Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.In the vasculature, physiological levels of nitric oxide (NO) protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS) at Ser The free radical nitric oxide (NO), produced by endothelial NO synthase (eNOS), is an important vasoactive substance in normal vascular biology and pathophysiology. In addition to its well-known vascular functions such as vessel dilation and angiogenesis via S-nitrosylation of N-Ethylmaleimide-sensitive Factor (NSF) While the molecular mechanisms underlying eNOS activation and NO production in arterial ECs in response to chemical stimuli and shear stress have been studied extensively, little is known about the mechanism in venous ECs under continuous stretch. Actually, continuous stretch of venous ECs caused by the abrupt and sustained dilation of veins is frequently observed in patients with portal vein embolization, venous congestion due to acute heart failure, and venous-arterial grafts 1177 phosphorylation leads to increased eNOS activity and NO production 1177 phosphorylation of eNOS 1177, eNOS has several other potential phosphorylation sites, including Ser615 and Ser633, the phosphorylation of which enhances the activity of eNOS. It has been shown that Ser615 is phosphorylated in a PKB/Akt-dependent manner while Ser633 in a PKA-dependent manner It is known that Ser1177 phosphorylation of eNOS and NO production in human umbilical vein endothelial cells (HUVECs) under continuous stretch by using kinase-specific inhibitors and gene-specific shRNAs.Therefore, we set out to determine whether AMPK, Akt, PKA, and MAPK regulate the Ser1177 phosphorylation of eNOS and NO production. Early reports suggest that under shear stress, sustained eNOS activation for as long as 30–60 min was detected 1177 phosphorylation of eNOS in a time-dependent manner without significant change in the amount of total eNOS protein and had no significant effect on cell death within 2 h . We found that stretch induced the Ser1177 phosphorylation in a magnitude-dependent manner  to assess the NO levels in HUVECs. The NO levels were significantly increased after stretch compared with the control. In addition, L-NAME (a NOS inhibitor) significantly inhibited the stretch-induced NO production  in a PKA-dependent manner. Interestingly, after stretch for >15 min Ser1177 phosphorylation of eNOS and NO production were not completely abolished but still slightly increased in the presence of 50 µM H89 or expressing the shRNAs targeting both PKA subunits, compared with unstretched cells , and then subjected to stretch for 15 min. The LY294002 treatment abolished the stretch-induced phosphorylation of Akt in a dose-dependent manner but had no significant effect on the phosphorylation of eNOS under these conditions , whereas inhibition of Akt pathway by the inhibitor as well as gene-specific shRNAs decreased Ser1177 phosphorylation of eNOS in the late phase (>15 min) (633 phosphorylation of eNOS by stretch occurred relatively earlier (from 2 to 15 min), compared to Akt-mediated Ser615 phosphorylation of eNOS (from 15 to 60 min) (Our conclusion that PKA and Akt kinases play distinctive roles in the activation of eNOS in a time-dependent manner is based on the following lines of evidence: (1) stretch-induced activation of PKA pathway occurred relatively earlier than Akt pathway ; (2) inh>15 min) , 5 ; (3) 60 min) . Taken tvia GPCRs. Some reports have shown that VEGFR2-mediated activation of the PI3K/Akt pathway by mechanical stress is relatively delayed (within minutes) 1177 phosphorylation of eNOS. A slight increase of Ser1177 phosphorylation of eNOS can be detected under stretch for 15 min in HUVECs after inhibition of the PKA pathway, while inhibition of the PI3K/Akt pathway does not attenuate stretch-induced Ser1177 phosphorylation of eNOS at this time point (1177 in the early phase (≤15 min) and when PKA is inhibited or deactivated (>15 min), Akt replaces PKA and plays the main role in maintaining Ser1177 phosphorylation of eNOS until the recruitment of Hsp90 in the eNOS complex One possible explanation for the biphasic response is that the difference of the two signaling pathways is caused different rates of activation by upstream pathways. Previous work has shown that VEGFR2 and GPCRs are components of the mechanosensor complex in ECs me point  and 5. T1177 phosphorylation in an AMPK-independent manner, although phosphorylation of AMPK was also significantly increased  and phospho-eNOS (Ser633) (for detecting eNOS activation) and eNOS were from BD Biosciences . Rabbit antibody to phospho-eNOS (Ser615) was from Upstate (Millipore). Rabbit polyclonal antibodies to phospho-PKA substrate, phospho-Akt (Ser473) (for detecting Akt activation), phospho-AMPKα (Thr172) (for detecting AMPKα activation), AMPKα and phosphor-MEK1/2 (Thr202/Tyr204) were from Cell Signaling Technology . DMSO (dimethyl sulfoxide), H89, LY294002 and 8-Br-cAMP were from Sigma . L-NAME, PP2 and Rp-cAMP were from Alexis . SNAP (S-nitroso-N-acetyl-DL-penicillamine) and PD98059 were form Caymen Chemical. Compound C was from Merck Millipore. DAF-FM diacetate was from Invitrogen . The vWF ELISA kit was described previously Rabbit polyclonal antibodies to Akt, PKACα, PKACβ and Erk2 were from Santa Cruz Biotechnology . Mouse monoclonal antibodies to phospho-eNOS (Serhttp://jura.wi.mit.edu/bioc/siRNAext/). The shRNA sequence targeting eNOS was 5′-GTGGCCAACGCCGTGAAGATC-3′; for PKA catalytic subunits α and β, 5′-GCTCCCTTCATACCAAAGTTT-3′ and 5′-CACAGCCCACTTGGATCAGTT-3′;for AMPKα1, 5′-GTACGACTAAGCCCAAATCTT-3′; for Akt1 and Akt2, 5′-GGAGGGTTGGCTGCACAAATT-3′ and 5′-CTCCTTGGCAAGGGAACCTTT -3′; and the control scrambled sequence was 5′-CCTAAGGTTAAGTCGCCCTCG-3′.To silence eNOS, AMPKα1, Akt1/2 and PKA , we used a commercial lentiviral system from Sigma to deliver short hairpin RNAs (shRNAs). The target and control scrambled sequences were selected according to an open program (2. Cells were used from passages 3 to 6. 293T and HL-60 cells were obtained from the ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum.The origin and the culture of human umbilical vein endothelial cells (HUVECs) used in this manuscript have been described in the literature HUVECs were stretched on a device established for the study of static continuous stretch, which has been described in detail Confluent HUVECs were starved in serum-free M199 medium supplemented with 2% BSA for 4 h, and then stimulated by stretch or other factors. The supernatant was harvested and centrifuged at 3600 rpm at 4°C. The concentration of vWF was assessed by the standard sandwich ELISA procedure according to the manufacturer’s instructions.Confluent ECs were washed twice with cold PBS, fixed with 4% PFA supplemented with 0.2 mol/L sucrose for 1 hr at 4°C. After washing three times with PBS, ECs were stained with Annexin V/PI kits (Invitrogen V13241) according to the protocol and incubated at room temperature for 15 min and then washed another three times. ECs were also stained with DAPI.Preparations of lentiviruses were made in 293T cells. Forty-eight hours after the cells were transfected, the virus-containing supernatant was harvested. Three milliliters of supernatant, mixed with 3 mL fresh M199 medium containing a final concentration of 8 µg/mL polybrene, was used to infect ECs as previously described 2, 1 mM EGTA, 5 mM EDTA, 0.27 g/mL Na4P2O7, 5 g/mL aprotinin, 1 g/mL prostatin A, 1 g/mL antipan, 10 g/mL leupeptin, 1 mg/mL PMSF, 2 mM beta-glycerol phosphate, 10 mM NaF, and 2 mM Na3VO4. The lysates were fractionated on 8% SDS-PAGE, followed by standard Western blot analysis.Confluent HUVECs were starved in serum-free M199 medium supplemented with 1% BSA for 16 h before stimulation. The stimulated cells were washed twice in ice-cold PBS and lysed in buffer containing 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgClConfluent HUVECs were starved in serum-free M199 medium supplemented with 2% BSA for 3 h, and incubated with 5 µM DAF-FM diacetate in phenol red-free DMEM for 30 min at 37°C. Then the cells were washed gently with PBS and stimulated by stretch. The real-time changes in DAF fluorescence were recorded though a 40×oil-immersion lens and analyzed by laser scanning confocal microscopy .6 HL-60 cells were added to the surface of the endothelial cells. After 30-min incubation, the unbound cells were washed off 3 times with M199 medium, the bound cells were fixed with 4% paraformaldehyde, and images were captured on a Nikon inverted microscope with a 20× objective lens. The numbers of bound HL-60 cells in each field were counted and analyzed by ANOVA. Representative results of three independent experiments are shown.Confluent HUVECs were starved in serum-free M199 medium supplemented with 2% BSA for 4 h, and then stimulated by stretch for 30 min. Then the supernatant was discarded and 2×10P<0.05 was considered statistically significant.Results are expressed as mean ± S.D. on the basis of triplicate experiments. Statistical analysis was done using ANOVA with Bonferroni’s correction. A value of Figure S1Effects of stretch on the survival and death of HUVECs. (A) Immunofluorescence staining of Annexin V/PI and DAPI in HUVECs stretched for 1 hr–8 hrs. HUVECs starved in empty M199 medium served as positive controls. (B) Quantitative analysis of trypanblue staining of HUVECs stretched for 1 hr–8 hrs. Results are representative of 3 individual experiments and expressed as mean ± SD (n = 4). *P<0.05; N.S., not significant.(TIF)Click here for additional data file.


In obesity, there is an increase in reactive oxygen species (ROS) within adipose tissue caused by increases in inflammation and overnutrition. Hormone sensitive lipase (HSL) is part of the canonical lipolytic pathway and critical for complete lipolysis. This study hypothesizes that ROS is a signal that integrates regulation of lipolysis by targeting HSL. Experiments were performed with human differentiated adipocytes from the subcutaneous depot. Antioxidants were employed as a tool to decrease ROS, and it was found that scavenging ROS with diphenyliodonium, N-acetyl cysteine, or resveratrol decreased lipolysis in adipocytes. HSL phosphorylation of a key serine residue, Ser552, as well as translocation of this enzyme from the cytosol to the lipid droplet upon lipolytic stimulation were both abrogated by scavenging ROS. The phosphorylation status of other serine residues on HSL were not affected. These findings are significant because they document that ROS contributes to the physiological regulation of lipolysis via an effect on translocation. Such regulation could be useful in developing new obesity therapies. Hormone Sensitive Lipase (HSL) is a key enzyme in the regulation of lipid, the largest energy reserve in the body. Recently, there has been a renewed interest in HSL as an attractive therapeutic candidate for obesity because of its crucial role in lipolysis. The lipolytic pathway has been described for quite some time; however, the regulation of this pathway is not as well defined. In this study, we examine regulation of lipolysis and specifically HSL modulation by reactive oxygen species (ROS), which are increased in obesity.Reduced activity of HSL improves metabolic homeostasis. Mice that lack a functional copy of the gene encoding HSL are resistant to both genetic and diet-induced obesity In vitro, phosphorylation of human HSL Ser649 and Ser650 are the major determinants of its hydrolytic activity As the name suggests, HSL hydrolyzes esters of neutral lipids, principally diacylglyceride (DG), in a manner activated by a variety of hormones that increase cAMP, including catecholamines, adrenocorticotropic hormone (ACTH) and glucagon Upon phosphorylation, HSL translocates to the lipid droplet to participate in lipolysis. PKA phosphorylation induces a conformational change to expose hydrophobic groups on HSL, which facilitates HSL binding to its substrate, lipid ROS is a candidate for the regulation of lipolysis, because there is a positive correlation between both ROS and lipolysis with obesity ROS levels are increased in obesity and decreased by ROS scavengers.2-DCFDA. It can be a challenge to increase ROS levels modestly in cell culture models, although scavenging with antioxidants is feasible. Various reactive species have different half-lives and may act in different compartments that are difficult to target in a cell culture system. Also, a portion of ROS is scavenged by components of the experimental media including pyruvate We next assessed lipolysis by measuring glycerol release from cells into the cell culture media. Next, we determined whether the effect of DPI on lipolysis was specific for DPI or if other antioxidants also decreased lipolysis. Taken together, the above data showed that scavenging ROS inhibited lipolysis. To determine the mechanism, we investigated the cAMP lipolytic pathway. cAMP is a common point of regulation of lipolysis To confirm the lack of effect on PKA, we next measured the phosphorylation state of individual proteins that PKA phosphorylates. Perilipin, a lipid droplet coating protein, is hyperphosphorylated upon lipolytic stimulation. Perilipin phosphorylation facilitates HSL-mediated lipolysis by its interaction with lipid droplet associated HSL Hormone sensitive lipase is another target of PKA, though it can also be activated by other kinases Further studies were undertaken to determine how deficient phosphorylation of HSL Ser552 decreased lipolysis in the presence of DPI. We next assessed the time course of decrease in this site’s phosphorylation in the presence of DPI under forskolin stimulation. Phosphorylation of HSL Ser552 was prevented by DPI as early as 15 minutes following compound exposure (our earliest time point) and was maintained at 1 hour of treatment  ROS has a physiological role on lipolysis. 2) lipolytic alterations that occur during obesity may be due to ROS. With respect to the physiological role of ROS, the results of the antioxidant studies suggest that ROS is a necessary component of the lipolytic signaling cascade and that when ROS is scavenged by DPI, both basal and stimulated lipolysis are decreased. The antioxidants NAC and resveratrol each also inhibited stimulated lipolysis, suggesting that this was a bona fide antioxidant effect. Experiments performed with freshly isolated rat adipocytes are in agreement with the findings of this study in vitroin vivoGMP non-canonical lipolytic pathway. Interestingly, HSL is phosphorylated in response to ANP, although the specific serine residue(s) on HSL that are phosphorylated are unknown. Of note, the reactive oxygen species, NO also activates this pathway A number of mechanistic possibilities were assessed to understand how ROS exerted an effect on lipolysis. The lipolytic pathway was unaltered by DPI treatment as evidenced by no alteration in cAMP, PKA substrate phosphorylation, or perilipin phosphorylation. This suggested that DPI inhibited lipolysis in an unconventional manner. One future direction is to assess the cGMP non-canonical lipolytic pathway. Natriuretic peptides (NPs), atrial (ANP) and brain NP stimulate lipolysis An examination of the DG lipase HSL led to some surprising and novel modes of regulation of lipolysis. The phosphorylation of the PKA site, HSL Ser552 was reduced in the presence of DPI. Since this effect was seen as early as 15 min, it is likely not due to an alteration in either translation or degradation of HSL protein. However, phosphorylation of the PKA target site HSL Ser650 did not change under these same experimental conditions. AMPK is not implicated in the abrogation of HSL Ser552 via increased phosphorylation of HSL Ser554, as phosphorylation of the latter site was unchanged in the presence of DPI. Of note, stimulation of lipolysis, which has been shown to activate AMPK in 3T3-L1 cells These findings certainly do not rule out the regulation of other lipases by antioxidants. Adipocyte triglyceride lipase (ATGL) is a candidate for future studies since this lipase is the rate determining enzyme of lipolysis in vitro, HSL Ser649 and Ser650 are the major determinants of hydrolytic activity, but not Ser552 Work from the laboratory of Cecilia Holm et al revealed that These data suggest that the ability of the enzyme to be localized near its substrate is an important layer of regulation. To date, this is the first study implicating human HSL Ser552 as a key residue for lipolysis. Evidence in 3T3L1 cells suggests that translocation precedes phosphorylation at HSL Ser563 (human HSL Ser552) This is also the first study to suggest that under some conditions, such as altered ROS, two PKA-target phosphorylation sites (HSL Ser650 and Ser552) do not always have the same pattern of phosphorylation. This could suggest that two distinct kinases phosphorylate these two serine residues. Older literature names glycogen synthase kinase 4 (GSK-4) (now renamed to GSK-3ß) as a kinase that phosphorylates HSL at Ser552 The testable model that evolves from these data is that ROS is an important signal that promotes lipolysis, and we propose that the mechanism by which this occurs is by altered translocation of HSL from the cytosol to the lipid droplet. This leads to the questions: 1) Under conditions of high ROS, as in obesity, is the increase in lipolysis mediated by ROS? 2) Could other lipolytic components and also lipid synthetic enzymes be regulated in a similar manner by ROS, i.e., translocation? Lastly, this paper aims to instill a renewed interest in HSL as a target of small molecule inhibitors and activators as a means of modulating lipid turnover under conditions of both excess and also reduced lipid storage.The differentiation of human primary preadipocytes into adipocytes was carried out essentially as previously described The following antibodies were purchased from Cell Signaling Technology : polyclonal antibody against HSL, polyclonal antibody against phosphorylated human HSL serine residue 552 (rat 563), polyclonal antibody against phosphorylated human HSL serine residue 554 (rat 565), polyclonal antibody against phosphorylated human HSL serine residue 650 (rat 660), monoclonal antibody against PKA substrates, and monoclonal antibody against tubulin. Polyclonal antibody against perilipin A was a kind gift from Dr. A. Greenberg (Tufts University), previously described 4 mM, 3.3 mM CaCl2, 24.5 mM NaHCO3, 1 mM KH2PO4, 5 mM glucose, incubated for 10–15 min with 5% CO2, 95% O2) for 30 minutes and then switched to KRB with 0.5 mM oleate and 150 µM BSA in the presence or in the absence of 5 µM forskolin for 4 hours. Glycerol release was measured by using a luciferase based enzyme-linked luminescent assay previously described Cells were preincubated in Krebs Ringer Buffer (KRB)  and transferred to a polyvinylidene fluoride 0.2 µm membrane (Biorad). Membranes were blocked with 5% nonfat milk in TBS-T buffer  for 1 hour. Membranes were then probed overnight at 4°C with primary antibodies followed by 1 hour incubation with secondary antibody conjugated to horseradish peroxidase. Protein bands were detected with an enhanced chemiluminescence substrate kit  using a FujiFilm LAS-4000 Luminescent Image Analyzer.cAMP was measured using a fluorescent assay kit  according to manufacturer’s instructions.2DCFDA, which cleaves to become DCF . Cells were grown and differentiated in 12-well plates (2.3 × 10∧5 cells per well). On the day of the experiment, media was changed to DMEM with 1% BSA. CM-H2DCFDA was added for a 30 minute preincubation at a final concentration of 5 µM. Cells were then washed and media was changed to Hank’s Balanced Salts (HBSS). Fluorescent readings were taken on a plate reader  every 30 seconds for 1.5 hours at 37°C at excitation 485, emission 538 nm. A preliminary experiment was performed to establish conditions and it was determined that BSA interferes with the assay and was therefore not included during the 1.5 hours of measurement although 0.1% is added during the loading of CM-H2DCFDA into the cells.ROS measurements were performed with the ROS sensitive dye CM-HCells were grown and differentiated in 35 mm coated glass bottom dishes  and subsequently imaged in these plates in an inverted microscope. At the commencement of the experiment, cells were fixed for 20 minutes in 4% formaldehyde and then blocked and concomitantly permeabilized for one hour in PBS with 0.3% Triton X-100 and 5% goat serum at room temperature. Cells were stained overnight at 4°C with the primary antibody, anti-HSL  at a 1∶200 dilution in PBS with 0.3% Triton X-100 and 1% BSA, followed by a one hour incubation at room temperature with Alexa Fluor 488 anti-rabbit secondary antibody  at a 1∶400 dilution, while concomitantly staining with Lipotox red stain  at a 1∶1000 dilution to visualize lipid droplets and Hoescht 33342  at a 1∶1000 dilution to visualize nuclei. The solution was then replaced with 100 µl of SlowFade Gold antifade reagent  and cells were examined by confocal fluorescent microscopy using a Zeiss LSM 710-Live Duo scan microscope. .http://imagej.nih.gov/ij/. Figure S1 is the macro that was generated and run in ImageJ to analyze HSL translocation from the cytosol to lipid droplets. Briefly, the goal was to measure the pixel intensity around each lipid droplet and also measure the pixel intensity within the cytosol. This ratio of average pixel intensity around the lipid droplet over average pixel intensity of the cytosol was termed the ‘translocation to the lipid droplet’ and was used as an index to compare experimental conditions to control.Data were analyzed using ImageJ software from the National Institute of Health (NIH)  and can be found at Student’s paired two-tailed t test was used to evaluate statistical significance of results. Values of p < 0.05 were considered to be significant differences.Figure S1Macro to analyze HSL translocation with Image J software. For the pixel intensity around the lipid droplet, the confocal picture of the lipid droplets, detected using Lipotox red stain, was converted into a mask. This mask was then duplicated and one copy was dilated twice. The unaltered lipid mask was then subtracted from the twice dilated lipid mask and the resulting mask outlined the perimeters of the lipid droplets. This ‘peri-lipid’ mask was then applied to the HSL confocal picture and the number of pixels and intensity at each pixel could be extracted from the histogram for this picture. For the cytosolic pixel intensity, a mask was generated from the HSL confocal picture. This mask was then subtracted from the twice dilated lipid mask generated for the ‘peri-lipid’ analysis. The resulting mask, termed ‘HSL mask’, included all of the cytosol except that which was directly around each lipid droplet. This HSL mask was then applied to the HSL confocal picture and the number of pixels and intensity at each pixel could be extracted from the histogram for this picture. The index ‘translocation to the lipid droplet’ was then calculated using the average pixel intensities from the peri-lipid and cytosol analyses.(TIF)Click here for additional data file.


The purpose of the present study was to investigate the type of receptor involved in ODN-induced inhibition of SOD and catalase in cultured rat astrocytes. We found that ODN induced a rapid stimulation of SOD and catalase gene transcription in a concentration-dependent manner. In addition, 0.1 nM ODN blocked H2O2-evoked reduction of both mRNA levels and activities of SOD and catalase. Furthermore, the inhibitory actions of ODN on the deleterious effects of H2O2 on SOD and catalase were abrogated by the metabotropic ODN receptor antagonist cyclo1-8[Dleu5]OP, but not by the CBR antagonist flumazenil. Finally, the protective action of ODN against H2O2-evoked inhibition of endogenous antioxidant systems in astrocytes was protein kinase A (PKA)-dependent, but protein kinase C-independent. Taken together, these data demonstrate for the first time that ODN, acting through its metabotropic receptor coupled to the PKA pathway, prevents oxidative stress-induced alteration of antioxidant enzyme expression and activities. The peptide ODN is thus a potential candidate for the development of specific agonists that would selectively mimic its protective activity.Astroglial cells possess an array of cellular defense systems, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damage caused by oxidative stress on the central nervous system. Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides including the octadecaneuropeptide (ODN). ODN is the ligand of both central-type benzodiazepine receptors (CBR), and an adenylyl cyclase- and phospholipase C-coupled receptor. We have recently shown that ODN is a potent protective agent that prevents hydrogen peroxide (H A receptor-chloride channel complex (i/0 protein-coupled receptor leading to the activation of phospholipase C (PLC) in astrocytes (The octadecaneuropeptide (ODN) is a peptide generated through the proteolytic cleavage of the 86-amino acid precursor diazepam-binding inhibitor DBI;  which is complex . It has trocytes . In additrocytes . ODN exetrocytes , reductitrocytes , or throtrocytes . Similartrocytes  and actitrocytes , or throtrocytes  and modutrocytes , 2004.2O2), is implicated in the pathology of several neurological disorders including cerebral ischemia and neurodegenerative diseases , such as hydrogen peroxide .N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), fetal bovine serum (FBS), the antibiotic-antimycotic solution, and trypsin-EDTA were obtained from Gibco . Bovine liver catalase, chelerythrine, DL-epinephrine, H89, Triton X-100, and insulin were purchased from Sigma Aldrich . Flumazenil was a generous gift from Hoffmann-La Roche . Rat ODN and the G protein-coupled receptor antagonist cyclo1-8[DLeu5]OP were synthesized by using the standard Fmoc procedure, as previously described (Dulbecco’s modified Eagle’s medium (DMEM), F12 culture me-dium, D(+)-glucose, L-glutamine, escribed . All oth2 flasks , and incubated at 37°C in a 5% CO2/95% O2 atmosphere. When cultures were confluent, astrocytes were isolated by shaking overnight the flasks with an orbital agitator and plated on 35-mm Petri dishes at a density of 0.3 × 106 cells/ml. All experiments were performed on 5- to 7-day-old secondary cultures.Secondary cultures of rat cortical astrocytes were prepared as previously described . Briefly2, SYBR green reporter dye, AmpliTaq Gold DNA polymerase, and forward (5′-CCTTCTTGTTCTGCAACC-TGCTA-3′) and reverse (5′-CCGGACTCTCCGGTATCTGA-3′) SOD (GenBank accession no. NM_012880) primers, or forward (5′-CCACAGTCGCTGGAGAGTCA-3′) and reverse (5′-GTTTC-CCACAAGGTCCCAGTT-3′) catalase (GenBank accession no. NM_012520) primers, or forward (5′-CAGCCTCGTCTCATAGA-CAAGATG-3′) and reverse (5′- CAATGTCCACTTTGTCACAAG-AGAA-3′) glyceraldehyde-3-phosphate dehydrogenase  primers , using the ABI Prism 7000 sequence detection system (Applied Biosystems). The amount of SOD and catalase cDNA in each sample was calculated by the comparative threshold cycle (Ct) method and expressed as 2-δδCt using GADPH as an internal control.Cultured cells were incubated at 37°C with fresh serum-free medium. At the end of the incubation, the culture medium was removed and the cells were washed twice with phosphate buffered saline . Total RNA was extracted by using Tri reagent  and purified using the NucleoSpin kit . cDNA was synthetized from 3–4 µg of total RNA with ImProm II Promega kit (Promega). Quantitative RT-PCR was performed on cDNA in the presence of a 1× Fast SYBR Green universal PCR Master mix  containing concentrations of dNTPs, MgClg, 4°C, 20 min) and the cellular extract contained in the supernatant was stored at -20°C until enzyme activity determinations.Cultured cells were incubated at 37°C with fresh serum-free medium. At the end of the incubation, cells were washed twice with PBS and total cellular proteins were extracted by using the lysis buffer containing 50 mM Tris–HCl (pH 8.0), 10 mM EDTA, 100 µM phenylmethylsulfonyl fluoride, and 1% Triton X-100. The homogenate was centrifuged . The oxidation of epinephrine was measured at 480 nm with a Bio-Rad spectrophotometer .The activity of SOD was measured using a spectrophotometric assay, which consists in measuring epinephrine autoxidation induced by superoxide anion. Samples, prepared as described above, were incubated for 3 min with a mixture containing bovine catalase (0.4 U/µl), DL-epinephrine (5 mg/ml), and Na2O2. Samples, prepared as described above, were mixed with 30 mM H2O2 in PBS. The disappearance of H2O2 was measured at 240 nm for 180 s at 30 s intervals. Catalase activity was calculated using the extinction coefficient of 40/mM/cm for H2O2.The activity of catalase was determined on the basis of the decrease of Ht-test, ANOVA, followed by Bonferroni’s test, and two-way ANOVA test. A p-value of 0.05 or less was considered as statistically significant.Data are presented as the mean ± SEM from three independent experiments performed in quadruplicate. Statistical analysis of the data was performed by using Student’s 2O2 on SOD and catalase activities in cultured rat astrocytes . Thereafter, the stimulatory effect of ODN on SOD and catalase expression gradually declined and vanished 60 and 30 min after the onset of ODN administration, respectively. Exposure of astrocytes to increasing concentrations of ODN (0.01 pM to 0.1 nM) induced a concentration-dependent increase of SOD and catalase mRNA levels . In contrast, incubation of astrocytes with graded concentrations of H2O2 (100–500 µM) dose-dependently decreased both SOD and catalase mRNA levels . We next examined the effects of ODN/H2O2 co-incubation on enzyme expression. For moderate concentrations of H2O2 (100–300 µM), ODN (0.1 nM) restored SOD and catalase mRNA levels above control, whereas for higher concentrations of H2O2 (400 and 500 µM), ODN only partially prevented the decrease of SOD and catalase gene expression .We have previously shown that picomolar concentrations of ODN suppress the inhibitory effects of 300 µM Htrocytes . To expl1-8[DLeu5]OP (1 µM) to cultured astrocytes did not induce any modification of SOD and catalase mRNA levels and activities, but totally abolished the effects of 0.1 nM ODN on H2O2-evoked inhibition of antioxidant enzyme gene transcription and activities. In contrast, the CBR antagonist flumazenil (1 µM) did not affect the protective action of ODN against the deleterious effect of H2O2 on endogenous antioxidant systems .We next examined the type of receptor of ODN involved in the stimulatory effects of ODN on endogenous antioxidant systems. Administration of the selective metabotropic receptor antagonist cyclo2O2 on SOD and catalase mRNA levels and activities. In contrast, administration of the protein kinase C (PKC) inhibitor chelerythrine (0.1 µM) did not modify the effects of ODN , indicating that only the PKA pathway is involved in the protective activity of ODN.Incubation of astrocytes with the selective protein kinase A (PKA) inhibitor H89 (20 µM) abrogated the effect of ODN on the inhibitory action of H2O2 in cultured astrocytes, through activation of a metabotropic receptor positively coupled to the AC/PKA signaling pathway.Alteration of endogenous antioxidant systems, especially decrease of SOD and catalase activities, causes exacerbation of oxidative damages leading to apoptosis in various cell types, including astroglial cells . Here, wWe have previously reported that, in cultured astrocytes, ODN at very low concentrations (in the picomolar range) stimulates SOD and catalase activities . The pre2O2-reduced SOD and catalase mRNA levels. Similar time-response curves have already been observed on SOD and catalase activities, in cultured astrocytes (2O2. That ODN-induced increase of transcription and activity of antioxidant enzymes is responsible, at least in part, for inhibition of cell death is consistent with previous data showing that SOD and catalase blockers suppress the protective effect of ODN against H2O2-induced astrocyte apoptosis (Kinetic experiments indicate that the action of ODN on antioxidant enzyme gene transcription is very rapid but transient. Nevertheless, ODN exerts a protective effect against Htrocytes . These dpoptosis . Furtherpoptosis .A receptor (2O2-evoked reduction of SOD and catalase mRNA levels and activities were suppressed by the ODN analog cyclo1-8[DLeu5]OP but were not affected by the specific CBR antagonist flumazenil. It has been reported that the cyclic analog of ODN exerts potent antagonistic activities on ODN-induced polyphosphoinositide turnover increase and intracellular calcium mobilization in rat astrocytes (Previous studies have shown that, in cultured astrocytes, ODN can interact with either CBR associated with the GABAreceptor  or with receptor . Here, wtrocytes . Thus, t2O2-evoked inhibition of SOD and catalase gene transcription and enzyme activities was totally abrogated by the PKA inhibitor H89, while the PKC inhibitor chelerythrine had no effect. That, the ODN G protein-coupled receptor could stimulate the AC/PKA transduction cascade is in agreement with recent data indicating that ODN increases the production of cAMP in astrocytes (2O2-induced oxidative stress can be specifically ascribed to the activation of the AC/PKA signaling pathway. Consistent with this notion, it has been shown that the SOD and catalase promoters contain a cAMP-responsive element-like sequence (We next investigated the signaling cascade involved in the effect of ODN on endogenous antioxidant systems. ODN blockage of Htrocytes . Altogetsequence  and thatsequence  and the sequence , respectsequence , strongl2O2-reduced antioxidant enzyme expression and activities might have a physiopathological significance in neurodegenerative diseases and stroke. CNS is sensitive to oxidative stress due to its high metabolic rate and high levels of unsaturated lipids so that up-regulation of antioxidant enzyme systems in astroglial cells could be beneficial against cell death observed during and after ischemia and neurodegenerative diseases. In agreement with this hypothesis, we have recently shown that the endozepine ODN exerts a potent protective action against apoptosis induced by oxidative stress in astrocytes (2O2 and ROS (The protective effect of ODN against Htrocytes , 2012 an and ROS . The fac and ROS . In part and ROS . Since c and ROS , the dev1-8[DLeu5]OP antagonist, exerts a potent antioxidant action against H2O2-induced oxidative stress in astrocytes. This antioxidant effect of ODN is attributable to activation of both gene expression and activities of enzymatic antioxidant systems and can be ascribed to the stimulation of the AC/PKA transduction pathway.In conclusion, the present study has demonstrated that the endozepine ODN, acting through a metabotropic receptor sensitive to the cycloThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Estrogens have well-recognized and complex cardiovascular effects, including altering myocardial contractility through changes in myofilament function. The presence of multiple estrogen receptor (ER) isoforms in the heart may explain some discrepant findings about the cardiac effects of estrogens. Most studies examining the impact of estrogens on the heart have focused on chronic changes in estrogen levels, and have not investigated rapid, non-genomic pathways. The first objective of this study was to determine how acute activation of ERα impacts cardiac myofilaments. Nongenomic myocardial estrogen signaling is associated with the activation of a variety of signaling pathways. p38 MAPK has been implicated in acute ER signaling in the heart, and is known to affect myofilament function. Thus, the second objective of this study was to determine if acute ERα activation mediates its myofilament effects through p38 MAPK recruitment. Hearts from female C57Bl/6 mice were perfused with the ERα agonist PPT and myofilaments isolated. Activation of ERα depressed actomyosin MgATPase activity and decreased myofilament calcium sensitivity. Inhibition of p38 MAPK attenuated the myofilament effects of ERα activation. ERα stimulation did not affect global myofilament protein phosphorylation, but troponin I phosphorylation at the putative PKA phosphorylation sites was decreased. Changes in myofilament activation did not translate into alterations in whole heart function. The present study provides evidence supporting rapid, non-genomic changes in cardiac myofilament function following acute ERα stimulation mediated by the p38 MAPK pathway. Although premenopausal women are protected from cardiovascular morbidity as compared with age-matched men, the risk of heart disease increases in women following menopause Estrogen action can be mediated through binding to one of three known estrogen receptors (ER). It has been shown that ERα, ERβ and GPR30 are expressed in adult cardiac myocytes Cardiac myofilaments constitute the central contractile apparatus in the heart, and changes to their biochemical properties, notably their interaction with calcium, can alter the mechanical properties of the whole heart. Chronic E2 withdrawal following ovariectomy results in hypersensitivity of myofilaments to calcium The purpose of the current study was to determine whether the acute and specific activation of ERα results in changes in myofilament function. Furthermore, we sought to determine if p38 MAPK is involved in the rapid activation of ERα in cardiac myocytes, and if it mediates the effects of ERα on cardiac myofilaments.Female C57Bl6 mice were obtained from Charles River Laboratories . All animals were cared for in accordance with the principles and guidelines provided by the Animal Care Committee at the University of Guelph.2 inhalation, and rinsed in ice-cold saline. The aorta was cannulated and hearts were perfused at 80 mmHg with oxygenated (95% O2/5% CO2) Krebs-Henseleit buffer (pH 7.4). A balloon attached to a pressure transducer was inserted into the left ventricle via the left atrium and inflated to give an end diastolic pressure of <5 mmHg. To activate ERα, some hearts were perfused with 4,4′,4′′- trisphenol  for up to 5 min. This concentration of PPT maximally activates ERα without stimulating ERβ Hearts were excised from mice following euthanasia by COMyofilaments were isolated from ventricles as described previously 2+ATPase activity was measured using a modified Carter assay as previously published 4 and 0.5% ammonium molybdate in 0.5 M H2SO4, and reading the absorbance of each solution at 630 nm.Actomyosin MgMyofilament proteins (40 µg) were separated on a 12% SDS-polyacrylamide gel. Gels were fixed in 50% methanol/10% acetic acid at room temperature overnight and stained with Pro-Q Phosphoprotein Diamond Stain  according to the manufacturer’s instructions. Imaging was done using a Typhoon gel scanner . Densitometric analysis was performed on bands representing cardiac myosin-binding protein C (MyBP-C), troponin T (TnT), tropomyosin (Tm) and troponin I (TnI) using ImageJ software . Protein loading was determined by Coomassie staining and phosphorylation measurements were normalized to protein load.Homogenized myocardium (150 µg) was separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane . Membranes were blocked with 5% bovine serum albumin (phospho-p38 MAPK) or 5% dry milk powder  and probed overnight at 4°C with antibodies against phosphorylated p38 MAPK (1∶500) or total p38 MAPK (1∶1000) . Secondary antibodies (1∶5000) were conjugated to horseradish peroxidase . Bands were detected using Western Lightning . Densitometric analysis was performed using Image J software, and the ratio of phosphorylated p38 MAPK to total p38 MAPK was calculated. Phosphorylation of cTnI at serines 23 and 24 (S23/S24) was assessed in a similar manner, except that 40 µg of isolated myofilaments were separated in a 12% SDS-polyacrylamide gel.3, 10.0 mM D-(+) Glucose, 5.0 mM KCl, 2.0 mM NaH2PO4, 1.2 mM MgSO4 and 0.2 mM CaCl2. Standard Buffer contained 60 mM KCl, 2 mM MgCl2, 30 mM Imidazole (pH 7.0). Skinning Buffer contained 10 mM EGTA, 8.2 mM MgCl2, 14.4 mM KCl, 60 mM Imidazole (pH 7.0), 5.5 mM ATP, 12 mM creatine phosphate, 10 U/mL bovine creatine phosphokinase. Activating buffer contained 23.5 mM KCl, 5 mM MgCl2, 3.2 mM ATP, 2 mM EGTA, 20 mM Imidazole (pH 7.0), 2.2 mM CaCl2. Relaxing buffer contained 26 mM KCl, 5.1 mM MgCl2, 3.2 mM ATP, 2 mM EGTA, 20 mM Imidazole (pH 7.0), 4.9 µM CaCl2. All solutions (except KH buffer) contained protease inhibitors  added just prior to use.Krebs-Henseleit (KH) Buffer contained 118.5 mM NaCl, 26.6 mM NaHCOAll values are presented as mean ± SEM. Statistical analysis was carried out using one-way ANOVA and a post-hoc Dunnet’s t-test (actomyosin ATPase data). Phosphorylation and immunoblot data were analyzed using Fisher’s Least Significant Difference (LSD) test. Values of P ≤ 0.05 were accepted as statistically significant.50 values. The ERα antagonist MPP abolished the effects of PPT, confirming the specificity of PPT for ERα. Together these results indicate that acute ERα activation rapidly decreases myofilament cross-bridge cycling and sensitivity to calcium.To determine whether myofilament activation can be altered through rapid, nongenomic ER signaling, we examined myofilament function of murine hearts following acute ERα activation with the selective ligand PPT. Maximum actomyosin MgATPase activity tended to be reduced following 1, 2.5 or 5 minutes of PPT treatment, but did not reach statistical significance , Table 1Changes in the phosphorylation status of myofilament proteins allow for the rapid regulation of cardiac myofilament dynamics. We sought to determine if the changes in myofilament function following acute myocardial ERα stimulation were associated with alterations in cardiac myofilament protein phosphorylation. Interestingly, there were no significant changes in the phosphorylation of MyBP-C, cTnT, Tm or cTnI following PPT treatment at various time points .Cardiac myofilament proteins each contain multiple phosphorylation sites that can be individually targeted by intracellular signaling molecules. The phosphorylation of S23/S24 in the N-terminal region of murine cTnI is well known to decrease myofilament calcium sensitivity. To determine if there were site-specific changes in cTnI phosphorylation following ERα activation, we probed myofilament preparations with an antibody that recognizes the phosphorylated S23/S24 form of cTnI. Our data show that S23/S24 phosphorylation is reduced following PPT treatment, although statistical significance was only seen at 5 min .50 at 5 min  as a model of menopause in pre-pubescent Estrogens acutely reduce myocardial contractility, a functional effect that is due at least in part to decreased calcium transients Previous studies have noted only a marginal effect of estrogen on p38 MAPK activation. In one study, 5 minute or longer treatment of adult cardiomyocytes with 1 nM E2 failed to show a significant increase in p38 MAPK phosphorylation either through Western blot analysis or phosphorylation assay, whereas activation of ERK1/2 and JNK was observed Changes in circulating estrogens tend to occur over prolonged periods of time. The rapid effects examined in the current study may not necessarily reflect physiological events in which circulating estrogen levels fluctuate quickly. However, there is evidence to suggest a local, acute, cardiac metabolic pathway, making this a feasible possibility The current study presents insight into the myocardial effects of acute ERα activation and the intracellular pathways by which this receptor may regulate nongenomic function and myofilament activation. We found that short-duration treatment of myocardium with the ERα isoform-selective agonist PPT results in the activation of p38 MAPK, and a decrease in myofilament calcium sensitivity. These changes were not associated with any observable alteration in total myofilament protein phosphorylation, but variations in the pattern of cTnI phosphorylation were detected. The complex and controversial nature of cardiovascular regulation by estrogens necessitates an in-depth investigation of how ER control cardiovascular function. This includes an understanding of the role ER-isoforms play in regulating heart function and their intracellular mechanisms of action. A clear picture of how ER affect myocardial function under physiological conditions provides a fundamental basis for future investigations into the role of estrogens in cardiovascular disease, including the controversies associated with hormone replacement therapy.


Ten thousand years before Neolithic farmers settled in permanent villages, hunter-gatherer groups of the Epipalaeolithic period  inhabited much of southwest Asia. The latest Epipalaeolithic phase (Natufian) is well-known for the appearance of stone-built houses, complex site organization, a sedentary lifestyle and social complexity—precursors for a Neolithic way of life. In contrast, pre-Natufian sites are much less well known and generally considered as campsites for small groups of seasonally-mobile hunter-gatherers. Work at the Early and Middle Epipalaeolithic aggregation site of Kharaneh IV in eastern Jordan highlights that some of these earlier sites were large aggregation base camps not unlike those of the Natufian and contributes to ongoing debates on their duration of occupation. Here we discuss the excavation of two 20,000-year-old hut structures at Kharaneh IV that pre-date the renowned stone houses of the Natufian. Exceptionally dense and extensive occupational deposits exhibit repeated habitation over prolonged periods, and contain structural remains associated with exotic and potentially symbolic caches of objects  that indicate substantial settlement of the site pre-dating the Natufian and outside of the Natufian homeland as currently understood. Archaeologists have conventionally associated the origins of stone-built architecture with the Late Epipalaeolithic (Natufian) c. 14,500 years ago, and suggest that they represent the first semi-sedentary settlements, marking a critical threshold in human evolution 2 drainage basin around the Azraq Oasis. Until the 1980s the oasis was a rich and extensive wetland area centered within a semi-arid steppe and desert landscape. The region has long been recognized for its dense concentration of Epipalaeolithic sites 2, making it the largest known Late Pleistocene site in the region. Rising almost 2 meters above the surrounding landscape, it is easily identifiable by its staggering concentration of stone tool debris and animal bones  throughout all occupational horizons. This depth and density of material suggests that people occupied this now-arid area on a protracted basis and gathered at the site regularly to produce such extensive artifact densities over such a large area and depth. While acknowledging that such qualitative terms as prolonged, continuous, or regular occupation are extremely difficult to quantify and discussions regarding the definition of sedentism continue, e.g., Since 2008 three seasons of excavation at Kharaneh IV have documented approximately one thousand years of repeated and sequential occupation. Thirteen Accelerator Mass Spectrometry dates obtained from the principle excavation areas allow us to confidently date the occupation of the site to between 19.9 and 18.6 ka cal BP . Excavations have been carried out in two main areas (Areas A and B) in which prolonged and intense phases of occupation were documented with no evidence for hiatuses in deposition In Area B our work focuses on a combination of horizontal excavation and vertical exposure of the complete stratigraphic profile of the site's deepest part. The sequence of deposits offers a fine-grained record of the formation of the site. Thin (2–3 cm) and compacted occupation surfaces alternate with thicker (10–15 cm) midden deposits characterized by very dense concentrations of chipped stone and faunal remains. Occupational deposits in Area B reach a depth of 1.35 m below the surface, below which is archaeologically sterile clay representing the deposits of an ancient lake. Charcoal taken directly from the exposed sections here has provided a continuous sequence of radiocarbon dates for the Early Epipalaeolithic spanning from c. 19.9 to 18.9 ka cal BP . The lithic industry of Area B is dominated by the production of narrow and gracile bladelets and non-geometric microliths traditionally referred to as belonging to the Kebaran industry. Muheisen reported several large pit features and two human burials in Area B in situ burning. Burning also reddened the structure's former floor, making its deposits notably different in color and texture from the sediments surrounding the dwelling.Structure 1 was exposed approximately 60 cm below modern surface in Area B. It is oval in plan, measuring 3.2 m by 2.2 m and consists of several distinct layers. The structure was placed into a shallow depression dug into the preceding occupation deposits. A thin (2–3 cm), compact, dark reddish-brown clayey deposit represents the former floor of the structure. It is overlain by an organic-rich, black layer (c. 5 cm thick) containing abundant charcoal fragments Figure 4Sitting on top of the structure's floor and covered by the black burnt layer were two fragments of groundstone, a large flat stone, red ochre, and five articulated wild aurochs lumbar vertebrae . These samples came from immediately above and immediately below Structure 1's floor and date the structure to between 19.2 and 18.8 ka cal BP . These dates fit into a stratigraphically coherent sequence of twelve newly obtained dates, which contains no major outliers. We will discuss this sequence in more detail elsewhere.Muheisen first suggested a possible hut floor in Area B (his Area R/S2/60) in the 1980's when his trench cut into the corner of a dark brown layer (Couche V) beneath which he recorded two human burials Chenopodiaceae sp. and tamarisk have been identified from associated contexts in Area B .By analogy with the hut structures from Ohalo II, we interpret the thin, burnt, charcoal-rich layer on top of the structure's floor as the remnants of the hut's former superstructure, which burned and collapsed onto the floor. Further examination of the macro- and micro-botanical remains taken from this layer is currently underway, but the types of charcoal documented in other contexts (see above) suggest it highly likely that this was probably constructed of locally-available vegetation. Shrub and tree charcoal from The three concentrations of ochre and marine shells are particularly intriguing. Together these produced as many pierced marine shells as found in the entirety of the remaining excavation areas. Pierced holes strongly suggest that these were pendants of some kind, either worn as necklaces, headdresses, sewn onto clothing or attached to other artifacts Although only partially excavated, Structure 2 has a similar stratigraphic profile to Structure 1. It consists of a shallow, semi-circular depression dug into existing cultural deposits. Filling the depression is compact, light-colored clay, overlain by several overlapping dark-colored, organic-rich layers that appear to represent several episodes of hut floor re-use. This structure does not show evidence of burning like Structure 1. Notably, this hut also has sterile orange-brown sand in its uppermost levels. The complete dimensions of this structure remain unknown until it is fully excavated; however, it appears similar in size to Structure 1. Although no shell/ochre caches have been found in Structure 2, a cache of gazelle and aurochsen horn cores was found on the edge of the structure  was a complete tortoise shell, a large rounded stone, and four articulated fox paws surrounding a flint core that are likely the remains of a pelt pouch  and social complexity , and its associated symbolic behavior, arose with the onset of the Natufian period One of EFAP's primary archaeological questions regarding occupation of Kharaneh IV, now in one of the driest areas of the region, is why people chose this location and repeatedly occupied the site at least a thousand years. Off-site and on-site geomorphological work demonstrates that the Late Pleistocene landscape was very different from that of today. Instead of a marginal environment, the conditions prevailing during the LGM appear to have facilitated a moister local climate than today with the likelihood of year-round rivers and bodies of standing water outside of the oasis, in a landscape that supported a diversity of plant and animal species and extensive and intensive human occupation To-date, Epipalaeolithic sites with hut structures have only been documented in the western and southern (northern Negev) parts of the southern Levant – the former of these areas sometimes referred to as the Mediterranean core see . We see The evidence for hut structures in the Early Epipalaeolithic, at Kharaneh IV and elsewhere, demonstrates that these were features of repeatedly-occupied, probably multi-season, sites. Furthermore, these structures pre-date Natufian stone architecture and were not confined to the supposedly lush Mediterranean zone of the Levant. Although detailed analyses of the huts, their sediments, and associated artifacts are ongoing they provide evidence that the Ohalo II structures are not isolated instances. Perhaps more importantly for this discussion, the Kharaneh IV huts show that the local environment could support long-term settlement which, together with palaeoenvironmental evidence for well-watered environments near the site The field work under discussion here was carried out during three excavation seasons between 2008 and 2010. All necessary permits were obtained from the Department of Antiquities of Jordan for the described field studies.A main focus of EFAP's work is to reconstruct the Late Pleistocene palaeoenvironment to contextualize hunter-gatherer behavior; therefore, field work necessarily integrates archaeological, biological, and geomorphological datasets. We have begun to reconstruct a picture of site occupation, subsistence behaviors, and some of the social aspects of repeated occupation. To provide a more holistic reconstruction of landscape and land use EFAP is undertaking a rigorous program of sampling for archaeobotanical remains and conduct flotation for 100% of all subsurface deposits. The excellent preservation conditions in this now-desert environment enable the collection of botanical remains that are not often preserved on archaeological sites of any period. The result is an extensive record of habitat change and plant use, as well as the ability to radiocarbon date all contexts.2 is identified by its SW corner alphanumeric designation such that the southwestern-most 1 m2 of the site is labelled Square A1. All other 1 m2 excavation units follow this same system.The site is excavated in a grid system 200 m (east-west) by 140 m (north-south) in size, covering an area slightly larger than the surface spread of artefacts to allow for future exploration (if required) off-site to the north and east. The grid is labelled with an alphanumeric system beginning in the southwestern-most square metre of the gridded universe, and has a letter designation running along the Y-axis (north-south) and a number designation running along the X-axis. Each 1 m2 have been excavated to-date. In both Area A and Area B, excavation was conducted on our basic site grid of 1×1 m excavation squares. However, sometimes these squares were subdivided into 50×50 cm quadrants or 25×25 cm sub-quadrants where dictated by the stratigraphic context . Structure 1 was excavated in 25×25 cm sub-quadrants. Vertical subdivisions of natural and cultural stratigraphic layers into 5–10 cm thick spits were used in order to record as much spatial information as possible. For the uppermost disturbed levels we screened 100% of excavated deposits on-site through 4 mm and 2 mm mesh. However, this proved to be very time-consuming on-site given the density of flint and bone from surface deposits. Given the potential for preservation of plant and animal remains that could greatly inform us on season and duration of occupation at the site, as well as on-site activities and subsistence practices, 100% of deposits were collected for flotation in the field lab. Flotation was conducted daily in the field lab along with sorting of the 4 mm heavy fraction from flotation. In addition, samples were systematically collected for micro-artefacts, micro-fauna, micromorphology, and soil analyses that aid in reconstructing activities at the site and general site-formation processes.Excavations have focussed in two main areas, Area A and Area B, but 1×1 m test trenches have been placed in several locations across the surface of the site and a 9×1 m geological trench traced the transition from on-site occupational deposits to the modern wadi margins. A total of 104 m


Background: The small molecule indirubin-3′-monoxime (I3MO) inhibits activation of STAT3 in vascular smooth muscle cells, with an unresolved mechanism.Results: Activation of 12/15-lipoxygenase (LO) is crucial for PDGF-induced Src and STAT3 activation and is impaired by I3MO.Conclusion: I3MO interferes with PDGFR-Src-STAT3 signaling via impaired 12/15-LO activation.Significance: 12/15-LO is an important signaling hub within the PDGF-STAT3 pathway. S)-hydroxyeicosatetraenoic acid (15(S)-HETE) production, indicating an inhibitory action of I3MO on 12/15-LO and consequently on STAT3. We identify 12/15-LO as a hitherto unrecognized signaling hub in PDGF-triggered STAT3 activation and show for the first time a negative impact of I3MO on 12/15-LO.We showed previously that the small molecule indirubin-3′-monoxime (I3MO) prevents vascular smooth muscle cell (VSMC) proliferation by selectively inhibiting signal transducer and activator of transcription 3 (STAT3). Looking for the underlying upstream molecular mechanism, we here reveal the important role of reactive oxygen species (ROS) for PDGF-induced STAT3 activation in VSMC. We show that neither NADPH-dependent oxidases (Noxes) nor mitochondria, but rather 12/15-lipoxygenase (12/15-LO) are pivotal ROS sources involved in the redox-regulated signal transduction from PDGFR to STAT3. Accordingly, pharmacological and genetic interference with 12/15-LO activity selectively inhibited PDGF-induced Src activation and STAT3 phosphorylation. I3MO is able to blunt PDGF-induced ROS and 15( The ant in vivo , was linferation , 4. Otheferation . How I3MPDGF-mediated STAT3 activation is still incompletely understood. PDGF is a family of five dimeric isoforms: PDGF-AA, -AB, -BB, -CC, and -DD, formed by four genetically different, but structurally related polypeptide chains. They exert their effects on cells through differential binding to two related tyrosine kinase receptors, PDGF receptor α and β, causing their homo- or heterodimerization. PDGF-BB, a ligand isoform used in this study, can trigger signal transduction mediated by all three receptor dimers, PDGF receptor-αα, -αβ, and -ββ . PDGF-inPDGF-induced phosphorylation of STAT proteins may be directly mediated by the PDGF receptor tyrosine kinase after recruitment of the Src homology 2 domain-containing STAT proteins to specific receptor autophosphorylation sites , 8. HoweSeveral publications have described an increased STAT3 phosphorylation and its translocation into the nucleus upon exposure to elevated levels of reactive oxygen species (ROS) –17. ROS tat (RKKRRQRRRCSTRIRRQL-NH2), and its scrambled counterpart, gp91ds-scr (RKKRRQRRRAGAGAGAGA-NH2), were ordered from Caslo Laboratory . Nordihydroguaiaretic acid (NDGA) and diphenyleneiodonium chloride (DPI) were purchased from Sigma. H2O2 and human recombinant PDGF-BB (in text referred to as PDGF) were ordered from Carl Roth  and Bachem , respectively. Purified hydroxy- and hydroperoxyeicosatetraenoic acid (15(S)-HETE and 15(S)-HpETE) were obtained from Biomol . The antibodies directed against phosphorylated Tyr579 or Tyr857 of PDGFRβ and against phosphorylated Tyr416 of Src were from Life Tech . Anti-actin and anti-mouse horseradish peroxidase-linked secondary antibody was from MP Biomedicals . All other primary antibodies used in this study as well as the secondary anti-rabbit antibody, were from Cell Signaling .I3MO was purchased from Enzo Life Sciences . PD 146176 and ebselen were from Cayman Chemicals , whereas gp91ds-ml-glutamine, 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 10% newborn calf serum  at 37 °C and 5% CO2. Passages 5 to 14 were used in this study. Primary human aortic smooth muscle cells (hAOSMCs) were purchased from Lonza. They were passaged twice a week and maintained at 37 °C and 5% CO2 in SmBM basal medium supplemented with SmGM®-2 Single Quots® , all from Lonza . Passages 5 to 12 were used for experiments. The heterozygous and homozygous 12-LO knock-out mouse embryonic fibroblasts (MEFs) were kindly provided by Dr. Marcus Conrad, German Research Center for Environmental Health, Munich, Germany. Cells were passaged twice a week and cultured in DMEM supplemented with 2 mml-glutamine, 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 10% fetal bovine serum  at 37 °C and 5% CO2. Cells were grown to subconfluence and serum starved for 24 h, if not stated otherwise, in the appropriate medium supplemented with 0.1% serum, before each experiment.Unless stated otherwise, the experiments presented in this study were performed in VSMCs isolated from thoracic aortas of Sprague-Dawley rats using a digestion method, as described previously . Cells wS)-H(p)ETE (1 μm), or H2O2 for the indicated periods of time. Afterward cells were lysed with an ice-cold lysis buffer , supplemented with 1 mm phenylmethylsulfonyl fluoride (PMSF), 1× CompleteTM (Roche Applied Science), and 1% (v/v) Triton X-100. Lysates were centrifuged at 5600 × g at 4 °C for 10 min and supernatants were used for protein denaturation in 3× SDS sample buffer for 8–10 min at 95 °C. Protein concentrations were determined using Rotiquant reagent according to the manufacturer's instructions (Carl Roth). If not stated otherwise, 20 μg of protein was separated by 7.5% polyacrylamide SDS-PAGE. Standard gel electrophoresis and blotting techniques were used (Bio-Rad Laboratories). Proteins were visualized after antibody incubation using an LAS-3000 luminescent image analyzer  and quantified with AIDATM software, version 4.06 . Densitometric values of phosphorylated proteins were always normalized to those of the corresponding total protein. If applicable, those normalized data were expressed as fold-change compared with control cells whose response was set 100%. Stripping of membranes was performed by incubation in 0.5 n NaOH for 10–20 min.Unless stated otherwise, for all Western blot experiments, serum-starved cells were preincubated with compounds for 30 min and subsequently stimulated with PDGF-BB (20 ng/ml), 15(VSMC were serum starved and pretreated as indicated for 30 min. Then cells were stimulated with PDGF (20 ng/ml) for 5 min and immediately lysed. Anti-PDFGRβ antibody (1:100 dilution) was added to 750 μg of protein extract and incubated on ice for 3 h. After another 45 min rolling end-over-end with protein-Sepharose A/G beads  at 4 °C the immunoprecipitates were collected, washed three times with cold lysis buffer, separated from the beads by boiling in SDS sample buffer for 5 min, and subjected to immunoblot analyses as indicated. Specificity of the pulldown was assured in pilot experiments using an isotype control antibody.TM . The luminescence was measured 15 min after the addition of the substrate with a Tecan GENiosTM Pro Microplate reader .The nonradioactive Src kinase assay was performed using the tyrosine kinase assay kit (chemiluminescent) according to the manufacturer's instructions and a recombinant Src (human) protein, both from Milipore2α coding sequence was cloned from pVL1393 plasmid  into pFastBacTM HT A containing a His6 tag coding sequence. The recombinant plasmid was transformed into DH10BacTMEscherichia coli. Sf9 cells were transfected with recombinant bacmid DNA using Cellfectin® reagent and the generated baculovirus was amplified. His-tagged cPLA2 was overexpressed in baculovirus-infected Sf9 cells and isolated using nickel-nitrilotriacetic acid-agarose beads. Multilamelar vesicles were prepared by drying 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycerol in a 2:1 ratio (in chloroform) under nitrogen in glass vials. After addition of 20 mm Tris buffer (pH 7.4), 134 mm NaCl and 1 mg/ml of fatty acid-free BSA were added. Multilamelar vesicle suspension was disrupted by several freeze-thaw cycles (liquid nitrogen). The suspension was extruded 11 times with a mini-extruder  through a polycarbonate membrane (100 nm pore diameter) at room temperature (above transition temperature of the lipids) to produce large unilamelar vesicles. The final total concentration of lipids was 250 μm in 200 μl. Test compounds and 1 mm Ca2+ were added to the vesicles, and the reaction was started by addition of 500 ng of His-tagged cPLA2α (in 10 μl of buffer). After 1 h at 37 °C, 1.6 ml of MeOH was added, and arachidonic acid was extracted by RP-18 solid phase extraction. Following derivatization with p-anisidinium chloride, the resulting derivate was analyzed by RP-HPLC at 249 nm , pre-treated with DMSO, I3MO, or DPI for 30 min and stimulated with PDGF-BB for 10 min. 2′,7′-Dihydrodichlorofluorescein-diacetate  was added 15 min before PDGF stimulation, and from this step the experiment was performed in the dark. Cells were trypsinized and re-suspended in 2% BSA/PBS solution. The fluorescence was measured using the flow cytometer FACS CaliburTM (BD Biosciences) at the excitation/emission wavelength of 488/530 nm.Serum-starved cells were covered with HBSS buffer , supplemented with 0.01 mmol/liter of Amplex® Red reagent and horseradish peroxidase, 1 unit/μl (both from Invitrogen), and equilibrated for 5 min at 37 °C and 5% CO2. Cells were subsequently stimulated with PDGF-BB (20 ng/ml) in the presence or absence of 150 units of catalase (Sigma) for 30 min. 80 μl of supernatants, in triplicate for each treatment condition, were transferred into a black 96-well plate  and fluorescence intensity was measured at 535/590 nm (excitation/emission) in the plate reader Tecan GENiosTM Pro . Fluorescence values from wells containing catalase were subtracted from those without catalase to obtain the values for H2O2 specific fluorescence. External addition of H2O2 (0.5 μm) to one set of cells served as a positive control in the experiment.Serum-starved VSMCs were treated with either DMSO or I3MO for 30 min, the medium was aspirated, and the cells were washed once with PBS. They were then covered with KRPG buffer (145 mS)-HETE enzyme immunoassay kit. Eicosanoid extracts were 12 times more concentrated compared with the original supernatants. The levels of 15(S)-HETE in the extracts were determined by using 15(S)-HETE enzyme immunoassay kit  as recommended in the product manual. All absorbance measurements were performed in duplicate.Cells were seeded in 10-cm dishes, grown to subconfluence, and serum-starved for 24 h. They were treated with vehicle or compounds for 30 min and stimulated with PDGF-BB for 10 min. Eicosanoid extractions out of supernatants were performed using HyperSep C18 SPE columns  as instructed in the manual of the 15 using OligofectamineTM Transfection reagent (Invitrogen) and left in Opti-MEM® reduced serum medium (Invitrogen) overnight. Opti-MEM was then replaced with the starvation medium to let cells recover from transfection complexes. 24 h after transfection, VSMCs were stimulated with PDGF for 5 min, trypsinized shortly thereafter and divided into two halves: one to be used for the determination of knockdown efficiency ) and the other for the detection of phospho-STAT3 levels upon PDGF stimulation . 12/15-LO knock-down was accomplished with 2 different oligonucleotide sequences: siRNA-1, sense, 5′-AACUGGAUUUCUGUGAAGG-3′; antisense, 5′-CCUUCACAGAAAUCCAGUUGC-3′  and siRNA-2, sense, 5′-CGGAUUUCUUCCUUCUGGA-3′, antisense, 5′-UCCAGAAGGAAGAAAUCCG-3′ (Sigma). More details for the siRNA knockdown of Nox4 in VSMCs, together with data showing the achieved transfection efficiency, have been described elsewhere (VSMCs were seeded at the density of 0.5 × 10lsewhere .TM First-strand Synthesis System (Invitrogen), respectively. The RT-qPCR was carried out using Light CyclerTM LC480 SYBR Green I Master reagent (Roche Diagnostics) in reaction volume of 15 μl. QuantiTect® Primer Assay for rat leukocyte-type 12/15-LO was ordered from Qiagen , diluted, and aliquoted as recommended by the manufacturer. Forward and reverse primers for the housekeeping gene 18S were from Invitrogen and their respective sequences were as follows: GAATTGACGGAAGGGCACCACCAG and GTGCAGCCCCGGACATCTAAGG. PCR contained one denaturation step (10 min at 95 °C) and up to 50 amplification cycles . Melting curves of the amplified DNA were analyzed to make sure that the PCR resulted in amplification of one specific product only. Data were analyzed using Light CyclerTM LC480 software and the 2−ΔΔCt method.RNA isolation and subsequent cDNA synthesis were performed according to the instructions of the manufacturer, using the PeqGOLD Total RNA kit  and SuperscriptTM, OptiluxTM, black/clear bottom, TC Surface, sterile with lid, BD Biosciences) to grow for the next 24 h and starved overnight. The starvation medium was freshly exchanged and cells were preincubated with either DMSO (0.1%) or increasing concentrations of I3MO (0.1–5 μm) for 30 min and stimulated with PDGF-BB (20 ng/ml) for the next 24 h. Cells were loaded with bromodeoxyuridine  4 h after PDGF stimulation. BrdU incorporation was measured according to the manufacturer's instructions using the BrdU Cell Proliferation ELISA kit (chemiluminescent) from Roche Diagnostics GmbH.hAOSMCs were seeded at the density of 15,000 cells/well in a 96-well plate . Graphs represent values of at least three independent experiments, mean ± S.D.Statistical analyses of experiments are based on one-way analysis of variance and Bonferroni's post-test of selected data sets (after log transformation when appropriate). The effect was considered significant when 2O2 can serve as a messenger transducing receptor tyrosine kinase signals to downstream targets .H targets . To testt Tyr705 a.705 was inhibited without an effect on the phosphorylation and activation of Akt and ERK1/2 kinases (b and c). These data are in concordance with other studies showing STAT3 activation as being highly redox sensitive  , 28 and  kinases , b and censitive –17. Theyin vitro assay, I3MO (3 μm) indeed inhibited Src kinase activity up to 50% (a). Moreover, SU6665, a known selective Src inhibitor . A look at the phosphorylation of Tyr579 in the PDGFR, known to constitute the binding site for the Src homology 2 domain of Src kinases (c). In line with existing knowledge about early PDGFR signaling (416 in I3MO-treated VSMC compared with control cells (d). These data indicate that Src is a major kinase mediating STAT3 phosphorylation in PDGF-stimulated rat VSMC, and that I3MO may act as a direct Src kinase inhibitor. In contrast to mere Src kinase inhibitors, I3MO also interfered with Src activation at the upstream step of recruitment of the kinase to the activated PDGFR. Notably also, the ROS-reducing DPI hampered phosphorylation of PDGFR at Tyr579 and Src at Tyr416 (e). This underlines the essential role of ROS for the signal transduction, PDGFR → Src → STAT3, which we focused on in the following experiments.Next, we investigated whether interference of I3MO with ROS-dependent STAT3 signaling involves Src kinase. Src is a redox-sensitive protein-tyrosine kinase capable of phosphorylating STAT3  and propp to 50% a. Moreovnhibitor , abolish of I3MO b. A look kinases , uncover kinases c. In linignaling , Src wasol cells d. These t Tyr416 e. This u2DCF-DA (a). The inhibition of PDGF-induced ROS formation by I3MO was confirmed by reduced extracellular H2O2 release as evident by measurements using the fluorogenic substrate Amplex® Red (b). I3MO itself did not show radical scavenging or reducing properties in the presence of 2,2-diphenyl-1-picrylhydrazyl and 3--2,5-diphenyltetrazolium bromide, respectively (data not shown). Thus, I3MO influences the activity of cellular ROS sources or detoxification systems.We determined whether I3MO may directly interfere with ROS formation induced by PDGF in VSMCs. Using H2DCF-DA , we obse2DCF-DA a. The inlex® Red b. I3MO itat that competes with the membrane bound gp91phox for the cytosolic p47phox and thus prevents Nox1 membrane assembly (c). In contrast to Nox1, Nox4 is considered to be constitutively active and is regulated on the transcriptional level (d). We conclude that neither Nox1 nor Nox4 are involved in PDGF-induced signal transduction toward STAT3 in rat VSMCs.In the next step we aimed to delineate the endogenous ROS source, which is crucial for PDGF-induced STAT3 phosphorylation. Nox proteins are usually considered as the most important ROS-producing enzymes and regulators of PDGF-induced signal transduction in VSMCs , 27, 35.assembly . The funassembly . Here, wassembly c. In conal level , 37. We al level . PDGF stal level d. We con2O2, can result from an inefficient/leaky electron transfer process in the MRC . Additionally, PDGF-induced phosphorylation of Akt and ERK1/2 remained intact or was only weakly affected (a and d), resembling the signaling pattern of PDGF-activated VSMCs treated with I3MO. Mammalian LO are non-heme iron-containing dioxygenases that insert molecular oxygen into polyunsaturated fatty acids, like arachidonic or linoleic acid (S)- and 15(S)-HpETE - and 15(S)-HETE) and are therefore termed as 12/15-LO. Although LOs are a group of enzymes comprising several different subtypes . Ebselen, a glutathione peroxidase mimetic selenoorganic compound and described to mainly counteract 12/15-LO signaling (c and d). However, the rather high concentration of PD 146176 (30 μm) may not guarantee truly specific inhibition of 12/15-LO, and ebselen has recently been reported to inhibit Nox1-mediated ROS production as well (a). Cells with silenced 12/15-LO and stimulated with PDGF showed an impaired STAT3 phosphorylation compared with cells transfected with the scrambled control (b). Furthermore, and in accordance with our data in VSMC, we were able to observe stronger STAT3 activation with increasing expression levels of leukocyte 12-LO, the murine representative of the 12/15-LO family, in embryonic fibroblasts (c). These findings underline an important role of 12/15-LO activity for STAT3 activation that may even be true for cell types other than VSMCs upon PDGF stimulation. In line with our observations in 579 and phosphorylation of Src at Tyr416 (d). Total PDGFR levels were reduced in MEF after a 5-min PDGF exposure, which is most likely due to rapid receptor internalization and degradation of the receptor in this cell type (but not due to uneven protein loading as seen by the actin blot). Ebselen reproducibly modestly reduced PDGF-induced Src phosphorylation, almost reaching significance after three performed experiments (p = 0.053).To pin down the source of ROS responsible for STAT3 activation, we administered commonly used inhibitors for additional different ROS sources in VSMCs and checked for reduction of PDGF-induced ROS (data not shown) and their (selective) inhibitory action on STAT3 over ERK1/2 and AKT. With this approach, we excluded a potential role of nitric-oxide synthases, cyclooxygenases, and xanthine oxidase in PDGF-induced STAT3 phosphorylation . Howeverm NDGA , a and daffected , a and deic acid . Accordieic acid . Furthersubtypes , the leusubtypes , 43. We,d ERK1/2 , b and dignaling , also shignaling , c and d as well . We ther as well a. Cells  control b. Furtheroblasts c. These t Tyr416 d. Total S)-HETE, which was significantly elevated within 10 min of PDGF stimulation, as quantified by an enzyme immunoassay (a). The decrease of PDGF-induced STAT3 phosphorylation by I3MO may, thus, in turn be a result of reduced 12/15-LO activity. Of note, addition of 15(S)-H(p)ETE was not able to rescue STAT3 phosphorylation in I3MO-treated VSMC within 10 min of PDGF stimulation in our cell system. This may suggest that another 12/15-LO-derived (by)product mediates the early signal transduction from PDGF to STAT3 (b).The next obvious question to answer then was whether I3MO interferes with 12/15-LO. Porcine aortic smooth muscle cells were shown to display elevated 12/15-LO products after PDGF stimulation . We, thuunoassay a. The deto STAT3 b.in vitro enzyme assay (data not shown). Also, I3MO failed to interfere with the supply of arachidonic acid, the substrate for 12/15-LO. Neither PDGF-induced phosphorylation/activation of cytosolic phospholipase A2 (cPLA2), that causes release of arachidonic acid from membrane lipids, nor the activity of the purified cPLA2 were markedly influenced by I3MO at concentrations used in our experiments (c and d).I3MO did not inhibit purified human 12/15-LO in an eriments , c and dm (a and b). The activation of Akt and ERK1/2 kinases was not affected by I3MO in hAOSMCs either (b). I3MO abolished the PDGF-induced intracellular ROS formation as measured by using H2DCF-DA and successfully blocked 15(S)-HETE production, as evident in the respective enzyme immunoassay (c and d). These results show that I3MO successfully inhibited cellular 15-LO product synthesis, the corresponding human isoform of 12/15-LO also in hAOSMCs. Furthermore, the results are in line with the notion that PDGF-induced STAT3 phosphorylation in hAOSMCs might require the activity of 15-LO.To examine whether 12/15-LO is also required for PDGF-induced STAT3 phosphorylation in human cells, we repeated key experiments with I3MO in hAOSMCs. I3MO inhibited PDGF-induced proliferation and PDGF-induced STAT3 phosphorylation concentration dependently, the latter with a significant effect at already 0.3 μm , a and bs either b. I3MO aunoassay , c and dS)-HETE production, and does not only directly inhibit the kinase activity but also the activation of Src by PDGF.Starting off with I3MO as a selective small molecule inhibitor of STAT3 in VSMCs we show here that (i) PDGF-induced signaling toward STAT3 in VSMCs requires ROS and (ii) that this ROS-dependent signaling pathway from the PDGF-receptor to STAT3 in VSMCs relies on 12/15-LO, which mediates optimal recruitment of Src to the activated receptor. We show for the first time that I3MO interferes with 12/15-LO activity in PDGF-stimulated VSMCs, blunts ROS and 15-HETE as a direct stimulus and observing late STAT3 phosphorylation (>15–30 min) in vascular smooth muscle and endothelial cells -HETE formation in PDGF-activated VSMCs, and this effect could be linked to the previously reported inhibition of STAT3. More importantly, we unravel the importance of 12/15-LO activity for the PDGF-induced and Src-mediated STAT3 activation in rat VSMC.Overall, our study shows for the first time that I3MO inhibits PDGF-induced ROS and 15(


Allergic fungal rhinosinusitis (AFRS) is characterized by the growth of fungi, mostly Aspergillus sp., in the paranasal sinuses together with the formation of nasal polyps, peanut-butter like "allergic mucin" with fungal hyphae and typical CT-findings, as well as increased serum total IgE and Aspergillus-specific IgE concentrations. We here hypothesize that the increase in serum total IgE is caused by the local symbiosis of Asp. sp. with Staphylococcus aureus, a germ which is known for the production of enterotoxins with superantigenic properties. We demonstrate the presence of S. aureus specific IgE antibodies in the sera of AFRS patients, correlating with total serum IgE concentrations, as well as the coexistence of both, A. fumigatus and S. aureus, in biofilm-like formations on the sinus mucosa. Similar mechanisms and findings may apply for Allergic Broncho-Pulmonary Aspergillosis/Mykosis (ABPA/M). This knowledge may result in new diagnostic and therapeutic approaches including anti-IgE strategies.