Monika Biniecka

Synovial tissue hypoxia and inflammation in vivo

Introduction Hypoxia is a microenvironmental feature in the inflamed joint, which promotes survival advantage for cells. The aim of this study was to examine the relationship of partial oxygen pressure in the synovial tissue (tPO2) in patients with inflammatory arthritis with macroscopic/microscopic inflammation and local levels of proinflammatory mediators. Methods Patients with inflammatory arthritis underwent full clinical assessment and video arthroscopy to quantify macroscopic synovitis and measure synovial tPO2 under direct visualisation. Cell specific markers (CD3 (T cells), CD68 (macrophages), Ki67 (cell proliferation) and terminal deoxynucleotidyl transferase dUTP nick end labelling (cell apoptosis)) were quantified by immunohistology. In vitro migration was assessed in primary and normal synoviocytes (synovial fibroblast cells (SFCs)) using a wound repair scratch assay. Levels of tumour necrosis factor α (TNFα), interleukin 1β (IL1β), interferon γ (IFNγ), IL6, macrophage inflammatory protein 3α (MIP3α) and IL8 were quantified, in matched serum and synovial fluid, by multiplex cytokine assay and ELISA. Results The tPO2 was 22.5 (range 3.2–54.1) mm Hg and correlated inversely with macroscopic synovitis (r=−0.421, p=0.02), sublining CD3 cells (−0.611, p<0.01) and sublining CD68 cells (r=−0.615, p<0.001). No relationship with cell proliferation or apoptosis was found. Primary and normal SFCs exposed to 1% and 3% oxygen (reflecting the median tPO2 in vivo) induced cell migration. This was coupled with significantly higher levels of synovial fluid tumour necrosis factor α (TNFα), IL1β, IFNγ and MIP3α in patients with tPO2 <20 mm Hg (all p values <0.05). Conclusions This is the first study to show a direct in vivo correlation between synovial tPO2, inflammation and cell migration, thus it is proposed that hypoxia is a possible primary driver of inflammatory processes in the arthritic joint.

Oxidative damage in synovial tissue is associated with in vivo hypoxic status in the arthritic joint

Objectives To assess levels of oxidative DNA damage (8-oxo-7,8-dihydro-2′-deoxyguanine; 8-oxo-dG) and lipid peroxidation (4-hydroxy-2-nonenal; 4-HNE) in serum, synovial fluid and tissue of patients with inflammatory arthritis in relation to in vivo hypoxia levels, disease activity and angiogenic markers. Methods Oxygen levels in synovial tissue were assessed using an oxygen/temperature probe. Nuclear and cytoplasmic 8-oxo-dG and 4-HNE levels were assessed in synovial tissue from 23 patients by immunohistochemistry. 8-Oxo-dG and 4-HNE levels in serum and synovial fluid were determined using 8-oxo-dG and hexanoyl-Lys (HEL) adduct ELISAs, respectively. Serum vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) levels were also measured by ELISA. Results The median oxygen tension in synovial tissue was profoundly hypoxic at 19.35 mm Hg (2.5%). Nuclear 8-oxo-dG levels were significantly higher than nuclear 4-HNE levels in the lining and sublining layers (all p<0.001). In contrast, cytoplasmic 4-HNE levels were higher than cytoplasmic 8-oxo-dG levels in both cell layers (all p<0.001). Reduced in vivo oxygen tension correlated with high lipid peroxidation in synovial fluid (p=0.027; r=0.54) and tissue (p=0.004; r=0.58). Serum VEGF levels were positively correlated with cytoplasmic 4-HNE expression (p=0.05; r=0.43) and intensity (p=0.006; r=0.59) in the lining layer. Serum Ang2 levels were positively correlated with nuclear 4-HNE expression and intensity in both cell layers (all p≤0.05). DAS28-C-reactive protein was correlated with nuclear 4-HNE expression in the sublining layer (p=0.02; r=0.48) and DAS28-erythrocyte sedimentation rate was correlated with nuclear 4-HNE expression in both cell layers (p≤0.03). Conclusions Lipid peroxidation is associated with low oxygen tension in vivo, disease activity and angiogenic marker expression in inflammatory arthritis.

Dysregulated bioenergetics: a key regulator of joint inflammation

Objectives This study examines the relationship between synovial hypoxia and cellular bioenergetics with synovial inflammation. Methods Primary rheumatoid arthritis synovial fibroblasts (RASF) were cultured with hypoxia, dimethyloxalylglycine (DMOG) or metabolic intermediates. Mitochondrial respiration, mitochondrial DNA mutations, cell invasion, cytokines, glucose and lactate were quantified using specific functional assays. RASF metabolism was assessed by the XF24-Flux Analyzer. Mitochondrial structural morphology was assessed by transmission electron microscopy (TEM). In vivo synovial tissue oxygen (tpO2 mmHg) was measured in patients with inflammatory arthritis (n=42) at arthroscopy, and markers of glycolysis/oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PKM2, GLUT1, ATP) were quantified by immunohistology. A subgroup of patients underwent contiguous MRI and positron emission tomography (PET)/CT imaging. RASF and human dermal microvascular endothelial cells (HMVEC) migration/angiogenesis, transcriptional activation (HIF1α, pSTAT3, Notch1-IC) and cytokines were examined in the presence of glycolytic inhibitor 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). Results DMOG significantly increased mtDNA mutations, mitochondrial membrane potential, mitochondrial mass, reactive oxygen species and glycolytic RASF activity with concomitant attenuation of mitochondrial respiration and ATP activity (all p<0.01). This was coupled with altered mitochondrial morphology. Hypoxia-induced lactate levels (p<0.01), which in turn induced basic fibroblast growth factor (bFGF) secretion and RASF invasiveness (all p<0.05). In vivo glycolytic markers were inversely associated with synovial tpO2 levels <20 mm Hg, in contrast ATP was significantly reduced (all p<0.05). Decrease in GAPDH and GLUT1 was paralleled by an increase in in vivo tpO2 in tumour necrosis factor alpha inhibitor (TNFi) responders. Novel PET/MRI hybrid imaging demonstrated close association between metabolic activity and inflammation. 3PO significantly inhibited RASF invasion/migration, angiogenic tube formation, secretion of proinflammatory mediators (all p<0.05), and activation of HIF1α, pSTAT3 and Notch-1IC under normoxic and hypoxic conditions. Conclusions Hypoxia alters cellular bioenergetics by inducing mitochondrial dysfunction and promoting a switch to glycolysis, supporting abnormal angiogenesis, cellular invasion and pannus formation.

Mitochondrial mutagenesis correlates with the local inflammatory environment in arthritis

Background To examine the association between mitochondrial mutagenesis and the proinflammatory microenvironment in patients with inflammatory arthritis. Methods Fifty patients with inflammatory arthritis underwent arthroscopy and synovial tissue biopsies, synovial fluid and clinical assessment were obtained. Fifteen patients pre/post-TNFi therapy were also recruited. Normal synovial biopsies were obtained from 10 subjects undergoing interventional arthroscopy. Macroscopic synovitis/vascularity was measured by visual analogue scale. Cell-specific markers CD3 (T cells) and CD68 (macrophages) were quantified by immunohistology. TNFα, IL-6, IFNγ and IL-1β were measured in synovial fluids by MSD multiplex assays. Synovial tissue mitochondrial mutagenesis was quantified using a mitochondrial random mutation capture assay (RMCA). The direct effect of TNFα on oxidative stress and mitochondrial function was assessed in primary cultures of rheumatoid arthritis synovial fibroblast cells (RASFCs). Mitochondrial mutagenesis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and mitochondrial mass (MM) were quantified using the RMCA and specific cell fluorescent probes. Results A significant increase in mtDNA mutation frequency was demonstrated in inflamed synovial tissue compared with control (p<0.05), an effect that was independent of age. mtDNA mutations positively correlated with macroscopic synovitis (r=0.52, p<0.016), vascularity (r=0.54, p<0.01) and with synovial fluid cytokine levels of TNFα (r=0.74, p<0.024) and IFNγ (r=0.72, p<0.039). mtDNA mutation frequency post-TNFi therapy was significantly lower in patients with a DAS<3.2 (p<0.05) and associated with clinical and microscopic measures of disease (p<0.05). In vitro TNFα significantly induced mtDNA mutations, ROS, MM and MMP in RASFCs (all p<0.05). Conclusion High mitochondrial mutations are strongly associated with synovial inflammation showing a direct link between mitochondrial mutations and key proinflammatory pathways.

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.

Tumor necrosis factor blocking therapy alters joint inflammation and hypoxia.

OBJECTIVE To examine the effect of tumor necrosis factor (TNF) blocking therapy on hypoxia in vivo, macroscopic and microscopic inflammation, and magnetic resonance imaging (MRI) results in patients with inflammatory arthritis. METHODS Patients with inflammatory arthritis (n = 20) underwent full clinical assessment, arthroscopy, synovial biopsy, and MRI before and after initiation of biologic therapy. Macroscopic synovitis/vascularity was assessed with a visual analog scale, and tissue PO(2) (tPO(2) ) was measured at arthroscopy using a Licox probe. Cell-specific markers (CD4, CD8, CD68, CD20, and CD19) and blood vessel maturity were quantified by immunohistologic analysis and dual-immunofluorescence factor VIII/α-smooth muscle actin staining, respectively. Contiguous gadoteric acid-enhanced MRI of the target knee was used to assess synovial enhancement. RESULTS Biologic therapy responders showed a significant increase of tPO(2) in vivo (P < 0.05). This response was associated with significant reductions in 28-joint Disease Activity Score using the C-reactive protein level (DAS28-CRP) (P = 0.012), macroscopic synovitis (P = 0.017), macroscopic vascularity (P = 0.05), CD4+ T cells (P < 0.041), and CD68+ macrophages (P < 0.011). Blood vessel numbers were also reduced in responders; however, this did not reach statistical significance. Strong inverse correlations were demonstrated between changes in tPo(2) levels and changes in DAS28-CRP (r = -0.53, P < 0.001), CD4 (r = -0.44, P < 0.026), CD68 (r = -0.46, P < 0.003), and macroscopic vascularity (r = -0.314, P = 0.049) after therapy. Furthermore, changes in inflammation as measured by MRI showed a strong inverse correlation with tPO(2) levels (r = -0.688, P < 0.002) and positive correlations with CRP levels (r = 0.707, P = 0.001), macroscopic synovitis (r = 0.457, P = 0.056), macroscopic vascularity (r = 0.528, P= 0.017), CD4 (r = 0.553, P < 0.032), and CD68 (r = 0.670, P < 0.002) after therapy. CONCLUSION This is the first study to show that successful biologic therapy significantly improves in vivo synovial hypoxia. Changes are strongly associated with changes in macroscopic and microscopic measures of joint inflammation and MRI improvement. These data further strengthen the concept that hypoxia is an important event driving synovial inflammation.

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.

Resolution of TLR2-induced inflammation through manipulation of metabolic pathways in Rheumatoid Arthritis

During inflammation, immune cells activated by toll-like receptors (TLRs) have the ability to undergo a bioenergetic switch towards glycolysis in a manner similar to that observed in tumour cells. While TLRs have been implicated in the pathogenesis of rheumatoid arthritis (RA), their role in regulating cellular metabolism in synovial cells, however, is still unknown. In this study, we investigated the effect of TLR2-activation on mitochondrial function and bioenergetics in primary RA-synovial fibroblast cells (RASFC), and further determined the role of glycolytic blockade on TLR2-induced inflammation in RASFC using glycolytic inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). We observed an increase in mitochondrial mutations, ROS and lipid peroxidation, paralleled by a decrease in the mitochondrial membrane potential in TLR2-stimulated RASFC. This was mirrored by differential regulation of key mitochondrial genes, coupled with alteration in mitochondrial morphology. TLR2-activation also regulated changes in the bioenergetic profile of RASFC, inducing PKM2 nuclear translocation, decreased mitochondrial respiration and ATP synthesis and increased glycolysis:respiration ratio, suggesting a metabolic switch. Finally, using 3PO, we demonstrated that glycolytic blockade reversed TLR2-induced pro-inflammatory mechanisms including invasion, migration, cytokine/chemokine secretion and signalling pathways. These findings support the concept of complex interplay between innate immunity, oxidative damage and oxygen metabolism in RA pathogenesis.

Inhibition of Dendritic Cell Maturation by the Tumor Microenvironment Correlates with the Survival of Colorectal Cancer Patients following Bevacizumab Treatment

Development of bevacizumab has improved survival in colorectal cancer, however, currently there are no biomarkers that predict response to bevacizumab and it is unknown how it influences the immune system in colorectal cancer patients. Dendritic cells are important for the induction of an antitumor immune response; however tumors are capable of disabling dendritic cells and escaping immune surveillance. The aim of this study was to assess the numbers of CD11c+ cells infiltrating tumor tissue and to examine the effects of tumor conditioned media (TCM) and bevacizumab conditioned media (BCM) on dendritic cell maturation and correlate our findings with patient survival. colorectal cancer explant tissues were cultured with or without bevacizumab, to generate BCM and TCM, which were used to treat dendritic cells. CD80, CD86, CD83, CD54, HLA-DR, and CD1d expression was measured by flow cytometry. Interleukin (IL)-10 and IL-12p70 were measured by ELISA. The Cox proportional hazards model was used to associate survival with dendritic cell inhibition. TCM and BCM inhibited lipopolysaccharide (LPS)-induced dendritic cell maturation and IL-12p70 secretion (P < 0.0001), while increasing IL-10 secretion (P = 0.0033 and 0.0220, respectively). Inhibition of LPS-induced CD1d (P = 0.021, HR = 1.096) and CD83 (P = 0.017, HR = 1.083) by TCM and inhibition of CD1d (P = 0.017, HR = 1.067), CD83 (P = 0.032, HR = 1.035), and IL-12p70 (P = 0.037, HR = 1.036) by BCM was associated with poor survival in colorectal cancer patients. CD11c expression was elevated in tumor tissue compared with normal tissue (P < 0.001), but this did not correlate with survival. In conclusion, TCM and BCM inhibit dendritic cells, and this inhibition correlates with survival of colorectal cancer patients receiving bevacizumab. Mol Cancer Ther; 11(8); 1829–37. ©2012 AACR.

Notch-1 mediates endothelial cell activation and invasion in psoriasis

Notch receptor–ligand interactions are critical for cell proliferation, differentiation and survival; however, the role of Notch signalling in psoriasis remains to be elucidated. Serum amyloid A (A‐SAA) is an acute‐phase protein with cytokine‐like properties, regulates cell survival pathways and is implicated in many inflammatory conditions. To examine the role of Notch‐1 signalling in the pathogenesis of psoriasis, Notch‐1, DLL‐4, Jagged‐1, Hrt‐1/Hrt‐2, A‐SAA, Factor VIII and vascular endothelial growth factor (VEGF) mRNA and/or protein expression in psoriasis skin biopsies, serum and dHMVEC were assessed by immunohistology, dual‐immunofluorescence, real‐time PCR, ELISA and Western blotting. A‐SAA‐induced angiogenesis and invasion in the presence of Notch‐1 siRNA was assessed by matrigel tube formation assays and Transwell invasion assay. Increased Notch‐1, its ligand DLL‐4 and Hrt‐1 expression were demonstrated in lesional skin compared with non‐lesional skin, with greatest expression observed in the dermal vasculature (P < 0.05). Dual‐immunofluorescent staining demonstrated co‐localization of Notch‐1 to endothelial cell marker Factor VIII. A significant increase in A‐SAA levels was demonstrated in psoriasis serum compared with healthy control serum (P < 0.05), and A‐SAA expression was higher in lesional skin compared with non‐lesional. In dHMVEC, A‐SAA significantly induced Jagged‐1, Hrt‐1 and VEGF mRNA expression (P < 0.05) and activated Notch‐1 IC indicative of transcriptional regulation. In contrast, A‐SAA significantly inhibited DLL‐4 mRNA expression (P < 0.05). Finally A‐SAA‐induced angiogenesis and invasion were inhibited by Notch‐1 siRNA (P < 0.05). Notch receptor–ligand interactions mediate vascular dysfunction in psoriasis and may represent a potential therapeutic target.