Novel tempeh (fermented soyabean) isoflavones inhibit in vivo angiogenesis in the chicken chorioallantoic membrane assay

Anti-angiogenic strategies are emerging as an important tool for the treatment of cancer and inflammatory diseases. In the present investigation we isolated several isoflavones from a tempeh (fermented soyabean) extract. The isolated isoflavones were identified as 5,7,4′-trihydroxyisoflavone (genistein), 7,4′-dihydroxyisoflavone (daidzein), 6,7,4′-trihydroxyisoflavone (factor 2), 7,8,4′-trihydroxyisoflavone (7,8,4′-TriOH) and 5,7,3′,4′-tetrahydroxyisoflavone (orobol). The effects on angiogenesis of these isoflavones were evaluated in the chicken chorioallantoic membrane assay; their capacity to inhibit vascular endothelial growth factor-induced endothelial cell proliferation and expression of the Ets 1 transcription factor, known to be implicated in the regulation of new blood vessel formation, were also investigated. We found that all isoflavones inhibited angiogenesis, albeit with different potencies. Compared with negative controls, which slightly inhibited in vivo angiogenesis by 6·30 %, genistein reduced angiogensis by 75·09 %, followed by orobol (67·96 %), factor 2 (56·77 %), daidzein (48·98 %) and 7,8,4′-TriOH (24·42 %). These compounds also inhibited endothelial cell proliferation, with orobol causing the greatest inhibition at lower concentrations. The isoflavones also inhibited Ets 1 expression, providing some insight into the molecular mechanisms of their action. Furthermore, the chemical structure of the different isoflavones suggests a structure-activity relationship. Our present findings suggest that the new isoflavones might be added to the list of low molecular mass therapeutic agents for the inhibition of angiogenesi

Alterations in the gut microbiome implicate key taxa and metabolic pathways across inflammatory arthritis phenotypes

Musculoskeletal diseases affect up to 20% of adults worldwide. The gut microbiome has been implicated in inflammatory conditions, but large-scale metagenomic evaluations have not yet traced the routes by which immunity in the gut affects inflammatory arthritis. To characterize the community structure and associated functional processes driving gut microbial involvement in arthritis, the Inflammatory Arthritis Microbiome Consortium investigated 440 stool shotgun metagenomes comprising 221 adults diagnosed with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and 219 healthy controls and individuals with joint pain without an underlying inflammatory cause. Diagnosis explained about 2% of gut taxonomic variability, which is comparable in magnitude to inflammatory bowel disease. We identified several candidate microbes with differential carriage patterns in patients with elevated blood markers for inflammation. Our results confirm and extend previous findings of increased carriage of typically oral and inflammatory taxa and decreased abundance and prevalence of typical gut clades, indicating that distal inflammatory conditions, as well as local conditions, correspond to alterations to the gut microbial composition. We identified several differentially encoded pathways in the gut microbiome of patients with inflammatory arthritis, including changes in vitamin B salvage and biosynthesis and enrichment of iron sequestration. Although several of these changes characteristic of inflammation could have causal roles, we hypothesize that they are mainly positive feedback responses to changes in host physiology and immune homeostasis. By connecting taxonomic alternations to functional alterations, this work expands our understanding of the shifts in the gut ecosystem that occur in response to systemic inflammation during arthritis

TLR expression profiles are a function of disease status in rheumatoid arthritis and experimental arthritis

The role of the innate immune system has been established in the initiation and perpetuation of inflammatory disease, but less attention has been paid to its role in the resolution of inflammation and return to homeostasis. Toll-like receptor (TLR) expression profiles were analysed in tissues with differing disease status in rheumatoid arthritis (RA), ankylosing spondylitis (AS), and in experimental arthritis. TLR gene expression was measured in whole blood and monocytes, before and after TNF blockade. In RA and osteoarthritis synovia, the expression of TLRs was quantified by standard curve qPCR. In addition, four distinct stages of disease were defined and validated in collagen-induced arthritis (CIA), the gold standard animal model for RA - pre-onset, early disease, late disease and immunised mice that were resistant to the development of disease. TLR expression was measured in spleens, lymph nodes, blood cells, liver and the paws (inflamed and unaffected). In RA whole blood, the expression of TLR1, 4 and 6 was significantly reduced by TNF blockade but the differences in TLR expression profiles between responders and non-responders were less pronounced than the differences between RA and AS patients. In RA non-responders, monocytes had greater TLR2 expression prior to therapy compared to responders. The expression of TLR1, 2, 4 and 8 was higher in RA synovium compared to control OA synovium. Circulating cytokine levels in CIA resistant mice were similar to naïve mice, but anti-collagen antibodies were similar to arthritic mice. Distinct profiles of inflammatory gene expression were mapped in paws and organs with differing disease status. TLR expression in arthritic paws tended to be similar in early and late disease, with TLR1 and 2 moderately higher in late disease. TLR expression in unaffected paws varied according to gene and disease status but was generally lower in resistant paws. Disease status-specific profiles of TLR expression were observed in spleens, lymph nodes, blood cells and the liver. Notably, TLR2 expression rose then fell in the transition from naïve to pre-onset to early arthritis. TLR gene expression profiles are strongly associated with disease status. In particular, increased expression in the blood precedes clinical manifestation

The hypoxia-inducible factor (HIF) pathway in varicose veins

Primary varicose vein wall weakness and dilatation are thought to be caused by various biochemical and structural changes. Hypoxia and mechanical stress have been postulated to contribute to primary varicose vein wall changes. Hypoxia-inducible factors (HIF) are nuclear transcriptional factors that regulate the transcription of genes of oxygen homoeostasis. The HIF pathway is regulated by factors including oxygen tension and mechanical stress. The study aimed to assess the expression of HIF and its target genes in varicose and non-varicose veins, and examine their regulation by hypoxia, mechanical stress and pharmacological agents. Structural variations between varicose and non-varicose veins were demonstrated using histological analysis with special stains. Increased mRNA and protein expression of HIF-1α, HIF-2α and their target genes was found in varicose compared to non-varicose veins. Immunohistochemistry demonstrated that HIF-1α was only expressed in some endothelial cells, whereas HIF-2α was more widely expressed in endothelial and smooth muscle cells of varicose and non-varicose veins. Furthermore, a vein organ culture model was prepared and validated. Exposure of varicose and non-varicose vein organ cultures to 16 hours of 1% oxygen or the hypoxia mimetic dimethyloxallyl glycine up-regulated the expression of HIF-1α, HIF-2α proteins, and their target genes. Micronised purified flavonoid fraction at a concentration corresponding to therapeutic dose appeared to reduce the increases in expression of HIF-1α, HIF-2α, and their target genes in varicose vein organ cultures exposed to hypoxia, although the reduction was not statistically significant. Meanwhile, doxycycline at a concentration corresponding to therapeutic dose did not alter the expression of HIF-1α, HIF-2α, and their target genes in varicose and non-varicose vein organ cultures exposed to hypoxia. In rat inferior vena cava model, prolonged increases in vein wall tension were associated with over expression of HIF-1α and HIF-2α. The up-regulation of HIF-1α and HIF-2α secondary to prolonged increases in vein wall tension was associated with elevated MMP-2 and MMP-9 expression and changes in venous tone. In conclusion, HIF-α and target genes expression is increased in varicose compared to non-varicose veins. The expression of HIF-α and target genes in venous tissues appeared to be regulated by hypoxia and mechanical stress. The data suggest that the HIF pathway may be an important regulator of various biochemical and structural changes in varicose vein wall.EThOS - Electronic Theses Online Servicesee acknowledgements page of thesisGBUnited Kingdo

Hypoxia-induced invadopodia formation:a role for β-PIX

During tumour progression, oxygen tension in the microenvironment surrounding tumour cells is reduced, resulting in hypoxia. It iswell established that cancer cells resist the negative effects of hypoxia by inducing angiogenesis predominantly via the activity of transcription factor hypoxia-inducible factor-1 (HIF-1). However, more recentlyHIF-1α has also been linked to increased invasive potential, although the molecular mechanisms remain to be defined. Invasive cancer cells are thought to employ membrane protrusions, termed invadopodia, to achieve matrix degradation. While many invadopodia components have been identified, signalling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. Indeed, the relationship between invadopodia formation and HIF-1α has not been explored. We now report that HIF-1α is a driver of invadopodia formation. Furthermore, we have identified an important, direct and novel link between the Rho family activator β-PIX, HIF-1α and invadopodia formation. Indeed, we find that β-PIX expression is essential for invadopodia formation. In conclusion, we identify a new HIF-1α mechanistic pathway and suggest that β-PIX is a novel downstream signalling mediator during invadopodia formation

The JAK inhibitor baricitinib inhibits Oncostatin M induction of proinflammatory mediators in ex-vivo synovial derived cells

Objectives: To investigate the ex vivo effect of the JAK1/2 inhibitor baricitinib on expression of pro-inflammatory mediators in rheumatoid arthritis (RA) fibroblast like synoviocytes (FLS) stimulated with TNFα, IL-1β and oncostatin M (OSM), and in RA synovial membrane cells (SMCs). Methods: RA and osteoarthritis (OA) SMCs, were isolated from arthroplasty specimens of RA (n=8) and OA (n=8) patients, respectively, using enzymatic digestion followed by cell propagation to obtain RA (n=5) and OA (n=3) FLS. Normal FLS and normal human foreskin fibroblasts (HSF) were purchased from commercial sources. Fibroblasts were stimulated with cytokines with or without baricitinib. RA SMCs were cultured in the presence of baricitinib without stimulation. JAK/STAT activation and levels of mRNA and proteins of the various inflammatory cytokines (IL-6, IL-8, MCP-1, RANTES and IP-10) were determined by qPCR, ELISA and MSD. Results: Baricitinib inhibited OSM-induced JAK signalling in RA synovial fibroblasts and effectively suppressed subsequent expression of the proinflammatory mediators IL-6, MCP-1 and IP-10. However, baricitinib was not effective in altering levels of spontaneously released TNFα, IL-6 and IL-8 in RA SMC. Although both TNFα and IL-1β signal independently of the JAK/STAT pathway, in HSF, but not in RA FLS, baricitinib significantly inhibited TNFα- and IL-1β-induced MCP-1 and IP-10 protein levels in a dose dependent manner. Furthermore, baricitinib did not inhibit TNFα- and IL-1β-induced expression of IL-6, IL-8 and MCP-1 in RA FLS. Conclusions: These findings are consistent with known signalling pathways employed by OSM, TNFα and IL-1β, but our data suggest that in HSF, baricitinib may have anti-inflammatory effects via downstream modulation of cytokines and chemokines produced in response to TNFα or IL-1β