Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants

Vascular endothelial growth factor (VEGF) promotes cartilage-degrading pathways, and there is evidence for the involvement of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been reported. Here, we investigate whether the expression of VEGF is modulated by ROS. Aspirates of synovial fluid from patients with osteoarthritis (OA) were examined for intra-articular VEGF using ELISA. Immortalized C28/I2 chondrocytes and human knee cartilage explants were exposed to phorbol myristate acetate (PMA; 0–20 μg/ml), which is a ROS inducer, or 3-morpholino-sydnonimine hydrochloride (SIN-1; 0–20 μM), which is a ROS donor. The levels of VEGF protein and nitric oxide (NO) production were determined in the medium supernatant, using ELISA and Griess reagent, respectively. Gene expression of VEGF-121 and VEGF-165 was determined by splice variant RT-PCR. Expression of VEGF and VEGF receptors (VEGFR-1 and VEGFR-2) was quantified by real-time RT-PCR. Synovial fluid from OA patients revealed markedly elevated levels of VEGF. Common RT-PCR revealed that the splice variants were present in both immortalized chondrocytes and cartilage discs. In immortalized chondrocytes, stimulation with PMA or SIN-1 caused increases in the levels of VEGF, VEGFR-1 and VEGFR-2 mRNA expression. Cartilage explants produced similar results, but VEGFR-1 was only detectable after stimulation with SIN-1. Stimulation with PMA or SIN-1 resulted in a dose-dependent upregulation of the VEGF protein (as determined using ELISA) and an increase in the level of NO in the medium. Our findings indicate ROS-mediated induction of VEGF and VEGF receptors in chondrocytes and cartilage explants. These results demonstrate a relationship between ROS and VEGF as multiplex mediators in articular cartilage degeneration

The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

<p>Abstract</p> <p>Background</p> <p>Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure) mediates activation of the immune response in bacterial infection of the central nervous system (CNS). The chemotactic G-protein-coupled receptor (GPCR) formyl-peptide-receptor like-1 (FPRL1) plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear.</p> <p>Methods</p> <p>Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide) expression after infection with bacterial supernatants of <it>Streptococcus pneumoniae </it>(SP) and <it>Neisseria meningitides </it>(NM). Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia) and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene) expression and signal transduction were determined.</p> <p>Results</p> <p>We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA) verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between NM- or SP-induced signal transduction.</p> <p>Conclusions</p> <p>We propose that NM and SP induce glial cell activation and rCRAMP expression also via FPRL1 and MARCO. Thus the receptors contribute an important part to the host defence against infection.</p

Involvement of formyl peptide receptors in receptor for advanced glycation end products (RAGE) - and amyloid beta 1-42-induced signal transduction in glial cells

<p>Abstract</p> <p>Background</p> <p>Recent studies suggest that the chemotactic G-protein-coupled-receptor (GPCR) formyl-peptide-receptor-like-1 (FPRL1) and the receptor-for-advanced-glycation-end-products (RAGE) play an important role in the inflammatory response involved in neurodegenerative disorders such as Alzheimer’s disease (AD).</p> <p>Therefore, the expression and co-localisation of mouse formyl peptide receptor (mFPR) 1 and 2 as well as RAGE in an APP/PS1 transgenic mouse model using immunofluorescence and real-time RT-PCR were analysed. The involvement of rat or human FPR1/FPRL1 (corresponds to mFPR1/2) and RAGE in amyloid-β 1–42 (Aβ1-42)-induced signalling were investigated by extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, the cAMP level in primary rat glial cells (microglia and astrocytes) and transfected HEK 293 cells was measured. Formyl peptide receptors and RAGE were inhibited by a small synthetic antagonist WRW4 and an inactive receptor variant delta-RAGE, lacking the intracytoplasmatic domains.</p> <p>Results</p> <p>We demonstrated a strong increase of mFPR1/2 and RAGE expression in the cortex and hippocampus of APP/PS1 transgenic mice co-localised to the glial cells. In addition, the Aβ1-42-induced signal transduction is dependant on FPRL1, but also on FPR1. For the first time, we have shown a functional interaction between FPRL1/FPR1 and RAGE in RAGE ligands S100B- or AGE-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition a possible physical interaction between FPRL1 as well as FPR1 and RAGE was shown with co-immunoprecipitation and fluorescence microscopy.</p> <p>Conclusions</p> <p>The results suggest that both formyl peptide receptors play an essential role in Aβ1-42-induced signal transduction in glial cells. The interaction with RAGE could explain the broad ligand spectrum of formyl peptide receptors and their important role for inflammation and the host defence against infections.</p

In vivo imaging of antioxidant response element activity during liver regeneration after partial hepatectomy

The nuclear factor-erythroid 2-related factor 2 (Nrf2) -antioxidant response element (ARE) pathway is important for the regulation of antioxidative stress response and detoxification. To activate the expression of its target genes, such as heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1), Nrf2 binds to the ARE within the promoter region of these genes. Partial hepatectomy and consecutive liver regeneration lead to oxidative stress with activation of the Nrf2-ARE pathway. The aim of this study was to investigate ARE activity in?vivo during liver regeneration after partial hepatectomy.Transgenic ARE-luc mice were used. In these mice, the luciferase reporter gene is under the control of an ARE promoter element. Following 2/3 partial hepatectomy (PHx), mice underwent in?vivo bioluminescence imaging up until the ninth postoperative day. In addition, liver tissue was analyzed by immunohistochemistry (Nrf2 and HO-1), quantitative reverse transcription-PCR (HO-1 and NQO1) and in?vitro luminescence assays.Bioluminescence imaging revealed a significant increase in Nrf2-ARE activity after PHx. The signal maximum was recorded on the third day after PHx. Seven days postoperatively, the signal almost reached baseline levels. In immunohistochemistry, significantly more hepatocytes were positive for Nrf2 and HO-1 on the third postoperative day compared with baseline levels. The mRNA expression of HO-1 and NQO1 were significantly increased on day 3 as measured by qRT-PCR.This study demonstrated the time-dependent activation of the Nrf2-ARE system during liver regeneration in?vivo. The transgenic ARE-luc mouse provided a convenient model for studying Nrf2-mediated gene expression noninvasively and may facilitate further experiments with therapeutic modulation of the antioxidative stress response

Expression and regulation of antimicrobial peptide rCRAMP after bacterial infection in primary rat meningeal cells

Bacterial meningitis is characterized by an inflammation of the meninges and continues to be an important cause of mortality and morbidity. Meningeal cells cover the cerebral surface and are involved in the first interaction between pathogens and the brain. Little is known about the role of meningeal cells and the expression of antimicrobial peptides in the innate immune system. In this study we characterized the expression, secretion and bactericidal properties of rat cathelin-related antimicrobial peptide (rCRAMP), a homologue of the human LL-37, in rat meningeal cells after incubation with different bacterial supernatants and the bacterial cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN). Using an agar diffusion test, we observed that supernatants from meningeal cells incubated with bacterial supernatants, LPS and PGN showed signs of antimicrobial activity. The inhibition of rCRAMP expression using siRNA reduced the antimicrobial activity of the cell culture supernatants. The expression of rCRAMP in rat meningeal cells involved various signal transduction pathways and was induced by the inflammatory cytokines interleukin-1, -6 and tumor necrosis factor alpha. In an experimental model of meningitis, infant rats were intracisternally infected with Streptococcus pneumoniae and rCRAMP was localized in meningeal cells using immunohistochemistry. These results suggest that cathelicidins produced by meningeal cells play an important part in the innate immune response against pathogens in CNS bacterial infections