoproteins (APO), LXR, sterol regulatory binding proteins (SREBPs), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) were analyzed by RT-PCR, Western blot, and ELISA. The release of radiolabeled PLs from FLS was determined, and statistical analysis was performed using R (N = 5-9). (3) Results: Like synthetic LXR agonists, IL-1ß induced a 1.4-fold greater release of PLs from FLS. Simultaneously, IL-1ß upregulated the level of the PL transporter ABCA1 and of cholesterol hydroxylases CH25H and CYP7B1. IL-1ß and T0901317 stimulated the expression of SREBP1c, whereas only T0901317 enhanced SREBP2, HMGCR, APOE, LXRα, and ABCG1 additionally. (4) Conclusions: IL-1ß partially controls PL levels in OA-SF by affecting the release of PLs from FLS. Our data show that IL-1ß upregulates cholesterol hydroxylases and thus the formation of oxysterols, which, as natural agonists of LXR, increase the level of active ABCA1, in turn enhancing the release of PLs.

A novel model of a biomechanically induced osteoarthritis-like cartilage for pharmacological in vitro studies.

Excessive pressure or overload induces and aggravates osteoarthritic changes in articular cartilage, but the underlying biomechanical forces are largely ignored in existing pharmacological in vitro models that are used to investigate drugs against osteoarthritis (OA). Here, we introduce a novel in vitro model to perform pathophysiological and pharmacological investigations, in which cartilage explants are subjected to intermittent cyclic pressure, and characterize its ability to mimic OA-like tissue reactivity. Mechanical loading time-dependently increased the biosynthesis, content and retention of fibronectin (Fn), whereas collagen metabolism remained unchanged. This protocol upregulated the production and release of proteoglycans (PGs). The release of PGs from explants was significantly inhibited by a matrix metalloproteinase (MMP) inhibitor, suggesting the involvement of such proteinases in the destruction of the model tissue, similar to what is observed in human OA cartilage. In conclusion, the metabolic alterations in our new biomechanical in vitro model are similar to those of early human OA cartilage, and our pharmacological prevalidation with an MMP-inhibitor supports its value for further in vitro drug studies.

Biomarkers of Hand Osteoarthritis Are Detectable after Mechanical Exercise.

BACKGROUND: Hand osteoarthritis (OA) is one of the most common joint diseases, but studies on biomarkers are rare. The aim of this explorative study was (a) to evaluate potential biomarkers of hand OA, (b) to identify an optimal time point to sample venous blood, and (c) to correlate biomarker levels with radiological and clinical scores. METHODS: Four female cohorts were investigated. One with a more Heberden-accentuated OA and one with a more Bouchard-accentuated hand OA, and two symptom-free control groups aged 20-30 or 50-75 years. The venous blood was sampled before and at eight time points after mechanical exercise of the OA hand. X-rays of OA hands were assessed using the Kellgren and Lawrence as well as Kallman scores. Participants were evaluated clinically using the AUSCAN™ Index, visual analog scale (VAS), and Health Assessment Questionnaire (HAQ). Serum levels of seven biomarkers were measured by ELISA. RESULTS: The concentrations of CPII, COMP, IL-15, sVCAM-1, NGAL, and PIIANP were significantly increased within 15 min after exercise. PIIANP was markedly elevated in the Heberden-accentuated OA group as compared to both control groups, but did not correlate with any radiological or clinical score. Analysis of the probabilistic index further revealed that CPII can distinguish between Bouchard's OA and premenopausal controls whereas COMP can discriminate between Bouchard's and Heberden's OA. CONCLUSIONS: This study demonstrates that even previously undetectable biomarkers can be quantified in serum after mechanical exercise. Future larger studies are needed to determine specificity and sensitivity of these markers and their ability to diagnose even pre-radiological OA.

The Effect of Dexamethasone, Adrenergic and Cholinergic Receptor Agonists on Phospholipid Metabolism in Human Osteoarthritic Synoviocytes.

Phospholipids (PLs) possess the unique ability to contribute to synovial joint lubrication. The aim of our study was to determine for the first time the effect of dexamethasone and some adrenergic and cholinergic agonists on the biosynthesis and release of PLs from human fibroblast-like synoviocytes (FLS). Osteoarthritic human knee FLS were treated with dexamethasone, terbutaline, epinephrine, carbachol, and pilocarpine, or the glucocorticoid receptor antagonist RU 486. Simultaneously PL biosynthesis was determined through the incorporation of stable isotope-labeled precursors into PLs. Radioactive isotope-labeled precursors were used to radiolabel PLs for the subsequent quantification of their release into nutrient media. Lipids were extracted and quantified using electrospray ionization tandem mass spectrometry or liquid scintillation counting. Dexamethasone significantly decreased the biosynthesis of phosphatidylcholine, phosphatidylethanolamine (PE), PE-based plasmalogen, and sphingomyelin. The addition of RU 486 abolished these effects. A release of PLs from FLS into nutrient media was not recognized by any of the tested agents. None of the adrenergic or cholinergic receptor agonists modulated the PL biosynthesis. We demonstrate for the first time an inhibitory effect of dexamethasone on the PL biosynthesis of FLS from human knees. Moreover, our study indicates that the PL metabolism of synovial joints and lungs are differently regulated.

Comparative Analysis of Peptide Composition and Bioactivity of Different Collagen Hydrolysate Batches on Human Osteoarthritic Synoviocytes.

Collagen hydrolysates (CHs) are heterogeneous mixtures of collagen peptides that are often used as nutraceuticals for osteoarthritis (OA). In this study, we compared the peptide composition and pharmacological effects of three different CH preparations (CH-Alpha®, Peptan® B 2000 and Mobiforte®) as well as their production batches. Our biochemical analysis using MALDI-TOF mass spectrometry and the ICPL™-isotope labelling method revealed marked differences between different CH preparations and even between some production batches of the same preparation. We also investigated the pharmacological effects of these CHs on human fibroblast-like synoviocytes (FLS). No significant effects on cultured FLS could be demonstrated for either production batch of CH-Alpha®, Peptan® B 2000, and Mobiforte® analyzing a small number of pharmacological relevant targets. Thus, our study already shows for the first time that different production batches of the same CH preparation as well as different CH preparations can differ significantly in their peptide composition. In this line, further studies are also needed to verify equal pharmacological efficacy of CH batches on a much broader range of (patho)physiological relevant targets. If OA patients are to be offered a safe and effective nutraceutical a better knowledge about all potential effects as well as ensuring the same active-substance levels are a prerequisite.

Pharmacological treatment of knee osteoarthritis: Special considerations of the new German guideline.

The pharmacological treatment of knee osteoarthritis (OA) is a purely symptomatic therapy, which often ensures that the mobility of the patient is successfully retained. This article refers to the recommendations and opinions regarding the pharmacotherapy of knee OA contained in the new guideline of the Association of the Scientific Medical Societies in Germany (AWMF), highlighting several important aspects and describing the considerations underlying the decision-making process. With this article it is hoped that therapeutic effectiveness can be realistically estimated, that any risks of medication errors and avoidable side effects can be reduced, and that further helpful measures can be taken into consideration.

Growth factors regulate phospholipid biosynthesis in human fibroblast-like synoviocytes obtained from osteoarthritic knees.

Elevated levels of growth factors and phospholipids (PLs) have been found in osteoarthritic synovial fluid (SF), although the metabolic regulation of PLs is currently unknown. This study aimed to determine the effects of growth factors on the biosynthesis of PLs by fibroblast-like synoviocytes (FLS) obtained from human osteoarthritic knee joints. Electrospray ionization tandem mass spectrometry was applied to analyse the newly synthesized PLs. In the presence of stable isotope-labelled PL precursors, cultured FLS were treated with either transforming growth factor-ß1 (TGF-ß1), bone morphogenetic protein (BMP)-2, BMP-4, BMP-7 or insulin-like growth factor-1 (IGF-1) alone or in combination with specific inhibitors of cell signalling pathways. TGF-ß1 and IGF-1 markedly stimulated the biosynthesis of phosphatidylcholine (PC) before sphingomyelin (SM) and lysophosphatidylcholine (LPC) species were stimulated. BMPs elaborated less pronounced effects. The BMPs tested have different potentials to induce the biosynthesis of phosphatidylethanolamine (PE) and PE-based plasmalogens. Our study shows for the first time that TGF-ß1 and IGF-1 substantially regulate the biosynthesis of PC, SM and LPC in human FLS. The functional consequences of elevated levels of PLs require additional study. The BMPs tested may be joint protective in that they upregulate PE-based plasmalogens that function as endogenous antioxidants against reactive oxygen species.

Metabolic Response of Human Osteoarthritic Cartilage to Biochemically Characterized Collagen Hydrolysates.

The most frequent disease of the locomotor system is osteoarthritis (OA), which, as a chronic joint disease, might benefit more from nutrition than acute illnesses. Collagen hydrolysates (CHs) are peptidic mixtures that are often used as nutraceuticals for OA. Three CHs were characterized biochemically and pharmacologically. Our biophysical (MALDI-TOF-MS, NMR, AFM) and fluorescence assays revealed marked differences between CHs of fish (Peptan® F 5000, Peptan® F 2000) and porcine (Mobiforte®) origin with respect to the total number of peptides and common peptides between them. Using a novel dual radiolabeling procedure, no CH modulated collagen biosynthesis in human knee cartilage explants. Peptan® F 2000 enhanced the activities of the aggrecanase ADMATS4 and ADMATS5 in vitro without loss of proteoglycan from cartilage explants; the opposite effect was observed with Mobiforte®. Interleukin (IL)-6, matrix metalloproteinase (MMP)-1, -3 and -13 levels were elevated in explants that were treated with Mobiforte® and Peptan® F 5000, but not with Peptan® F 2000. In conclusion, the heterogeneous peptide composition and disparate pharmacological effects between CHs suggest that the effect of a CH preparation cannot be extrapolated to other formulations. Thus, the declaration of a CH as a safe and effective nutraceutical requires a thorough examination of its pleiotropic effects.

Acetylation reduces SOX9 nuclear entry and ACAN gene transactivation in human chondrocytes.

Changes in the content of aggrecan, an essential proteoglycan of articular cartilage, have been implicated in the pathophysiology of osteoarthritis (OA), a prevalent age-related, degenerative joint disease. Here, we examined the effect of SOX9 acetylation on ACAN transactivation in the context of osteoarthritis. Primary chondrocytes freshly isolated from degenerated OA cartilage displayed lower levels of ACAN mRNA and higher levels of acetylated SOX9 compared with cells from intact regions of OA cartilage. Degenerated OA cartilage presented chondrocyte clusters bearing diffused immunostaining for SOX9 compared with intact cartilage regions. Primary human chondrocytes freshly isolated from OA knee joints were cultured in monolayer or in three-dimensional alginate microbeads (3D). SOX9 was hypo-acetylated in 3D cultures and displayed enhanced binding to a -10 kb ACAN enhancer, a result consistent with higher ACAN mRNA levels than in monolayer cultures. It also co-immunoprecipitated with SIRT1, a major deacetylase responsible for SOX9 deacetylation. Finally, immunofluorescence assays revealed increased nuclear localization of SOX9 in primary chondrocytes treated with the NAD SIRT1 cofactor, than in cells treated with a SIRT1 inhibitor. Inhibition of importin ß by importazole maintained SOX9 in the cytoplasm, even in the presence of NAD. Based on these data, we conclude that deacetylation promotes SOX9 nuclear translocation and hence its ability to activate ACAN.

Articular Joint Lubricants during Osteoarthritis and Rheumatoid Arthritis Display Altered Levels and Molecular Species.

BACKGROUND: Hyaluronic acid (HA), lubricin, and phospholipid species (PLs) contribute independently or together to the boundary lubrication of articular joints that is provided by synovial fluid (SF). Our study is the first reporting quantitative data about the molecular weight (MW) forms of HA, lubricin, and PLs in SF from cohorts of healthy donors, patients with early (eOA)- or late (lOA)-stage osteoarthritis (OA), and patients with active rheumatoid arthritis (RA). METHODS: We used human SF from unaffected controls, eOA, lOA, and RA. HA and lubricin levels were measured by enzyme-linked immunosorbent assay. PLs was quantified by electrospray ionization tandem mass spectrometry. Fatty acids (FAs) were analyzed by gas chromatography, coupled with mass spectrometry. The MW distribution of HA was determined by agarose gel electrophoresis. RESULTS: Compared with control SF, the concentrations of HA and lubricin were lower in OA and RA SF, whereas those of PLs were higher in OA and RA SF. Moreover, the MW distribution of HA shifted toward the lower ranges in OA and RA SF. We noted distinct alterations between cohorts in the relative distribution of PLs and the degree of FA saturation and chain lengths of FAs. CONCLUSIONS: The levels, composition, and MW distribution of all currently known lubricants in SF--HA, lubricin, PLs--vary with joint disease and stage of OA. Our study is the first delivering a comprehensive view about all joint lubricants during health and widespread joint diseases. Thus, we provide the framework to develop new optimal compounded lubricants to reduce joint destruction.

Detecting cathepsin activity in human osteoarthritis via activity-based probes.

INTRODUCTION: Lysosomal cathepsins have been reported to contribute to Osteoarthritis (OA) pathophysiology due to their increase in pro-inflammatory conditions. Given the causal role of cathepsins in OA, monitoring their specific activity could provide means for assessing OA severity. To this end, we herein sought to assess a cathepsin activity-based probe (ABP), GB123, in vitro and in vivo. METHODS: Protein levels and activity of cathepsins B and S were monitored by immunoblot analysis and GB123 labeling in cultured primary chondrocytes and conditioned media, following stimuli with tumor necrosis factor alpha (TNFα) and/or Interleukin 1 beta (IL-1ß). Similarly, cathepsin activity was examined in sections of intact cartilage (IC) and degraded cartilage (DC) regions of OA. Finally, synovial fluid (SF) and serum from donors with no signs of diseases, early OA, late OA and rheumatoid arthritis (RA) patients were analyzed with GB123 to detect distinct activity levels of cathepsin B and S. RESULTS: Cathepsin activity in cell lysates, conditioned media explants and DC sections showed enhanced enzymatic activity of cathepsins B and S. Further histological analysis revealed that cathepsin activity was found higher in superficial zones of DC than in IC. Examining serum and SF revealed that cathepsin B is significantly elevated with OA severity in serum and SF, yet levels of cathepsin S are more correlated with synovitis and RA. CONCLUSIONS: Based on our data, cathepsin activity monitored by ABPs correlated well with OA severity and joint inflammation, directing towards a novel etiological target for OA, which possesses significant translational potential in developing means for non-invasive detection of early signs of OA.

Sphingolipids in human synovial fluid--a lipidomic study.

Articular synovial fluid (SF) is a complex mixture of components that regulate nutrition, communication, shock absorption, and lubrication. Alterations in its composition can be pathogenic. This lipidomic investigation aims to quantify the composition of sphingolipids (sphingomyelins, ceramides, and hexosyl- and dihexosylceramides) and minor glycerophospholipid species, including (lyso)phosphatidic acid, (lyso)phosphatidylglycerol, and bis(monoacylglycero)phosphate species, in the SF of knee joints from unaffected controls and from patients with early (eOA) and late (lOA) stages of osteoarthritis (OA), and rheumatoid arthritis (RA). SF without cells and cellular debris from 9 postmortem donors (control), 18 RA, 17 eOA, and 13 lOA patients were extracted to measure lipid species using electrospray ionization tandem mass spectrometry--directly or coupled with hydrophilic interaction liquid chromatography. We provide a novel, detailed overview of sphingolipid and minor glycerophospholipid species in human SF. A total of 41, 48, and 50 lipid species were significantly increased in eOA, lOA, and RA SF, respectively when compared with normal SF. The level of 21 lipid species differed in eOA SF versus SF from lOA, an observation that can be used to develop biomarkers. Sphingolipids can alter synovial inflammation and the repair responses of damaged joints. Thus, our lipidomic study provides the foundation for studying the biosynthesis and function of lipid species in health and most prevalent joint diseases.

A lipidomic study of phospholipid classes and species in human synovial fluid.

OBJECTIVE: Membrane phospholipid species contribute to boundary lubrication that is provided by synovial fluid (SF). Altered levels of lubricants can be associated with increased friction, leading to articular cartilage damage. This study was undertaken to determine whether the composition of phospholipid species is altered in diseases of human knee joints. METHODS: The study was performed using SF from unaffected controls and patients with early osteoarthritis (OA), late OA, or rheumatoid arthritis (RA). Lipids were extracted from cell- and vesicle-free SF from 9 control donors postmortem and from 17 patients with early OA, 13 patients with late OA, and 18 patients with RA. Phospholipid species were quantified by electrospray ionization tandem mass spectrometry. RESULTS: We conducted lipidomic analysis to provide the first detailed overview of phospholipid species in human SF. We identified 130 lipid species belonging to 8 lipid classes (phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, plasmalogens, phosphatidylserine, phosphatidylglycerol, sphingomyelin, and ceramide). Compared to SF from controls, SF from patients with early OA and those with late OA had higher levels of most phospholipid species. Moreover, the concentrations of 64 and 27 phospholipids differed between RA and early OA SF and between RA and late OA SF, respectively. Also, the levels of 66 phospholipid species were altered in early OA versus late OA. CONCLUSION: Our data indicate disease- and stage-dependent differences in the relative composition and levels of phospholipid species in human SF. Such alterations might affect articular joint lubrication. Because certain phospholipids scavenge reactive oxygen species (ROS) and are pro- or antiinflammatory, any altered phospholipid level might influence ROS-scavenging activity of SF and the inflammatory status of joints. Thus, phospholipids may be associated with the pathogenesis of OA.

Towards elucidating the role of SirT1 in osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease particularly affecting the elderly population. Although several genetic features have been characterized as risk factors for OA susceptibility, a growing body of evidence indicates that epigenetic effectors may also modulate gene expression and thus contribute to OA pathology. One such epigenetic regulator of particular relevance to OA is Silent Information Regulator 2 type 1 (SirT1) which has been linked to aging and caloric intake, Consistently, SirT1 has been also connected with various age-associated diseases such as diabetes type II, Alzheimers and osteoporosis. Recent reports show that OA is linked to changes in SirT1 activity or levels within cartilage. In human chondrocytes, SirT1 plays a role in cartilage extracellular matrix (ECM) synthesis and promotes cell survival, even under proinflammatory stress. It appears that SirT1 fine tunes many cellular biochemical processes through its capacity to interact and modify various histone and non-histone proteins. Taken together these investigations demonstrate that SirT1 is involved in cartilage biology and could potentially serve as novel drug target in treating OA even at its premature stages, thereby possibly reversing mechanical-stress induced cartilage degeneration.

Cytokines in osteoarthritis-current status on the pharmacological intervention.

Cytokines and their broad spectrum of effects have been investigated since the 1980s. The already existing preliminary scientific results have been highly suggestive of the idea that cytokines play an essential role in the pathogenesis of OA. Nevertheless, the extent to which cytokines participate in the origin of OA, or are taken as a consequence of the OA process, remain unanswered questions. Unlike the case with rheumatoid arthritis, studies on the application of anti-cytokine medications with OA remain in their infancy. At the present time no clinical studies relating to OA have confirmed that anti-cytokine medications are antiphlogistically effective and/or prevent the origin of morphologically recognizable cartilage defects or at least decelerate the increasing destruction of joint cartilage. As with every other therapeutic approach the risk-benefit scenario is decisive for deciding on the current application of anti cytokine medications for rheumatoid arthritis as well as their potential future use against OA. Considering the long-term consequences of an anti-cytokine based therapy, our poor state of knowledge should be seen in a very critical light, since the discussed approach represents an immunesuppressive therapy that entails consequences with regard to defence against infections and tumour suppression. Also, little is currently known about the interplay between pro- and anti-inflammatory cytokines and growth factors in OA; the resulting specific and fundamental therapeutic possibilities for performing a structure-modifying basic therapy in OA are worthy of further study on the part of academic and industrial institutions.