Emese Balogh

Toll-Like Receptor 2 Induced Angiogenesis and Invasion Is Mediated through the Tie2 Signalling Pathway in Rheumatoid Arthritis

Background Angiogenesis is a critical early event in inflammatory arthritis, facilitating leukocyte migration into the synovium resulting in invasion and destruction of articular cartilage and bone. This study investigates the effect of TLR2 on angiogenesis, EC adhesion and invasion using microvascular endothelial cells and RA whole tissue synovial explants ex-vivo. Methods Microvascular endothelial cells (HMVEC) and RA synovial explants ex vivo were cultured with the TLR2 ligand, Pam3CSK4 (1 µg/ml). Angiopoietin 2 (Ang2), Tie2 and TLR2 expression in RA synovial tissue was assessed by immunohistology. HMVEC tube formation was assessed using Matrigel matrix assays. Ang2 was measured by ELISA. ICAM-1 cell surface expression was assessed by flow cytometry. Cell migration was assessed by wound repair scratch assays. ECM invasion, MMP-2 and -9 expression were assessed using transwell invasion chambers and zymography. To examine if the angiopoietin/Tie2 signalling pathway mediates TLR2 induced EC tube formation, invasion and migration assays were performed in the presence of a specific neutralising anti-Tie2mAb (10 ug/ml) and matched IgG isotype control Ab (10 ug/ml). Results Ang2 and Tie2 were localised to RA synovial blood vessels, and TLR2 was localised to RA synovial blood vessels, sub-lining infiltrates and the lining layer. Pam3CSK4 significantly increased angiogenenic tube formation (p<0.05), and upregulated Ang2 production in HMVEC (p<0.05) and RA synovial explants (p<0.05). Pam3CSK4 induced cell surface expression of ICAM-1, from basal level of 149±54 (MFI) to 617±103 (p<0.01). TLR-2 activation induced an 8.8±2.8 fold increase in cell invasion compared to control (p<0.05). Pam3CSK4 also induced HMVEC cell migration and induced MMP-2 and -9 from RA synovial explants. Neutralisation of the Ang2 receptor, Tie2 significantly inhibited Pam3CSK4-induced EC tube formation and invasion (p<0.05). Conclusion TLR2 activation promotes angiogenesis, cell adhesion and invasion, effects that are in part mediated through the Tie2 signalling pathway, key mechanisms involved in the pathogenesis of RA.

Hypoxia and STAT3 signalling interactions regulate pro-inflammatory pathways in rheumatoid arthritis.

OBJECTIVE To examine the effect of hypoxia on Signal Transducer and Activator of Transcription 3 (STAT3)-induced pro-inflammatory pathways in rheumatoid arthritis (RA). METHODS Detection of phospho-STAT3 was assessed in RA synovial tissue and fibroblasts (RASFC) by immunohistology/immunofluorescence. Primary RASFCs and a normal synoviocyte cell line (K4IM) were cultured under hypoxic and normoxic conditions±Stat3-siRNA, HIF-siRNA or WP1066 (JAK2-inhibitor). HIF1α, p-STAT3, p-STAT1 and Notch-1IC protein expression were analysed by western blot. Functional mechanisms were quantified by invasion chamber, matrigel and migration assays. IL-6, IL-8, IL-10 and matrixmetalloproteinases (MMP)-3 were quantified by ELISA. Notch-1 receptor, its DLL-4 ligand and downstream target genes (hrt-1, hrt-2) were quantified by real-time PCR. The effect of WP1066 on spontaneous secretion of pro/anti-inflammatory cytokines and Notch signalling was examined in RA synovial explants ex vivo. RESULTS p-STAT3 was increased in RA synovium compared with control (p<0.05). Hypoxia induced p-STAT3, p-STAT1 and HIF1α expression, an effect blocked by Stat3-siRNA and WP1066. Hypoxia-induced cell invasion, migration and cytokine production were inhibited by Stat3-siRNA (p<0.05) and WP1066 (p<0.05). While HIF1α siRNA inhibited hypoxia-induced p-STAT3 detection, Stat3-siRNA also inhibited hypoxia-induced HIF1α. Furthermore, hypoxia-induced Notch-1IC, DLL4, hrt-1 and -2 expression were significantly inhibited by WP1066 (p<0.05). Finally, in RA synovial explant cultures ex vivo, WP1066 decreased spontaneous secretion of IL-6, IL-8 and MMP3 (p<0.05), Notch-1 mRNA (p<0.05) and induced IL-10 (p<0.05). CONCLUSIONS This is the first study to provide evidence of a functional link between HIF1α, STAT3 and Notch-1 signalling in the regulation of pro-inflammatory mechanisms in RA, and further supports a role for STAT blockade in the treatment of RA.

Successful tumour necrosis factor (TNF) blocking therapy suppresses oxidative stress and hypoxia-induced mitochondrial mutagenesis in inflammatory arthritis

IntroductionTo examine the effects of tumour necrosis factor (TNF) blocking therapy on the levels of early mitochondrial genome alterations and oxidative stress.MethodsEighteen inflammatory arthritis patients underwent synovial tissue oxygen (tpO2) measurements and clinical assessment of disease activity (DAS28-CRP) at baseline (T0) and three months (T3) after starting biologic therapy. Synovial tissue lipid peroxidation (4-HNE), T and B cell specific markers and synovial vascular endothelial growth factor (VEGF) were quantified by immunohistochemistry. Synovial levels of random mitochondrial DNA (mtDNA) mutations were assessed using Random Mutation Capture (RMC) assay.Results4-HNE levels pre/post anti TNF-α therapy were inversely correlated with in vivo tpO2 (P < 0.008; r = -0.60). Biologic therapy responders showed a significantly reduced 4-HNE expression (P < 0.05). High 4-HNE expression correlated with high DAS28-CRP (P = 0.02; r = 0.53), tender joint count for 28 joints (TJC-28) (P = 0.03; r = 0.49), swollen joint count for 28 joints (SJC-28) (P = 0.03; r = 0.50) and visual analogue scale (VAS) (P = 0.04; r = 0.48). Strong positive association was found between the number of 4-HNE positive cells and CD4+ cells (P = 0.04; r = 0.60), CD8+ cells (P = 0.001; r = 0.70), CD20+ cells (P = 0.04; r = 0.68), CD68+ cells (P = 0.04; r = 0.47) and synovial VEGF expression (P = 0.01; r = 063). In patients whose in vivo tpO2 levels improved post treatment, significant reduction in mtDNA mutations and DAS28-CRP was observed (P < 0.05). In contrast in those patients whose tpO2 levels remained the same or reduced at T3, no significant changes for mtDNA mutations and DAS28-CRP were found.ConclusionsHigh levels of synovial oxidative stress and mitochondrial mutation burden are strongly associated with low in vivo oxygen tension and synovial inflammation. Furthermore these significant mitochondrial genome alterations are rescued following successful anti TNF-α treatment.

Targeting of proangiogenic signalling pathways in chronic inflammation

Angiogenesis is de novo capillary outgrowth from pre-existing blood vessels. This process not only is crucial for normal development, but also has an important role in supplying oxygen and nutrients to inflamed tissues, as well as in facilitating the migration of inflammatory cells to the synovium in rheumatoid arthritis, spondyloarthritis and other systemic autoimmune diseases. Neovascularization is dependent on the balance of proangiogenic and antiangiogenic mediators, including growth factors, cytokines, chemokines, cell adhesion molecules and matrix metalloproteinases. This Review describes the various intracellular signalling pathways that govern these angiogenic processes and discusses potential approaches to interfere with pathological angiogenesis, and thereby ameliorate inflammatory disease, by targeting these pathways.

Redox‐Mediated Angiogenesis in the Hypoxic Joint of Inflammatory Arthritis

Inflammatory arthritis is associated with joint inflammation, synovial tissue proliferation, and degradation of articular cartilage and bone. Angiogenesis is an early and fundamental component of synovial inflammation. Oxygen metabolism is recognized as an important mediator of joint vascular remodeling. The aim of this study was to determine whether in vivo synovial hypoxia (tissue PO2 [tPO2]) and tumor necrosis factor (TNF) blocking therapy alter synovial vascular expression of NADPH oxidase (NOX) and how this action regulates angiogenic mechanisms.

Polymorphism of clotting factors in Hungarian patients with Raynaud's phenomenon

Patients with primary Raynauds phenomenon may have a genetically determined risk for clotting factors that predispose them to aberrant microvascular thrombosis. We investigated the prevalence of factor V substitution of G to A at position 1691 (FVLeiden), prothrombin G20210A, and methyltetrahydrofolate reductase C677T mutations in these patients. Two hundred (158 women, 42 men, mean age of 42.4 ± 13.7 years) consecutive patients with primary Raynauds phenomenon and 200 age-sex-matched healthy controls of Hungarian origin were included in a case–control study. The prevalence of methyltetrahydrofolate reductase C677T homozygous among patients was significantly lower than in the control group (odds ratio 0.4, 95% confidence interval 0.2–0.9, P < 0.05). The prevalence of other thrombosis-associated alleles did not differ between patients with primary Raynauds phenomenon and control subjects. FVLeiden, prothrombin G20210A, and polymorphism, prothrombin G20210A mutations have no apparent effect on the etiology of primary Raynauds phenomenon.

[Cardiovascular actions of a standardized polyphenol concentrate on patients undergoing coronary bypass grafting: a randomized, double-blind, placebo-controlled study].

In this study the authors analyzed the action of Flavon Max product on the cardiovascular system of patients with severe coronary disease. Two randomized, double-blind, placebo controlled trials were carried out using impedance-cardiography, arteriography, vascular Doppler and biochemical laboratory methods. The results demonstrate that Augmentation Index measured with arteriography and C reactive protein (CRP) levels were significantly ameliorated after 2 x 2 months Flavon Max therapy. In conclusion, this product is beneficial as adjuvant in the treatment of atherosclerotic coronary disease.

Oxidative stress impairs energy metabolism in primary cells and synovial tissue of patients with rheumatoid arthritis

BackgroundIn this study, we examined the effect of oxidative stress on cellular energy metabolism and pro-angiogenic/pro-inflammatory mechanisms of primary rheumatoid arthritis synovial fibroblast cells (RASFC) and human umbilical vein endothelial cells (HUVEC).MethodsPrimary RASFC and HUVEC were cultured with the oxidative stress inducer 4-hydroxy-2-nonenal (4-HNE), and extracellular acidification rate, oxygen consumption rate, mitochondrial function and pro-angiogenic/pro-inflammatory mechanisms were assessed using the Seahorse analyser, complex I–V activity assays, random mutation mitochondrial capture assays, enzyme-linked immunosorbent assays and functional assays, including angiogenic tube formation, migration and invasion. Expression of angiogenic growth factors in synovial tissue (ST) was assessed by IHC in patients with rheumatoid arthritis (RA) undergoing arthroscopy before and after administration of tumour necrosis factor inhibitors (TNFi).ResultsIn RASFC and HUVEC, 4-HNE-induced oxidative stress reprogrammed energy metabolism by inhibiting mitochondrial basal, maximal and adenosine triphosphate-linked respiration and reserve capacity, coupled with the reduced enzymatic activity of oxidative phosphorylation complexes III and IV. In contrast, 4-HNE elevated basal glycolysis, glycolytic capacity and glycolytic reserve, paralleled by an increase in mitochondrial DNA mutations and reactive oxygen species. 4-HNE activated pro-angiogenic responses of RASFC, which subsequently altered HUVEC invasion and migration, angiogenic tube formation and the release of pro-angiogenic mediators. In vivo markers of angiogenesis (vascular endothelial growth factor, angiopoietin 2 [Ang2], tyrosine kinase receptor [Tie2]) were significantly associated with oxidative damage and oxygen metabolism in the inflamed synovium. Significant reduction in ST vascularity and Ang2/Tie2 expression was demonstrated in patients with RA before and after administration of TNFi.ConclusionsOxidative stress promotes metabolism in favour of glycolysis, an effect that may contribute to acceleration of inflammatory mechanisms and subsequent dysfunctional angiogenesis in RA.

04.20 Oxidative stress alters cellular bioenergetics in inflammatory arthritis

Background Hypoxia and oxidative stress are involved in mediating angiogenic processes in the inflamed synovial tissue. In this study we examine the expression of angiogenic markers in inflammatory arthritis and the effects of tumour necrosis factor alpha inhibitors (TNFi) on angiogenesis in relationship to synovial hypoxia in vivo and oxidative stress. In vitro oxidative stress-induced angiogenic responses are also investigated. Materials and methods 44 IA patients pre/post-TNFi underwent knee joint arthroscopy and in vivo oxygen levels (tpO2) were measured. Expression of angiogenic growth factors in paired serum, synovial fluid and synovial tissue was accessed by ELISA and immunohistochemistry. Pro-angiogenic processes, mitochondrial function and metabolism were assessed in RA Synovial Fibroblast Cells (RASFCs) and Human Microvascular Endothelial Cells (HMVECs) under normoxia or 3% hypoxia in the presence of oxidative stress inducer - 4-HNE using the tube formation and invasion assays and the XFe Extracellular Flux Seahorse Analyzer. Results VEGF and Ang2 were expressed in the joint synovial fluid and Ang1 and PDGF-B were expressed at a systemic level. The highest synovial tissue (ST) VEGF, Ang2 and Tie2 positivity was observed in vascular region compared to lining and sublining layers (all p<0.05). Significant reduction in synovial vascularity and ST Ang2/Tie2 expression, paralleled by an increase in in vivo tpO2 was found after TNFi (all p<0.05). ST oxidative stress expression (8-oxo-dG and 4-HNE) was colocalized with VEGF, Ang2 and Tie2 and showed an association pre/post anti TNF-α therapy. In vitro 4-HNE promoted RASFCs secretion of pro-angiogenic mediators, ROS production, and cell proliferation (all p<0.05). In HMVECs 4-HNE elevated angiogenic tube-formation and cell invasiveness (all p<0.05), effects that were further potentiated by 3% hypoxia. Furthermore, 4-HNE significantly increases glycolytic activity of RASFC and HMVECs with concomitant attenuation of mitochondrial respiration. Conclusions Differential local and systemic expression of angiogenic factors may indicate a constant remodelling of synovial vasculature. Reduced levels of Ang2 and Tie2 following TNFi demonstrate a relationship between angiogenesis, hypoxia and TNFα pathway. Redox signalling can alter angiogenic responses of synovial cells. Oxidative stress alters cellular bioenergetics by promoting switch to glycolysis mitochondrial respiration supporting cellular invasion and dysregulated angiogenesis.

A6.12 Effects of anti-tnf therapy on markers of angiogenesis and vascular pathology in arthritis: a comparative approach

Background and objectives Accelerated atherosclerosis, increased cardiovascular (CV) morbidity and mortality, as well as the perpetuation of angiogenesis and abundant production of angiogenic factors have been associated with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Biologics may influence both vascular function and angiogenesis. However, the effects of targeted therapies on vascular function and angiogenesis have been poorly studied in a comparative manner. Therefore, vascular function, markers of atherosclerosis and angiogenesis, as well as the effects of anti-TNF therapy on these biomarkers were assessed in the very same arthritis patient cohort. Patients and methods Altogether 31 arthritis patients including 18 RA patients treated with either etanercept (ETN) or certolizumab pegol (CZP) and 13 AS patients treated with ETN were included in a 12-month follow-up study. Ultrasonography was performed to determine flow-mediated vasodilation (FMD), a marker of endothelial dysfunction; common carotid intima-media thickness (ccIMT), a marker of atherosclerosis and pulse-wave velocity (PWV), an arterial stiffness parameter in all patients. Furthermore, circulating markers of angiogenesis including vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), angiopoetin 1 and 2(Ang1, Ang2) and thrombospondin 1 (TSP-1) were assessed in the sera by ELISA. DAS28, BASDAI and CRP, markers of disease activity, were also determined. All assessments were performed at baseline, as well as 6 and 12 months after treatment initiation. Results Anti-TNF treatment was highly effective in both diseases, as the mean DAS28 decreased from 5.00 to 2.97 (p < 0.001) in RA, mean BASDAI decreased from 5.99 to 1.82 (p < 0.001) in AS and CRP decreased from 13.8 to 3.9 mg/l (p = 0.021) in RA+AS over a 12-month period. Anti-TNF treatment resulted in significant improvement in FMD (from 7.15% to 9.11%; p = 0.009) and a tendency of improvement in PWV (from 7.57 to 6.82 m/sec; p = 0.190). Among markers of angiogenesis, mean VEGF (from 268.3 to 222.6 pg/ml; p = 0.006) and PDGF-BB (from 8187 to 6020 pg/ml; p = 0.012) levels significantly decreased after 12 months of therapy. Moreover, PDGF levels after 12 months, as well as Ang2 levels at all time points correlated with disease duration (p < 0.05) and Ang1, Ang2 and TSP1 levels all correlated with CRP at baseline (p < 0.05). When markers of vascular function and angiogenesis were compared, baseline PDGF levels correlated with baseline ccIMT (p < 0.05). Conclusions In a mixed cohort of RA and AS patients, anti-TNF therapy improved endothelial function and decreased the circulating levels of some angiogenic markers. Both impaired vascular function and the perpetuation of angiogenesis may be due to active systemic inflammation associated with these arthritides.