Notch-1 mediates hypoxia-induced angiogenesis in rheumatoid arthritis.

OBJECTIVE: To examine the effect of hypoxia on Notch-1 signaling pathway components and angiogenesis in inflammatory arthritis. METHODS: The expression and regulation of Notch-1, its ligand delta-like protein 4 (DLL-4) and downstream signaling components (hairy-related transcription factor 1 [HRT-1], HRT-2), and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions (1-3%) were assessed in synovial tissue specimens from patients with inflammatory arthritis and controls and in human dermal microvascular endothelial cells (HDMECs) by immunohistology, dual immunofluorescence staining (Notch-1/factor VIII), Western blotting, and real-time polymerase chain reaction. In vivo synovial tissue oxygen levels (tissue PO2) were measured under direct visualization at arthroscopy. HDMEC activation under hypoxic conditions in the presence of Notch-1 small interfering RNA (siRNA), the γ-secretase inhibitor DAPT, or dimethyloxalylglycine (DMOG) was assessed by Matrigel tube formation assay, migration assay, invasion assay, and matrix metalloproteinase 2 (MMP-2)/MMP-9 zymography. RESULTS: Expression of Notch-1, its ligand DLL-4, and HRT-1 was demonstrated in synovial tissue, with the strongest expression localized to perivascular/vascular regions. Localization of Notch-1 to synovial endothelium was confirmed by dual immunofluorescence staining. Notch-1 intracellular domain (NICD) expression was significantly higher in synovial tissue from patients with tissue PO2 of <20 mm Hg (<3% O2) than in those with tissue PO2 of >20 mm Hg (>3% O2). Exposure of HDMECs to 3% hypoxia induced HIF-1α and NICD protein expression and DLL-4, HRT-1, and HRT-2 messenger RNA expression. DMOG directly induced NICD expression, while Notch-1 siRNA inhibited hypoxia-induced HIF-1α expression, suggesting that Notch-1/HIF-1α signaling is bidirectional. Finally, 3% hypoxia-induced angiogenesis, endothelial cell migration, endothelial cell invasion, and proMMP-2 and proMMP-9 activities were inhibited by Notch-1 siRNA and/or the γ-secretase inhibitor DAPT. CONCLUSION: Our findings indicate that Notch-1 is expressed in synovial tissue and that increased NICD expression is associated with low in vivo tissue PO2. Furthermore, Notch-1/HIF-1α interactions mediate hypoxia-induced angiogenesis and invasion in inflammatory arthritis.

Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy.

OBJECTIVE: To investigate the relationship between acute-phase serum amyloid A (A-SAA) and joint destruction in inflammatory arthritis. METHODS: Serum A-SAA and C-reactive protein (CRP) levels, the erythrocyte sedimentation rate (ESR), and levels of matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-9, MMP-13, tissue inhibitor of metalloproteinases 1 (TIMP-1), vascular endothelial growth factor (VEGF), and type I and type II collagen-generated biomarkers C2C and C1,2C were measured at 0-3 months in patients with inflammatory arthritis commencing anti-tumor necrosis factor α (anti-TNFα) therapy and were correlated with 1-year radiographic progression. The effects of A-SAA on MMP/TIMP expression on RA fibroblast-like synoviocytes (FLS), primary human chondrocytes, and RA/psoriatic arthritis synovial explant cultures were assessed using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, antibody protein arrays, and gelatin zymography. RESULTS: Serum A-SAA levels were significantly (P < 0.05) correlated with MMP-3, the MMP-3:TIMP-1 ratio, C1,2C, C2C, and VEGF. The baseline A-SAA level but not the ESR or the CRP level correlated with the 28-joint swollen joint count and was independently associated with 1-year radiographic progression (P = 0.038). A-SAA increased MMP-1, MMP-3, MMP-13, and MMP/TIMP expression in RA FLS and synovial explants (P < 0.05). In chondrocytes, A-SAA induced MMP-1, MMP-3, and MMP-13 messenger RNA and protein expression (all P < 0.01), resulting in a significant shift in MMP:TIMP ratios (P < 0.05). Gelatin zymography revealed that A-SAA induced MMP-2 and MMP-9 activity. Blockade of the A-SAA receptor SR-B1 (A-SAA receptor scavenger receptor-class B type 1) inhibited MMP-3, MMP-2, and MMP-9 expression in synovial explant cultures ex vivo. Importantly, we demonstrated that A-SAA has the ability to induce TNFα expression in RA synovial explant cultures (P < 0.05). CONCLUSION: A-SAA may be involved in joint destruction though MMP induction and collagen cleavage in vivo. The ability of A-SAA to regulate TNFα suggests that A-SAA signaling pathways may provide new therapeutic strategies for the treatment of inflammatory arthritis.

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

INTRODUCTION: To examine the effects of tumour necrosis factor (TNF) blocking therapy on the levels of early mitochondrial genome alterations and oxidative stress. METHODS: Eighteen inflammatory arthritis patients underwent synovial tissue oxygen (tpO(2)) 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. RESULTS: 4-HNE levels pre/post anti TNF-α therapy were inversely correlated with in vivo tpO(2) (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 tpO(2) 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. CONCLUSIONS: High 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.

Macrophages in synovial inflammation.

Synovial macrophages are one of the resident cell types in synovial tissue and while they remain relatively quiescent in the healthy joint, they become activated in the inflamed joint and, along with infiltrating monocytes/macrophages, regulate secretion of pro-inflammatory cytokines and enzymes involved in driving the inflammatory response and joint destruction. Synovial macrophages are positioned throughout the sub-lining layer and lining layer at the cartilage-pannus junction and mediate articular destruction. Sub-lining macrophages are now also considered as the most reliable biomarker for disease severity and response to therapy in rheumatoid arthritis (RA). There is a growing understanding of the molecular drivers of inflammation and an appreciation that the resolution of inflammation is an active process rather than a passive return to homeostasis, and this has implications for our understanding of the role of macrophages in inflammation. Macrophage phenotype determines the cytokine secretion profile and tissue destruction capabilities of these cells. Whereas inflammatory synovial macrophages have not yet been classified into one phenotype or another it is widely known that TNFα and IL-l, characteristically released by M1 macrophages, are abundant in RA while IL-10 activity, characteristic of M2 macrophages, is somewhat diminished. Here we will briefly review our current understanding of macrophages and macrophage polarization in RA as well as the elements implicated in controlling polarization, such as cytokines and transcription factors like NFκB, IRFs and NR4A, and pro-resolving factors, such as LXA4 and other lipid mediators which may promote a non-inflammatory, pro-resolving phenotype, and may represent a novel therapeutic paradigm.

Angiogenesis and blood vessel stability in inflammatory arthritis.

OBJECTIVE: To assess blood vessel stability in inflammatory synovial tissue (ST) and to examine neural cell adhesion molecule (NCAM), oxidative DNA damage, and hypoxia in vivo. METHODS: Macroscopic vascularity and ST oxygen levels were determined in vivo in patients with inflammatory arthritis who were undergoing arthroscopy. Vessel maturity/stability was quantified in matched ST samples by dual immunofluorescence staining for factor VIII (FVIII)/alpha-smooth muscle actin (alpha-SMA). NCAM and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were examined by immunohistochemistry. Angiogenesis was assessed in vitro, using human dermal endothelial cells (HDECs) in a Matrigel tube formation assay. RESULTS: A significant number of immature vessels (showing no pericyte recruitment) was observed in tissue from patients with inflammatory arthritis (P < 0.001), in contrast to osteoarthritic and normal tissue, which showed complete recruitment of pericytes. Low in vivo PO(2) levels in the inflamed joint (median [range] 22.8 [3.2-54.1] mm Hg) were inversely related to increased macroscopic vascularity (P < 0.04) and increased microscopic expression of FVIII and alpha-SMA (P < 0.04 and P < 0.03, respectively). A significant proportion of vessels showed focal expression of NCAM and strong nuclear 8-oxodG expression, implicating a loss of EC-pericyte contact and increased DNA damage, levels of which were inversely associated with low in vivo PO(2) (P = 0.04 for each comparison). Circulating cells were completely negative for 8-oxodG. Exposure of HDEC to 3% O(2) (reflecting mean ST in vivo measurements) significantly increased EC tube formation (P < 0.05). CONCLUSION: Our findings indicate the presence of unstable vessels in inflamed joints associated with hypoxia, incomplete EC-pericyte interactions, and increased DNA damage. These changes may further contribute to persistent hypoxia in the inflamed joint to further drive this unstable microenvironment.

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 < or = 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 < or = 0.03). CONCLUSIONS: Lipid peroxidation is associated with low oxygen tension in vivo, disease activity and angiogenic marker expression in inflammatory arthritis.