Astrid Jullumstrø Feuerherm

Elevated levels of osteoprotegerin (OPG) and hepatocyte growth factor (HGF) in rheumatoid arthritis

Rheumatic diseases are often associated with changes in bone metabolism. Excessive production and release of cytokines and other growth factors due to inflammation, e.g. tumor necrosis factor-α (TNF-α), receptor activator of NF-κB ligand (RANKL), interleukins such as IL-1and IL-6, may cause alterations in bone homeostasis leading to bone degradation. Other components such as osteoprotegerin (OPG) and possibly the ligand-receptor pair hepatocyte growth factor (HGF) and c-met may counteract this destruction. We have measured the levels of OPG, and HGF/c-met, in serum, synovial fluid (SF), and cartilage from patients with rheumatoid arthritis (RA) and other arthritides. We found a) elevated levels of both OPG and HGF in SF from RA patients relative to arthritides of other causes, b) increased levels of both OPG and HGF in SF from seropositive RA patients (RA+ ) compared to seronegative RA patients (RA-), c) elevated levels of both OPG and HGF in serum from RA patients compared to healthy controls, d) no correlation between severity of inflammation and levels of OPG or HGF, and e) presence of HGF/c-met in both cartilage and synovial tissue. The most significant elevations of OPG and HGF were found in patients with RA, the rheumatic disease most frequently associated with the development of secondary osteoporosis.Rheumatic diseases are often associated with changes in bone metabolism. Excessive production and release of cytokines and other growth factors due to inflammation, e.g. tumor necrosis factor-alpha (TNF-alpha), receptor activator of NF-kappaB ligand (RANKL), interleukins such as IL-1 and IL-6, may cause alterations in bone homeostasis leading to bone degradation. Other components such as osteoprotegerin (OPG) and possibly the ligand-receptor pair hepatocyte growth factor (HGF) and c-met may counteract this destruction, we have measured the levels of OPG, and HGF c-met, in serum, synovial fluid (SF), and cartilage from patients with rheumatoid arthritis (RA) and other arthritides. We found a) elevated levels of both OPG and HGF in SF from RA patients relative to arthritides of other causes, b) increased levels of both OPG and HGF in SF from seropositive RA patients (RA+) compared to seronegative RA patients (RA-), c) elevated levels or both OPG and HGF in serum from RA patients compared to healthy controls, d) no correlation between severity of inflammation and levels of OPG or HGF, and e) presence of HGF c-met in both cartilage and synovial tissue. The most significant elevations of OPG and HGF were found in patients with RA, the rheumatic disease most frequently associated with the development of secondary osteoporosis.

Multiple phospholipase A2 enzymes participate in the inflammatory process in osteoarthritic cartilage.

Objective: The aim of this study was to determine the involvement of pro-inflammatory phospholipase A2 (PLA2) enzymes in human chondrocytes from patients with osteoarthritis (OA). Methods: PLA2 involvement in OA chondrocytes was analysed by (a) arachidonic acid (AA) and oleic acid release, (b) PLA2 mRNA analysis, and (c) prostaglandin E2 (PGE2) production in cultured OA chondrocytes in response to various cytokines and platelet activating factor (PAF). Results: Pro-inflammatory cytokines and PAF stimulation led to increased AA release, interleukin (IL)-1β and tumour necrosis factor (TNF) being the strongest inducers. The pattern of oleic acid release was similar to but less prominent than AA release, suggesting that predominantly arachidonyl selective enzymes were activated. IL-1β, TNF, IL-6, and IL-8 upregulated secretory group IIA, IID, and V phospholipase A2 (sPLA2-IIA, -IID, -V) and cytosolic group IVA phospholipase A2 (cPLA2-IVA) expression, where induction of chondrocyte sPLA2-IID is a novel finding. Furthermore, IL-1β, TNF, and IL-6 also induced COX2 expression. PAF induced expression of group IIA, IID and IVA PLA2, and COX2. In line with its anti-inflammatory properties, IL-4 was unable to induce either AA release or expression of PLA2s or COX2. IL-1β and TNF strongly increased PGE2 production, with IL-1β as the most prominent inducer. Conclusion: Multiple PLA2 isoforms are expressed and influenced by pro-inflammatory stimuli in OA chondrocytes. Hence, several PLA2 enzymes may contribute to chondrocyte function by their upregulation and activation, and increased AA release and PGE2 production may therefore be important effectors in OA pathophysiology. PLA2 enzymes and cPLA2-IVA in particular are thus possible therapeutic targets in OA.

Cytosolic phospholipase A2 regulates TNF-induced production of joint destructive effectors in synoviocytes

Introduction Rheumatoid arthritis (RA) is an inflammatory disease of the joint characterized by chronic synovitis causing pain, swelling and loss of function due to destruction of cartilage and bone. The complex series of pathological events occurring in RA is largely regulated via excessive production of pro-inflammatory cytokines, the most prominent being tumor necrosis factor (TNF). The objective of this work was to elucidate possible involvement of group IVA cytosolic phospholipase A2 (cPLA2α) in TNF-induced regulation of synovitis and joint destructive effectors in RA, to evaluate the potential of cPLA2α as a future therapeutic target. Methods The involvement of cPLA2α in tumor necrosis factor (TNF)-induced intracellular signaling cascades in synoviocytes (synovial fibroblast-like cells) was analyzed by arachidonic acid (AA) release assay, synoviocyte enzyme activity assay, gene expression analysis by real-time PCR and ELISA immunoassay for the detection of prostaglandin E2 (PGE2), interleukin 8 (IL8) and stromelysin-1 (MMP3), respectively. Results Inhibitors of cPLA2α enzyme activity (AVX002, ATK) significantly reduced TNF-induced cellular release of AA, PGE2, IL8 and MMP3. This reduction was evident both at transcriptional, protein or metabolite levels. Interestingly, cPLA2α inhibition affected several key points of the arachidonyl cascade; AA-release, cyclooxygenase-2 (COX2) expression and PGE2 production. Furthermore, the results suggest that cPLA2α is subject to transcriptional auto-regulation as inhibition of cPLA2α resulted in reduced PLA2G4A gene expression in TNF-stimulated synoviocytes. Conclusions cPLA2α appears to be an important regulator of central effectors of inflammation and joint destruction, namely MMP3, IL8, COX2, and PGE2. Decreased transcription of the PLA2G4A and COX2 genes in response to cPLA2α enzyme inhibition further suggest a self-reinforcing effect of cPLA2α inhibition in response to TNF. Collectively, these results support that cPLA2α is an attractive therapeutic target candidate as its inhibition reduces the production of multiple key pro-inflammatory factors involved in RA pathogenesis.

The ω3‐polyunsaturated fatty acid derivatives AVX001 and AVX002 directly inhibit cytosolic phospholipase A2 and suppress PGE2 formation in mesangial cells

ω3‐polyunsaturated fatty acids (ω3‐PUFAs) are known to exert anti‐inflammatory effects in various disease models although their direct targets are only poorly characterized.

Inhibition of Group IVA Cytosolic Phospholipase A2 by Thiazolyl Ketones in Vitro, ex Vivo, and in Vivo

Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is the rate-limiting provider of pro-inflammatory mediators in many tissues and is thus an attractive target for the development of novel anti-inflammatory agents. In this work, we present the synthesis of new thiazolyl ketones and the study of their activities in vitro, in cells, and in vivo. Within this series of compounds, methyl 2-(2-(4-octylphenoxy)acetyl)thiazole-4-carboxylate (GK470) was found to be the most potent inhibitor of GIVA cPLA2, exhibiting an XI(50) value of 0.011 mole fraction in a mixed micelle assay and an IC50 of 300 nM in a vesicle assay. In a cellular assay using SW982 fibroblast-like synoviocytes, it suppressed the release of arachidonic acid with an IC50 value of 0.6 μM. In a prophylactic collagen-induced arthritis model, it exhibited an anti-inflammatory effect comparable to the reference drug methotrexate, whereas in a therapeutic model, it showed results comparable to those of the reference drug Enbrel. In both models, it significantly reduced plasma PGE2 levels.

Presence of secretory group IIa and V phospholipase A2 and cytosolic group IVα phospholipase A2 in chondrocytes from patients with rheumatoid arthritis

Abstract Both secretory and cytosolic phospholipase A2 enzymes have been implicated in the pathogenesis of arthritis in animal models, but the exact expression patterns of the enzymes in diseased human joint tissue are uncertain. We investigated the messenger RNA expression of group IIa, IVα and V phospholipase A2 and localized the presence of group IIa and IVα phospholipase A2 at protein levels in articular cartilage from patients with rheumatoid arthritis, osteoarthritis and patients with non-arthritic joints. Both group IIa phospholipase A2 messenger RNA and protein were detected in all samples independent of diagnosis, but were far more prominent in cartilage from rheumatoid arthritis samples. In cartilage with rheumatoid arthritis, the enzyme was detected both within the chondrocytes and in the extracellular matrix, whereas only few osteoarthritic cartilage samples showed positive staining in the matrix. In the cartilage matrix of non-arthritic controls, group IIa phospholipase A2 was totally absent. Messenger RNA for the group IVα and V phospholipase A2 was, except for one osteoarthritic cartilage sample, exclusively detected in rheumatoid arthritic cartilage. For group IVα phospholipase A2 this was also confirmed at the protein level. These results suggest that each phospholipase A2 enzyme has distinct roles in both healthy and diseased joint tissue, and that the diversity and amount of enzyme correlate with the grade of inflammation and disease severity.

Cytosolic phospholipase A2 modulates TLR2 signaling in synoviocytes.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis leading to destruction of cartilage and bone. PLA2 enzymes are key players in inflammation regulating the release of unsaturated fatty acids such as arachidonic acid (AA), a precursor of pro-inflammatory eicosanoids. Several lines of evidence point to toll-like receptors (TLRs) as drivers of synovitis and joint destruction in RA. However, few studies have addressed the implication of PLA2 activity downstream TLR activation in the synovium. Here, we aimed to characterize PLA2 enzyme involvement in TLR2-induced signaling in synovial fibroblast-like cells. TLRs1-7 and a range of sPLA2, iPLA2 and cPLA2 enzymes were found to be transcriptionally expressed in cultured synoviocytes. Activation of TLR2/1 and TLR2/6 led to phosphorylation of cPLA2α at Ser505, and induced AA release and PGE2 production; effects that were attenuated by cPLA2α inhibitors. In contrast, sPLA2 inhibitors did not affect AA or PGE2 release. cPLA2α inhibitors furthermore attenuated TLR-induced expression of IL-6, IL-8 and COX2. COX1/2 inhibitors attenuated TLR2/6-induced IL-6 transcription and protein production comparable to cPLA2α inhibition. Moreover, exogenously PGE2 added alone induced IL-6 production and completely rescued IL-6 transcription when added simultaneously with FSL-1 in the presence of a cPLA2α inhibitor. Our results demonstrate for the first time that cPLA2α is involved in TLR2/1- and TLR2/6-induced AA release, PGE2 production and pro-inflammatory cytokine expression in synoviocytes, possibly through COX/PGE2-dependent pathways. These findings expand our understanding of cPLA2α as a modulator of inflammatory molecular mechanisms in chronic diseases such as RA.

Platelet-activating factor induces proliferation in differentiated keratinocytes.

Abstract Increased levels of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) are found in several inflammatory dermatoses, but PAF’s exact role in epidermis is uncertain. In order to better understand the physiological consequences of excess PAF production in epidermis, we examined the gene regulatory effects of PAF short-term stimulation in differentiated HaCaT keratinocytes by transcriptional profiling. Even though PAF induces COX2 expression, we found that PAF regulates only few genes associated with inflammation in differentiated keratinocytes. Rather, we show that natural PAF rapidly regulates genes involved in proliferation, (anti)-apoptosis and migration, all sub-processes of re-epithelialization and wound healing. Moreover, profiling of phosphorylated kinases, cellular wound-scratch experiments, resazurin assay and flow cytometry cell cycle phase analysis all support a role for PAF in keratinocyte proliferation and epidermal re-epithelialization. In conclusion, these results suggest that PAF acts as an activator of proliferation and may, therefore, function as a connector between inflammation and proliferation in differentiated keratinocytes.

A novel compound isolated from Sclerochloa dura has anti-inflammatory effects

The activation of PLA 2 by means of pro-inflammatory cytokines results in the subsequent release of arachidonic acid (AA) and generates eicosanoids, which further propagate inflammation. By 6the cyclooxygenases (COX1/2) responsible for the enzymatic conversion of AA to eicosanoids, the non-steroidal anti-inflammatory drugs are effective in relieving the pain and discomfort of inflammation. By using AA release assay as a guide for biological and anti-inflammatory activity, novel compound 1- O -(3- O -linolenoyl-6-deoxy-6-sulfo- α -D-glucopyranosyl)-glycerol (1) together with five known compounds isovitexin, byzantionoside B, tricin 4 ’ - O -(erythro- β -guaiacylglyceryl) ether 7- O - β -glucopyranoside, 1- O -feruloyl glycerol and tricin 7-glucoside were isolated from the methanol extract of the aerial parts of Sclerochloa dura using LC techniques (Sephadex LH-20 column chromatography, preparative HPLC and semi-preparative HPLC). All isolated compounds were identified using spectroscopic NMR spectroscopy and MS spectrometry. Novel compound (1) was found to be an effective inhibitor of AA release with an IC 50 value of 0.09 ± 0.03 mg mL -1 .