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

Multiple Regulatory Mechanisms to Inhibit Untimely Initiation of DNA Replication Are Important for Stable Genome Maintenance

Genomic instability is a hallmark of human cancer cells. To prevent genomic instability, chromosomal DNA is faithfully duplicated in every cell division cycle, and eukaryotic cells have complex regulatory mechanisms to achieve this goal. Here, we show that untimely activation of replication origins during the G1 phase is genotoxic and induces genomic instability in the budding yeast Saccharomyces cerevisiae. Our data indicate that cells preserve a low level of the initiation factor Sld2 to prevent untimely initiation during the normal cell cycle in addition to controlling the phosphorylation of Sld2 and Sld3 by cyclin-dependent kinase. Although untimely activation of origin is inhibited on multiple levels, we show that deregulation of a single pathway can cause genomic instability, such as gross chromosome rearrangements (GCRs). Furthermore, simultaneous deregulation of multiple pathways causes an even more severe phenotype. These findings highlight the importance of having multiple inhibitory mechanisms to prevent the untimely initiation of chromosome replication to preserve stable genome maintenance over generations in eukaryotes

Mathematical Modelling of DNA Replication Reveals a Trade-off between Coherence of Origin Activation and Robustness against Rereplication

Eukaryotic genomes are duplicated from multiple replication origins exactly once per cell cycle. In Saccharomyces cerevisiae, a complex molecular network has been identified that governs the assembly of the replication machinery. Here we develop a mathematical model that links the dynamics of this network to its performance in terms of rate and coherence of origin activation events, number of activated origins, the resulting distribution of replicon sizes and robustness against DNA rereplication. To parameterize the model, we use measured protein expression data and systematically generate kinetic parameter sets by optimizing the coherence of origin firing. While randomly parameterized networks yield unrealistically slow kinetics of replication initiation, networks with optimized parameters account for the experimentally observed distribution of origin firing times. Efficient inhibition of DNA rereplication emerges as a constraint that limits the rate at which replication can be initiated. In addition to the separation between origin licensing and firing, a time delay between the activation of S phase cyclin-dependent kinase (S-Cdk) and the initiation of DNA replication is required for preventing rereplication. Our analysis suggests that distributive multisite phosphorylation of the S-Cdk targets Sld2 and Sld3 can generate both a robust time delay and contribute to switch-like, coherent activation of replication origins. The proposed catalytic function of the complex formed by Dpb11, Sld3 and Sld2 strongly enhances coherence and robustness of origin firing. The model rationalizes how experimentally observed inefficient replication from fewer origins is caused by premature activation of S-Cdk, while premature activity of the S-Cdk targets Sld2 and Sld3 results in DNA rereplication. Thus the model demonstrates how kinetic deregulation of the molecular network governing DNA replication may result in genomic instability

Activation of Sirt1 by Resveratrol Inhibits TNF-α Induced Inflammation in Fibroblasts

Inflammation is one of main mechanisms of autoimmune disorders and a common feature of most diseases. Appropriate suppression of inflammation is a key resolution to treat the diseases. Sirtuin1 (Sirt1) has been shown to play a role in regulation of inflammation. Resveratrol, a potent Sirt1 activator, has anti-inflammation property. However, the detailed mechanism is not fully understood. In this study, we investigated the anti-inflammation role of Sirt1 in NIH/3T3 fibroblast cell line. Upregulation of matrix metalloproteinases 9 (MMP-9), interleukin-1beta (IL-1β), IL-6 and inducible nitric oxide synthase (iNOS) were induced by tumor necrosis factor alpha (TNF-α) in 3T3 cells and resveratrol suppressed overexpression of these pro-inflammatory molecules in a dose-dependent manner. Knockdown of Sirt1 by RNA interference caused 3T3 cells susceptible to TNF-α stimulation and diminished anti-inflammatory effect of resveratrol. We also explored potential anti-inflammatory mechanisms of resveratrol. Resveratrol reduced NF-κB subunit RelA/p65 acetylation, which is notably Sirt1 dependent. Resveratrol also attenuated phosphorylation of mammalian target of rapamycin (mTOR) and S6 ribosomal protein (S6RP) while ameliorating inflammation. Our data demonstrate that resveratrol inhibits TNF-α-induced inflammation via Sirt1. It suggests that Sirt1 is an efficient target for regulation of inflammation. This study provides insight on treatment of inflammation-related diseases

Synthesis and Properties of Random Carbazole/1,3,4-Oxadiazole/Aniline Copolymers for Organic Light-Emitting Devices

Random copolymers, poly[(M1-M2)-ran-(M1-M3)], have been synthesized by N-(3,6-dibromo)carbazole (M1), aniline (M2) and 2-p-methylphenyl-5-(4- aminophenyl)-1,3,4-oxadiazole (M3). The random copolymers were obtained with high solubility and good film forming properties. Optical properties of these copolymers were investigated. As increasing the content of M3, the maximum absorption peaks (max,UV) of copolymers at 360nm increased gradually, which was the typical max,UV of poly(M1-alt-M3). Compared with maximum photoluminescence emission peak (max,PL) at 452nm of poly(M1-alt-M2), all the random copolymers displayed max,PL at about 519nm, similar with that of poly(M1-alt-M3). It was notable that max,PL of the random copolymers shifted to that of poly(M1-alt-M3) with the addition of M3 in the copolymers, and then remained almost unchanged irrespective of further increasing the content of M3, implying the emission from the lower band gap segment of poly(M1-alt-M3). The band gap energy of the copolymers was measured in the range of 2.822.91eV, and HOMO energy in the range of-4.95-5.11eV. Hole transporting properties of the synthesized copolymers in OLEDs were measured with the configuration of ITO/copolymer/Alq3/LiF/Al, where Alq3 was used as an emitting layer. It appeared that the random copolymers (P4P5) with the compositions of M1:M2:M3 (10:4:610:6:4 mole ratio) showed better hole-transporting property.

Red Phosphorescent Dopant/Polymeric Host System as an Emitting Layer: Effect of Hole Blocking Materials

Phosphorescent OLEDs with the configuration of ITO/PEDOT/EML/HBL/Alq3/LiF/ Al were fabricated and their electroluminescent performance was investigated. Synthesized red phosphorescent iridium (III) bis(2-phenyl-isoquinoine) (2-acetyl-cyclohexane) (Ir-PIQCH) dopant and p{[N-(2-ethylhexyl)-3,6-bis(styryl) carbazole]-alt-[2,5-(4-biphenyl)-1,3,4-oxadiazole]} [P(CZ-OXD)] host were used as emitting layer in OLEDs. The devices showed red emission (max.EL about 660nm), similar with PL emission of Ir-PIQCH dopant. EL properties of the phosphorescent OLEDs were measured to investigate the effect of hole blocking layers such as 2-(4-biphenylyl)-5-(4-tert-butylphenyl)-1,3,4-oxadiazole (PBD), bathocuproine (BCP), and bis(2-methyl-8-quinolinate)-4-phenylphenolate (BAlq) on luminescence and color purity of the devices. It was found that Device 3 using BCP as HBL showed pure red emission with CIE coordinates value as (x=0.6673, y=0.3231), due to higher energy barrier (1.03eV) between P(CZ-OXD) host polymer and hole blocking BCP.

Development of novel red phosphorescent iridium complex with phenyl-isoquinoline ligand for organic electrolumine scent device

Novel red phosphorescent compound (IR-PIQC-1), iridium complex with two phenyl-isoquinoline ligands and one 2-acetyl-1,3-cyclohexanedione ligand was designed and synthesized. The electroluminescent (EC) characteristics of IR-PIQC-1 in the EL device were evaluated. The thin layers of EL devices were fabricated by the vacuum deposition method, and the layer-structures of the devices were ITO/NPD/IR-PIQC-1 or IR-BTPAC (10%) in CBP/Balq/Alq/LiF/Al. Brightness of the IR-PIQC-1 device was 574 cd/m(2) at 11 mA/cm(2), and the external quantum efficiency was estimated as 5.28% at the same current density. The EL spectrum of the IR-PIQC-1 device was detected at about 620 nm as the maximum peak with red phosphorescence. (c) 2005 Elsevier B.V. All rights reservedclose2