Expression and regulation of HIF-1alpha in macrophages under inflammatory conditions; significant reduction of VEGF by CaMKII inhibitor

<p>Abstract</p> <p>Background</p> <p>Macrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue. Aim of the present study was to investigate intracellular signal transduction regulation of pro-inflammatory HIF-1 alpha expression in macrophages to identify possible new intervention strategies. We investigated the effects of CaMKII-inhibitors amongst other kinase inhibitors, on HIF-1 alpha expression and downstream production of pro-angiogenic factors in macrophages.</p> <p>Methods</p> <p>Differentiated THP-1 cells and synovial fluid (SF) macrophages were stimulated with 1 μg/ml LPS with or without pretreatment with specific inhibitors of the ERK pathway (PD98059), the PI3K pathway (LY294002), and the CaMKII pathway (KN93 and SMP-114). mRNA and protein expression of HIF-1 alpha, VEGF, MMP-9, and IL-8 was measured in cell lysates and cell supernatants.</p> <p>Results</p> <p>HIF-1 alpha protein expression in LPS-stimulated THP-1 macrophages could be blocked by ERK- and PI3K-inhibitors, but also by the CaMKII inhibitor KN93. THP-1 and SF macrophages produced high levels of VEGF, IL-8, and MMP-9, and VEGF protein production was significantly inhibited by PI3K-inhibitor, and by both CaMKII inhibitors. LPS stimulation in an hypoxic environment did not change VEGF levels, suggesting that LPS induced VEGF production in macrophages is more important than the hypoxic induction.</p> <p>Conclusions</p> <p>Expression of HIF-1 alpha and downstream effects in macrophages are regulated by ERK-, PI3K, but also by CaMKII pathways. Inhibition of HIF-1α protein expression and significant inhibition of VEGF production in macrophages was found using CaMKII inhibitors. This is an unknown but very interesting effect of the CaMKII inhibitor SMP-114, which has been in clinical trial as DMARD for the treatment of RA. This effect may contribute to the anti-arthritic effects of SMP-114.</p

Regional glutamine deficiency in tumours promotes dedifferentiation through inhibition of histone demethylation

Poorly organized tumour vasculature often results in areas of limited nutrient supply and hypoxia. Despite our understanding of solid tumour responses to hypoxia, how nutrient deprivation regionally affects tumour growth and therapeutic response is poorly understood. Here, we show that the core region of solid tumours displayed glutamine deficiency compared with other amino acids. Low glutamine in tumour core regions led to dramatic histone hypermethylation due to decreased α-ketoglutarate levels, a key cofactor for the Jumonji-domain-containing histone demethylases. Using patient-derived ^(V600E)BRAF melanoma cells, we found that low-glutamine-induced histone hypermethylation resulted in cancer cell dedifferentiation and resistance to BRAF inhibitor treatment, which was largely mediated by methylation on H3K27, as knockdown of the H3K27-specific demethylase KDM6B and the methyltransferase EZH2 respectively reproduced and attenuated the low-glutamine effects in vitro and in vivo. Thus, intratumoral regional variation in the nutritional microenvironment contributes to tumour heterogeneity and therapeutic response

The geographical distribution and first molecular analysis of Culicoides Latreille (Diptera: Ceratopogonidae) species in the Southern and Southeastern Turkey during the 2012 outbreak of bovine ephemeral fever

This study investigated the geographical distribution and molecular analysis of Culicoides species in the Southern and Southeastern Turkey during the 2012 outbreak of bovine ephemeral fever (BEF). The midge specimens caught by Onderstepoort-type light traps from livestock farms were tested for molecular evidence of existence of viral genome. Blood specimens were collected from clinically BEF-suspected acute febrile cattle. Total nucleic acid samples obtained from field specimens were checked against the BEF virus G gene and Culicoides internal transcribed spacer 1 (ITS-1) gene. A total of 20,845 Culicoides specimens (20,569 a (TM) Euroa (TM) Euro, 276 a (TM),a (TM),) comprising 11 species (Culicoides badooshensis, Culicoides circumscriptus, Culicoides gejgelensis, Culicoides imicola, Culicoides kibunensis, Culicoides longipennis, Culicoides newsteadi, Culicoides nubeculosus, Culicoides odiatus, Culicoides punctatus, Culicoides schultzei, Culicoides spp.) were collected. C. schultzei (18,032) was found as the dominant species and followed by C. imicola (1,857), C. nubeculosus complex (545), and C. circumscriptus (259), respectively. C. kibunensis was identified as new species for this region. PCR positivity of BEF was found 37.14 % (13/35) in blood samples whereas no viral genome was obtained from Culicoides specimens. Culicoides spp. ITS-1 gene sequences were analyzed phylogenetically with GenBank ITS-1 sequences. Molecular homology of Culicoides ITS-1 gene was ranged between 62.74 and 71.39 %. The results described first molecular detection and phylogenetic analysis of Culicoides ITS-1 gene with reference to the 2012 BEF outbreak in Turkey.Selcuk University Scientific Research CouncilSelcuk University [12401031]This research was partially funded by Selcuk University Scientific Research Council, res. no: 12401031. Researchers thank to Prof. Dr. Osman Erganis supply of BEFV as positive control

Hypoxia. The role of hypoxia and HIF-dependent signalling events in rheumatoid arthritis.

An adequate supply of oxygen and nutrients is essential for survival and metabolism of cells, and consequentially for normal homeostasis. Alterations in tissue oxygen tension have been postulated to contribute to a number of pathologies, including rheumatoid arthritis (RA), in which the characteristic synovial expansion is thought to outstrip the oxygen supply, leading to areas of synovial hypoxia and hypoperfusion. Indeed, the idea of a therapeutic modality aimed at 'starving' tissue of blood vessels was born from the concept that blood vessel formation (angiogenesis) is central to efficient delivery of oxygen to cells and tissues, and has underpinned the development of anti-angiogenic therapies for a range of cancers. An important and well characterized 'master regulator' of the adaptive response to alterations in oxygen tension is hypoxia-inducible factor (HIF), which is exquisitely sensitive to changes in oxygen tension. Activation of the HIF transcription factor signalling cascade leads to extensive changes in gene expression, which allow cells, tissues and organisms to adapt to reduced oxygenation. One of the best characterized hypoxia-responsive genes is the angiogenic stimulus vascular endothelial growth factor, expression of which is dramatically upregulated by hypoxia in many cells types, including RA synovial membrane cells. This leads to an apparent paradox, with the abundant synovial vasculature (which might be expected to restore oxygen levels to normal) occurring nonetheless together with regions of synovial hypoxia. It has been shown in a number of studies that vascular endothelial growth factor blockade is effective in animal models of arthritis; these findings suggest that hypoxia may activate the angiogenic cascade, thereby contributing to RA development. Recent data also suggest that, as well as activating angiogenesis, hypoxia may regulate many other features that are important in RA, such as cell trafficking and matrix degradation. An understanding of the biology of the HIF transcription family may eventually lead to the development of therapies that are aimed at interfering with this key signalling pathway, and hence to modulation of hypoxia-dependent pathologies such as RA

GC-MS analysis and antileishmanial activities of two Turkish propolis types

PubMedID: 20842509Propolis is a honeybee product with a very complex chemical composition and various pharmacological properties. This study was aimed to investigate antileishmanial activities of "Bursa" and "Hatay" propolis samples against Leishmania infantum and Leishmania tropica strains. Propolis samples were analysed with the gas chromatography-mass spectrometry technique. Promastigotes were incubated in Roswell Park Memorial Institute culture medium in the absence and presence of several concentrations (50, 100, 250, 500, 750, and 1,000 µg/mL) of each propolis sample. The viability and cell morphology of promastigotes in each concentration were examined after 24, 48, 72, and 96 h of incubation. The growth of leishmania parasites was significantly suppressed in the presence of 500, 750, and 1,000 µg/mL of Hatay propolis. Bursa propolis was found to be efficient in inhibiting the growth of leishmania promastigotes in culture media at these concentrations, 250, 500, 750, and 1,000 µg/mL. Thus, the in vitro results showed that the Hatay and Bursa propolis samples decreased significantly the proliferation of L. infantum and L. tropica parasites (p<0.001); however, Bursa propolis was found to be more effective than Hatay propolis against leishmania promastigotes. These two natural products may be useful agents in the prevention of leishmanial infections. © 2010 Springer-Verlag

Prolyl hydroxylase domain enzyme 2 is the major player in regulating hypoxic responses in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by hypoxia and the expression of hypoxia-inducible transcription factors (HIFs), which coordinate cellular responses to hypoxia. The objective of this study was to analyze the expression and regulation of prolyl hydroxylase domain (PHD) enzymes and factor-inhibiting HIF-1α (FIH-1), which regulate cellular HIF levels, and to study the roles of these enzymes in RA fibroblast-like synoviocytes (RA FLS). METHODS: The expression of PHD and FIH and downstream target genes was assessed by quantitative polymerase chain reaction and Western blotting. A small interfering RNA (siRNA) approach and an in vitro endothelial cell angiogenesis assay were used to analyze the roles of HIF hydroxylases. RESULTS: In human RA FLS, knockdown of PHD-2, but not knockdown of PHD-1 or FIH-1, dramatically augmented HIF-1α expression, modestly increased HIF-2α protein expression under normoxic conditions, and up-regulated HIF-dependent gene expression. In contrast, silencing of PHD-3 up-regulated HIF-2α but reduced HIF-1α, thereby decreasing the expression of HIF-regulated genes. A similar effect of PHD-2 knockdown was observed in osteoarthritis FLS (OA FLS) but not in nondiseased primary human dermal fibroblasts. These findings correlated with the induction of in vitro angiogenesis by supernatants from RA FLS and OA FLS transfected with siPHD-2 but not by supernatants from nondiseased fibroblasts or from siPHD-3-transfected cells. CONCLUSION: Our data suggest that PHD-2 is the major hydroxylase regulating HIF levels and the expression of angiogenic genes in arthritic cells. PHD-2 appears to regulate responses relevant to arthritis via HIF-α, highlighting the major importance of this enzyme in hypoxia- and angiogenesis-dependent inflammatory diseases such as RA