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

Cluster Lenses

Clusters of galaxies are the most recently assembled, massive, bound structures in the Universe. As predicted by General Relativity, given their masses, clusters strongly deform space-time in their vicinity. Clusters act as some of the most powerful gravitational lenses in the Universe. Light rays traversing through clusters from distant sources are hence deflected, and the resulting images of these distant objects therefore appear distorted and magnified. Lensing by clusters occurs in two regimes, each with unique observational signatures. The strong lensing regime is characterized by effects readily seen by eye, namely, the production of giant arcs, multiple-images, and arclets. The weak lensing regime is characterized by small deformations in the shapes of background galaxies only detectable statistically. Cluster lenses have been exploited successfully to address several important current questions in cosmology: (i) the study of the lens(es) - understanding cluster mass distributions and issues pertaining to cluster formation and evolution, as well as constraining the nature of dark matter; (ii) the study of the lensed objects - probing the properties of the background lensed galaxy population - which is statistically at higher redshifts and of lower intrinsic luminosity thus enabling the probing of galaxy formation at the earliest times right up to the Dark Ages; and (iii) the study of the geometry of the Universe - as the strength of lensing depends on the ratios of angular diameter distances between the lens, source and observer, lens deflections are sensitive to the value of cosmological parameters and offer a powerful geometric tool to probe Dark Energy. In this review, we present the basics of cluster lensing and provide a current status report of the field.Comment: About 120 pages - Published in Open Access at: http://www.springerlink.com/content/j183018170485723/ . arXiv admin note: text overlap with arXiv:astro-ph/0504478 and arXiv:1003.3674 by other author

The influence of habitat structure on genetic differentiation in red fox populations in north-eastern Poland

The red fox (Vulpes vulpes) has the widest global distribution among terrestrial carnivore species, occupying most of the Northern Hemisphere in its native range. Because it carries diseases that can be transmitted to humans and domestic animals, it is important to gather information about their movements and dispersal in their natural habitat but it is difficult to do so at a broad scale with trapping and telemetry. In this study, we have described the genetic diversity and structure of red fox populations in six areas of north-eastern Poland, based on samples collected from 2002–2003. We tested 22 microsatellite loci isolated from the dog and the red fox genome to select a panel of nine polymorphic loci suitable for this study. Genetic differentiation between the six studied populations was low to moderate and analysis in Structure revealed a panmictic population in the region. Spatial autocorrelation among all individuals showed a pattern of decreasing relatedness with increasing distance and this was not significantly negative until 93 km, indicating a pattern of isolation-by-distance over a large area. However, there was no correlation between genetic distance and either Euclidean distance or least-cost path distance at the population level. There was a significant relationship between genetic distance and the proportion of large forests and water along the Euclidean distances. These types of habitats may influence dispersal paths taken by red foxes, which is useful information in terms of wildlife disease management

VEGF release is associated with reduced oxygen tensions in experimental inflammatory arthritis.

OBJECTIVE: The contribution of local VEGF production and subsequent angiogenesis within the synovial membrane to the propagation of arthritis is unclear. The relationship between synovial oxygenation and blood flow in the development of arthritic disease is unknown. We have therefore measured oxygen levels and perfusion rates in the synovial space in a murine model of arthritis. METHODS: Arthritis was induced in DBA/1 mice by immunisation with type II collagen. Oxygen and perfusion levels were measured polarographically using silver needle microelectrodes within the knee joints prior to and 10 days after the onset of arthritis. In addition, synovial cells were isolated from knee joints of naive, pre-arthritic and arthritic mice. RESULTS: Onset of arthritis was associated with a marked reduction in synovial oxygen tensions (pO2). The perfusion rates in naive and arthritic animals were not significantly different: in naive mice, the rate was 0.58 +/- 0.11 ml/min/g and in arthritic joints, 0.64 +/- 0.17 ml/min/g. Furthermore, synovial cells isolated from the knee joints of naive animals did not express mRNA for VEGF, but significant levels were detected in cells from non-arthritic mice immunised with collagen. The onset of arthritis was associated with expression of VEGF mRNA and protein, and correlated negatively with pO2 levels. CONCLUSION: These data demonstrate that decreases in intra-articular pO2 occur in established arthritic conditions and may be the stimulus for local VEGF production. However, perfusion was not increased in arthritic animals and vascular density was unaltered, suggesting that the neovascularisation associated with inflammatory arthritis, is insufficient to restore oxygen homeostasis in the joint

Electrochemical measurement of transport into scoliotic intervertebral discs in vivo using nitrous oxide as a tracer.

STUDY DESIGN: An in vivo study measuring nitrous oxide concentrations in scoliotic intervertebral discs during surgery. OBJECTIVES: To determine pathways for nutrient transport into scoliotic human discs in vivo. SUMMARY OF BACKGROUND DATA: The intervertebral disc is the largest avascular structure in the body. Disc cells in the nucleus rely on the blood supply from the vertebral bodies for supply of nutrients and removal of waste. Loss of nutrient supply is thought to lead to disc degeneration, but solute transport has not been measured in vivo in humans. METHODS: We measured solute transport into the disc using N2O as a tracer, in 19 human discs from five patients with neuromuscular scoliosis (6-19 years of age) during surgery for correction of scoliotic deformities. During anesthesia N2O diffuses into the disc at a rate governed by effective permeability of the vertebral body-disc interface. Intradiscal N2O concentrations were measured amperometrically using silver needle microelectrodes, which were inserted into the discs once they were exposed by an anterior approach. RESULTS: For all spines N2O concentrations were very low in the disc at the curve apex (6% those expected from unimpeded diffusion) and, although still low, were significantly higher 2 discs below or above the apex. CONCLUSIONS: Because flux into the apical disc is most restricted, the decrease in solute transport is possibly induced by changes in mechanical stress on the disc; microfocal radiographs of a scoliotic spine suggest that increased endplate calcification could be partly responsible for limiting solute diffusion

Measurements of oxygenation and perfusion in skeletal muscle using multiple microelectrodes.

This paper describes an apparatus to measure tissue oxygenation and perfusion (as measured by the wash-in rate of gaseous hydrogen) simultaneously at multiple points in muscle using needle microelectrodes. The development of suitable electrodes and apparatus is described, as well as the development of the method and its validation. In particular, the potential for tissue damage secondary to electrode insertion, the need for in vivo voltammetric determination of the operating potential and the extent of any electrode-tissue and of electrode-electrode interactions are explored, and are shown to be insufficient in magnitude to affect the technique. Its subsequent use to characterise oxygenation and perfusion in rabbit skeletal muscle at rest is also described. In resting tibialis anterior muscle of the rabbit the mean pO2 was 18 +/- 13.3 mm Hg and the mean perfusion was 4.4 +/- 1.3 ml s-1 100 g-1. There was a heterogeneity in simultaneously-measured values of pO2 and perfusion at different points within muscle, and also a temporal variation at the same site. The spans between the highest and lowest simultaneously-measured values of pO2 in muscle ranged from 14 to 80 mm Hg, and for perfusion, from 1 to 12 mls-1 100 g-1. No significant correlation was evident from histological examination between either pO2 or perfusion and surrounding fibre type or capillary density